36th Annual Meeting of the European Environmental Mutagen Society

Transcription

36th Annual Meeting of the European
Environmental Mutagen Society
“From Genes to Molecular Epidemiology”
under auspices of
Jiří Havel
Vice Prime Minister for Economic Affaires
of the Czech Republic
and
Václav Pačes
President of the Academy of Sciences
of the Czech Republic
FROM GENES TO MOLECULAR EPIDEMIOLOGY
eems2006-abstract-inside.indd 1
26.6.2006 16:04:15
WELCOME ADDRESS
Vice Prime Minister for Economic Affairs
Jiří Havel
Ladies and gentlemen,
It is a great pleasure for me to welcome you in Prague on the occasion of the 36th Annual Meeting of the
European Environmental Mutagen Society while being honoured to accept the auspices over this event.
During the following several days there will be a great opportunity for meeting the people who are involved
in the study of molecular and genetic interactions of a human with the mutagens in the human’s environment.
It should not be surprising that it is the human, who increases the amount of mutagens in his own environment. It is the human’s desire for permanent growth of welfare, which brings more and more chemicals
and ionizing radiation to the prospering neighbourhoods. The governments in the world permanently try to
support enterprises and entrepreneurs for producing more and more useful commodities. But next to the
production of commodities there are always some unwanted externalities produced meanwhile. Therefore
the governments need to adopt measures to eliminate the harming externalities from the human’s environment. The government needs to assure a sustainable growth of people’s welfare.
To take appropriate measures the government needs substantial knowledge about the impacts of unwanted externalities on the health of people and about the ways how to prevent the harmful impacts. Scientific
research creates and discovers the knowledge which can be afterwards extracted from the research and
utilized by the government to take an appropriate action. It is therefore important that the government
supports besides the applied research in industrial sector also the basic research at universities and
research institutes, while the importance of the basic research is revealed in the long-run.
The scientists have been at least since 1970, the first year of EEMS, taking action in order to deeply explore
the impacts of chemicals in the environment. It appears that their effort has brought benefits to all people.
I am very proud that Czech scientists joined and contributed to the mission and results of the EEMS.
Czech government supports the activities of Czech scientists at home as well as abroad in various fields
and academic activities. Therefore the variety of the knowledge to be discovered can be assured, and the
society can be prepared for new challenges on the way to the prosperity with security. The government
supports general secondary education for a higher number of tertiary study enrolment. The government
permanently increases the financial support for scientific research and helps to extend the opportunities
for obtaining and exchanging the knowledge of undergraduate and graduate programs students, teachers
and researchers. The government supports the programs of study abroad for Czech students and afterwards the students are offered a program for their integration within the Czech scientific bodies in project
research. Thereby a new capacity of project leaders is created from relatively young but experienced
researchers. I believe that the enthusiasm of young people will persist for the future.
Let me please wish you a fruitful and inspiring discussions and presentations during the following
days, and also a pleasant stay in the capital of the Czech Republic, Prague.
Jiří Havel
Vice Prime Minister for Economic Affairs of the Czech Republic
EUROPEAN ENVIRONMENTAL MUTAGEN SOCIETY
26.6.2006 16:04:57
WELCOME TO PRAGUE
Dear conference participants,
On behalf of the Local Organizing and Scientific Programme Committees we welcome you to the 36th Annual
Meeting of the European Environmental Mutagen Society. This is co-organized with ECNIS (Environmental
Cancer Risk, Nutrition and Individual Susceptibility, a Network of Excellence operating in the context of
the 6th EU Framework Programme for Research and Development). The conference is held under the title
“From Genes to Molecular Epidemiology” and covers most of the current topics of genetic toxicology and
environmental mutagenesis. This meeting seems to be one of the largest annual meetings in the history
of EEMS with almost 450 participants from 40 countries worldwide, which makes it truly international. We
believe that the outstanding scientific programme, combined with the warm atmosphere of the historical
city of Prague will be stimulating for all delegates.
We wish you a good time in Prague.
Jan Topinka
Chair of the Local Organizing
Committee
Radim J. Šrám
Chair of the Scientific Programme
Committee
26.6.2006 16:04:57
EEMS PRESIDENT INTRO
Dear Colleagues,
It is my pleasure to warmly welcome you to Prague, the Czech Republic, and the 36th annual meeting of
the European Environmental Mutagen Society.
We have now learned a great deal about mechanisms responsible for the induction of genomic changes,
repair of DNA damage, cell survival and death and appropriate methods to assess the effects of genotoxic
chemicals and radiations at molecular and cellular levels, and about their threats to human health.
Recent advances in genetic research unravelled new fascinating aspects of the complexity of gene functions. This will modify our understanding of the genotype-phenotype-disease relationships, and open new
perspectives for prevention of diseases related to gene-environment interactions. In the relatively close
future, one may also hope that our understanding of individual susceptibility to genotoxicants will allow us
to perform risk assessment with increased accuracy, in particular for susceptible populations, and to design
personalised therapies. Therefore, we need scientifically valuable information at the epidemiological level
combining genotypes, phenotypes, biomarkers for exposure and effects, and disease. The theme of this
meeting „From genes to molecular epidemiology“ addresses these questions within an international and
interdisciplinary context.
The outstanding scientific programme set up by Radim Sram, Jan Topinka, and the Programme Committee
is comprehensive and very stimulating : 3 keynote lectures, 17 symposia, 4 workshops, presentations by
awardees and several poster discussion sessions. Particular attention was given to young scientists by
offering travel awards, poster prizes and platforms for poster discussions.
May this meeting be an opportunity for all of us to develop and maintain new and fruitful friendships in
scientific collaboration.
On behalf of the European Environmental Mutagen Society, I wish you a pleasant and scientific excellent
stay in Prague.
Micheline Kirsch-Volders
President of EEMS
26.6.2006 16:04:58
ECNIS NETWORK OF EXCELLENCE
Cancer constitutes a major health problem in modern society and its statistics are appalling: nearly one of three
Europeans will develop cancer at some time in a lifetime, while 1.7 million people in the European Union alone
die from cancer every year. Hence there is an urgent need for a concerted action to reduce cancer burden. To
overcome the fragmented nature of research in this area, ECNIS (Environmental Cancer Risk, Nutrition and
Individual Susceptibility) Network of Excellence operating in the context of FP6 was launched in May 2005. ECNIS
brings together partners from 21 of the best European research groups and 3 SMEs, located in 14 countries.
The workplan is organized into 15 workpackages, classified in four groups of activities, including Integration,
Joint Research, Spreading of Excellence, and Management. The overall and specific objectives of the project are
to be achieved through the development of harmonized and integrated resources, databases, procedures and
quality standards, as well as scientific meetings, joint research and training and mobility programmes for scientists within and outside the Network. The first year of ECNIS activity has witnessed a dynamic development of
the Network in all its activity areas. All together 50 scientific and organizational meetings, workshops, symposia, and training courses, with hundreds of participating scientists were organized or supported by ECNIS. The
Integration activities started with developing an inventory of techniques and research capacities available within ECNIS and of the related research within and outside Europe. This was essential for assessment of the initial
research potential and future research planning activities of the Network. Shortly afterwards, ECNIS website was
also created, with links to EU and world activities relevant to ECNIS. The website (www.ecnis.org) is intended
both for the ECNIS partners and wider audience. In ECNIS, we pay much attention to the education and training
activities. Accordingly, an exchange training program was established and seven fellowships for young scientists
were awarded. Four training courses and two workshops were organized. Also, the exchange of experts was
initiated. An important activity was the “Construction of a knowledge database on molecular epidemiology and
cancer (MEC)”to compile data from studies in molecular epidemiology of cancer for further use in meta-analyses
and data pooling. Much of the work within the Joint Research activity area was dedicated to the assessment
of existing resources, abilities and collaborative research. As a result, twenty-six ongoing collaborative projects
were identified and 19 were submitted for evaluation by the consortium. A significant achievement was the
series of the state-of-the art reviews on issues most relevant to ECNIS research. These included biomarkers of
exposure and bioindicators of disease, biomarkers of dietary exposure to anticarcinogenic food components, and
validation of biomarkers for molecular epidemiology. Another important outcome is the position paper on carcinogenic mechanisms and their impact on hazard and risk assessment. We also initiated work on a protocol for
exposure data analysis and a framework for integrating biomarkers of exposure and early effects into cancer risk
assessment. A draft report on genotype vs. phenotype studies was prepared and a framework is being set up
for future genetic polymorphism studies. Since ECNIS research involves human biomonitoring, special emphasis
has been laid on strict compliance with the rules and principles pertaning to this approach. Therefore, a report
was developed on the methods for recruitment of study participants and collection of samples as well as on
the gender and ethical issues in ECNIS research. The Spreading of Excellence activities are focused on the
dissemination of knowledge within the scientific community and distribution of outcomes to different stakeholders. Both of these objectives were successfully implemented within the reported period. Among other activities,
ECNIS was involved in organizing the EC Workshop on Community-Sponsored Research on Environmental Cancer
Risks which was combined with the ECNIS kick-off meeting. Both the events were held in Warsaw in May 2005.
In the context of the 35th Annual EEMS Meeting on Kos, Greece, ECNIS sponsored the opening Plenary Session,
the Symposium on the role of environmental genotoxins in human carcinogenesis and an Interactive Workshop
on training in genetic toxicology. ECNIS also supported a number of events within the 42nd Annual Congress
of EUROTOX, which was held in Krakow, Poland, in September 2005. These included the Continuing Education
Course on molecular mechanisms of food contaminants toxicity, the opening Plenary Session and the Workshop
on „Genetics and Individual Susceptibility“. ECNIS research planning was developed through several workshops
organized in Athens in January 2006 where reports from particular WP workshops were discussed to have insight
into research activities carried out within the Network. ECNIS is also actively involved in making EEMS meeting
in Prague (2-6Jun, 2006) a great success, organizing six scientific sessions. To spread the scientific knowledge
among the different stakeholders, the Science Portal with the Science News sub-section on research advances
and a Library of technical or other documents of wider interest was established. Also ECNIS Newsletter no. 1 was
issued, with news about ECNIS as well as items of more general interest in the area of ECNIS research. To sum
up, within the first year of the Network’s existence, the various activities of ECNIS were carried out according to
schedule and much progress has been made in all of its activity areas. This successful start-up is a good predictor of further development of our consortium and the research conducted under ECNIS and we can certainly be
hopeful of the future cooperation and partnership in our common effort to reduce cancer burden in Europe.
Konrad Rydzynski
ECNIS Coordinator
26.6.2006 16:04:58
© 2005 Johnson & Johnson Pharmaceutical Research & Development, Division of Janssen Pharmaceutica N.V.
CO M B I N I N G I N N O VAT I O N
AND EXPERIENCE
Across the globe, around the clock,
Johnson & Johnson Pharmaceutical Research & Development
is committed to improving the lives of millions.
www.jnjpharmarnd.com
26.6.2006 16:04:58
CONTENTS
COMMITTEES
2
VENUE FLOOR PLANS
3
PROGRAMME AT A GLANCE
4-7
CONFERENCE PROGRAMME
10-27
LIST OF POSTERS
30-57
INSTRUCTION FOR SPEAKERS AND AUTHORS
58
EXHIBITION
59
LIST OF EXHIBITORS
60
CONFERENCE INFORMATION
61-63
SOCIAL PROGRAMME
64
GUIDED TOURS
65
USEFUL INFORMATION ON PRAGUE
ORAL PRESENTATIONS
66-69
71-139
POSTER PRESENTATIONS
141-241
AUTHORS INDEX
242-247
LIST OF PARTICIPANTS
249-277
26.6.2006 16:05:03
COMMITTEES
SCIENTIFIC PROGRAMME COMMITTEE
President:
Radim J. Šrám, Institute of Experimental Medicine AS CR, Prague, Czech Republic
Bjorn Akesson, University of Lund, Lund, Sweden (ECNIS)
William W. Au, University of Texas, Galveston, TX, USA
Herman Autrup, Aarhus University, Aarhus, Denmark
Douglas A. Bell, National Institute of Environmental Health Sciences, NC, USA
Paolo Boffetta, International Agency for Research on Cancer, Lyon, France (ECNIS)
Stefano Bonassi, National Institute of Cancer Research, Genova, Italy
Eugenia Dogliotti, Instituto Superiore di Sanita, Rome, Italy
Peter B. Farmer, University of Leicester, Leicester, United Kingdom (ECNIS)
Aage Haugen, National Institute of Occupational Health, Oslo, Norway
Lisbeth E. Knudsen, University of Copenhagen, Copenhagen, Denmark (ECNIS)
Soterios Kyrtopoulos, National Hellenic Research Foundation, Athens, Greece (ECNIS)
Takehiko Nohmi, National Institute of Health Sciences, Tokyo, Japan
Kirsti Husgafvel-Pursiainen, Finnish Institute of Occupational Health, Helsinki, Finland
Konrad Rydzinski, Nofer Institute of Occupational Medicine, Lodz, Poland (ECNIS)
Bernadette Schoket, National Institute of Public Health, Budapest, Hungary (ECNIS)
Frederik J. Van Schooten, University of Maastricht, the Netherlands (ECNIS)
Peter J. Stambrook, University of Cincinnati, OH, USA
Krysztof Szyfter, Polish Academy of Sciences, Poznan, Poland
Jan Topinka, Institute of Experimental Medicine AS CR, Prague, Czech Republic
Harri Vainio, Finnish Institute of Occupational Health, Helsinki, Finland
Paolo Vineis, Imperial College of London, United Kingdom (ECNIS)
Yasuhito Yuasa, Graduate School of Medicine and Dentistry, Tokyo, Japan
LOCAL ORGANISING COMMITTEE
President:
Jan Topinka, Institute of Experimental Medicine AS CR, Prague
Milena Černá, 3rd Medical faculty of Charles University, Prague
Eva Dejmková, Institute of Experimental Medicine AS CR, Prague
Lubomír Dobiáš, Health Institute Ostrava and University of Ostrava
Miroslav Dostál, Institute of Experimental Medicine AS CR, Prague
Tomáš Gichner, Institute of Experimental Botany AS CR, Prague
Jana Křížová, Institute of Experimental Medicine AS CR, Prague
Hana Lehocká, Health Institute Ostrava and University of Ostrava
Božena Novotná, Institute of Experimental Medicine AS CR, Prague
Pavel Rössner, Institute of Experimental Medicine AS CR, Prague
Pavel Rössner Jr., Institute of Experimental Medicine AS CR, Prague
Jiří Rubeš, Veterinary Research Institute, Brno
Pavel Souček, National Institute of Public Health, Prague
Marie Stiborová, Charles University, Prague
Radim J. Šrám, Institute of Experimental Medicine AS CR, Prague
Pavel Vodička, Institute of Experimental Medicine AS CR, Prague
2
eems2006-abstract-inside.indd Odd1:2
26.6.2006 16:05:06
VENUE FLOOR PLANS
3
26.6.2006 16:05:07
Sunday, July 2
Plenary Lecture Hall
08
Room I
8.30-12.30
Monday, July 3
Room I
Room II
9.30-13.00
9.30-13.00
9.30-13.00
Symposium
Symposium
Symposium
10.30 Coffee break
10.30 Coffee break
10.20 Coffee break
NEW MATTERS IN DNA
REPAIR AND MUTATION
RESEARCH
FOOD MUTAGENS
IMPACT ON HEALTH
(ECNIS)
MOLECULAR
EPIDEMIOLOGY: NEW
KNOWLEDGE FROM
BIOMARKERS OF
EFFECTS (ECETOC)
13.00 Lunch break
13.00 Lunch break
13.00 Lunch break
13.30-14.15
13.30-14.15
13.30-14.15
Poster viewing
Poster viewing
Poster viewing
14.15-15.00
14 15 15 00
14.15-15.00
14 15 15 00
14.15-15.00
14 15 15 00
Poster discussion
Poster discussion
Poster discussion
15.00-18.00
15.00
18.00
15.00-18.00
15.00
18.00
15.00-18.00
15.00
18.00
Symposium
Symposium
Symposium
16.30 Coffee break
16.30 Coffee break
16.40 Coffee break
8.30-9.20
Keynote Lecture I
Workshop
W
k h 1
09
10
10.30 Coffee break
HUMN PROJECT –
AUTOMATED
MICRONUCLEUS
WORKSHOP
11
12
13.00-16.00
13
Workshop 2
14
TRANSCRIPTOMICS IN
GENETIC TOXICOLOGY
AND MOLECULAR
EPIDEMIOLOGY
15
16
17
16.30-17.00
Opening Ceremony
17.00-18.00
F it S
Fritz
Sobels
b l A
Award
d
Lecture
Herman N. Autrup
NEW MATTERS: FROM
MECHANISMS TO HUMAN NUTRIGENOMICS (ECNIS)
DISEASE
BIOMARKERS FOR THE
EVALUATION OF
CHILDREN’S HEALTH
(ECETOC)
18.00-20.00
18.00
20.00
18
18.30-20.30
Welcome Reception
Workshop 3
19
20
TEACHING IN GENETIC
TOXICOLOGY
21
4
26.6.2006 16:05:08
Tuesday, July 4
Room I
Room II
08
8.30-9.20
Keynote Lecture II
9.20-12.30
9.30-13.00
9.30-13.00
Symposium
Symposium
Symposium
10.30 Coffee break
10.30 Coffee break
10.30 Coffee break
NEW KNOWLEDGE FROM
BIOMAKERKERS OF
EXPOSURE (ECNIS)
NEW ASPECTS OF
REGULATORY TESTING
AND RISK ASSESSMENT
GENOTOXICITY AND
MUTAGENICITY OF
COMPLEX MIXTURES
09
10
11
12
12.30-13.00
Prof. Lars Ehrenberg
13.00 Lunch break
13.00 Lunch break
13.00 Lunch break
13.30-14.15
13.30-14.15
13.30-14.15
Poster viewing
Poster viewing
Poster viewing
14.15-15.00
14 15 15 00
14.15-15.00
14 15 15 00
14.15-15.00
14 15 15 00
Poster discussion
Poster discussion
Poster discussion
15.00
18.00
15.00-18.00
15.00 18.00
15.00-18.00
15.00 17.30
15.00-17.30
13
14
15
Symposium
Symposium
Symposium
16.30 Coffee break
16.30 Coffee break
16.30 Coffee break
GENE-ENVIRONMENT
INTERACTION IN THE
RISK OF COMMON NON
CANCER DISEASE
EPIGENETIC
MECHANISMS IN
CARCINOGENESIS
(ECNIS)
BIOTRANSFORMATION
OF CARCINOGENS
16
17
18
19
20:00-22:00
20:00
22:00
20
Black Light Theatre Performance
21
5
26.6.2006 16:05:09
Wednesday, July 5
Room I
Room II
8.30-11.00
8.30-11.00
8.30-10.30
Symposium
Symposium
08
09
10
11
12
Poster discussion
session
OXIDATIVE STRESS
RESPONSES (ECNIS)
11.00
Coffee break
11.30
Young Scientist
Award Lecture
11.00
Coffee break
GERM CELL
MUTAGENESIS /
EPIDEMIOLOGY
11.00
Coffee break
Room 2.2
9.00-11.00
Workshop 4
GENETIC
ECOTOXICOLOGY
11.00
Coffee break
12.10
GENERAL ASSEMBLY
13
13.10
FUTURE MEETINGS
13.20 Lunch break
19
20
21
19:30-22:30
Conference Dinner at Zofin Palace
22
23
6
26.6.2006 16:05:09
Thursday, July 6
Room I
Room II
08
8.30-9.20
Keynote Lecture III
9.20-13.00
9.30-13.00
9.30-13.00
Symposium
Symposium
Symposium
10.30 Coffee break
10.30 Coffee break
10.30 Coffee break
IMPACT OF GENETIC
VARIATION ON
INDIVIDUAL
SUSCEPTIBILITY
RISK ASSESSMENT
(ECNIS)
OCCUPATIONAL
EXPOSURE TO
MUTAGENS AND
CARCINOGENS
09
10
11
12
13.00-13.30
CLOSING CEREMONY
13.30
Lunch break
13.30
Lunch break
13.30
Lunch break
13
7
26.6.2006 16:05:09
NOTES:
8
26.6.2006 16:05:10
CONFERENCE
PROGRAMME
SUNDAY, JULY 2, 2006
9
26.6.2006 16:05:10
PRECONFERENCE WORKSHOPS
11.20
O-009
Room I
8.30 - 12.30
8.30
8.40
O-001
9.00
O-002
9.20
O-003
9.40
O-004
10.00
O-005
10.20
O-006
Workshop 1
HUMN PROJECT - AUTOMATED
MICRONUCLEUS WORKSHOP
Chairpersons:
Vrije Universiteit Brussels, Brussels, Belgium
Michael Fenech
CSIRO Human Nutrition, Adelaide, Australia
Michael Fenech
Brief introduction to the HUMN project
objectives
Roberto Barale
Pisa University, Pisa, Italy
Validation of micronuclei assay in peripheral
blood lymphocytes as early
cancer risk biomarkers by a nested case-control
study in Italy
Marlies DeBoeck
Johnson & Johnson Pharmaceutical Research &
Development, Beerse, Belgium
Comparison of automated analysis methods for
micronucleus assays in human and rodent cells
Stephen Dertinger
Litron Laboratories Ltd., Rochester, NY, USA
Flow cytometric methods for scoring micronuclei
in erythrocytes and nucleated cells
Lilianne Abramsson-Zetterberg
National Food Administration, Uppsala, Sweden
The micronucleus assay in human erythrocytes
Ash Lal
NGC, Oakland, CA, USA
Immunomagnetic separation technique with
single-laser flow cytometry to isolate and
analyze micronuclei in reticulocytes
Zoryana Cammerer
Novartis Pharma AG, Basel, Switzerland
Comparison of the peripheral blood
micronucleus test in rat and mouse exposed
to aneugens: involvement of the spleen in
the selective removal of the micronucleated
erythrocytes
10.30
Coffee Break
10.40
O-007
Christian Schunck
MetaSystems
Automated in-vitro and in-vivo micronucleus
scoring using the Metafer MicroNuclei slide
scanning system
Francoise Soussaline
IMSTAR S.A., Paris, France
A new integrated quantitative imaging method
for automated micronuclei detection and scoring
using pathfinder™ image cytometer
11.00
O-008
10
11.40
O-010
12.00
O-011
12.20
O-012
12.30
Wilfried Frieauff
Novartis Pharma AG, Basel, Switzerland
Automated micronucleus scoring using image
analysis at Novartis
Ida-Maria Sintorn
Centre for Image Analysis, Uppsala, Sweden
Towards automatic scoring of the CytokinesisBlock MicroNucleus Cytome assay
Vrije Universiteit Brussels, Brussels, Belgium
Advantages and disadvantages of different
automated systems and current knowledge gaps
Michael Fenech
Developing a collaborative network focussed on
automated systems and their validation as part
of the HUMN project
CLOSURE
Room I
13.00 - 16.00 Workshop 2
13.00
O-013
13.30
O-014
14.00
O-015
14.30
O-016
15.00
O-017
15.30
O-018
TRANSCRIPTOMICS IN GENETIC
TOXICOLOGY AND MOLECULAR
EPIDEMIOLOGY
Chairpersons:
Jos Kleinjans
Maastricht University, Maastricht, The Netherlands
Jiri Aubrecht
Safety Sciences, Pfizer US, Groton, USA
Jiri Aubrecht
Safety Sciences, Pfizer US, Groton, USA
Toxicogenomic analysis of gene expression an
emerging approach for differentiating genotoxic
mechanisms
Jos Kleinjans
Discrimination of genotoxic from non-genotoxic
carcinogens by expression profiling in vitro
Raffaella Corvi
ECVAM-Joint Research Centre of European
Commission, Ispra. Italy
Can we integrate transcriptomics in regulatory
genetic toxicology?
Hans J. Ahr
Bayer HealthCare AG, Wuppertal, Germany
Toxicogenomics of carcinogens: Identification
of characteristic expression signals in liver and
kidney
Jochen König
Genedata AG, Basel, Switzerland
Leveraging complementary data:
Toxicogenomics based on combined proteomics
and transcriptomics reference compendia
Leire Arbillaga
University of Navarra, Pamplone, Spain
New data supporting indirect genotoxicity
damage as a possible mechanism of Ochratoxin
A carcinogenicity by analysing modulation
of gene expression in HK-2 cells
26.6.2006 16:05:10
OPENING SESSION
16.30 - 17.00
OPENING CEREMONY
17.00
FRITZ SOBELS AWARD LECTURE
Eugenia Dogliotti
Vice President of EEMS
Introduction
17.05
Herman N. Autrup
Aaarhus University, Aaarhus, Denmark
DNA adducts as risk indicators
18.00 - 20.00
WELCOME RECEPTION
(FOYER OF THE PLENARY HALL)
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CONFERENCE
PROGRAMME
MONDAY, JULY 3, 2006
26.6.2006 16:05:12
8.30 - 9.20
9.30 - 13.00
9.30
O-019
10.00
O-020
Keynote Lecture I
Chairperson:
Paolo Boffetta
IARC, Lyon, France
Pierre Hainaut
IARC, Lyon, France
TP53 mutations as biomarkers of carcinogen
exposure and tumorigenesis
Symposium
NEW MATTERS IN DNA REPAIR
AND MUTATION RESEARCH
Chairpersons:
Eugenia Dogliotti
Istituto Superiore di Sanita, Rome, Italy
Takehiko Nohmi
National Institute of Health Sciences, Tokyo, Japan
Eugenia Dogliotti
Istituto Superiore di Sanita, Rome, Italy
The effect of cellular type and state in the
response to DNA damage
Hans Krokan
Norwegian University of Science and Technology,
Trondheim, Norway
DNA repair proteins and acquired immunity
10.30
Coffee Break
11.00
O-021
Takehiko Nohmi
Control of multiple DNA polymerases dealing
with lesions induced by environmental mutagens
Robert Schiestl
UCLA Schools of Medicine and Public Health,
Los Angeles, CA, USA
Radiation induces microhomology mediated
recombination in trans at undamaged sites
Nejla Gungor
Inhibition of nucleotide excision repair by
neutrophils: role of myeloperoxidase
Claudia Aiub
Salzburg University, Salzburg, Austria
DNA lesions induced by N-Nitrosodiethylamine:
Does methyltransferases play a role?
Kazuo Fujikawa
Kinki University, Higashiosaka, Japan
Accumulation and persistence of mutations
induced in somatic stem cells of mice during
irradiation with low dose-rate gamma rays for
483 days, a preliminary report
Michael Routledge
University of Leeds, Leeds, U.K.
SupF mutations induced by urban particulate
matter in a metabolism free system
11.30
O-022
12.00
O-023
12.15
O-024
12.30
O-025
12.45
O-026
13:00
15.00 - 18.00 Symposium
15.00
O-027
15.30
O-028
16.00
O-029
NEW MATTERS: FROM MECHANISMS
TO HUMAN DISEASE
Chairpersons:
Alain Sarasin
CNRS-Goustave Roussy Institute, Villejuif, France
Jean-Sébastien
Hoffmann IPBS-CNRS, Toulouse, France
Alain Sarasin
CNRS-Goustave Roussy Institute, Villejuif, France
High efficient DNA repair pathways can be
detrimental to human health
Eisaburo Sueoka
Saga University, Japan
Lung carcinogenesis mediated through hnRNP
B1: A cross talk between RNA splicing regulators
and DNA repair system
Keith Caldecott
University of Sussex, Brighton, U.K.
Mammalian chromosomal single-strand break
repair and neurodegenerative disease
16.30
Coffee Break
17.00
O-030
Jean-Sébastien Hoffmann
IPBS-CNRS, Toulouse, France
Upregulation of adaptive DNA polymerases beta
and kappa impedes fork progression without
activating the replication checkpoint
Yusuke Hiraku
Mie University Graduate School of Medicine, Mie,
Japan
Nitroguanine as a biomarker for inflammationrelated carcinogenesis
Luis Blanco
CSIC-University of Madrid, Madrid, Spain
Characterisation of a natural mutator variant of
human DNA polymerase lambda, and its impact
in DNA repair and mutagenesis
17.30
O-031
17.45
O-032
Lunch Break
13.30 - 14.15 Poster viewing
14.15 - 15.00 Poster discussion
14
26.6.2006 16:05:14
Room I
Room I
9.30 - 13.30
Symposium
9.30
O-033
FOOD MUTAGENS IMPACT ON HEALTH
(ECNIS)
Chairpersons:
Beatrice Pool-Zobel
F.Schiller University, Jena, Germany
Konrad Rydzinski
Nofer Institute of Occupatinal Medicine, Lodz,
Poland
Beatrice Pool-Zobel
F.Schiller University, Jena, Germany
Evaluation of genotoxicity and antigenotoxicity
by food-derived compounds in human colon,
prostate, breast and blood cells using novel
genomics and transcriptomics-base methods
Rashmi Sinha
NIH-National Cancer Institute, Rockville, MD, USA
Cooked and preserved meat carcinogens and
cancer
10.00
O-034
15.00 - 18.00 Symposium
15.00
O-041
15.30
O-042
16.00
O-043
NUTRIGENOMICS (ECNIS)
Chairpersons:
Fred F. Kadlubar
National Center for Toxicological Research,
Jefferson, AR, USA
Björn Akesson
Lund University, Lund, Sweden
Gunter Kuhnle
MRC-Dunn Human Nutrition Unit, Cambridge, U.K.
Interaction of nutritional and genetic factors in
colorectal cancer
Björn Akesson
Lund University, Lund, Sweden
The effects of cereal components on the
properties and proliferation of colon cancer cells
Lynnette Ferguson
University of Auckland, Auckland, New Zealand
Nutrigenomics and inflammatory bowel disease
10.30
Coffee Break
16.30
Coffee Break
11.00
O-035
Jakob Linseisen
Technical University of Munich, Munich, Germany
Meat cooking, heterocyclic amines and cancer
risk
Joseph Rafter
Karolinska Institutet, Novum, Stockholm, Sweden
Anticarcinogenic activity of probiotic bacteria
Miriam Poirier
National Cancer Institute,NIH, Bethesda, MD, USA
Formation of polycyclic aromatic hydrocarbon
- DNA adducts in human leukocytes
David DeMarini
US Environmental Protection Agency, Research
Triangle Park, NC, USA
Inhibition of fried meat-induced DNA damage:
Use of cruciferous vegetables, yougurt, and
chlorophyllin in a dietary intervention study in
humans
Kazuaki Kawai
University of Occupational and Environmental
Health, Kitakyushu, Japan
Detection of 8-OH-dG, ribonucleoside 8-OH-Gua
and free base 8-OH-Gua in fish food products
Suzanne Jeurissen
Wageningen University, Wageningen, Netherlands
Identification of the human cytochrome
P450enzymes involved in the bioactivation
of the herb-based carcinogens estragole and
methyleugenol
17.00
Sven Pettersson (presented by Joseph
Rafter)
Host-microbe crosstalk
Theo de Kok
Profiling of gene expression modulation in
human lymphocytes is not sensitive marker for
exposure to Ah-receptor agonists
11.30
O-036
12.00
O-037
12.15
O-038
12.30
O-039
12.45
O-040
13:00
O-044
17.30
O-045
18.30 - 20.30 Workshop 3
TEACHING IN GENETIC TOXICOLOGY
Chairpersons:
Ricardo Marcos
University of Barcelona, Barcelona, Spain
Daniel Marzin
Institut Pasteur de Lille, Lille, France
Lunch Break
15
26.6.2006 16:05:14
Room II
9.30 - 13.00
9.30
9.40
O-046
Room II
Symposium
MOLECULAR EPIDEMIOLOGY:
NEW KNOWLEDGE FROM BIOMARKERS
OF EFFECTS (ECETOC)
Chairperson:
Soterios A. Kyrtopoulos
National Hellenic Research Foundation, Athens,
Greece
Awni Sarrif
ECETOC, DuPont, Germany
Awni Sarrif
ECETOC, DuPont, Germany
Introduction
Stefano Bonassi
National Institute of Cancer Research, Genoa, Italy
Predictivity of micronuclei in cancer risk and
chromosomal aberrations for quantitative
analysis for cancer risk
10.20
Coffee Break
10.50
O-047
Radim J. Sram
Institute of Experimental Medicine AS CR, Prague,
Czech Rep.
The interpretation of cytogenetic analysis by
FISH
Bennard Van Ravenzwaay
BASF AG, Ludwigshafen, Germany
Use of metabionomics as a new biomarker of
effect
Ricardo Marcos
University of Barcelona, Barcelona, Spain
Telomere length shortening
Randa A El-Zein
The University of Texas MD Anderson Cancer
Center, Houston, TX, U.S.A
The cytokinesis-blocked micronucleus assay as a
novel biomarker for lung cancer risk
Antonina Cebulska-Wasilewska
H.Niewodniczanski Institute of Nuclear Physics,
Cracow, Poland
Repair competence and Comet assay as
biomarker of effect and susceptibility EXPAH
studies
Soterios A. Kyrtopoulos
Concluding remarks
11.20
O-048
11.50
O-049
12.20
O-050
12.35
O-051
12.50
13:00
15.00 - 18.00 Symposium
15:00
15.10
O-052
15.40
O-053
16.10
O-054
BIOMARKERS FOR THE EVALUATION
OF CHILDREN’S HEALTH ECETOC
Chairpersons:
Lisbeth E. Knudsen
University of Copenhagen, Copenhagen, Denmark
Introduction
Hans-Juergen Wiegand
Degussa AG, Düsseldorf, Germany
Nina Holland
University of Berkeley, Berkeley, CA, USA
Biomarkers during prenatal and early postnatal
life: examples from USA studies
Jos Kleinjans
Mother-child comparison in genomic response to
air pollution exposure
Domenico Franco Merlo
National Cancer Research Institute, Genoa, Italy
Baseline chromosome aberrations in children
16:40
Coffee Break
17.10
O-055
Ilse Decordier
Vrije Universiteit Brussel, Brussels, Belgium
Inherent susceptibility of children
Jan Topinka
Czech Rep.
Impact of PAHs and genetic polymorphisms on
respiratory morbidity in children
Lisbeth E. Knudsen
Concluding remarks
17. 40
O-056
17.55
Lunch Break
16
26.6.2006 16:05:14
CONFERENCE
PROGRAMME
TUESDAY, JULY 4, 2006
17
26.6.2006 16:05:14
8.30 - 9.20
Keynote Lecture II
Chairperson:
Lynette Fergusson
University of Auckland, New Zealand
Fred F. Kadlubar
National Center for Toxicological Research,
Jefferson, USA
Nutrigenomics: Applications to the molecular
epidemiology of food-borne mutagens and
carcinogens
9.20 - 12.30
Symposium
9.30
O-057
GENOTOXICITY AND MUTAGENICITY
OF COMPLEX MIXTURES
Chairpersons:
Brinda Mahadevan
Oregon State University, Corvallis, OR, USA
J. Topinka
Czech Rep.
Brinda Mahadevan
Oregon State University, Corvallis, OR, USA
Responses to mutagenic complex mixtures: An
overview
Albrecht Seidel
Biochemical Institute for EnvironmentalCarcinogen,
Grosshansdorf, Germany
Determination of urinary PAH metabolites
as biomarkers to assess the exposure of the
general population to PAH mixtures
10.00
O-058
10.30
Coffee Break
11.00
O-059
Volker Arlt
Institute of Cancer Research, Sutton, U.K.
3-Nitrobenzanthrone, a potential human cancer
hazard in diesel exhaust and urban air pollution
Adyapalam T.Natarajan
Leiden University Medical Center, Leiden, The
Netherlands
Cytogenetic biomarkers for monitoring human
populations exposed to physical or chemical
mutagens
Miroslav Machala
Veterinary Research Institute, Brno, Czech Rep.
Comparison of genotoxic and nongenotoxic
potencies of complex environmental mixtures in
airborne and river sediment extracts
Alena Gabelova
Cancer Research Institute, Bratislava, Slovak Rep.
Interactions of aromatic hydrocarbons in their
binary mixtures; an in vitro study
11. 30
O-060
12.00
O-061
12.15
O-062
12.30
O-063
13:00
Lunch Break
15.00 - 18.00 Symposium
15.00
O-064
15.30
O-065
16.00
O-066
GENE-ENVIRONMENT INTERACTION
IN THE RISK OF COMMON NON CANCER
DISEASE
Chairperson:
Curtis C. Harris
NIH-National Institute of Health, Bethesda, MD,
USA
Alberto Izzotti
University of Genoa, Genoa, Italy
Curtis C.Harris
NIH-National Institute of Health, Bethesda, MD,
USA
Mechanisms of inflammatory diseases
Alberto Izzotti
Gene-environment interactions in atherosclerosis
and glaucoma
Tarja Laitinen
Geneos Ltd., Helsinki, Finland
What we know about molecular genetic
subclasses of asthma and atopy related
phenotypes so far?
16.30
Coffee Break
17.00
O-067
Ari Hirvonen
Finnish Institute of Occupational Health, Helsinki,
Finland
Role of EPHX1 genotypes in the development of
asbestos and smoking induced non-malignant
pulmonary diseases
Krzysztof Szyfter
Institute of Human Genetics, Polish Academy of
Sciences, Poznan, Poland
Tobacco smoke-associated laryngeal cancer.
Differentiation between multiple primary
tumours and tumor reccurrence
Rudolf Stetina
Faculty of Military Health Sciences, Charles
University, Hradec Kralove, Czech Rep.
DNA damage and DNA repair capacity in
children with type 1 diabetes mellitus
17.30
O-068
17.45
O-069
18
PRESENTATION TO COMMEMORATE
THE SCIENTIFIC CONTRIBUTION
OF PROF. LARS EHRENBERG
Chairperson:
Adyapalam T. Natarajan
Leiden University Medical Center, Leiden,
The Netherlands
Margareta Littorin Törnqvist
Stockholm University, Stockholm, Sweden
Cancer risk model based on in vivo dosimetry:
studies on acrylamide and butadiene
26.6.2006 16:05:15
Room I
Room I
9.30 - 13.00
Symposium
9.30
O-070
NEW KNOWLEDGE FROM BIOMARKERS
OF EXPOSURE (ECNIS)
Chairpersons:
Peter B. Farmer
Leicester University, Leicester, U.K.
Radim J. Sram
Czech Rep.
David Phillips
The use and limitations of DNA adducts in
determining human exposure to carcinogens
and the aetiology of human cancers
Günther Speit
University of Ulm, Ulm, Germany
Systemic genotoxic effects of formaldehyde
detected in biomonitoring studies and evaluation
of their plausibility in an ex vivo approach
10.00
O-071
10.30
Coffee Break
11.00
O-072
Peter B. Farmer
University of Leicester, Leicester, U.K.
The potential of mass spectrometry for the
detection of DNA damage
Frederik-Jan Van Schooten
Biomonitoring of inhalatory exposures: from
invasive to non-invasive methods
Johanna Haglund
Stockholm University, Stockholm, Sweden
Phosphate adducts - a potential biomarker?
Sophia Pavanello
University of Padova, Padova, Italy
Gene-exposure interaction on anti-benzo[a]pyre
nediolepoxide-B[a]PDE-DNA adduct formation in
lymphocytes of humans
Sarolta Gundy
National Institute of Oncology, Budapest, Hungary
Cytogenetic biomarkers to assess the genetic
damage induced in chronic alcoholics with
malignant, premalignant, and non-malignant
diseases
11.30
O-073
12.00
O-074
12.30
O-075
12.45
O-076
13:00
15.00 - 18.00 Symposium
15.00
O-077
15.30
O-078
16.00
O-079
EPIGENETIC MECHANISMS
IN CARCINOGENESIS (ECNIS)
Chairpersons:
Zdenko Herceg
IARC, Lyon, France
Peter J. Stambrook
University of Cincinnatti, Cincinnatti, OH, USA
Zdenko Herceg
IARC, Lyon, France
Epigenetic mechanisms of regulation ofccellular
processes and tumorigenesis
Steven Belinsky
Lovelace Respiratory Research Institute,
Albuquerque, NM, USA
Targeting of genes for silencing by promoter
hypermethylation: Influence of environmental
exposures
Peter J. Stambrook
Mutation, cell cycle and apoptosis: Preserving
genomic integrity in somatic and embryonic
stem cells
16.30
Coffee Break
17.00
O-080
Yasuhito Yuasa
Tokyo Medical and Dental University, Tokyo, Japan
Epigenetic epidemiology of gastric cancer
Jan Vondracek
Institute of Biophysics, Brno, Czech Rep.
The dual role of AhR in genotoxic and
nongenotoxic mechanisms of toxicity of
environmental PAHs in liver epithelial cells
Katarzyna Urbanek
National Institute of Hygiene, Warsaw, Poland
Effect of phenobarbital on the level methylation
of promoter region
og P16 gene in rat liver
17.30
O-081
17.45
O-082
Lunch Break
19
26.6.2006 16:05:15
Room II
9.30 - 13.00
9.30
O-083
10.00
O-084
Room II
Symposium
NEW ASPECTS OF REGULATORY TESTING
AND RISK ASSESSMENT
Chairpersons:
David Kirkland
Covance Laboratories Ltd., Harrogate, U.K.
Lutz Mueller
F.Hoffmann-La Roche Ltd., Basel, Switzerland
Robert Kroes
Utrecht University, Utrecht, The Netherlands
The threshold of toxicological concern TTC
approach in health risk assessment
Lutz Mueller
F.Hoffmann-La Roche Ltd., Basel, Switzerland
A rationale for controlling genotoxic impurities in
pharmaceuticals - the staged TTC concept
10.30
Coffee Break
11.00
O-085
Veronique Thybaud
Sanofi Aventis, Vitry sur Seine, France
IWGT and ILSI initiatives on the interpretation
of and follow-up to positive in vitro genotoxicity
results
David Tweats
University of Wales, Swansea, U.K.
The bone marrow micronucleus test: positive
compounds inactive in vitro and ‘positive’
compounds that do not indicate genotoxic
hazards: Report of the IWGT working group
David Kirkland
Covance Laboratories Ltd., Harrogate, U.K.
How to reduce false positive results with in
vitro genotoxicity testing and avoid unnecessary
follow-up animal tests: Report of an ECVAM
Workshop
Richard Walmsley
University of Manchester, Manchester, U.K.
A high specificity genotoxicity assay measuring
the response of the human GADD45 alpha gene
11.30
O-086
12.00
O-087
12.30
O-088
13:00
15.00 - 17.30 Symposium
15.00
O-089
15.30
O-090
16.00
O-091
BIOTRANSFORMATION OF CARCINOGENS
Chairpersons:
Fred Guengerich
Vanderbilt University School of Medicine, Nashville,
USA
Thomas Wolff
GSF-Institute of Toxicology, Neuherberg, Germany
Hansruedi Glatt
German Institute of Human Nutrition, Postdam,
Germany
Human and rodent enzyme systems for
activating amino- and nitroarenes in
genotoxicity tests
Fred Guengerich
Vanderbilt University School of Medicine, Nashville,
USA
Biotransformation of carcinogens, enzymes, DNA
adducts, relevance and validation
Franz Oesch
University of Mainz, Mainz, Germany
Carcinogen-activated transcription factors
controlling both, carcinogen metabolizing
enzymes and cell cycle
16.30
Coffee Break
17.00
O-092
Marie Stiborova
Charles University, Prague, Czech Rep.
Activation and detoxication of anticancer drug
ellipticine as a model genotoxicant: enzymes,
DNA, adducts, effects
Lunch Break
20
26.6.2006 16:05:15
CONFERENCE
PROGRAMME
WEDNESDAY, JULY 5, 2006
21
26.6.2006 16:05:15
Room I
8.30 - 11.00
8.30 - 11.00
POSTER DISCUSSION SESSION
Chairpersons:
James M. Parry
University of Wales, Swansea, U.K.
David H. Philipps
Emanuela Taioli
University of Pittsburgh Cancer Institute, USA
11:00
Coffee Break
11.30
YOUNG SCIENTIST AWARD LECTURE
Eugenia Dogliotti
Vice President of EEMS
Introduction
Volker Arlt
Aristolochic acid a potential risk factor for
Balkan endemic nephropathy and associated
urothelial cancer: potential mechanisms
12.10
GENERAL ASSEMBLY
13.10
FUTURE MEETINGS
13:20
Lunch Break
8.30
O-093
9.00
O-094
9.30
O-095
10.00
O-096
10.30
O-097
11:00
22
Symposium
OXIDATIVE STRESS RESPONSES (ECNIS)
Chairpersons:
Regina M. Santella
Columbia University, New York, USA
Silvio de Flora
Jagadeesan Nair
German Cancer Research Centre, Heidelberg,
Germany
Oxidative stress response: Lipid peroxidation
induced DNA damage and repair in cancer-prone
inflammatory diseases
Regina M. Santella
Columbia University, New York, CO, USA
Oxidative stress and breast cancer
Steffen Loft
University of Copenhagen, Copenhagen, Denmark
Oxidative DNA damage and risk of cancer
Silvio de Flora
Oxidants and antioxidants in the prevention of
mutation-related diseases
Hiroshi Ide
Hiroshima University, Hiroshima, Japan
Repair of DNA lesions induced by oxidative and
nitrosative stress
Coffee Break
26.6.2006 16:05:16
Room II
8.30 - 10.30
8.30
O-098
9.00
O-099
9.30
O-100
10.00
O-101
11:00
Meeting room 2.2
Symposium
GERM CELL MUTAGENESIS/EPIDEMIOLOGY
Chairpersons:
Taisei Nomura
Osaka University, Osaka, Japan
Jiří Rubeš
Veterinary Research Institute, Brno, Czech Rep.
Donald P. Evenson
South Dakota State University, Brookings, SD, USA
Sperm DNA fragmentation as a marker in
reproductive toxicology
Taisei Nomura
Transmissible germ cell mutagenesis and
carcinogenesis in mice and humans
Jiří Rubeš
Veterinary Research Institute, Brno, Czech Rep
Semen quality of young fertile men in the Czech
Republic
Rosalie Elespuru
FDA/CDRH, Rockville, MD, USA
Integrating new technologies into the
assessment of heritable genetic effects
Coffee Break
9.00 - 11.00
9.00
O-102
9.30
O-103
10.00
O-104
10.30
O-105
11:00
Workshop 4
GENETIC ECOTOXICOLOGY
Chairpersons:
Jette Rank
NINR, Tronsheim, Norway
Aase Krokje
NTNU, Trondheim, Norway
Aase Krokje
Chromosome aberrations in lymphocytes from
grey seal Halichoerus grypus pups from Estonia
and Norway
Jette Rank
NINR, Trondheim, Norway
Biomonitoring studies of DNA damage in
mussels exposed to polluted coastal waters in
Denmark
Renate Haldsrud
Induction of DNA double strand Breaks in the
H4IIE cell line caused by co exposure to copper,
cadmium and zinc singly and in combinations
Jerome Cachot
University of Le Havre, Le Havre, France
The use of embryosof the Lambda cII transgenic
medaka for environmental risk assessment.
Application to sediments of the Seine estuary
Coffee Break
23
26.6.2006 16:05:16
OLYMPUS FLUOVIEW FV1000
A new concept of laser confocal microscopy by light of stimulated processes
The OLYMPUS FV1000 laser confocal microscope offers
a unique set of two independent laser scanners. The system
allows exact synchronization of the process of laser stimulation
in cells with simultaneous confocal display. The feedback
Furthermore, the system excels in:
■ Spectrum analysis with 2 nm accuracy.
Spectral resolution of fluorochromes
with very close emission maximums.
laser ray intensity control provides excellent high excitation
■ High-speed spectroscopy, 100 nm/ms.
stability. The original design makes FV 1000 an optimal
microscopic device for methods such as FRAP, FLIP, FRET and
■ High imaging speed, 16 images/second
at 256 x 256 resolution (4,000 Hz).
other experiments using photon activation.
■ Wide spectrum range from UV to IR area.
The system of fluorescent microscopy
of dynamic processes in live cells
The main advantages of the Cell-R
system consist in the combination
of a unique fluorescent source
and an advanced control unit.
This combination results in highly
stable high-speed excitation with
accurate control permitting optimum timing and synchronizing.
Thanks to this, the system is
especially suitable for a wide
spectrum
of
applications
including highly demanding
methods such as deconvolution,
Foerster Resonance Energy
Transfer (FRET), multiple GFP,
etc. For more information, see
www.olympus.cz.
OLYMPUS C&S spol. s r.o., Czech Republic: Evropská 176, 160 41 Praha 6, tel.: +420 221 985 227, fax: +420 221 985 579
Slovakia: Trnavská 84, 821 02 Bratislava, tel.: +421 244 457 933-34, fax: +421 244 457 935 ● E-mail: [email protected], www.olympus.cz
26.6.2006 16:05:16
CONFERENCE
PROGRAMME
THURSDAY, JULY 6, 2006
25
26.6.2006 16:05:18
8.30 - 9.20
9.30 - 13.00
9.30
O-106
10.00
O-107
Keynote Lecture III
Chairperson
Radim J. Sram
Institute of Experimental Medicine AS CR,
Prague, Czech Rep.
David DeMarini
US Environmental Protection Agency, Research
Triangle Park, USA
New insights from an old assay: Salmonella
revisited
Symposium
IMPACT OF GENETIC VARIATION
ON INDIVIDUAL SUSCEPTIBILITY
Chairpersons:
Douglas Bell
NIEHS, Research Triangle Park, NC, USA
Emanuela Taioli
University of Pittsburgh Cancer Institute,
Pittsburgh, PA, USA
Douglas Bell
NIEHS, Research Triangle Park, NC, USA
Discovery and functional analysis of variation
in human damage response pathways
David Hein
University of Louisville School of Medicine,
Louisville, KY, USA
Role of N-Acetyltransferase genetic
polymorphisms in cancer risk
10.30
Coffee Break
11.00
O-108
Emanuela Taioli
University of Pittsburgh Cancer Institute,
Pittsburgh, PA, USA
Gene - gene environment interaction and cancer
risk
Wiliam W. Au
University of Texas Medical Branch, Galveston, TX,
USA
Genetic susceptibility, genomic instability and
usefulness in cancer therapy
Kei Nakachi
Radiation Effect Research Foundation, Hiroshima,
Japan
Molecular epidemiology of immune defense
against cancer therapy
Pavel Soucek
National Institute of Public Health, Prague,
Czech Rep.
Biotransformation enzymes as modifiers in
breast cancer
Pavel Vodicka
Czech Rep.
Genetic epidemiology of the sporadic colorectal
cancer in the Czech Republic
11.30
O-109
12.00
O-110
12.30
O-111
12.45
O-112
Room I
9.30 - 13.00
9.30
O-113
10.00
O-114
Symposium
RISK ASSESSMENT (ECNIS)
Chairpersons:
Rob Baan
IARC, Lyon, France
Paolo Vineis
Imperial College London, London, U.K.
Paolo Vineis
Imperial College London, London, U.K.
The contribution of the ECNIS network to
the validation of markers for risk assessment
Yasunobu Aoki
National Institute for Environmental Studies,
Tsukuba, Japan
Evaluation of in vivo mutagenicity using
transgenic animals
10.30
Coffee Break
11.00
O-115
Hans Kromhout
University of Utrecht, Utrecht, The Netherlands
Progress with integrated risk assessment?
Lee Byung-Mu
Sungkyunkwan University, Suwon, Korea
Toxikokinetic pattern of phtalic acid for risk
assessment approach
Ryeom Tai-Kyung
Korea Food and Drug Administration, Seoul, Korea
Human exposure assessment of heterocyclic
amines in foods
James E.Trosko
Michigan State University, East Lansing, MI, USA
Crises in the chemical genotoxicity paradigm:
Stem cells, cell-cell communication and systems
biology as ignored concepts
Christopher Portier
National Toxicology Program, Research Triangle
Park, NC, USA
Looking to the future: Where will “Omics” and
systems biology lead risk assessment
11.30
O-116
11.45
O-117
12.00
O-118
12.30
O-119
13.00 - 13.30 CLOSING CEREMONY
13:30
Lunch Break
26
26.6.2006 16:05:21
Room II
9.30 - 12.45
9.30
O-120
10.00
O-121
Symposium
OCCUPATIONAL EXPOSURE TO MUTAGENS
AND CARCINOGENS
Chairpersons:
Richard J. Albertini
University of Vermont, Burlington, VT, USA
Paolo Boffeta
IARC, Lyon, France
Richard J. Albertini
University of Vermont, Burlington, VT, USA
Molecular epidemiological studies in 1,3butadiene exposed Czech workers
Paolo Boffeta
IARC, Lyon, France
Burden of cancer attributable to occupational
exposures
10.30
Coffee Break
11.00
O-122
Tomonori Hayashi
Radiation Effects Research Foundation, Hiroshima,
Japan
Individuals at high risk of stomach cancer
among atomic-bomb survivors identified in RERF
immunogenome study
Pavel Rössner, Jr.
Czech Rep.
Oxidative stress markers in bus drivers
Hiroshi Kasai
University of Occupational and Environmental
Health, Kitakyushu, Japan
4-Oxo-2-hexenal, a volatile mutagen formed by
omega-3 fat peroxidation
Hannu Norppa
Finnish Institute of Occupational Health, Helsinki,
Finland
Chromosomal aberrations in workers exposed to
nitrotoluenes: effect of genetic polymorphism in
xenobiotic-metabolizing enzymes
Alessio Naccarati
Czech Rep.
Genotoxic effects and secondary oxidative stress
in workers occupationally exposed to styrene
Bozena Novotna
Czech Rep.
DNA damage in policemen from Prague
11.30
O-123
11.45
O-124
12.00
O-125
12.15
O-126
12.30
O-127
27
26.6.2006 16:05:21
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Siemens Medical Solutions that help
26.6.2006 16:05:21
LIST OF POSTERS
26.6.2006 16:05:23
LIST OF POSTERS
P-001
BIOMARKERS OF SUSCEPTIBILITY AND EFFECTS IN HUMANS OCCUPATIONALLY EXPOSED TO
URBAN AIR POLLUTION
Sabrina Angelini 1,2
Fabio Carbone 1; Francesca Maffei 1; Giorgio Cantelli-Forti 1; Rajiv Kumar 2; Giorgio Cantelli-Forti 1; Kari
Hemminki 2; Patrizia Hrelia 1; Kari Hemminki 2
Department of Pharmacology, University of Bologna, Bologna, Italy1
Division of Molecular Epidemiology, German Cancer Center, Heidelberg, Germany2
P-002
TP53 MUTATIONS AND ARG72PRO GENE POLYMORPHISM IN A HUNGARIAN PRIMARY LUNG
CANCER STUDY POPULATION
Livia Anna 1
Bernadette Schoket 1; Erika Gyorffy 1; Zoltan Gyori 2; Judit Segesdi 2; Janos Minarovits 2; Ibolya Soltesz 3;
Szilard Kostic 3; Attila Csekeo 3; Reetta Holmila 4; Kirsti Husgafvel-Pursiainen 4
Fodor Jozsef National Center for Public Health, Budapest, Hungary1; Johan Bela National Center for
Epidemiology, Budapest, Hungary2; Koranyi National Institute for Pulmonology, Budapest, Hungary3;
Finnish Institute of Occupational Health, Helsinki, Finland4
P-003
THE POSSIBILITY OF APPLICATION OF COMET ASSAY IN ASSESSMENT OF IN VIVO
CARDIOTOXICITY OF OXIDATIVE STRESS INDUCERS
Agnieszka Bartoszek 1
Anna Cieślak 1; Anita Piasek 1; Jolanta Łukowicz 1,2; Grażyna Peszyńska-Sularz 1,2; Stefan Popadiuk 2;
Włodzimierz Grajek 3;
Dept. of Pharmaceutical Technology and Biochemistry, Gdańsk University of Technology, Gdańsk, Poland1;
Medical University of Gdańsk, Gdańsk, Poland2; Dept. of Biotechnology and Food Microbiology, The August
Cieszkowski Agricultural University of Poznań, Poznań, Poland3
P-004
CYTOGENETIC BIODOSIMETRY: THE ROLE OF THE MICRONUCLEUS TEST IN MONITORING LOW
LEVEL EXPOSURE TO IONIZING RADIATION
Claudia Bolognesi 1
Cristina Balia 1; Elena Giordano 1; Paola Roggieri 1; Maria Concetta Nucci2; Vittorio Lodi 2; Francesco
Saverio Violante3
Environmental Carcinogenesis Unit, National Institute for Research on Cancer, Genova, Italy1; Servizio di
Sicurezza Igiene e Medicina del Lavoro, Universitŕ, Bologna, Italy2; Unitŕ Operativa Medicina del Lavoro,
Universita, Bologna, Italy3
P-005
SEPARATION AND DETECTION OF FLUORESCENCE LABELED RNA MODIFICATIONS BY
CAPILLARY ELECTROPHORESIS WITH LASER-INDUCED FLUORESCENCE DETECTION
Michael G. Cornelius 1,2
Manfred Wiessler 1; Heinz H. Schmeiser 1
German Cancer Research Center, Division of Molecular Toxicology, Heidelberg, Germany1; Karolinska
Institute, Novum, Department of Biosciences and Nutrition, Huddinge, Sweden2
P-006
INFLUENCE OF XRCC1 ARG399GLN POLYMORPHISM ON BASAL AND RADIATION-INDUCED
MICRONUCLEUS FREQUENCIES IN HEAD AND NECK CANCER PATIENTS AND THEIR FIRST
DEGREE RELATIVES
Erdem Coskun1
Gonca Demirgcigil Cakmak1; Neslihan Aygun Kocabas1; Sema Burgaz1 ; Faik Cetindag2; Osman Sunter 2;
Hayriye Edinsel 2
Gazi University, Faculty of Pharmacy, Dept. of Toxicology , Ankara, Turkey1; Abdurrahman Yurtaslan
Oncology Hospital, Dept.of Radiation Oncology, Head and Neck Group, Ankara, Turkey2 ;
30
26.6.2006 16:05:25
LIST OF POSTERS
P-007
ROLE OF CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL LYMPHOCYTES IN POPULATIONBASED BIOMONITORING OF THE CZECH GENERAL POPULATION
Milena Cerna 1,2
Dana Ocadlikova 1; Hana Bavorova 1; Jiri Smid 1; Pavel Rossner 1
National Institute of Public Health, Prague, Czech Republic1; Charles Univ., 3rd Fac. Med., Prague, Czech
Republic2
P-008
POLYMORPHISMS IN DNA REPAIR GENES, BIOMARKERS OF DNA DAMAGE AND PROCESS OF
AGING
Zuzana Džupinková
Ladislava Wsólová ; Mária Dušinská
Research Base of Slovak Medical University, Bratislava, Slovakia
P-009
ASSESSMENT OF BIOMARKERS IN CERVICAL CANCER PATIENTS
Timea Farkasová 1
Soňa Gurská 1; Alena Gábelová 1; Zuzana Macháčková 2; Pavol Lukačko 2
Cancer Research Institute, Bratislava, Slovakia1; National Cancer Institute, Bratislava, Slovakia2
P-010
RELATIONSHIP BETWEEN DNA REPAIR GENETIC POLYMORPHISMS AND SMOKING-RELATED
DNA ADDUCT LEVELS IN HUMAN BRONCHIAL TISSUES
Erika Gyorffy 1
Livia Anna 1; Bernadette Schoket 1; Zoltan Gyori 2; Judit Segesdi 2; Janos Minarovits 2; Ibolya Soltesz 3;
Szilard Kostic 3; Attila Csekeo 3
Jozsef Fodor National Center for Public Health, Budapest, Hungary1; Bela Johan National Center for
Epidemiology, Budapest, Hungary2; Koranyi National Institute of Pulmonology, Budapest, Hungary3
P-011
PAPILLARY THYROID CARCINOGENESIS AMONG ATOMIC-BOMB SURVIVORS
Kiyohiro Hamatani
Hidetaka Eguchi; Masataka Taga; Kei Nakachi
Radiation Effects Research Foundation, Hiroshima, Japan
P-012
PROTEIN ADDUCTS FORMED IN VITRO BETWEEN HUMAN HAEMOGLOBIN AND DEUTERIUM
LABELLED PHTHALIC ACID ANHYDRIDE, CHARACTERIZED WITH MASS SPECTROMETRY
Marina C Jeppsson
Bo A.G Jönsson; Christian H Lindh
Department of occupational and environmental medicine, Lund, Sweden
P-013
THE ABILITY OF Γ-TOCOPHEROL TO DECREASE OXIDATIVE DNA DAMAGE
Clara Johansson 1
Lennart Möller 1; Jonas Nygren 2; Bengt Vessby 3
Unit for Analytical Toxicology, Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge,
Sweden1; Laboratory of Molecular and Cellular Toxicology, Department of Industrial Hygiene and
Toxicology, Finnish Institute of Occupational Health, Helsinki, Finland2; Department of Public Health and
Caring Sciences, Clinical Nutrition and Metabolism, Uppsala University, Uppsala, Sweden3
P-014
THE EFFECTS OF GSTM1 AND GSTT1 POLYMORPHISMS ON MICRONUCLEUS FREQUENCIES IN
HUMAN LYMPHOCYTES IN VIVO
Raluca Mateuca 1
Mathieu Roelants 2; Michael Fenech 3; Micheline Kirsch-Volders 4
Vrije Universiteit Brussel (VUB), Brussels, Belgium1; Vrije Universiteit Brussel (VUB), Brussels, Belgium2;
CSIRO Health Sciences and Nutrition, Adelaide, Australia3; Vrije Universiteit Brussel (VUB), Brussels,
Belgium4
31
26.6.2006 16:05:25
LIST OF POSTERS
P-015
LONG-TERM ELEVATION OF INFLAMMATORY MARKERS AMONG ATOMIC-BOMB SURVIVORS:
ACCELERATED IMMUNOLOGICAL AGING
Yukari Morishita
Tomonori Hayashi; Hiroko Nagamura; Mayumi Maki; Yoshiko Kubo; Yoichiro Kusunoki; Kei Nakachi
P-016
DNA-ADDUCT COMPARISON BETWEEN 3- AND 2-NITROBENZANTRONES IN RATS
Nagy Eszter 1
Duan Jianxin 1; Zeisig Magnus 1; Möller Lennart1; Adachi Shuichi 2
Takamura-Enya Takeji 3
Karolinska Institutet, Huddinge, Sweden1; Sagami Women's University, Kanagawa, Japan2; National
Cancer Center Research Institute, Tokyo, Japan3
P-017
THE USE OF BIOMARKERS OF EXPOSURE TO POLYCYCLIC AROMATIC HYDROCARBONS (PAHs)
IN PSORIATIC PATIENTS UNDERGOING COAL-TAR THERAPY
Anna Pastorková 1
Marek Brabec 1; Milena Černá 1,2; Lenka Borská 3; Zdeněk Fiala 3
National Institute of Public Health, Prague, Czech Republic1; Charles Univ., 3rd. Fac. Med., Prague, Czech
Republic2; Charles Univ., Fac. Med., Hradec Králové, Czech Republic3
P-018
ASSESSMENT OF GENOTOXICITY IN RUBBER INDUSTRY WORKERS
Beatriz Pérez-Cadahía 1,2
Josefina Mendez 1; Blanca Laffon 1,2; Eduardo Pásaro 2; Joao Paulo Teixeira 3; Susana Silva 3; Joana RomaTorres 3; Olga Mayan 3
Department of Cell and Molecular Biology, University of A Coruna, La Coruna, Spain1; Toxicology Unit,
University of A Coruna, La Coruna, Spain2; Environmental Health and Toxicology Department, National
Institute of Health, Porto, Portugal3;
P-019
MOLECULAR AND GENETIC CHARACTERISTICS OF SPORADIC COLORECTAL CANCER IN
THE CZECH REPUBLIC
Veronika Polakova 1
Monika Hanova 1; Elena Tulupova 1; Jana Slyskova 1; Alessio Naccarati 1; Barbara Pardini 1; Pavel Erik
Vodicka 1; Ludmila Vodickova 1,2; Jan Novotny 3; Kari Hemminki 4
Institute of Experimental Medicine, Academy of Sciences of Czech Republic, Praha, Czech Republic1;
National Institute of Public Health, PRAHA, Czech Republic2; First Medical Faculty, Charles University,
Praha, Czech Republic3; German Cancer Research Centre, Heidelberg, Germany4
P-020
DETECTION OF A METHYLGLYOXAL DNA ADDUCT BY HPLC COUPLED TO TANDEM MASS
SPECTROMETRY
Jean-Luc Ravanat
Lucie Mollard; Carine Badouard; Alain Favier
CEA Grenoble, Grenoble, France
P-021
CHROMOSOMAL ABERRATIONS MEASURED BY FISH PAINTING AS BIOMARKER OF AIR
POLLUTION EXPOSURE – PROSPECTIVE STUDY OF POLICEMEN IN PRAGUE
Andrea Rössnerová
Olena Beskyd; Pavel Rössner; Ivo Solanský; Radim J. Sram
Institute of Experimental Medicine, Prague, Czech Republic
P-022
32P-POSTLABELLING ANALYSIS OF THYMINE DIMER IN URINE FROM CHILDREN
OF DIFFERENT SKIN TYPES
T I Sandberg 1
D.Segerbäck 1; N. Kotova 2; P.C.Turner 3; C.P.Wild 3; A.Sylla 4; M.S.Diallo 4
Karolinska Institute, Huddinge, Sweden1; Stockholm University, Stockholm, Sweden2; University of Leeds,
Leeds, United Kingdom3 ; Institut Pasteur de Guinée, Kindia, Republic of Guinea4
32
26.6.2006 16:05:26
LIST OF POSTERS
P-023
DEVELOPMENT OF A MICRONUCLEUS ASSAY FOR MOUSE ALVEOLAR TYPE II CELLS
Hanna Sandvik 1
Ghita Falck 1; Tiina Santonen 1; Hannu Norppa 1; Julia Catalán 1,2
Finnish Institute of Occupational Health, Helsinki, Finland1; University of Zaragoza, Zaragoza, Spain2
P-024
DEVELOPMENT OF A LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY METHOD
FOR THE DETECTION AND QUANTITATION OF BENZO[A]PYRENE DERIVED DNA ADDUCTS
Rajinder Singh 1
Peter B Farmer 1; Margaret Gaskell 1; Rachel C Le Pla 1; Balvinder Kaur 1; Ali Azim-Araghi 1; Jonathan
Roach 1; George Koukouves 2; Vassilis L Souliotis 2; Soterios A Kyrtopoulos 2
Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester, University Road, Leicester,
LE1 7RH, United Kingdom1, Institute of Biological Research and Biotechnology, National Hellenic Research
Foundation, Athens, Greece2
P-025
INTER-STRAND DNA CROSS-LINKS IN THE PIG SKIN TREATED WITH SULPHUR MUSTARD
IN VIVO
Rudolf Stetina 1
Frantisek Oplustil 2
Faculty of Military Health Sciences, Hradec Kralove, Czech Republic , Hradec Kralove, Czech Republic1,
Military Technical Institute for Protection, Brno, Czech Republic, Brno, Czech Republic2
P-026
CARCINOGENICITY TESTING BY A FLUORESCENCE-BASED RECOMBINATION ASSAY IN HUMAN
CELLS.
Lisa Wiesmueller
Nuray Akyuz; Cindy Baumann
Department of Obstetrics and Gynaecology at the University of Ulm, Ulm, Germany
P-027
THE FORMATION OF HYDROXYETHYL VALINE IN HOSPITAL STERILIZATION WORKERS:
EFFECTS OF GENETIC POLYMORPHISMS OF GLUTATHIONE S-TRANSFERASE THETA AND
MICROSOMAL EPOXIDE HYDROLYSE
Kuen-Yuh1
Kuen-Yuh Wu 1; Chia-Fang Wu 1; Ming-Fong Chen 1; Lan-Tyi Duann 2; Tsung Jen Cheng 3
Division of Environmental Health and Occupational Medicine, National Health Research Institutes,
Zhunan Town, Miaoli County, Taiwan1; National Taichung Nursing College, Taichung, Taiwan2; Institute of
Occupational Medicine and Hygiene, School of Public Health, Taiwan University, Taipei, Taiwan3
P-028
ACCELERATED 32P-HPLC FOR LIPOPHILIC DNA ADDUCTS
Magnus Zeisig 1
Eszter Nagy 1; Lennart Moller 1
Karolinska Institutet, Dept. of Biosciences and Nutrition, Huddinge, Sweden
P-029
COMPARISON BETWEEN THE FREQUENCIES OF STABLE AND UNSTABLE CHROMOSOMAL
ABERRATIONS IN GENERAL POPULATION OF THE REPUBLIC CROATIA
Davor Zeljezic 1
Aleksandra Fucic 1; Nevenka Kopjar 1; Vilena Kasuba 1; Ruzica Rozgaj 1; Snjezana Ramic 1; Joe Nathan
Lucas 2
Institute for Medical Research and Occupational Health, Zagreb, Croatia1; ChromoTrax, Inc., Frederick
Innovative Technology Center, Federick, United States2
P-031
FACTORS AFFECTING BACKGROUND AND INDUCED LEVELS OF SCE AND MICRONUCLEI
IN FROZEN HUMAN LYMPHOCYTES
Andrea Zijno
Francesca Saini; Ester Siniscalchi; Riccardo Crebelli
Istituto Superiore di Sanitŕ, Rome, Italy
33
26.6.2006 16:05:26
LIST OF POSTERS
P-032
AN IN VITRO INVESTIGATION INTO LEVELS OF DNA DAMAGE IN LYMPHOCYTES FROM
MOTHERS AND BABIES’ CORD BLOOD
Diana Anderson 1
Natalie P Wyatt 1; Cesar Falque-Gonzalez 1; Diane Farrar 2; Derek Tuffnell 2; Donald Whitelaw 2
Dept. of Biomedical Sciences / The University of Bradford, Bradford, United Kingdom1; NHS Trust /
Bradford Royal Infirmary, Bradford, United Kingdom2
P-033
NEWGENERIS: A NEW FP6 INTEGRATED PROJECT ON NEWBORNS AND GENOTOXIC EXPOSURE
RISKS
Maria Botsivali
NewGeneris Communication and Dissemination Office, National Hellenic Research Foundation, Institute of
Biological Research and Biotechnology, Athens, Greece
P-034
THE INFLUENCE OF DNA REPAIR GENES POLYMORPHISMS ON DNA DAMAGE AND ITS REPAIR
IN CHILDREN ENVIRONMENTALLY EXPOSED TO LEAD
Lucyna Kapka 1
Danuta Mielzynska 1; Marcin Kruszewski 2
Institute of Occupational Medicine and Environmental Health, Sosnowiec, Poland1; Institute of Nuclear
Chemistry and Technology, Warsaw, Poland2
P-035
EUROPEAN NETWORK ON CHILDREN’S SUSCEPTIBILITY AND EXPOSURE TO ENVIRONMENTAL
GENOTOXICANTS CHILDRENGENONETWORK (QLK4-CT-2002-02198)
Lisbeth E. Knudsen
Marie Pedersen
University of Copenhagen, Denmark
P-036
ETHICAL ISSUES RELATED TO INDIVIDUALISED BIOMONITORING STUDIES OF CHILDREN
Lisbeth E. Knudsen
Marie Pedersen
P-037
INVESTIGATION OF ANTIOXIDANT STATUS AND GST POLYMORPHISM ACCORDING TO DEGREE
OF AUTISM IN CHILDREN WITH DEVELOPMENTAL DELAY
Eunju Park 1
Su-Yeon Kim 1; Kyung-Im Jeon 1; Bo-Young Seo 1; Hyun-Jin Lee 1; Han-Woo Lee 2; Jung-Hwa Choi 3
Dept. of Food and Nutrition, Kyungnam Univeristy, Masan, Korea1; Div. of Social Welfare, Jinju
International University, Jinju, Korea2; Div. of Food Science, Jinju International University, Jinju, Korea3
P-038
GENOTOXIC EFFECTS IN THE EASTERN MUDMINNOW (UMBRA PYGMAEA L.) AFTER EXPOSURE
TO RHINE WATER USING THE SCE AND COMET ASSAY; A COMPARISON BETWEEN 1978
AND 2005
Gerrit M. Alink 1
Bert Spenkelink 1; Joris T. K. Quik 1,2; Serge G. P. Rotteveel 2; Hannie L. Maas 2; Wim Hoogenboezem 3;
Eric J. M. Penders 3
Chair Toxicology, Wageningen University, Wageningen, Netherlands1; RIZA Institute for Inland Water
Management and Waste Water Treatment, Lelystad, Netherlands2 ; Het Waterlaboratorium, RIWA Rhine
Water Works, Nieuwegein, Netherlands3
P-039
EVALUATION OF DNA DAMAGE IN MURINE FIBROBLASTS TREATED WITH CIGARETTE SMOKE
CONDENSATE
Cristina Andreoli 1
Floriana Flamma 1; Francesco Mercati 1; Angela Martino 1; Maurizio Mauro 2; Fabio Caradonna 2; Giulia
Sciandrello 2
Research Department, BAT-Italia SpA, Naples, Italy1 ; Cellular and Developmental Biology Department,
University of Palermo, Palermo, Italy2
34
26.6.2006 16:05:26
LIST OF POSTERS
P-040
INCREASED GENOMIC INSTABILITY IN PERIPHERAL LYMPHOCYTES OF DOWN SYNDROME
PATIENTS DUE TO INCREASED RATE OF CHROMOSOME NON-DISJUNCTION
Konstadinos Andrianopoulos
Georgia Stephanou; Michael Tyrakis; Asimina Kouloumenta; Constantinos Andrianopoulos;
Nikos A. Demopoulos
Division of Genetics, Cell and Developmental Biology, Department of Biology, University of Patras, Patras,
Greece
P-041
POLY(ADP-RIBOSE) POLYMERASE-1 (PARP-1) ANTAGONIZES TOPOISOMERASE I-DEPENDENT
RECOMBINATION STIMULATION BY P53
Cindy Baumann 1
Lisa Wiesmüller 1; Gisa S Boehden 2; Alexander Bürkle 3
Universitätsfrauenklinik, Ulm, Germany1; Heinrich-Pette-Institut für Experimentelle Virologie und
Immunologie , Hamburg, Germany2; Molecular Toxicology Group, Department of Biology, Konstanz,
Germany3
P-042
ASSESSMENT OF ANTIDIABETIC AGENT PIOGLITAZONE GENOTOXICITY IN RATS BY COMET
ASSAY
Abdulkerim Bedir 1
Yuksel Aliyazicioglu 1; Zafer Yurdakul 1; Mehmet Uysal 1; Duygu Erol Suvaci 1; Ali Okuyucu 1; Zeliha Cansel
Özmen 1; Birşen Bilgici 1; Muhlise Alvur 1; Hakki Kahraman 2; Murat Hökelek 3
Ondokuz Mayis University, Faculty of Medicine, Department of Biochemistry, Samsun, Turkey1; Ondokuz
Mayis University, Faculty of Medicine, Department of Internal medicine, Samsun, Turkey2; Ondokuz Mayis
University, Faculty of Medicine, Department of Microbiology, Samsun, Turkey3
P-043
THE EUKARYOTIC PSO2P/SNM1P FAMILY REVISITED: IN SILICO ANALYSES OF PSO2P A, B
AND PLASMODIUM GROUPS
Diego Bonatto 1
Martin Brendel 2; João Antonio Pęgas Henriques 3
Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil 1; Universidade Estadual de Santa Cruz,
Ilheus, Brazil2; Universidade de Caxias do Sul, Caxias do Sul, Brazil3
P-044
DEGREE RELATIVES
Erdem Coskun 1
Neslihan Aygun Kocabas 1; Gonca Demircigil Cakmak 1; Sema Burgaz 1; Faik Cetindag 2; Osman Sunter 2;
Hayriye Edinsel 2
Gazi University Faculty of Pharmacy Department of Toxicology, Ankara, Turkey1; Abdurrahman Yurtaslan
Oncology Hospital Department of Radiation Oncology Head and Neck Group, Ankara, Turkey2
P-045
THE HIGH LEVEL OF SPONTANEOUS ENDOREDUPLICATION IN EM9 MUTANT CELL LINE
CORRELATES WITH A LOW LEVEL OF DNA METHYLATION
Inmaculada Dominguez1
Gloria Cantero 1; Santiago Mateos 1; Nuria Pastor 1; Manuel Luis Orta 1; Felipe Cortés 1
Department of Cell Biology, Faculty of Biology, University of Sevilla, Sevilla, Spain
P-046
OXIDATIVE DAMAGE TO DNA AND OXIDATIVE STRESS IS INVOLVED IN EARLY STAGES OF
COLON CANCER DEVELOPMENT
Daniel Gackowski 1
Rafal Rozalski 1; Agnieszka Siomek 1; Anna Szpila 1; Tomasz Dziaman 1; Jolanta Guz 1; Karol Bialkowski 1;
Marek Foksinski 1; Ryszard Olinski 1; Zbigniew Banaszkiewicz 2; Arkadiusz Jawien 2
Department of Clinical Biochemistry, Collegium Medicum Nicolaus Copernicus University, Bydgoszcz,
Poland1; Clinic of Surgery, Collegium Medicum Nicolaus Copernicus University, Bydgoszcz, Poland2
35
26.6.2006 16:05:26
LIST OF POSTERS
P-047
CORRELATION BETWEEN DNA ADDUCT FORMATION AND CYTOGENETIC DAMAGE INDUCTION
IN MAMMALIAN CELLS EXPOSED TO ACRYLAMIDE AND GLYCIDAMIDE
Gonçalo Gamboa da Costa 1
Nuno G. Oliveira 2; Célia Martins 3; Marta Pingarilho 3; Vanda Martins 3; José Rueff 3; Jorge F. Gaspar 3;
M. Matilde Marques 4; Frederick A. Beland 5; Mona I. Churchwell 5; Daniel R. Doerge 5
The Institute of Cancer Research, Sutton, Surrey, United Kingdom1; Faculty of Pharmacy, University of
Lisbon, Lisbon, Portugal2; Department of Genetics, Faculty of Medical Sciences, UNL, Lisbon, Portugal3;
Centro de Química Estrutural, Technical University of Lisbon, Lisbon, Portugal4; National Center for
Toxicological Research, Jefferson, Arkansas, United States5
P-048
DEVELOPMENT OF A HIGH-THROUGHPUT IMMUNOCHEMICAL ASSAY FOR THE ASSESSMENT
OF FUNCTIONAL METHYLGUANINE-DNA-METHYLTRANSFERASE (MGMT) LEVELS
Panagiotis Georgiadis
Vassiliki Pletsa; Stella Kaila; Soterios A Kyrtopoulos
National Hellenic Research Foundation, Athens, Greece
P-049
CHROMOSOMAL INSTABILITY IN SALIVARY GLAND TUMORS – PRELIMINARY RESULTS
Maciej Giefing 1
Malgorzata Rydzanicz 1; Roksana Cegla 1; Maciej Kujawski 1; Krzysztof Szyfter 1,2; Malgorzata Wierzbicka 1,2
Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland1 ; Department of
Otolaryngology, Medical University of Poznan, Poznan, Poland2
P-050
NEW SYSTEM FOR THE PRODUCTION OF MAMMALIAN DNA POLYMERASE KAPPA IN E. COLI:
PURIFICATION, CHARACTERIZATION AND IMMUNOCHEMISTRY OF THE HUMAN AND MOUSE
POLK PROTEINS
Petr Grúz
Naoko Niim; Akira Sassa; Takehiko Nohmi
P-051
ROLE OF DNA POLYMERASE DELTA IN THE REPAIR OF PSORALEN-PHOTOINDUCED DNA
INTERSTRAND CROSS-LINKS IN SACCHAROMYCES CEREVISIAE
Joao Antonio Pegas Henriques 1,4
Jaqueline M Cardone 1; Diego Bonatto 2; Martin Brendel 3
Federal University of Rio Grande do Sul/Biotechnology Center, Porto Alegre, Brazil1; University of Caxias
do Sul/Biotechnology Institute, Caxias do Sul, Brazil2; State University of Santa Cruz, Ilhéus, Brazil3;
University of Caxias do Sul/Biotechnology Institute, Caxias do Sul, Brazil4
P-052
GENETIC INSTABILITY IN MIGRANT/SEASONAL FARMWORKER (MSF) CHILDREN OF MEXICAN
ORIGIN RESIDING IN TWO TEXAS REGIONS.
María A. Hernández-Valero 1
Lovell A. Jones 2; Kevin J. Wolfe 3; Adele Guerin 3; Sherif Z. Abdel-Rahman 3
Department of Epidemiology & Center for Research on MInority Health, UT M.D. Anderson Cancer Center,
Houston, United States1; Department of Health Disparities Research & Center for Research on Minority
Health, UT M.D. Anderson Cancer Center, Houston, United States2; Department of Preventive Medicine &
Community Health, UT Medical Branch, Galveston, United States3
P-053
INDUCTION OF COINCIDENT MITOTIC RECOMBINATION AT UNLINKED LOCI IN YEAST
AT FREQUENCIES HIGHER THAN PREDICTED FOR INDEPENDENT EVENTS
George R. Hoffmann
Kathryn M. Freeman
College of the Holy Cross, Worcester, MA, United States
36
26.6.2006 16:05:26
LIST OF POSTERS
P-054
ERROR-PRONE AND ERROR-FREE NONHOMOLOGOUS END-JOINING FOR REPAIRING DNA
DOUBLE STRAND BREAKS IN HUMAN CELLS
Masamitsu Honma
Mayumi Sakuraba; Tomoko Koizumi; Yoshio Takashima; Hiroko Sakamoto; Makoto Hayashi
P-055
GENOTOXIC POTENTIAL OF ORGANOPHOSPHOROUS PESTICIDES PARATHION, PARAOXON
AND DIMEFOX
Irena Hreljac
Irena Zajc; Bojana Žegura; Tamara Lah Turnšek; Metka Filipič
National Institute of Biology, Department of Genetic Toxicology and Cancer Biology, Ljubljana, Slovenia
P-056
THE RELEVANCE AND SIGNIFICANCE OF N7-METHYLGUANINE AND N3-METHYLADENINE DNA
ADDUCTS ORIGINATING BY THE TOBACCO SMOKE METHYLATING CARCINOGENS
Jiří Chadt 1
Pavel Vodička 1; Robert Nilsson 2
Institute of Experimental Medicine, AS CR, Prague, Czech Republic1; Department of Genetics, Stockholm
University, Stockholm, Sweden2
P-057
EFFECT OF ALKB MUTATION ON MMS-INDUCED MUTAGENESIS IN E.COLI AB1157 STRAINS
Albert Jaworski
Jadwiga Nieminuszczy; Anna Sikora; Elzbieta Grzesiuk
Institute of Biochemistry and Biophysics PAS, Warszawa, Poland
P-058
GENOTOXICITY OF IRINOTECAN IN HUMAN LYMPHOCYTES ASSESSED USING
THE CYTOKINESIS-BLOCK MICRONUCLEUS ASSAY IN VITRO
Nevenka Kopjar 1
Snježana Ramie 1; Mirta Milie 1; Vesna Pavlica 2
Institute of Medical Research and Occupational Health, ZAGREB, Croatia1; University Hospital for Tumors,
Zagreb, Croatia2
P-059
EVALUATION OF TRYPTOPHOL TOXICITY IN HUMAN LYMPHOCYTES USING THE CYTOKINESISBLOCK MICRONUCLEUS ASSAY IN VITRO
Amalina Šafranie 1
Višnja Baeun-Družna1; Ivan Kosalec 2; Snježana Ramie3; Nevenka Kopjar 3
Faculty of Food Technology and Biotechnology, University of Zagreb, Croatia1; Faculty of Pharmacy and
Biochemistry, University of Zagreb, Zagreb, Croatia2; Institute of Medical Research and Occupational
Health, Zagreb, Croatia3
P-060
INFLUENCE OF THE DIETARY FACTORS ON THE RATE OF MODIFICATION OF OXIDATIVE DNA
DAMAGE REPAIR IN NEWBORN PIGS
Pawel Kowalczyk 1
Aleksandra Bielen 1; Barbara Tudek 1; Daniel Laubitz 2; Romuald Zabielski 3
Institute of Biochemistry and Biophysics PAS, Warsaw, Poland 1; The Kielanowski Institute of Animal
Physiology and Nutrition, Jablonna, Poland2; Department of Physiological Sciences, Faculty of Veterinary
Medicine, Warsaw Agricultural University, Warsaw, Poland3
P-061
EFFECTS OF VITAMIN C ON OXIDATIVE DNA DAMAGE TO HUMAN LYMPHOCYTES AND HELA
CELLS
Adela Lopez de Cerain 1
Amaya Azqueta 1; Andrew R Collins 2
Food Sciences & Toxicology, University of Navarra, Pamplona, Spain1 ; Department of Nutrition, University
of Oslo, Oslo, Norway2
37
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LIST OF POSTERS
P-062
SPONTANEUS AND BLEOMYCIN-INDUCED MICRONUCLEI IN HUMAN LYMPHOCYTES:
CORRELATING POLYMORPHISMS IN DNA REPAIR GENES WITH MUTAGEN SENSITIVITY
Francesca Maffei 1
Sabrina Angelini 1; Fabio Carbone 1; Giorgio Cantelli-Forti 1; Patrizia Hrelia 1;
Rajiv Kumar 2; Kari Hemminki 2
Department of Pharmacology, University of Bologna, Bologna, Italy1; Division of Molecular Genetic
Epidemiology, German Cancer Research Center, Heidelberg, Germany2
P-063
DNA DOUBLE-STRAND BREAKS REPAIR AS A FUNCTION OF THE SUBSTRATE ENDS IN NORMAL
AND CANCER HUMAN CELLS
Mariusz Malinowski 1,2
Tomasz Poplawski 1; Janusz Blasiak 1; Elzbieta Pastwa 3
Department of Molecular Genetics, University of Lodz, Lodz, Poland1; Department of Molecular and
Medical Biophysics, Medical University of Lodz, Lodz, Poland2; Molecular Genetics Department, Medical
University of Lodz, Lodz, Poland 3
P-064
INDUCTION OF GENETICS VARIABILYTY IN OFFSPRING OF MICE, GAMMA-IRRADIATED IN
DOSE RANGE FROM 1 GY TO 3 GY
Andrey E. Myazin
Lylyvati K. Ramayia; Gadjiramazan O. Shaikhaev; Vladimir A. Shevchenko; Marina D. Pomerantseva
Institute of general genetics, Moscow, Russian Federation
P-065
THE FREQUENCY OF GC TO AT BASE SUBSTITUTIONS ARISING IN CAA - TREATED DNA IS ALKB
-DEPENDENT
Jadwiga Nieminuszczy
Agnieszka M Maciejewska; Jaroslaw T Kusmierek; Elzbieta Grzesiuk
P-066
INFLUENCE OF OCCUPATIONAL EXPOSURE TO ENVIRONMENTAL POLYCYCLIC AROMATIC
HYDROCARBONS ON DNA REPAIR
Agnieszka Panek 1
Antonina Cebulska-Wasilewska 1,2; Ivan Kalina 3; Jozef Zidzik 3; Peter B Farmer 4
Department of Radiation and Environmental Biology, The H.Niewodniczański Institute of Nuclear Physics
PAN, Kraków, Poland1; Chair of the Epidemiology and Preventive Medicine, CM UJ, Kraków, Poland2;
Department of Molecular Biology of the P.J. Šafárik University, Košice, Slovakia3; Cancer Biomarkers and
Prevention Group, University of Leicester, Leicester, United Kingdom4
P-067
ROLE OF O6 METHYLGUANINE DNA METHYLTRANSFERASE (MGMT) IN METHYLATING AGENT–
INDUCED APOPTOSIS
Vassiliki Pletsa 1
Meropi Patrinou-Georgoula 1; Apostolia Guialis 1; Soterios Kyrtopoulos 1; Wynand Roos 2; Filippo Rosselli 3
National Hellenic Research Foundation, Athens, Greece1; Division of Applied Toxicology-Institute of
Toxicology, Mainz, Germany 2; Institut Gustave Roussy, Villejuif, France3
P-068
INHIBITION OF HOMOLOGOUS RECOMBINATION (HR) BY P53 IS DEPENDENT ON
PHOSPHORYLATION ON SERINE 15
Anja Restle
Christine Janz; Lisa Wiesmüller
Universitätsfrauenklinik, Ulm, Germany
38
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LIST OF POSTERS
P-069
ANTICANCER PML PROTEIN DIFFERENTIALLY REGULATES GENE PROMOTERS AND PARTIALLY
COLOCALIZES WITH WRN HELICASE
Marek Rusin 1
Rasa Vaitiekunaite1,2; Dorota Butkiewicz1; Agata Baginska1; Malgorzata Krzesniak1
Department of Tumor Biology, Center of Oncology, Maria Skłodowska-Curie Memorial Institute, Gliwice,
Poland 1; Institute of Oncology, Vilnius University, Vilnius, Lithuania2
P-070
INCREASED SPEED AND EFFICIENCY OF DNA STRAND BREAK REJOINING BY TEMPORARY
NON-GENOTOXIC STIMULATION OF POLY(ADP-RIBOSE) SYNTHESIS IN HUMAN CELLS IN
VITRO
Nadezhda I. Ryabokon 1,2
Joanna Rzeszowska-Wolny 1; Rose I. Goncharova 2; Gunars Duburs 3
Centre of Oncology, M. Sklodowska-Curie Memorial Institute, Gliwice, Poland1; Institute of Genetics
and Cytology, National Academy of Sciences of Belarus, Minsk, Belarus2; Latvian Institute of Organic
Synthesis, Riga, Latvia3
P-071
INFLUENCE OF POLYMORPHISMS IN DNA REPAIR GENES ON REPAIR KINETICS, MEASURED BY
THE COMET ASSAY
Charlotta M Ryk 1
Sai-Mei Hou 1; Bo Lambert 1; Rajiv Kumar 1,3; Christopher P Wild 2; Michael N Routledge 2;
Department of Biosciences and Nutrition / Karolinska Institute, Stockholm, Sweden, Sweden1; Molecular
Epidemiology Unit, The LIGHT Laboratories / University of Leeds, Leeds, England, England2; Division of
Molecular Genetic Epidemiology, German Cancer Research Centre, Heidelberg, Germany, Germany3
P-072
CELLULAR RESPONSE TO DNA LESIONS INDUCED BY DOXORUBICIN IN HUMAN FIBROBLASTS
PROFICIENT AND DEFICIENT IN NUCLEOTIDE EXCISION REPAIR
Jenifer Saffi 1,3
Mateus H Agnoletto 1; Temenouga N Guecheva 1; Joao Antonio Pegas Henriques 1,3; Luis FZ Batista 2;
Heleotonio Carvalho 2; G P Amarante-Mendes 2; Carlos F Menck 2
Federal University of Rio Grande do Sul, Porto Alegre,Brazil1; University of Sao Paulo, Sao Paulo, Brazil1,2;
Lutheran University of Brazil, Canoas, Brazil3
P-073
PERSISTENT GENOMIC INSTABILITY BY ARSENIC EXPOSURE IN V79 CHINESE HAMSTER CELLS
Giulia Sciandrello
Maurizio Mauro; Irene Catanzaro; Fabio Caradonna; Giusi Barbata
University of Palermo, Palermo, Italy
P-074
THE DEL ASSAY DETECTS CARCINOGENS
Robert H. Schiestl
University of California, Los Angeles, Los Angeles, United States
P-075
THE EFFECT OF ANTIOXIDANTS ON GENETIC INSTABILITY AND CANCER IN ATAXIA
TELANGIECTASIA
Robert H. Schiestl
Ramune Reliene
P-076
INFLUENCE OF CMF CHEMOTHERAPY ON THE LEVEL OF DNA DAMAGE IN LEUKOCYTES
OF WHOLE BLOOD IRRADIATED IN VITRO BY X-RAYS
Nikolai P. Sirota 1
Elena A. Kuznetsova 1; Irina G. Zakharova 2
Institute of Theoretical and Experimental Biophysics, RAS, Pushchino, Russian Federation1; Hospital of
Pushchino Research Center, Pushchino, Russian Federation2
39
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LIST OF POSTERS
P-077
MEASUREMENT OF P53-SPECIFIC DAMAGE FORMATION/REPAIR IN PERIPHERAL BLOOD
MONONUCLEAR CELLS FOLLOWING IN VITRO EXPOSURE TO MELPHALAN MAY PREDICT
CLINICAL OUTCOME IN PATIENTS WITH MULTIPLE MYELOMA
Vassilis L Souliotis 1
Soterios A Kyrtopoulos 1; Meletios A Dimopoulos 2; Athanasios Anagnostopoulos 2; Petros P Sfikakis 3
Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens,
Greece1 ; Department of Clinical Therapeutics, University of Athens School of Medicine, Athens, Greece2 ;
First Department of Propedeutic Medicine, University of Athens School of Medicine, Athens, Greece3
P-078
DEVELOPMENT AND VALIDATION OF A MULTIPLEX LONG QUANTITATIVE POLYMERASE CHAIN
REACTION TO MEASURE P53-SPECIFIC DAMAGE FORMATION AND REPAIR
Vassilis L Souliotis
Dimitris Typas; Margarita Bekyrou; Soterios A Kyrtopoulos
Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece
P-079
THE GLOBAL GENOME REPAIR SUBPATHWAY OF NUCLEOTIDE EXCISION REPAIR PLAYS A
CRUCIAL ROLE IN THE REPAIR OF MELPHALAN-INDUCED LESIONS
Hara G Episkopou1; Soterios A Kyrtopoulos1; Meletios A Dimopoulos2; Maria J Fousteri3; L HF Mullenders3;
Petros P Sfikakis4
Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens,
Greece1; Department of Clinical Therapeutics, University of Athens School of Medicine, Athens, Greece2;
Department of Toxicogenetics, Leiden University Medical Center, Leiden University, Leiden, Netherlands3;
First Department of Propedeutic Medicine, University of Athens School of Medicine, Athens, Greece4
P-080
THE INDUCTION OF MICRONUCLEI, SINGLE-STRAND BREAKS IN DNA AND DNA REPAIR IN
MICE EXPOSED TO 1,3-BUTADIENE BY INHALATION
Rudolf Stetina 1
Pavel Vodička 2; Ludmila Vodičková 2; Petr Šmerák 3; Ivo Barta 3
Faculty of Military Health Sciences, Hradec Kralove, Czech Republic, Hradec Kralove, Czech Republic1;
Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic,
Prague, Czech Republic2; Charles University, 3rd Medical Faculty,Prague, Czech Republic, Czech Republic3
P-081
SEASONAL VARIABILITY IN GENOTOXIC POTENTIAL OF URBAN AIR PARTICULATE MATTER
Oksana Sevastyanova
Jan Topinka; Zuzana Novakova; Katerina Hanzalova; Blanka Binkova; Radim J. Sram
Institute of Experimental Medicine AS CR, Prague, Czech Republic
P-082
GENOTOXICITY OF VANTEX-A PYRETROID INSECTICIDE IN MAMMALIAN CELLS
Fatma Unal 1
Serkan Yilmaz 2; Deniz Yuzbasıoglu 3; Hüseyin Aksoy 4; Mustafa Çelik 5
Gazi University, Ankara, Turkey1; Gazi University, Ankara, Turkey 2; Gazi University, Ankara, Turkey3;
Gazi University, Ankara, Turkey4; Sutcu Imam University, Kahramanmara, Turkey5
P-083
IN VIVO AND IN VITRO GENOTOXICITY TESTING OF THE ANTIFUNGAL DRUG TRIFLUCAN
Deniz Yuzbasıoglu
Hüseyin Aksoy; Mustafa Çelik; Fatma Unal ; Serkan Yilmaz
Gazi University, Ankara, Turkey
P-084
SCREENING OF FOOD ADDITIVE BENZOIC ACID FOR ITS GENOTOXIC PROPERTIES IN HUMAN
LYMPHOCYTES IN CULTURE
Serkan Yilmaz
Fatma Unal; Deniz Yuzbasıoglu
40
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LIST OF POSTERS
P-085
ANTIGENOTOXIC EFFECT OF XANTHOHUMOL DETECTED BY THE MODIFIED COMET ASSAY
IN PRECISION-CUT RAT LIVER SLICES
Janja Plazar
Metka Filipič, Geny M. M. Groothuis
Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Večna pot 111,
1000 Ljubljana, Slovenia; Pharmacokinetics and Drug Delivery, Groningen University Institute for Drug
Exploration, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands
P-086
PHOTOCHEMICALLY INDUCED DNA EFFECTS IN THE COMET ASSAY WITH EPIDERMAL CELLS
OF THE SKH-1 MICE AFTER SINGLE ADMINISTRATION OF DIFFERENT FLUOROQUINOLONES
AND 8-MOP PLUS UVA EXPOSURE
Uta Wirnitzer
Bayer HealthCare, Wuppertal, Germany
P-087
PROSTATECTOMY AFFECTS MRNA EXPRESSION OF DNA REPAIR GENES
Andreas Woelfelschneider 1,2
Peter Schmezer 1,2; Claudia Mayer 1,2; Helmut Bartsch 1,2; Odilia Popanda 1,2; Carmen Lilla 1,5;
Jenny Chang-Claude 1,5; Juergen Debus 3,4
German Cancer Research Center (DKFZ), Heidelberg, Germany1; Toxicology and Cancer Risk Factors,
Germany2; University of Heidelberg, Heidelberg, Germany3; Department of Clinical Radiology, Germany4;
Clinical Epidemiology, Germany5
P-088
THE INFLUENCE OF MICROCYSTIN-LR ON CELL PROLIFERATION, DNA DAMAGE AND
INTRACELLULAR REACTIVE OXYGEN SPECIES FORMATION IN DIFFERENT CELL LINES
Bojana Žegura
Meta Volčič; Tamara Turnšek Lah; Metka Filipič
Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia
P-089
STANDARD REFERENCE MATERIAL SRM1649A TOXICANTS MAY CONTRIBUTE TO
CARCINOGENESIS THROUGH BOTH GENOTOXIC AND NON-GENOTOXIC MECHANISMS OF
ACTION
Zdenek Andrysik 1,2
Sona Marvanova 1; Pavel Krcmar 1; Katerina Pencikova 1; Jiri Neca 1; Miroslav Ciganek 1; Miroslav
Machala 1; Jan Vondracek 1,2; Alois Kozubik 2; Brinda Mahadevan 3; William M. Baird 3
Veterinary Research Institute, Brno, Czech Republic1; Institute of Biophysics, Brno, Czech Republic2;
Oregon State University, Corvallis, United States3
P-090
ANALYSIS OF HYPERMETHYLATION IN TUMOUR SUPPRESSOR GENES FOR MOLECULAR
CHARACTERISATION OF BLADDER CANCER
Sonata Jarmalaite 1,2
Kristina Kurgonaite 1; Kestutis Suziedelis 1,3; Pertti Mutanen 2; Kirsti Husgafvel-Pursiainen 2; Dainius
Characiejus 3; Genovefa Chvatovic 3; Feliksas Jankevicius 3
Nature Faculty, Vilnius University, Vilnius, Lithuania1; Finnish Institute of Occupational Health, Helsinki,
Finland 2; Institute of Oncology, Vilnius University, Vilnius, Lithuania3
P-091
IN VITRO EVALUATION OF GENOTOXIC AND NONGENOTOXIC EFFECTS AND CHEMICAL
ANALYSIS OF RIVER SEDIMENT EXTRACTS AND THEIR FRACTIONS
Sona Marvanova 1
Katerina Pencikova 1; Jiri Neca 1; Miroslav Ciganek 1; Miroslav Machala 1; Anton Kocan 2; Jan Vondracek 1,3
Veterinary Research Institute, Brno, Czech Republic1; Institute of Preventive and Clinical Medicine,
Bratislava, Slovakia2; Institute of Biophysics, Brno, Czech Republic3
41
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LIST OF POSTERS
P-092
TIME COURSE STUDY OF THE CHANGES IN LEUKOCYTIC AND COLONOCYTIC DNA DAMAGE AND
PRENEOPLASTIC ABERRANT CRYPT FOCI OF DMH-INDUCED COLON CARCINOGENESIS IN F344
RATS
Eunju Park1
Kyung-Im Jeon 1; Bo-Young Seo 1; Hyun-Jin Lee 1; Su-Yeon Kim 1; Sun-Woo Jung 2; Kyung-Hea Lee 3
Dept. of Food and Nutrition, Kyungnam Univeristy, Masan, Korea1; Dept. of Biology, Changwon University,
Changwon, Korea2; Dept. of Food and Nutrition, Changwon Univeristy, Changwon, Korea3
P-093
DIETARY FLAVONOIDS, THEIR TOXICITY AND INFLUENCE ON DETOXYFING SYSTEM
Jasna Franekić Čolic
Ksenija Durgo 1; Jasna Franekic Čolic 1; Lidija Vukovic 2; Gordana Rusak 3; Maja Osmak 3
Faculty of Natural Science, Zagreb, Croatia1; Faculty of Food Technology and Biotechnology, Zagreb,
Croatia2; Ruđer Bošković Institute, Zagreb, Croatia3;
P-094
ANTIGENOTOXIC EFFECT OF THE POLYSACCHARIDE BETA-GLUCAN EXTRACTED FROM
AGARICUS BLAZEI AGAINST DAMAGE INDUCED BY BENZO(A)PYRENE
José Pedro Friedmann Angeli 1
Mario Sergio Mantovani 1; Vitor Basso Schul 1; Lucia Regina Ribeiro 2; Marilanda Ferreira Bellini 3;
Sandra de Aguiar Soares 4
Universidade Estadual de Londrina, Londrina, Brazil1; UNESP, Rio Claro, Brazil2; UNESP, Sao José do Rio
Preto, Brazil3; Universidade Federal do Ceara, Fortaleza, Brazil4
P-095
ATYPICAL AND HIDDEN WAYS OF CONTAMINATION OF ENVIRONMENT BY GENOTOXIC
COMPOUNDS
Andrej Gajdoš
Dagmar Gajdošová; Lívia Lucová; Zuzana Dietzová; Alexander Hudák
Regional Public Health Authority, Košice, Slovakia
P-096
THE ANTIMUTAGENIC EFFECT OF PHENETHYL ISOTHIOCYANATE
Martina Langova 1
Zdenka Polivkova 1; Petr Smerak 1; Jirina Bartova 1; Helena Sestakova 2
3rd Faculty of Medicine, Charles University, Prague, Czech Republic1; National Institute of Public Health in
Prague, Prague, Czech Republic2
P-097
CYTOTOXICITY AND GENOTOXICITY OF THE FOOD FLAVOURINGS EUGENOL AND ESTRAGOLE
IN V79 AND CHO CELL LINES
Celia Martins 1
Ana Filipa Monteiro 1; Alexandra Maralhas 1; Patricia Saraiva 1; Antonio Laires 1,2; José Rueff 1;
Antonio Sebastião Rodrigues 1
Genetics, Faculty of Medical Sciences-UNL, Lisbon, Portugal1; SABT, Faculty Sciences and Technology-UNL,
Lisbon, Portugal2
P-098
THE ROLE OF NATURAL POLYSACCHARIDE AS AGENT WITH ANTIMUTAGENIC/
ANTICARCINOGENIC ACTIVITY
Eva Miadoková 1
Slavomíra Naďová 1; Eva Pražmáriová 1; Viola Dúhová 1; Viera Vlčková 1; Grigorij Kogan 2; Peter Rauko 3
Comenius University, Faculty of Natural Sciences, Department of Genetics, Bratislava, Slovakia1; Istitute
of Chemistry, SAV, Bratislava, Slovakia2; Institute of Experimental Oncology, SAV, Bratislava, Slovakia3
P-099
WHEY PROTEIN INHIBITS OXIDATIVE STRESS INDUCED BY IRON OVERLOAD IN RATS
Eunju Park1
Hyun-Jin Lee 1; Kyung-Im Jeon 1; Bo-Young Seo 1; Su-Yeon Kim 1; Hyun-Dong Paik 2; Yoe-Chang Yoon 2
Dept. of Food and Nutrition, Kyungnam Univeristy, Masan, Korea1 ; Dept. of Food Science and
Biotechnology of Animal Resources, Konkuk University, Seoul, Korea2
42
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LIST OF POSTERS
P-100
HAEMOGLOBIN ADDUCTS AS BIOMARKER OF BACKGROUND EXPOSURE TO ACRYLAMIDE IN
FIVE EUROPEAN COUNTRIES – A PILOT STUDY
Birgit Paulsoon
Anna Vikström 1; Ronnie Davies 1; Per Rydberg 1; Margareta Törnqvist 1; Paul Aston 2; Paul Scheepers 3
Dept. of Environmental Chemistry, Stockholm University, Stockholm, Sweden1; Dept. of Environ.
Chemistry, Stockholm university, Stockholm, Sweden1; AB Biomonitoring Ltd, Cardiff, United Kingdom2;
University Nijmegen Medical Centre, Dept. of Epidemiol. & Biostatistics, Nijmegen, Netherlands3
P-101
ANTIMUTAGENIC AND IMMUNOPROTECTIVE EFFECTS OF LYCOPENE
Zdenka Polivkova 1
Martina Langova 1; Petr Smerak 1; Jirina Bartova 1; Helena Sestakova 2
3rd Faculty of Medicine, Charles University, Prague, Czech Republic1; National Institute of Public Health,
Prague, Czech Republic2
P-102
CONTRIBUTION OF STEROLS TO BREAST CANCER CELLS PROLIFERATION.
Maria Rybczynska
Blazej Rubis
University of Medical Sciences, Poznan, Poland
P-103
GENOTOXIC POTENTIALS OF SOME VEGETABLES GROWN OVER CHROMIUM RICH SOIL
Manjit Inder Singh Saggoo
Arneet Grewal
Department of Botany, Punjabi University, Patiala, India
P-104
INDUCTION OF MICRONUCLEI IN VERO CELLS BY CYANOBACTERIAL EXTRACTS
Elsa Dias 1
Paulo Pereira 2; Maria Joao Silva 3
Water Quality Center, National Institute of Health, Lisbon, Portugal1; Water Quality Center, National
Institute of Health, Lisbon, Portugal2; Center of Human Genetics, National Institute of Health, Lisbon,
Portugal3
P-105
URINARY MUTAGENICITY IN HEALTHY NON-SMOKING SUBJECTS
Sofia Pavanello 1
Alessandra Pulliero 1; Erminio Clonfero 1; Silvia Lupi 2; Pasquale Gregorio 2
Occupational Health Section, Department of Environmental Medicine and Public Health, University of
Padova, Padova, Italy1; Section of Hygiene and Occupational Medicine, Department of Clinical and
Experimental Medicine, University of Ferrara, Ferrara, Italy2
P-106
A NOVEL ENDOGENOUS MUTAGEN, AMINOPHENYLNORHARMAN FORMED FROM ABUNDANT
FOOD COMPONENTS, NORHARMAN AND ANILINE
Yukari Totsuka 1
Rena Nishigaki 1; Takashi Sugimura 1; Keiji Wakabayashi 1; Hiroyuki Kataoka 2
National Cancer Center Research Institute, Tokyo, Japan1; Shujitsu University, Okayama, Japan2
P-107
DNA DAMAGE AND REPAIR IN GERM CELLS OF PARP1-/-MALE MICE AFTER X-RAY
IRRADIATION
Eugenia Cordelli
Annamaria Fresegna; Alessia D'Alessio; Roberto Ranaldi; Patrizia Eleuter ; Paola Villani; Francesca
Pacchierotti
ENEA, Rome, Italy
P-108
REPRODUCTIVE TOXICITY AFTER EXPOSURE OF YOUNG MALE MICE
TO DI(2-ETHYLHEXYL)PHTHALATE
Malgorzata Dobrzynska
National Institute of Hygiene, Dept of Radiation Protection and Radiobiology, Warsaw, Poland
43
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LIST OF POSTERS
P-109
BULKY DNA-ADDUCTS AND POLYMORPHISMS IN METABOLIC GENES IN THE EPIC-SPAIN
COHORT
Antonio Agudo 1
Carlos A González 1; Gabriel Capella 1; Peluso Marco 2; Arnelle Munnia 2; Sala Nuria 3; Nadia Garcia 3; Pilar
Amiano 4; M Dolores Chirlaque 5; Eva Ardanaz 6; M José Sánchez 7; J Ramon Quirós 8
IDIBELL - Catalan Institute of Oncology, Hospitalet de Llobregat, Spain1; CSPO - Scientific Institute of
Tuscany Region, Florence, Italy2; IDIBELL - Oncology Research Institute, Hospitalet de Llobregat, Spain3;
Department of Public Health of Gipuzkoa, San Sebastian, Spain4; Consejería de Sanidad y Consumo,
Murcia, Spain5; Public Health Institute, Navarra, Spain6; Escuela Andaluza de Salud Pública, Granada,
Spain7; Consejería de salud y Servicios Sanitarios, Oviedo, Spain8
P-110
INFLUENCE OF GENETIC FACTORS ON TOLUENE DIISOCYANATE-RELATED SYMPTOMS
Karin Broberg 1
Hakan Tinnerberg 1; Anna Axmon 1; Margareta Littorin 1; Margareta Warholm 2; Agneta Rannug 2
Department of Occupational and Environmental medicine, Lund University, Lund, Sweden1; Department
of Work Environment Toxicology, Institute of Environmental Medicine, Karolinska Institute, Stockholm,
Sweden2
P-111
GENETIC PREDISPOSITION TO LUNG CANCER IN WOMEN FROM UPPER SILESIA, POLAND - A
PRELIMINARY REPORT FROM AN ONGOING STUDY
Dorota Butkiewicz 1
Marek Rusin 1; Malgorzata Krzesniak 1; Barbara Wlodarczyk 2; Jadwiga Rachtan 3;
Department of Tumor Biology Center of Oncology, M. Sklodowska-Curie Memorial Institute, Gliwice
Branch, Glwice, Poland1; Department of Cancer Epidemiology, Center of Oncology, M. SklodowskaCurie Memorial Institute, Gliwice Branch, Gliwice, Poland2; Epidemiology Unit, Center of Oncology, M.
Sklodowska-Curie Memorial Institute, Cracow, Poland3
P-112
MUTAGEN SENSITIVITY OF WORKERS EXPOSED TO LOW LEVELS OF IONIZING RADIATION
ASSESSED BY MICRONUCLEUS ANALYSIS AND FISH
Fabio Carbone 1,2
Patrizia Hrelia 1; Francesca Maffei 1; Sabrina Angelini 1; Giorgio Cantelli Forti 1; Hannu Norppa 2
Department of Pharmacology, University of Bologna, Bologna, Italy1; New Technologies and Risks, Finnish
Institute of Occupational Health, Helsinki, Finland2
P-113
MTHFR GENE POLYMORPHISM AS RISK FACTOR OF VASCULAR DISEASE
Zuzana Cermakova 1
T.Lhotanova1; A.Maluskova1,2
Dept. Of Blood Banking, Faculty Hospital Ostrava1, Medical - Social Faculty Ostrava, Czech Republic2
P-114
CHROMOSOME INSTABILITY AND FOLATE GENE POLYMORPHISMS IN YOUNG MOTHERS OF
DOWN SYNDROME INDIVIDUALS
Fabio Coppede 1
Gabriele Siciliano 1; Lucia Migliore 2; Ilaria Fontana 2; Alessia Bonelli 2; Renato Colognato 2; Stefania
Bargagna 3; Guia Astrea 3;
Department of Neurosciences, University of Pisa, Pisa, Italy 1; Dept. of Human and Environmental
Sciences, University of Pisa, Pisa, Italy2, Scientific Institute "Stella Maris", Calambrone, Pisa, Italy3
P-115
EFFECT OF POLYMORPHISM IN THE DNA REPAIR GENE HOGG1 (SER326CYS) ON PHENOTYPE
Soňa Gurská
Timea Farkašová; Alena Gábelová
Cancer Research Institute, Bratislava, Slovakia
44
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LIST OF POSTERS
P-116
GENETIC POLYMORPHISMS IN FOLATE METABOLISM AND THEIR ASSOCIATION WITH
CHROMOSOMAL ABERRATIONS AND SISTER CHROMATID EXCHANGES
Iiris Heilimo 1
Katja Metsola 1; Päivi Rosenström 1; Heidi Maunu 1; Hilkka Järventaus 1; Ari Hirvonen 1; Hannu Norppa 1;
Jarno Tuimala 2; Anne Kiuru3; Carita Lindholm 3
Finnish Institute of Occupational Health, Helsinki, Finland1; CSC, the Finnish IT Center for Science,
Espoo, Finland2; STUK Radiation and Nuclear Safety Authority, Helsinki, Finland3
P-117
LIFESTYLE FACTORS MODULATE GENETICALLY-PREDISPOSED CANCER
IMMUNOSURVEILLANCE
Kazue Imai
Tomonori Hayashi; Yukari Morishita; Kei Nakachi
P-118
LEVELS OF 2-THIOTHIAZOLIDINE-4-CARBOXYLIC ACID (TTCA) AND EVIDENCE OF EFFECT
MODIFICATION OF POLYMORPHISMS OF GLUTATHIONE-RELATED GENES IN VULCANIZATION
WORKERS IN THE SOUTHERN SWEDEN RUBBER INDUSTRIE
Lena S Jönsson
Karin Broberg; Ulf Bergendorf; Anna Axmon; Margareta Littorin; Bo AG Jönsson
Dep. of Laboratory Medicine, Division of Occupational and Envionmental Medicine, Lund University, Lund,
Sweden
P-119
XRCC1 ARG399GLN GENETIC POLYMORPHISM IN A TURKISH POPULATION
Bensu Karahalil
Neslihan Aygun Kocabas
Gazi University Pharmacy Faculty Toxicology Department, Ankara, Turkey
P-120
GENETIC VARIATION IN XRCC1 AND XRCC3 GENOTYPES AND RISK OF GLIOMA AND
MENINGIOMA
Anne Kiuru 1
Carita Lindholm 1; Sirpa Heinävaara 1; Taina Ilus 1; Päivi Jokinen 1,2; Anssi Auvinen 1,2; Tiina Salminen 2;
Beatrice Malmer 3; Maria Feychting 4; Christoffer Johansen 5; Minouk Schoemaker 6
STUK-Radiation and Nuclear Safety Authority, Helsinki, Finland1; Tampere School of Public Health/
University of Tampere, Tampere, Finland2; Umeĺ University Hospital, Umeĺ, Sweden3; Karolinska
Institute, Stockholm, Sweden4; Danish Cancer Society, Copenhagen, Denmark5; Institute of Cancer
Research, Surrey, United Kingdom6
P-121
INHERITED BREAST CANCERS
Petra Kopecká 1
Josef Kopecký 1; Eva Šilhánová 2; P. Plevová 2
Mamocentrum Femina s.r.o., Ostrava, Czech Republic1; FNsP Ostrava, Ostrava, Czech Republic2
P-122
GST GENE POLYMORPHISMS AND SUSCEPTIBILITY TO ASBESTOS AND SMOKING RELATED
LUNG DISEASES
Mari Kukkonen 1
Simo Kaleva 1; Tapio Vehmas 1; Matti Huuskonen 1; Harri Vainio 1; Ari Hirvonen 1; Päivi Piirilä 2
Finnish Institute of Occupational Health, Helsinki, Finland1; Helsinki University Hospital, Helsinki, Finland2
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P-123
GENETIC POLYMORPHISMS IN THE MDR-1 GENE ENCODING FOR P-GLYCOPROTEIN DRUG
TRANSPORTER AND SUSCEPTIBILITY TO COLORECTAL CANCER
Barbara Pardini 1,2
Roberto Barale 1; Pavel Vodicka 2; Elena Tulupova 2; Alessio Naccarati 2; Ludmila Vodickova 2,4;
Jan Novotny 3
Department of Biology, University of Pisa, Pisa, Italy1; Institute of Experimental Medicine, Academy of
Sciences of Czech Republic, Prague, Czech Republic2 ; First Medical Faculty, Charles University, Prague,
Czech Republic3; National Institute of Public Health, Prague, Czech Republic4
P-124
IMPACT OF GENETIC VARIATION IN GLUTATHIONE-RELATED GENES ON BODY BURDEN
OF METHYLMERCURY
Karin Schläwicke 1
Ulf Strömberg 1; Staffan Skerfving 1; Karin Broberg 1; Bengt Vessby 2; Göran Hallmans 3;
Department of Laboratory Medicine, Division of Occupational and Environmental Medicine, Lund
University, Lund, Sweden1; Department of Public Health and Caring Sciences, Uppsala University,
Uppsala, Sweden2 ; Department of Public Health and Clinical Medicine, Umeĺ University, Umea, Sweden3
P-125
POLYMORPHISMS IN NON HOMOLOGOUS END-JOINING GENES ASSOCIATED WITH BREAST
CANCER RISK AND CHROMOSOMAL RADIOSENSITIVITY
Petra Willems 1
Kathleen Claes 1; Ans Baeyens 1; Kim De Ruyck 1; Hubert Thierens 1; Anne Vral 1; Rudy Van Den Broecke 2;
Amin Makar 3
University of Ghent, Gent, Belgium1; Ghent University Hospital, Ghent, Belgium2; Middelheim Hospital,
Antwerpen, Belgium3
P-127
MICRONUCLEI INDUCTION IN TRADESCANTIA TETRADS BY PYRETHROIDS
Rafael Villalobos-Pietrini 1
Ana Rosa Flores-Márquez 1; Omar Amador-Mu?oz 1; Sandra Gómez-Arroyo 1; Miguel Angel
Rico-Rodríguez 2; Stefan M. Waliszewski 3
Instituto de Medicina Forense / Universidad Veracruzana, Veracruz, Mexico 1; Centro de Ciencias de la
Atmosfera / Universidad Nacional Autónoma de Mexico, Mexico, Mexico 1; Centro de Estudios Academicos
sobre Contaminacion Ambiental / Universidad Autonoma de Queretaro, Queretaro, Mexico 2; Instituto de
Medicina Forense / Universidad Veracruzana, Veracruz, Mexico3
P-128
MODULATION OF B[A]P-INDUCED EXPRESSION OF CYTOCHROME P450 AND PHASE II
ENZYMES BY PLANT PHENOLS IN MOUSE EPIDERMIS
Wanda Baer-Dubowska
Violetta Krajka-Kuzniak; Hanna Szaefer
Department of Pharmaceutical Biochemistry University of Medical Sciences, Poznan, Poland
P-129
THE EFFECT OF ORAL ADMINISTRATION OF BEETROOT JUICE ON THE ACTIVITY
AND EXPRESSION OF ENZYMES METABOLIZING XENOBIOTICS IN DMBA-INDUCED RAT LIVER
AND MAMMARY GLAND
Hanna Szaefer
Wanda Baer-Dubowska; Violetta Krajka-Kuzniak
P-130
EVALUATION OF THE DNA DAMAGE BY CHEMICAL CARCINOGENS AND ITS MODULATION BY
CLOUDY APPLE JUICE.
Ewa Ignatowicz
Zaneta Kazmierczak
Department of Pharmaceutical Biochemistry, University of Medical Sciences, Poznan, Poland
46
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P-131
ANTIGENOTOXIC APPLE POLYPHENOLS MODULATE GENE EXPRESSION IN HUMAN COLON
ADENOMA CELLS AS DETERMINED WITH A CUSTOM-MADE CDNA MICROARRAY FOR
TOXICOLOGICAL DEFENSE AND STRESS RESPONSE
Selvaraju Veeriah 1
Nina Habermann 1; Thomas Hofmann 1; Stefanie Klenow 1; Julia Sauer 1; Frank Böhmer 1; Stefan Wölfl 2;
Beatrice Louise Pool-Zobel 3
Institute of Molecular Cell Biology, Medical Faculty, Friedrich-Schiller-University, Drackendorfer Str. 1,
D-07747 Jena, Germany1; Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg,
Im Neuenheimer Feld 364, 69120 Heidelberg, Germany2; Department of Nutritional Toxicology, Institute
for Nutrition, Friedrich-Schiller-University, Dornburger Str. 25, D-07743 Jena, Germany3
P-132
ASSESSMENT OF THE GENOTOXICITY ASSOCIATED WITH KILNS USED FOR THE MANUFACTURE
OF RED BRICK AND OF PESTICIDES
Guillermo Cabrera
R.M.G. Durán; A.G. Perales; L.M. Pérez; G.L. Martínez
Universidad Autonoma de Querétaro, Querétaro, Mexico
P-133
Elsa Dias 1
Water Quality Center, National Institute of Health, Lisbon, Portugal1; Water Quality Center, National
institute of Health, Lisbon, Portugal2; Center of Human Genetics, National Institute of Health, Lisbon,
Portugal3
P-134
GENOTOXICITY OF NANOSIZED TITANIUM DIOXIDE
Ghita C-M Falck
Hanna Sandvik; Satu Suhonen; Hannu Norppa
Finnish Institute of Occupational Health, Helsinki, Finland
P-135
INVESTIGATION OF GENOTOXIC EFFECTS OF HOSPITAL WASTE WATERS IN BACTERIA AND
PRIMARY RAT HEPATOCYTES
Franziska Ferk 1
Armen Nersesyan 1; Robert Mader 1; Wolfram Parzefall 1; Christoph Buchmann 1; Siegfried Knasmüller 1;
Maria Fuerhacker 2
Medical University of Vienna, Institute of Cancer Research, Vienna, Austria1; Boku University of Natural
Resources and Applied Life Sciences, Vienna, Austria2
P-136
URINARY NAPHTHALENE AND PHENANTHRENE AS BIOMARKERS OF EXPOSURE TO POLYCYCLIC
AROMATIC HYDROCARBONS IN THE STEEL INDUSTRY
Steve Rappaport 1
J. Sobus 1; S. Waidyanatha 1; Andrej Gajdoš 2; Daniela Tarabeaková 2
School of Public Health, University of North Carolina, North Carolina, United States1; Regional Public
Health Authority, Košice, Slovakia2
P-137
CHROMOSOMAL ABERRATIONS ASSOCIATED WITH EXPOSURE TO PAHs
Dagmar Gajdošová 1
Daniela Tarabčáková 1; Lívia Lucová 1; Vladimír Rimár 1; Keneth Mund 2; Thomas Birk 2
Regional Public Health Authority, Košice, Slovakia1; Applied Epidemiology, Inc., Amherst MA,
United States2
47
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P-138
VALIDATION OF URINARY EXCRETION OF CYCLOPHOSPHAMIDE AS A BIOMARKER
OF EXPOSURE BY STUDYING ITS RENAL CLEARANCE AT HIGH AND LOW PLASMA
CONCENTRATIONS IN CANCER PATIENS
Maria Hedmer 1
Maria Albin 1; Bo AG Jönsson 1; Peter Höglund 2
Occupational and Environmental Medicine, Lund University, Lund, Sweden1
Clinical and Experimental Pharmacology, Lund University, Lund, Sweden2
P-139
INVESTIGATION ON THE RELATIONSHIP BETWEEN IL-4 OR IL-4 RECEPTOR GENETIC
POLYMORPHISMS AND ALLERGY-RELATED IMMUNOLOGIC STATUS IN ADULT EMPLOYEES
AT SOCIAL WELFARE FACILITIES
Yong Heo 1
Kyu Dong Ahn 2; Hyoung Ah Kim 3; Sang Hoon Kim 4
Catholic University of Daegu, College of Natural Sciences, Dept. Occupational Health, Kyongsan si,
Kyongbuk, Korea1; Soonchunhyang University, College of Medicine, Dept. Preventive Medicine, Asan si,
Chungnam, Korea2; The Catholic Univeristy of Korea, College of Medicine, Dept. Preventive Medicine,
Seoul, Korea3; Eulji University, School of Medicine, Dept. Internal Medicine, Seoul, Korea4
P-140
OCCUPATIONAL EXPOSURE TO POLYCYCLIC AROMATIC HYDROCARBONS (PAHs) IN
THE RUBBER INDUSTRY
Bo AG Jonsson 1
Christian H Lindh 1; Hans Kromhout 2; Roel Vermeulen 2
Division of Occupational and Environmental Medicine, Lund, Sweden1; Division of Environmental
and Occupational Health, Institute of Risk Assessment Sciences, Utrecht, Netherlands2
P-141
DEVELOPMENT OF FOUNDRY TECHNOLOGIES: GENOTOXIC RISK OF FURANE MOULDING
MIXTURES
Jaromira Kusová1
Lubomír Dobiáš1; Irena Mikulenková1; Hana Tomášková1; Milan Adamčík2
Institute of Public Health, Ostrava, Czech Republic1; Institute of Hygiene, Ostrava, Czech Republic2
P-142
QUARTZ AND CARCINOGENICITY RISK ON THE WORKPLACES: BIOMONITORING RESULTS
Hana Lehocká 1,2
Tomáš Adamus 1; Ivona Závacká 1; Lubomír Dobiáš 1,2; Jaromíra Kůsová 2
University of Ostrava, Ostrava, Czech Republic1; Institute of Public Health, Ostrava, Czech Republic2
P-143
MUTAGENICITY TESTS AS A TOOL FOR EFFECTIVE EVALUATION OF BIODEGRADATION
OF POLLUTANTS
Kateřina Malachová 1
Zuzana Pavličková 1; Čeněk Novotný 2; Tomáš Cajthaml 2
Faculty of Science, University of Ostrava, Ostrava, Czech Republic1; Institute of Microbiology, Academy
of Sciences, Praha, Czech Republic2
P-144
MEASUREMENT OF DNA ADDUCTS AS BIOMARKERS OF GENOTOXIC EXPOSURE
TO BENZO[A]PYRENE USING A LC-MS/MS TECHNIQUE
Caroline Marie 1
Jean-Luc Ravanat 1; Alain Favier 1; Emilie Vieu 2; Marie Marques 2; Anne Maitre 2
CEA Grenoble, Grenoble, France1; Faculté de Médecine de Grenoble, Grenoble, France2
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P-145
GENOTOXIC EVALUATION OF THE CO-EXPOSITION TO INDUSTRIAL AIR POLLUTANTS
BY THE IN VITRO COMET ASSAY ON MACROPHAGES ALVEOLAR CELLS AND LUNG CELLS
OF SPRAGUE-DAWLEY RAT
Daniel Marzin
Fabrice Nesslany 1; Ludovic le Hegarat 1,2; Daniel Marzin 1,4; Frank le Curieux 3,4
Institut Pasteur de Lille, Lille, France1; Faculté des Sciences Pharmaceutiques et Biologiques, Lille,
France2; Institut Pasteur de Lille, Lille, France3; Faculté des Sciences Pharmaceutiques et Biologiques,
Lille, France4
P-146
CHROMOSOME LESIONS IN FISHERMEN WHO PARTICIPATED IN THE CLEAN-UP
OF THE PRESTIGE OIL SPILL
Gemma Monyarch
MA Rigola; JP Zock; Y Torralba; L Bouso; F Gómez; H Verea; F Pozo Rodríguez; MD Coll; JA Barbera;
C Fuster; G Rodríguez Trigo
Grupo SEPAR-Prestige, Sociedad Espańola de Neumología y Cirugía Toracíca (SEPAR), Spain
P-147
Beatriz Pérez-Cadahia 1,2
Josefina Méndez 1; Blanca Laffon 1,2; Eduardo Pásaro 2; Joao Paulo Teixeira 3; Susana Silva 3;
Joana Roma-Torres 3; Olga Mayan 3
Department of Cell and Molecular Biology, University of A Coruńa, A Coruńa, Spain1; Toxicology
Unit, University of A Coruńa, A Coruńa, Spain2; National Institute of Health, Environmental Health
and Toxicology Department, O Porto, Portugal3
P-148
INDUCTION OF MICRONUCLEI IN LYMPHOCYTES OF FOUNDRY WORKERS DUE TO CHRONIC
EXPOSURE TO EXTREMELY-LOW FREQUENCY ELECTROMAGNETIC FIELDS
Claudia Schwarz 1
Elisabeth Kratochvil 1; Marietta Weninger 1; Petra Hartbauer 1; Hugo W Rüdiger 1; Otto Zierer 2
Medical University of Vienna, Division of Occupational Medicine, Vienna, Austria1; Vertical Galva,
Augsburg, Germany2
P-149
AN EVALUATION OF CYTOGENETIC DAMAGE IN A POPULATION EXPOSED TO URANIUM MINES
RESIDUES
Maria Joao Silva 1
Maria Guida Boavida 1; Anabela Dias 1; Ana Carla Sousa 1; Paula Costa 1; Octávia Monteiro Gil 2; Patricia
Cardoso Painço 2; Luisa Pedro 2; João Cardoso 3; Luis Santos 3
Paulo Nogueira 4; José Marinho-Falcão 4
Center of Human Genetics, National Institute of Health Dr. Ricardo Jorge, Lisbon, Portugal1; Department
of Radiological Protection and Nuclear Safety, Nuclear and Technological Institute, Sacavém, Portugal2;
Laboratory of Ionizing Radiation Metrology, Nuclear and Technological Institute, Sacavém, Portugal3;
National Health Observatory, National Institute of Health Dr. Ricardo Jorge, Lisbon, Portugal4
P-150
CYTOGENETIC MONITORING OF WORKERS OCCUPATIONALLY EXPOSED TO IONISING
RADIATION
Grazina Slapsyte 1
Jurate Mierauskiene 1; Inga Januskeviciute1; Birute Griciene 1,2
Vilnius University, Vilnius, Lithuania1; Radiation Protection Centre, Vilnius, Lithuania2
P-151
CYTOGENETIC ANALYSIS OF BUCCAL, NASAL AND UROTELIAL HUMAN CELLS
Ljudmila P. Sycheva
A.N. Sysin Research Institute of Human Ecology and Environmental Health, Moscow, Russian Federation
49
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P-152
MODIFICATION OF SENSITIVITY OF CELLS FROM HEALTHY DONORS TO MUTAGEN EXPOSURE
IN VITRO AFTER VITAMINS INTAKE
Ekaterina S. Voronina 1
Viktoria A. Nikitina 1; Ali K. Zhanataev 2; Andrey D. Durnev 2
Research Center for Medical Genetics, Russian Academy of Medical Sciences, Moscow, Russian
Federation1; State Zakusov Institute of Pharmacology, Russian Academy of Medical Sciences, Moscow,
Russian Federation2
P-153
A NEW MECHANISM FOR THE DIFFERENTIAL ANTICARCINOGENIC EFFECTS OF N-3 AND N-6
POLYUNSATURATED FATTY ACIDS
Gerrit Alink1
Vincent A. van Beelen 1; Jac M. M.J. G. Aarts 1; Astrid Reus 1; Ivonne M. C. M. Rietjens 1; Hans
Mooibroek 2; Lolke Sijtsma 2; Dirk Bosch 3
Chair Toxicology, Wageningen University, Wageningen, Netherlands1; Agrotechnology and Food
Innovations B.V, Wageningen, Netherlands2; Plant Research International, Wageningen, Netherlands 3
P-154
ANTIGENOTOXIC EFFECT OF BASIL (OCIMUM BASILICUM L.) IN MICROBIAL SHORT-TERM
TESTS
Branka Vukovic-Gacic
Tanja Beric ; Biljana Nikolic ; Jasna Stanojevic ; Draga Simic ; Jelena Knezevic-Vukcevic
Laboratory for Microbiology, Faculty of Biology, Serbia and Montenegro
P-155
OXIDATIVE STRESS-RELATED DNA DAMAGE AND P53 MODULATION IN BENZO[A]PYRENE
EXPOSED HUMAN HEPATOMA HEPG2 CELLS
Jinhee Choi
Sun-Yong Park; Ji-Yeon Rho
University of Seoul, Seoul, Korea
P-156
OXIDATIVE STRESS CAUSED BY SUBWAY PARTICLES
Hanna Karlsson
Lennart Moller
Department of Biosciences and Nutrition, Huddinge, Sweden
P-157
EFFECT OF ACTIVE AND PASSIVE SMOKING ON OXIDATIVE DNA DAMAGE MEASURING BY
8-OXO-7,8-DIHYDRO-2’-DEOXYGUANOSINE LEVELS IN HUMAN LEUKOCYTE
Maura Lodovici
P-158
PROTECTIVE EFFECT OF PLANT ANTIOXIDANTS AGAINST T-BOOH INDUCED DNA DAMAGE AND
MUTAGENESIS IN PROKARYOTIC AND EUKARYOTIC TESTS IN VITRO
Dragna Mitic-Culafic1
Biljana Nikolic 1; Branka Vukovic-Gacic 1; Jelena Knezevic-Vukcevic 1; Bojana Zegura 2; Metka Filipic 2
Faculty of Biology University of Belgrade, Belgrade, Serbia and Montenegro1; National Institute of Biology,
Department of Genetic Toxicology and Cancer Biology, Ljubljana, Slovenia2
P-159
-866 G/A POLYMORPHISM OF THE UNCOUPLING PROTEIN 2 IN RELATION TO CANCER RISK
Susan Nowell1
Kelly Mercer 1; Carolyn Wise 2; Luke Ratnasinghe 2; Fred F. Kadlubar 2; Christine B. Ambrosone 3; Nicholas
P Lang 4
University of Arkansas for Medical Sciences, Little Rock, United States1; Natiional Center for Toxicological
Research, Jefferson, United States 2; Roswell Park Cancer Institute, Buffalo, United States3; Central
Arkansas Veteran's Healthcare System, Little Rock, United States4
50
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P-160
SIX WEEKS OF CHLORELLA SUPPLEMENTATION HAS PARTIAL EFFECT ON BIOMARKERS
OF ANTIOXIDANT STATUS IN KOREAN MALE SMOKERS
Sun-Hee Lee 1
Jeong Woo Jeon 1; Yoo Kyoung Park 1;Hae Jin Kang 1; Hye-Jin Lee 2; Eun-Jae Jeon 2; Chang-sook Kim 2;
Myung-Hee Kang 2
Kyung-Hee University, Seoul, Korea1; Hannam University, Deajeon, Korea2
P-161
IRON OVERLOAD INDUCES OXIDATIVE DNA DAMAGE IN RAT LEUKOCYTES AND COLONOCYTES
KYUNG IM JEON 1
Su-Yeon Kim 1; Bo-Young Seo 1; Hyun-Jin Lee 1; Eunju Park 1; Beatrice L Pool-Zobel 2
Dept. of Food and Nutrition, Kyungnam Univeristy, Masan, Korea1; Dept. of Nutritional Toxicology,
Friedrich-Schiller-University, Jena, Germany2
P-162
ANTIOXIDANT AND ANTICANCER ACTIVITIES OF ACETON EXTRACT FROM STYELA CLAVA
Bo-Young Seo 1
Eunju Park 1; Eun-Sil Jung 2; Hae-Ryong Park 2; Seung-Chul Lee 2;
Dept. of Food and Nutrition, Kyungnam Univeristy, Masan, Korea 1; Dept. of Food Science and
Biotechnology, Kyungnam University, Masan, Korea2
P-163
ANTIOXIDATIVE AND CYTOTOXIC ACTIVITY OF RED BEET (BETA VULGARIS) IN COMBINATION
WITH INDUCERS OF OXIDATIVE STRESS
Piotr Szweda 1,2
T. Friedberg 2; M. J. Paine 2; L. McLaughlin 2; E. Polson 2; A. Stenhouse 2; J. Lewandowska ; A. Bartoszek 3
Dept. Food Chemistry, Technology and Biotechnology, Gdansk University of Technology, Gdansk, Poland 1;
Biomedical Research Centre, University of Dundee, Level 5, Ninewells Hospital and Medical School,
Dundee, United Kingdom2 ; Dept. of Pharmaceutical Technology and Biochemistry, Gdansk University of
Technology, Gdansk, Poland3
P-164
A COMPARATIVE STUDY ON THE EFFECT OF ALGAL AND FISH OIL ON VIABILITY AND CELL
PROLIFERATION OF HUMAN CACO-2 CELLS, AND ABERRANT CRYPT FOCI FORMATION IN RAT
Vincent A. van Beelen 1
Johannes Roeleveld 1; Ivonne M. C. M. Rietjens 1; Gerrit M. Alink 1; Hans Mooibroek 2; Lolke Sijtsma 2;
Raoul J. Bino 3; Dirk Bosch 3
Chair of Toxicology, Wageningen University, Wageningen, Netherlands1; Agrotechnology and Food
Innovations B. V., Wageningen, Netherlands2 ; Plant Research International, Wageningen, Netherlands3
P-165
NOVEL SALMONELLA TESTER STRAINS HIGHLY SENSITIVE TO PHOTOCHEMICAL SOURCES AND
OXIDATIVE DAMAGE
Masami Yamada
Keiko Matsui; Takehiko Nohmi
P-166
A BACTERIAL MUTATION (AMES) ASSAY DESIGN FOR COMPARATIVE ASSESSMENT
OF MUTAGENS
Mark Ballantyne
Ann Gradwell
Covance Laboratories Ltd., Harrogate, United Kingdom
P-167
AUTOMATED MICRONUCLEUS DETECTION IN VIVO AND IN VITRO, DIFFERING PERSPECTIVES
Ann T Doherty
Julie E Hayes; Mike R. O. Donovan
AstraZeneca, Cheshire, United Kingdom
51
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P-168
CONTINUED EVALUATION OF THE LITRON XCELL MN KITTM FOR THE FLOW CYTOMETRIC
ENUMERATION OF MICRONUCLEI WITH L5178Y MOUSE LYMPHOMA CELLS. TREATMENT WITH
DEXAMETHASONE AND STAUROSPORINE
Patricia Ellis 1
Joanne Collins 1; Antonia Booth 1; Claire Moore 1; James Harvey 1; Robert Rees 1; Anthony Lynch 1;
Svetlana Avlasevich 2; Steve Dertinger 2
Glaxosmithkline, Ware, United Kingdom1; Litron Laboratories, New York, United States2
P-169
POSITIVES IN IN-VITRO-CLASTOGENICITY-TESTS AND THEIR BIOLOGICAL RELEVANCE.
ANALYSIS OF TESTS SUBMITTED WITH DRUG SUBSTANCES TO THE BFARM IN THE PERIOD
1998–2005
Roland Froetschl
Peter Kasper
Federal Institute for Drugs and Medical Devices, Bonn, Germany
P-170
STATISTICAL ANALYSIS OF MOUSE LYMPHOMA ASSAY DATA AND ITS CORRELATION
TO CLASTOGENICITY PREDICTION BY THE CHROMOSOMAL ABERRATION TEST
Kristin Gritzko
Michael Kraft; Gregory Rodrigo; Thomas Becker
BSL Bioservice Scientific Laboratories GmbH, Planegg (Munich), Germany
P-171
THE AMES MPF 98/100 ASSAY: NOVEL MUTAGENICITY TESTING IN LIQUID MICROPLATE
FORMAT USING S. TYPHIMURIUM TA98 AND TA100
Sini Flückiger-Isler
Markus Kamber
Xenometrix, Allschwil, Switzerland
P-172
CLASTOGENICITY, PHOTO-CLASTOGENICITY OR PSEUDO-PHOTO-CLASTOGENICITY:
GENOTOXIC EFFECTS OF ZINC OXIDE IN THE DARK, IN PRE-IRRADIATED
OR SIMULTANEOUSLY-IRRADIATED CHINESE HAMSTER OVARY CELLS
David Kirkland1
Eric K Dufour2; Gerhard J Nohynek2; Hervé Toutain2; Tirukalikundram Kumaravel1
Covance Laboratories Ltd, Otley Road, Harrogate HG3 1PY, United Kingdom, Harrogate HG3 1PY, United
Kingdom1; L'Oreal Research & Development, Worldwide Safety Department, 92600 Asnieres, France,
92600 Asnieres, France2
P-173
MUTAGENICITY OF THE AMBIENT AIR IN OSTRAVA
Jaromira Kusová
Hana Miturová; Hana Tomášková; Lubomír Dobiáš
Institute of Public Health, Ostrava
P-174
GENOTOXICOLOGICAL STUDY AIMED AT ANTIMUTAGENIC/ANTICARCINOGENIC
AND BIOMODULATORY EFFECTS OF EXTRACT FROM ARTICHOKE CYNARA CARDUNCULUS
L. EVALUATION
Slavomíra Naďová 1
Viera Vlčková 1; Viola Dúhová 1; Mária Trebatická 1; Ján Grolmus 1; Eva Miadoková 1; Peter Rauko 2;
Ľuboš Čipák 2; Pavel Mučaji 3; Daniel Grančai 3
Comenius University, Faculty of Natural Sciences, Department of Genetics, Bratislava, Slovakia1;
Institute of Experimental Oncology, SAV, Bratislava, Slovakia2; Comenius University, Faculty of Pharmacy,
Department of Pharmacognosy and Botany, Bratislava, Slovakia3
52
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P-175
THE DETECTION OF IN VIVO MICRONUCLEI BY FLOW CYTOMETRY AND MICROSCOPIC
ANALYSIS IN PERIPHERAL BLOOD
Ingrid Pontén 1
George Bolcsfoldi 1; Eva-Lena Härnwall 1; Ann Doherty 2; Julie Hayes 2; Jo Cunliffe 2; Mike O'Donovan 2;
Jan Grawé 3
AstraZeneca R&D Södertälje, Safety Assessment Sweden, Södertälje, Sweden1; AstraZeneca R&D
Alderley Park, Safety Assessment UK, Mereside, United Kingdom2; Cellanalyslab, Rudbecklaboratoriet/C5,
Uppsala Universitet, Uppsala, Sweden3
P-176
THE NEWLY DEFINED ACCEPTANCE AND EVALUATION CRITERIA FOR THE MOUSE LYMPHOMA
ASSAY AND THEIR IMPACT AND RELEVANCE
Albrecht Poth
Hans-Eric Wollny; Susanne Kunz; Wolfgang Völkner
RCC Cytotest Cell Research GmbH, Rossdorf, Germany
P-177
AUTOMATION OF SCORING PROCEDURE BY FLOW CYTOMETRY FOR THE IN VITRO
MICRONUCLEUS TEST.
Gregory Rodrigo
Margit Oppong; Thomas Becker
BSL Bioservice Scientific Laboratories GmbH, Planegg (Munich), Germany
P-178
EVALUATION OF AN AUTOMATED SCORING SYSTEM (CELLOMICS) FOR THE IN VITRO
MICRONUCLEUS ASSAY IN CHO-K1 CELLS
Andrew Scott 1
Sophie Malcomber 1; Sharon Maskell 1; Claire Moore 1; Sam Windebank 1; Paul Carmichael 1; Dolores Diaz 2
Safety & Environmental Assurance Centre, Unilever, Sharnbrook, Bedfordshire, MK44 1LQ, United
Kingdom1; Cerep Inc., 15318 NE 95th Street, Redmond, Washington 98052, United States2
P-179
THE IMPORTANCE OF PREMATURE CENTROMERE DIVISION IN ASSESSING GENTOTOXIC RISK
OF CYTOSTATICS
Vladan Peter Bajic 1
Biljana Spremo Potparevic 2; Lada Gordana Zivkovic 2; Ninoslav Djelic Djelic 2; Zorka Milicevic Milicevic 3
Institute for Biomedical Research, Galenika pharmaceuticals, Belgrade, Serbia and Montenegro1;
Department of Biology, Faculty of Pharmacy, Belgrade, Serbia and Montenegro2; Institute for Nuclear
Research Vinca, Lab.mol.biol.endocrinology, Belgrade, Serbia and Montenegro3
P-180
INDUCTION OF LACZ MUTATIONS IN MUTAMOUSE CAN DISTINGUISH CARCINOGENIC FROM
NON-CARCINOGENIC ANALOGUES OF DIAMINOTOLUENES AND NITRONAPHTHALENES
Mark Ballantyne
Carol Beevers; David Kirkland
Covance Laboratories Ltd, Harrogate, United Kingdom
P-181
SMOKING AND CANCER RELATIONSHIP
Liana Monica Deac 1
Dana Coza 1; Fl. Nicula 2
“Professor Dr. Iuliu Moldovan“ Institute of Public Health Cluj-Napoca, Romania1; “Professor Dr. I.
Chiricuta“ Oncological Institute Cluj-Napoca, Romania2
P-182
GENOTOXICITY AND HEAVY METALS ASSESSMENT IN MARINE SEDIMENTS OF FOLLONICA
GULF (THYRRENIAN SEA, CENTRAL ITALY)
Stefania Frassinetti 1
Marco Cini 1; Clara Della Croce 1; Leonardo Caltavuturo 1; Marco Carlo Mascherpa 1;
Leonardo Lampugnani 2
National Research Council, 1Institute of Biology and Agricultural Biotechnology (IBBA); Institute of
Chemical and Physical Processes (IPCF) Research Area of Pisa, Italy2
53
26.6.2006 16:05:30
LIST OF POSTERS
P-183
ASSESSMENT OF DNA DAMAGE IN WHITE BLOOD CELLS OF HEALTH HUMAN DONORS USING
THE ALKALINE COMET ASSAY AND CHROMOSOME ABERRATION ANALYSIS
Verica Garaj-Vrhovac
Nevenka Kopjar; Snježana Ramic; Davor Želježic
Institute for Medical research and Occupational Health, Zagreb, Croatia
P-184
INDIVIDUAL GENETIC VARIABILITY IN BIOTRANSFORMATION ENZYMES CAN MODULATE RISK
FOR EXPOSED PEOPLE
Alexandra Horská
Jana Tulinská; Ladislava Wsólová; Mária Dušinská
P-185
TRANSPLACENTAL GENOTOXICITY AND HEALTH RISK ASSESSMENT OF PROCHLORAZ AND
TRICHLORFON
Su-Yin Chiang 1
Kuen-Yuh Wu 2; GP Chang-Chien 3; Ching-Tang Kuo 4; Hsiu-Ching Wu 5
Graduate Institute of Chinese Medical Science/China Medical University, Taichung, Taiwan 1; Institute of
Environmental Health and Occupational Medicine/National Health research Institute, Zhunan, Taiwan 2;
Super Micro Mass research and technology center/Cheng-Shiu University, Kaohsiung, Taiwan3; Graduate
Institute of Environmental Medicine/China Medical University, Taichung, Taiwan4; School of Post
Baccalaureate Chinese Medicine/China Medical University, Taichung, Taiwan5
P-187
EFFECTS OF ANIMAL AND PLANT METABOLISM IN THE MUTAGENICITY INDUCED
BY ORGANOPHOSPHORUS INSECTICIDES IN SALMONELLA TYPHIMURIUM
Sandra Gómez-Arroyo 1
Liliana Sánchez-Estrada 1; Josefina Cortés-Eslava 1; Rafael Villalobos-Pietrini 1;
Concepción Moreno-Zenteno1; Saúl Flores-Maya 2
Centro de Ciencias de la Atmosfera / Universidad Nacional Autonoma de Mexico, Mexico, Mexico1;
Facultad de Estudios Superiores-Iztacala / Universidad Nacional Autonoma de Mexico, Mexico, Mexico2
P-188
A FULLY AUTOMATED HIGH-CONTENT-SCREENING IMAGING SYSTEM FOR COMET ASSAY
Françoise Soussaline 1
Jérôme Sallette 1; Alexandre Papine 1; Jean-Philippe Belaidi 2; Laurent Marrot 2; Jean-Roch Meunier 2
IMSTAR S.A., PARIS, France1; Safety Department, Life Science Research, L'Oréal, Aulnay sous Bois,
France2
P-189
THE CYTOTOXIC EFFECT OF BEE VENOM ON HUMAN LYMPHOCYTES
Martina Djurinec
P-191
THE MOUSE SPLENOCYTE – A POTENTIAL TOOL FOR DETERMINING THRESHOLDS
FOR ANEUGENIC RESPONSES
Guy Steiblen 1
Catherine Pallen 1; Thierry Orsičre 2; Alain Botta2,3; Daniel Marzin 3
Bayer CropScience (Sophia Antipolis), Sophia Antipolis, France1 ; Medical school of Marseille, Marseille,
France2; Institut Pasteur de Lille, Lille, France3
P-192
INDUCTION OF CYP1A1 MRNA AND PROTEIN EXPRESSION BY THE ANTICANCER DRUG
ELLIPTICINE
Dagmar Aimova 1
Marie Stiborova 1; Pavel Soucek 2; Eva Frei 3
Charles University in Prague, Prague, Czech Republic1; National Institute of Public Health, Prague, Czech
Republic2; German Cancer Research Center, Heidelberg, Germany3
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LIST OF POSTERS
P-193
THE ENVIRONMENTAL POLLUTANT AND CARCINOGEN 3 NITROBENZANTHRONE AND ITS
HUMAN METABOLITE 3 AMINOBENZANTHRONE ARE POTENT INDUCERS OF RAT HEPATIC
CYTOCHROMES P450 1A1/2 AND NQO1
Helena Dracinska 1
Marie Stiborová 1; Volker M. Arlt 2; David H. Phillips 2; Eva Frei 3; Heinz H. Schmeiser 3
Charles University in Prague, Prague, Czech Republic1; Institute of Cancer Research, Sutton,
United Kingdom2; German Cancer Research Center, Heidelberg, Germany3
P-194
COMPARATIVE ANALYSIS OF GENE EXPRESSION IN THE TISSUES OF MICE EXPOSED TO
ARSENATE OR DIMETHYLARSINIC ACID IN DRINKING WATER.
Marafante Erminio1
Cimino Reale G 1; Casati B 1; Collotta A 1; Brustio R 1; Vhater M 2
EC Joint Research Centre, ispra, Italy1; Karolinska Instituet, Stockholm, Sweden2
P-195
CHARACTERISATION OF THE P53 GENE EXPRESSION RESPONSE INDUCED BY
BENZO(A)PYRENE-DIOL-EPOXIDE IN MAMMALIAN CELLS
Sarah L Hockley
Ian Giddings ; Volker M Arlt; David H Phillips
Institute of Cancer Research, Sutton, United Kingdom
P-196
GLOBAL ANALYSIS OF GENE EXPRESSION IN ACETAMINOPHEN-INDUCED MICE
HEPATOTOXICITY
Jin Seok Kang
Ki Kyung Jung; Soo Kyung Suh ; Joohwan Kim; Tae Gyun Kim; Bang Hyun Kim; Woo Sun Lee; Youn
Kyoung Jeong; Ye Mo Koo; Hyun-Joo Kim; Hai Kwan Jung; Sue Nie Park
National Institute of Toxicological Research, Korea Food and Drug Administration, Seoul, Korea
P-197
CYTOTOXICITY OF THE ANTICANCER DRUG ELLIPTICINE IN HUMAN LEUKEMIA AND
NEUROBLASTOMA CELL LINES
Jitka Poljakova 1
Marie Stiborova 1; Jan Hrabeta 1,2; Tomas Eckschlager 1,2; Eva Frei 3
Charles University in Prague, Prague, Czech Republic1; University Hospital Motol, Prague, Czech Republic2;
German Cancer Research Center, Heidelberg, Germany3
P-198
BIOACTIVATION OF BENZO(A)PYRENE IN AHR KNOCK OUT MICE
Steen Mollerup
Carlos Sagredo; Steinar Ovrebo; Ingrid V Botnen; Rita Baera; Einar Eilertsen; Aage Haugen
Section for Toxicology, National Institute of Occupational Health, Oslo, Norway
P-199
POLY (ADP-RIBOSE) POLYMERASE DEFICIENCY DOES NOT INFLUENCE THE MUTAGENESIS
OF ETHYLNITROSUREA IN LIVER AND TESTIS FROM TRANSGENIC MICE
Maria J. Silva
Henriqueta Louro; Ines Faustino; Ana Carla Sousa; Anabela Dias; Maria Guida Boavida
Center of Human Genetics, National Institute of Health Dr. Ricardo Jorge, Lisbon, Portugal
P-200
AN EXPERIMENTAL MODEL OF POTENTIAL TRANSPLACENTAL GENOTOXICITY
OF FLUCONAZOLE
Darko Markovic 1
Boris Mildner 1; Zeljko Ferencic 1; Glojnaric Ines 1; Ranko Stojkovic 2; Suzana Borovic-Sunjic 2; Ana Marija
Jazbec 3; Tomislav Dobranic 4; Aleksandra Fucic 5
PLIVA Research Institute Ltd, Zagreb, Croatia1; Rudjer Boskovic Institute, Zagreb, Croatia2; Faculty of
Forestry, Zagreb, Croatia3; Faculty of Veterinary Medicine, Zagreb, Croatia4; Institute for Medical Research
and Occupational Health, Zagreb, Croatia5
55
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LIST OF POSTERS
P-201
THE GENOTOXIC POTENTIAL AND CHEMOPREVENTIVE EFFECTS OF TWO COMMONLY USED
ANTI-ABORTION HERBAL MEDICINES ON ENU-INUDCED TRANSPLACENTAL GENOTOXICITY
IN MICE
Chiang Su-yin
Hsiu-Ching Wu 1; Mei-Yi Lin 2; Jun-Ming Chen 3; Chien-Liang Fang 4; Hui-Feng Huang 5; Su-Yin Chiang 5
School of Post Baccalaureate Chinese Medicine/China Medical University, Taichung, Taiwan1; Department
of Traditional Chinese Medicine/Chia-Yi Christian Hospital, Chiayi, Taiwan2; Chen Jun-Ming TCM clinics,
Taipei, Taiwan3; Department of Traditional Chinese Medicine/China Medical University Hospital at
Pei-Kang, Pei-Kang, Taiwan4; Graduate Institute of Chinese Medical Science/China Medical University,
Taichung, Taiwan5
P-202
PREGNANT WOMEN WHO SMOKE HAVE HIGHER LEVELS OF PLACENTAL POLYCYCLIC
AROMATIC HYDROCARBON (PAH)-DNA ADDUCTS COMPARED TO NON-SMOKERS:
AN IMMUNOHISTOCHEMICAL EVALUATION
Miriam C. Poirier 1
Paul Sirajuddin 1; Margaret Pratt 1; Radim J Sram 2; David K Manchester 3
National Cancer Institute, NIH, Bethesda, MD, United States1; Laboratory of Genetic Ecotoxicology,
Institute of Experimental Medicine, Prague, Czech Republic 2; University of Colorado School of Medicine,
Denver, Colorado, United States3
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LIST OF POSTERS
LATE ABSTRACTS
P-203
EVALUATION OF THE RESPONSE TO LOW DOSE IONIZING RADIATION BY GENE EXPRESSION
PROFILE
A. Colacci1
M. Vaccari1, E. Morandi1, D. Quercioli1 , C. Severini1, W. Horn P. Silingardi1, M. C. Nucci2, V. Lodi2,
F. Violante4, S. Grilli5
Excellence Environmental Carcinogenesis, Environmental Protection and Health Prevention AgencyEmilia-Romagna Region (ER-EPA), Bologna, Italy1; Occupational Health Unit, S. Orsola-Malpighi Hospital,
Bologna, Italy2; Alma Mater Studiorum-University of Bologna Occupational Health Unit Sant’Orsola
Malpighi Hospital4; Department of Experimental Pathology-Cancer Research Section, School of Medicine,
University of Bologna, Bologna, Italy5
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INSTRUCTION FOR SPEAKERS AND AUTHORS
The speakers center/ audiovisual equipment
The Speakers´Service Center is located in the Prague Congress Centre on the second floor and will be
available for speakers making an oral presentation.
All lecture rooms will be equipped with a computer and a data projector (beamer) for PowerPoint
presentations. PowerPoint presentation must be handed in via floppy disc, CD-ROM, USB compatible
memory stick, or via the own laptop at the Speakers´Service Center at least one hour before the begining of the session. Speakers will have the opportunity to check their presentations on PCs available in the
Speakers Service Center.
Poster session
Posters will be displayed continuously throughout whole Conference July 2-6, 2006.
Posters mounting
Sunday, July 2, 2006, 08:00-18:00 or July 3, 2006, 08:00-13:00
Posters removal
Thursday, July 6, 2006 not later than 13:00
Poster viewing and discussions are scheduled scheduled on:
Monday July 3, 2006
13:30-14:15 (viewing)
14:15-15:00 (discussion)
Tuesday July 4, 2006
13:30-14:15 (viewing)
14:15-15:00 (discussion)
Authors are encouraged to be present by their poster during these sessions.
Selected posters will be shortly presented during Poster discussion session held on Wednesday, July 5,
8:30-11:00 in Plenary Lecture Hall. Presenting authors are asked to prepare 2-3 slides summarizing the
poster.
Pins for mounting posters will be available at the Poster desk at Congress Hall Foyer on 2nd Floor.
Poster dimensions
Height: 97 cm
Width: 180 cm
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EXHIBITION
During the conference an Industrial Exhibition will take place at the Prague Congress Centre (PCC) in the
foyer of the 2nd Floor.
Exhibition dates and hours
Sunday
Monday
Tuesday
Wednesday
Thursday
July
July
July
July
July
2,
3,
4,
5,
6,
2006
2006
2006
2006
2006
16:00
08:30
08:30
08:30
08:30
-
20:00
18:00
18:00
13:00
13:00
FLOOR PLAN
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LIST OF EXHIBITORS
(in alphabetical order)
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CONFERENCE INFORMATION
Date
July 2- 6, 2006
Conference Venue
Prague Congress Centre (PCC)
5. Kvetna 65
140 21 Prague 4
Czech Republic
www.kcp.cz
The Prague Congress Centre (PCC) is located in a unique position on top of one of the Prague hills, offering
a beautiful view of the famous skyline of Prague, with the silhouette of the Prague Castle. The PCC has its
own metro station (VYSEHRAD) only three stops from downtown Prague.
Official conference language
Official Conference language is English, there will be no simultaneous translation provided.
Registration
All documents including registration packs (symposium bag, personal badge, Final Programme and
Abstracts, vouchers) will be handed out to registered participants during the following opening hours:
Sunday
Monday
Tuesday
Wednesday
Thursday
July
July
July
July
July
2,
3,
4,
5,
6,
2006
2006
2006
2006
2006
08:00-20:00
08:00-18:00
08:00-18:00
08:00-13:00
08:00-13:00
Onsite Registration Desk: +420 261 174 309.
Registration Fee
Registration Fees
(Including 19% VAT)
Regular
Student
Accompanying Person
Paid before April 30,
2006)
EUR 450
EUR 300
EUR 150
Paid between April 30 Onsite Registration
and June 20, 2006
EUR 550
EUR 380
EUR 150
EUR 600
EUR 420
EUR 180
Registration fee includes:
Conference bag
Final Programme/Abstract Book
Admission to all parts of the conference including Workshops
Opening Ceremony and Welcome reception
Performance of the Black Light Theatre
Coffee Breaks
Sandwich Lunch (Mon, Tue, Wed, Thu)
Conference Dinner
Municipal transport ticket
Accompanying person fee includes:
Opening ceremony and Welcome reception
Performance of the Black Light Theatre
Conference Dinner
Municipal transport ticket
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Payment
All fees for the Conference registration should be paid in EURO (EUR), free of all bank charges
-clearly stating the participant‘s name and PRN (Participants Registration Number)
Registration
Beneficiary: Institute of Experimental Medicine Academy of Sciences of the Czech Republic
Beneficiary address: Vídeňská 1083, CZ 142 20 Prague 4
Bank: Komerční banka, a.s., Na příkopě 33, CZ 114 07 Prague 1
IBAN (for international bank transfers only): CZ 04 0100 0000 3555 6465 0247
Account number (for domestic bank transfers only): 35-5564650247/0100
SWIFT Code: KOMBCZPPXXX
by credit card (Visa, Eurocard/Mastercard)
Accommodation, Social Programme
For the bank transfer (free of bank charges):
Name of the Bank: KOMERCNI BANKA a.s.,
Bank Address: Na Prikope 33, 114 07 Prague 1, Czech Republic
IBAN Number: CZ 5901 0000 0051 0903 490207
BIC Code: KOMBCZPPXXX
Account Number (for domestic bank transfers only): 51-0903490207/100
Beneficiary: CZECH-IN s.r.o., 5. Kvetna 65, CZ 140 21 Prague 4
by credit card (Visa, Eurocard/Mastercard)
Responsibility
The participant acknowledges that he/she has no right to lodge damage claims against the organisers
should the holding of the Conference be hindered or prevented by unexpected political or economic events
or generally by force majeure, or should the non-appearance of speakers or other reasons necessitate
programme changes. With registration, the participant accepts this proviso.
Confirmation
Upon receipt of the correct registration fee, each participant will receive a confirmation of registration.
Please bring this confirmation to the registration desk at the Prague Congress Centre as proof of your
registration
Badges
Acces to all scientific and social events will only be possible with your personal badge which you will receive
at the registration desk. Please always wear your badge! Lost badges will only be replaced with proof of
your original registration
Coffee breaks
From Sunday through Thursday , coffee breaks will be served in the exhibition and poster area.
Internet corner
Internet terminals wil be available for all conference participants at the Exhibition Area, 2nd Floor from
Sunday, July 2, 2006 until Thursday 6, 2006.
Health insurance
The conference organisers cannot accept liability for personal injuries sustained, or for loss or damage of
property belonging to conference participants (or their accompanying presons), either during or as result
of symposium. Please check validity of your health insurance.
Hotel accommodation
All delegates booked in official symposium hotels are kindly asked to present their accommodation voucher
at their hotels upon arrival. Those delgates that have their booking guaranteed by the credit card should
settle the accommodation at the hotel upon departure. All delegates are reminded to settle their extra
hotel charges (phones, minibar, etc.) upon departure at the hotel reception desk.
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Official housing agent
CZECH-IN has been appointed the official housing agent for the 36th Annual Meeting of the European
Environmental Mutagen Society “From Genes to Molecular Epidemiology”.
CZECH-IN
Prague Congress Centre
5. Kvetna 65
140 21 Prague 4
Czech Republic
Tel.: +420 261 174 305
Fax: +420 261 174 307
E-mail: [email protected]
Official Hotels
Address
Corinthia Towers Hotel*****
Kongresová 1, Prague 4
Holiday Inn Prague Congress Centre****
Na Pankráci 15/1684, Prague 4
Radisson SAS Alcron Prague****
Stepanska 40, Prague 1
Coronet Hotel****
Marie Cibulkove 8, Prague 4
Green Garden Hotel***
Fugnerovo namesti 4, Prague 2
Hotel Oya***
Na Pankraci 1337/109, Prague 4
Ibis Praha City***
Kateřinská 38, Prague 2
Hotel Pankrác
Na Pankráci 76, Prague 4
Podolí Dorms (University Campus)**
Na Lysine 12, Prague 4
Chodov Dorms (University Campus)**
K Verneraku 950, Prague 4
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SOCIAL PROGRAMME
Opening ceremony / welcome reception
Sunday, July 2, 2006 from 18:00
After the opening ceremony participants, accompanying persons and exhibitors are cordially invited to
attend the welcome reception, which will be held in the Prague Congress Centre in the foyer of the 2nd
floor.
Black Light Theatre Performance
Tuesday, July 4, 2006 from 20:00
A non-verbal, black-light theatre, multi-visual performance, freely based on the themes of the world
famous novels by Lewis Carroll “Alice in Wonderland” and Jonathan Swift, “Gulliver‘s Travellers”. With all
the distinctive magic of the National Black-light theatre Prague and its unique stage effects, animated and
acted films, we will take you on the Journey amongst Giants and Lilliputians, dancing octopuses, singing
flowers and live chess figures. We allow Alice to fly, shrink, or disappear, while Gulliver visits the most
bizarre continents, full of unknown objects and creatures. The performance features world patented stage
mechanisms allowing the actors to fly in close proximity to the spectator.
Transportation is not provided.
Address:
Divadlo Blaník (Theatre Blaník)
Václavské náměstí (Wenceslas Square) 802/56
Prague 1
Conference Dinner at Žofín Palace
Wednesday, July 5, 2006 from 19:30
The EEMS 2006 “From Genes to Molecular Epidemiology” will not only be
about science, but also about having a good time. Wednesday evening
will be party time!
We will offer an exquisite blend of excellent food and entertainment at
the Žofín Palace, a wonderful neo-renaissance palace located on the
most beautiful island in Prague “Slovanský Ostrov” (Slavonic Island).
Transportation is not provided.
Address:
Žofín Palace
Slovanský Ostrov 226
Prague 1
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GUIDED TOURS
A number of interesting guided tours have been prepared for all participants and accompanying persons.
Please find the respective schedules and short description below. All participants are kindly asked to meet
at the registration area of the Prague Congress Centre 15 minutes before scheduled times. The organizer
reserves the right to cancel any tour in case there are less than 15 registered persons. On-site requests
for guided tours are welcome but cannot be guaranteed.
Grand Tour of Prague
Saturday, July 1, 2006, 13:00-17:00
Tuesday, July 4, 2006, 09:00-13:00
Thursday, July 6, 2006, 13:00-17:00
This insightful historical tour lets you enjoy the city’s most famous sights. The tour
starts at PragueCastle (Hradčany), registered as the biggest castle complex in the
world. The tour follows the “Royal Route”, former promenade of the kings and part
of the official coronation by foot. Winding down to Lesser Town and picturesque
KampaIsland you will cross the famous Charles Bridge and continue to discover
remarkable monuments of the OldTown area like CharlesUniversity founded in 14th
century. The tour passes by the Estates Theatre where Mozart personally conducted
the world premiere of his Don Giovanni. This remarkable journey ends in front of the
Old TownCity Hall with its extraordinary astronomical clock.
Price: EUR 37, - per person
The price includes air-conditioned coach transportation, English speaking guide and
entrance fees.
Jewish Quarter
Monday, July 3, 2006, 09:00-13:00
Wednesday, July 5, 2006, 13:00-17:00
You will discover an astonishing history of the Jewish community in Prague, which
can be traced back to the middle of the 10th century. Miraculously many outstanding
monuments have remained intact through WWII. The visit includes the Old Jewish
Cemetery, the main synagogues (Old-New Synagogue, the oldest one in Europe, founded in 13th century,
the Pinkas Synagogue) as well as the Jewish Museum that stores a heart-breaking collection of children‘s
art from the Nazis concentration camp, Terezín. Before departing take some time to visit the museum of
the famous writer Franz Kafka.
Price: EUR 43,- per person
The price includes air-conditioned coach transportation, English speaking guide and entrance fees.
Prague’s Famous Characters: Mozart, Mucha, Kafka
Sunday, July 2, 2006, 13:00-17:00 hrs
Tuesday, July 4, 2006, 09:00-13:00 hrs
During this tour you will have a chance to learn more about three Prague’s Famous artist: W. A. Mozart,
Alfons Mucha, Franz Kafka. Tour will start at Bertramka, where W. A. Mozart completed in the autumn of
1787 his opera Don Giovanni. Bertramka is used as a venue for regular concerts of chamber music nowadays. Romantic garden has preserved the atmosphere of the Mozart period to this day. Tour will continue to
the world’s first Mucha Museum which is dedicated to the life and work of the world-acclaimed Czech ART
NOUVEAU artist Alphonse Mucha (1860-1939). The museum is housed in the Baroque Kaunický Palace in
the very heart of Prague. At the end Franz Kafka Permanent Exhibition will be waiting for you. Franz Kafka
(1883-1924) is the undoubtedly most famous Czech writer, who, with a circle of other German-speaking
Jewish writers in Prague, played a major role in the literary scene at the beginning of past century. The
memorial hall commemorates the life and work of Franz Kafka.
Price: EUR 42,- per person
The price includes air-conditioned coach transportation, English speaking guide and entrance fees
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USEFUL INFORMATION ON PRAGUE
(in alphabetical order)
City of Prague
Prague, the capital city of the Czech Republic, situated in the heart of Europe ranks amongst the most
impressive historical cities in the world. Prague was established as a town when Prague Castle was built in
870. In the 11th century, the first Czech king, Vratislav I, chose Prague as his capital. The city has always
played an important part in the history of the nation, country and Europe. Since medieval times, Prague
has been distinguished as one of the most beautiful cities in the world. Prague is not only a centre of
cultural movements dating back for centuries (Art from every period in history can be found here), it also
exhibits a unique collection of historical monuments, dominated by Prague Castle. Prague occupies both
banks of the Vltava River and consists of 6 districts: the Old Town, which includes Josefov (the Jewish
Quarter, which remains intact despite World War II), New Town, Lower Town, Hradcany and Vysehrad.
These districts house the majority of historical landmarks, museums and galleries. Charles University,
founded in 1348 is one of the oldest universities in Europe. In 1992, the historical core of the city (866
hectares) was listed in the UNESCO World Cultural and Natural Heritage Register as a town with a unique
and lively blend of Roman, Gothic, Renaissance, Baroque, Art Nouveau and Cubist architecture. Prague
was one of nine European cities awarded the title European City of Culture in 2000. Prague has around 1.2
million inhabitants. For more information, visit the website www.pis.cz
Climate
Prague is a city with a continental climate. At the beginning of July the average temperature varies
between 20°C and 30°C.
Currency
The official currency in the Czech Republic is the Czech Crown (CZK/Kc). 1 Czech crown (CZK/Kc) = 100
hellers (hal.). Coins: 1, 2, 5, 10, 20 and 50 crowns; 50 hellers. Bank notes: 20, 50, 100, 200, 500, 1000,
2000 and 5000 crowns. Exchange offices and ATM machines are easily available throughout the town and
at Prague International Airport. It is advisable to exchange money in banks rather than in the high street
exchange offices. The approximate exchange rate as per December 2005 is: 1 EUR = 28,9 CZK For up-todate exchange rates please visit www.cnb.cz
Language
The official language in the Czech Republic is Czech.
Restaurants - Czech cuisine
Czech cuisine is typical of Middle European gastronomy, yet clearly reflects a number of Czech elements
- e.g. bread or fruit dumplings, various kinds of soups, sauces, numerous potato dishes, cakes and a
wide range of festive dishes. In general, Czech gastronomy means roasted pork with dumplings and sauerkraut, potato pancakes, plum dumplings and bilberry cakes … and, of course, Czech drinks - primarily
beer and first-rate wines from South Moravia, not to mention “Slivovice”, a clear Czech plum brandy and
“Becherovka” a delicious herbal elixir with legendary aphrodisiac qualities. People usually have lunch
between 12:00 and
14:00 and, sit down for dinner between 18:00 and 21:00. However, it is possible to dine throughout the
whole day and every visitor’s needs can be met.
Shopping
Shops in Prague are normally open Monday-Friday from 09:00-18:00, Saturday from 09:00-12:00. Most
of the shops in the city centre are open every week day from 09:00-20:00. All major credit cards are
accepted.
Taxi
When taking a taxi; be sure that the taxi is equipped with a permanently installed yellow roof lamp with
the TAXI sign in black letters. The registration number, company name and the price list including the basic
rate, rate per kilometre and one-minute-waiting rate must be displayed on both front doors of the cab.
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These prices must correspond with the prices set on the meter in the cab. Customers are recommended
to order a taxi with non-stop dispatching offices where the information on fares is available in advance.
Time
The Czech Republic lies within the Central European Time zone, i.e. GMT+1 hour. Summer time (GMT+2
hours) is in effect from the end of March until the end of September.
Tipping
Service is almost always included in hotel and restaurant bills. A further tip of a few coins is appropriate.
Some typical percentages for tipping are: Restaurants 5-10%, Tourist guide 10%.
Transportation in Prague
Prague Public Transit Co. Inc. is the main public transport operator in the Czech Republic. Almost two thousand metro trains, trams and buses are dispatched every day in Prague and the surrounding region.
Metro:
The Metro network is the backbone of the 50 km of tracks. The Metro operates daily from
5:00 to 24:00. We recommend that you use this kind of transport as the fastest and cheapest form of
moving around the city.
Tram:
Trams operate 24 hours a day. The Tram Service manages over 270 km of tracks.
Bus:
The Bus Service operates 206 bus lines; with a total network length of almost 800 km. Buses operate 24
hours a day.
Visa
All foreign visitors to the Czech Republic must possess a passport valid for at least the next 3 months.
Participants who require a visa should apply in advance to the consular offices of the Czech Republic or
diplomatic missions in their country in order to avoid delays in travel arrangements to the meeting. Please
note that the visa application procedure can take up to two months. For more information about visa
requirements please www.mzv.cz. For more information about travelling to the Czech Republic please visit
web site www.czech.cz.
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UNDERGROUND MAP
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26.6.2006 16:05:41
ECETOC-EEMS 2006 SYMPOSIA
ECETOC and EEMS, co-sponsored by CEFIC-LRI, are organising two Symposia, on ‚Molecular epidemiology:
New knowledge from biomarkers of effects‘ and on ‚Biomarkers for the evaluation of children‘s health‘.
The Symposia will be held on Monday 3 July 2006 in Prague during the annual general meeting of EEMS,
and will be open to all conference participants. A detailed programme with invited speakers and topics is
available from the ECETOC Secretariat. It can be viewed shortly at the EEMS2006 website, which contains
updated information about the whole conference. Subsequent papers from the two Symposia will be published in Mutation Research.
The two symposia follow on from a similar activity at the last EEMS meeting (Kos, Greece, July 2005) with
the objectives (i) to review the state of the science in the fast developing area of biomarkers in identifying exposure to environmental stressors and subsequently in the field of molecular epidemiology, and (ii)
address our current understanding of the role of genotoxic environmental agents in childhood disease.
The Long-range Research Initiative (LRI)
Over the years, there has been growing awareness and concern about the potential impact of human activity and man-made substances on the environment and human health. The chemical industry is conscious
of the need to address societal concerns and take responsibility in understanding the long-term impacts
of its products and processes.
The idea for LRI began in the USA in 1996, with the goal of responding to public and stakeholder concerns
through scientific investigation. The focus is on gaps in industry’s knowledge and understanding that are
critical for risk assessment. The broad aim is a validated infrastructure of scientific advice on which the
entire industry and regulatory bodies will draw to respond more quickly and accurately to the public’s
questions. Today‘s LRI is jointly managed by the American Chemical Council, Japanese Chemical Industry
Association, and European Chemical Council (CEFIC).
LRI sponsors research to help address some of the priorities of the European public health strategy:
improving risk assessment of chemicals; and more specifically monitoring effects of chemicals on health;
understanding the environmental factors in human health; establishing endocrine disruption references;
and coordinating research, data and activities at a European level. LRI also addresses many of the environmental objectives of the Eeuropean Union, including: linking environmental factors to health effects;
understanding and reducing chemical risks to environment; and improving animal testing in risk assessment.
ECETOC has been a key partner to CEFIC from the earliest stage of the LRI process. It provides scientific
support into the LRI, and input into the Research Programme.
Further information on the LRI may be found on the Internet, at http://www.cefic-lri.org/.
The European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC)
ECETOC was established in 1978 as a scientific, non-profit making, non-commercial association, financed
by 49 of the leading companies with interests in the manufacture and use of chemicals. A stand-alone
organisation, it was established to provide a scientific forum through which the extensive specialist expertise in the European chemical industry could be harnessed to research, review, assess and publish studies
on the ecotoxicology and toxicology of chemicals.
The Association‘s main objective is to identify, evaluate and through such knowledge help industry minimise, any potentially adverse effects on health and the environment that may arise from the manufacture
and use of chemicals. To achieve this mission, ECETOC facilitates the networking of suitably qualified scientists from its member companies and co-operates in a scientific context with intergovernmental agencies,
health authorities and professional institutions.
ECETOC is managed by a Board of Directors comprising up to twelve senior executives from member
companies. The Board is responsible for the overall policy and finance of the organisation and appoints
the members of the Scientific Committee which defines, manages and peer reviews the ECETOC work
programme.
The output of the ECETOC work programme is delivered in published reports and papers, through scientific
representation in the activities of international organisations and regulatory groups and through presentations and organisation of specialised workshops and fora.
Further information on ECETOC and its Science Programme can be found on the Internet, at
http://www.ecetoc.org/.
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ORAL
PRESENTATIONS
Fritz Sobels Award Lecture
Young Scientists Award Lecture
Keynote Lectures
KL-1 - KL-3
HUMN Project - Automated Micronucleus Workshop
O-001 - O-012
Transcriptomics in Genetic Toxicology and Molecular Epidemiology
O-013 - O-018
New Matters in DNA Repair and Mutation Research
O-019 - O-026
New Matters: From Mechanisms to Human Disease
O-027 - O-032
Food Mutagens Impact on Health (ECNIS)
O-033 - O-040
Nutrigenomics
O-041 - O-045
Molecular Epidemiology: New Knowledge
from Biomarkers of Effects (ECETOC)
O-046 - O-051
Biomarkers for the Evaluation of Children’s Health (ECETOC)
O-052 - O-056
Genotoxicity and Mutagenicity of Complex Mixtures
O-057 - O-062
Gene-Environment Interaction in the Risk of Common non Cancer Disease
O-064 - O-069
New Knowledge from Biomarkers of Exposure (ECNIS)
O-070 - O-076
Epigenetic Mechanisms in Carcinogenesis (ECNIS)
O-077 - O-082
New Aspects of Regulatory Testing and Risk Assessment
O-083 - O-088
Biotransformation of Carcinogens
O-089 - O-092
Oxidative Stress Responses
O-093 - O-097
Germ Cell Mutagenesis/Epidemiology
O-098 - O-101
Genetic Ecotoxicology
O-102 - O-105
Impact of Genetic Variation on Individual Susceptibility
O-106 - O-112
Risk Assessment
O-113 - O-119
Occupational Exposure to Mutagens and Carcinogens
O-120 - O-127
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ORAL PRESENTATIONS
FRITZ SOBELS AWARD LECTURE
DNA ADDUCTS AS RISK INDICATORS
Herman Autrup
Institute of Public Health, University of Aarhus, Aarhus, Denmark
More than 50% of chemicals classified as carcinogens by IARC form adducts with DNA, either directly or after metabolic
activation, thus it is anticipated that these chemical modifications of play a major role in mutagenesis and carcinogenesis. The carcinogen binding index has been used to list the potency of chemical carcinogens. In experimental systems
an association between adducts and mutagenic events was demonstrated, an observation that formed the basis for
molecular dosimetry and the Sobel´s parallelogram approach. Until recently, there was only limited information on the
role of adducts in development of cancer in humans.
Early studies have demonstrated that human tissues can metabolise chemical carcinogens into DNA binding metabolites
and the products formed were qualitatively similar to those formed in animal tissues. A large inter-individual variation
in adduct levels were observed under similar exposure conditions, and quantitative differences between animals and
humans was noted.
Development of sensitive analytical procedures, e.g. P32 postlabelling and immunoassays allowed the detection of
adducts in humans following occupational and environmental exposure. These techniques have been extensively used
in ecological studies, and more recently in case control studies. An association between adduct levels in non-target cells
and cancer risk has been established for several cancer sites, mostly lung.
The genetic basis for the large inter-individual variation observed in the original in vitro studies have been explored with
the focus on genetic polymorphisms in carcinogen metabolizing enzymes and DNA repair enzymes. It appears as polymorphisms in CYP1A1 and GST enzymes both influence adduct levels and cancer risk, but the analysis is complicated
by variation in nutritional status and exposure levels.
Although overall adduct level has been linked to cancer risk, it is still a crude measure and more clear link between
gene and site specific adduction is required to clearly establish the biological significance. Adducts are increasing been
used in risk assessment, e.g. classification of carcinogenicity, and attempts are made to use adduct data in quantitative
risk assessment.
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ORAL PRESENTATIONS
YOUNG SCIENTIST AWARD LECTURE
ARISTOLOCHIC ACID A POTENTIAL RISK FACTOR FOR BALKAN ENDEMIC NEPHROPATHY AND
ASSOCIATED UROTHELIAL CANCER: POTENTIAL MECHANISMS
Volker Manfred Arlt
Aristolochic acid nephropathy (AAN) is a unique type of renal disease that predisposes patients to a high risk of urothelial malignancy. There is clear evidence that aristolochic acid (AA), a known human nephrotoxin, is a mutagen and rodent
carcinogen forming covalent DNA adducts. The most abundant DNA adduct detected in various tissues of AAN patients
and AA-treated rats is 7-(deoxyadenosin-N6-yl)aristolactam I (dA-AAI). In AAN patients urothelial atypia are associated
with the overexpression of the P53 protein, suggesting that p53 is mutated in AAN-associated tumours. Interestingly, in
one AAN patient available a characteristic A to T transversion mutation was found in the p53 gene in urothelial tumour
cells. To examine experimentally induced p53 mutation pattern and mutational specificity of AA in the human p53
gene in laboratory animals a human p53 knock-in (Hupki) mouse has been constructed. When the Hupki p53 gene of
immortalized cell lines derived from primary Hupki embryonic fibroblasts (HUFs) exposed to AA were sequenced, several mutations in HUFs carried a specific A to T transversion mutation in the p53 gene. A to T transversions are typical
mutations observed in the H-ras oncogene of tumours in rodents treated with AA and correspond with DNA adduct formation at adenine residues. These data may indicate the probable molecular mechanism whereby AA causes urothelial
tumours. On both clinical and morphological grounds, similarities between AAN and another fibrosing nephropathy, the
Balkan endemic nephropathy (BEN), including the association with urothelial tumours, have been observed. It has been
suggested that the mycotoxin ochratoxin A is associated with the development of BEN, however, the aetiology remains
still unclear. A role of AA in the aetiology of BEN was under debate, because AA was found in flour obtained from wheat
contaminated with seeds of Aristolochia clematitis in endemic areas. Using the 32P-postlabelling method we identified
AA-DNA adducts in patients with end-stage renal disease living in endemic areas for BEN, suggesting that AA may be
a risk factor in development of BEN and associated urothelial cancer.
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ORAL PRESENTATIONS
KEYNOTE LECTURES
KL-1
TP53 MUTATIONS AS BIOMARKERS OF CARCINOGEN EXPOSURE AND TUMORIGENESIS
Pierre Hainaut
International Agency for Research on Cancer,150 Cours Albert Thomas, 69372 Lyon, France
Mutations in the tumour suppressor TP53 are common in almost all forms of cancer. These mutations are mostly missense and show variations among cancers in their distribution and types. A database of these mutations is maintained
at IARC and contains over 20,000 entries on somatic or germline mutations (http://www-p53.iarc.fr). However, despite
many studies, the value of mutation detection for cancer detetion, diagnosis or prognosis, remains to be fully assessed.
Mutations can be found ahead of detectable cancer, in particular in tissues of subjects exposed to high levels of environmental mutagens, demonstrating that in some cases TP53 mutation precedes or accompanies the development of
precursor lesions. This is the case in subjects exposed to aflatoxins in area of high incidence of hepatocellular carcinoma
(HCC) such as sub-Saharan Africa and parts of South-East Asia. A particular mutation at codon 249 (R249S), considered as a mutagenic signature of aflatoxin metabolites, is common in HCC in these regions, in conjunction with chronic
Hepatitis B Virus infection. Studies using free DNA fragments isolated from plasma have shown that this mutation is
detectable in cancer subjects, in patients with non-cancer liver diseases, and in healthy subjects exposed to aflatoxin.
In a cohort study in West Africa, the use of a sensitive and quantitative mass spectrometric detection method allows to
detect R249S in plasma DNA with a seasonal variation that recapitulates patterns of aflatoxin exposure. Furthermore,
the amount of mutant DNA in the plasma is higher in HB chronic carriers, suggesting that chronic infection has an
impact on the mutagenicity of aflatoxin in liver cells. Thus, the syngergistic effect between chronic carriage and aflatoxin
exposure is manisfest at the early stages of the process leading ot hepatocarcinogenesis.
KL-2
NUTRIGENOMICS: APPLICATIONS TO THE MOLECULAR EPIDEMIOLOGY OF FOOD-BORNE
MUTAGENS AND CARCINOGENS
Fred Kadlubar
National Center for Toxicological Research, Jefferson, USA
Content: Nutrigenomics or nutrigenetics, the study of the interaction between genes (and gene variants) and food
constituents, including nutrients and by-products, has become synonymous with personalized nutrition. Metabolism
allows the utilization of nutrients and the detoxification of potentially harmful compounds/metabolites. Food-borne
mutagens/carcinogens, including heterocyclic amines (HCAs), polycyclic aromatic hydrocarbons (PAHs), nitrosamines,
and mycotoxins, form DNA adducts causing nucleotide alterations and chromosomal aberrations and have been associated with colon, breast, prostate, lung, or liver cancer. The genes responsible for their metabolism often have a number
of polymorphisms (SNPs) leading to distinct genotypes/haplotypes; and although the HapMap Project has been useful,
it is necessary to use functional genomic approaches for re-sequencing both coding and promoter regions of genes in
order to understand nutrient-genotype-phenotype interactions. In this regard, CYP1A2, which N-hydroxylates HCAs,
was re-sequenced over 39.6 kb from 32 individuals selected from 285 phenotyped individuals to show unequivocally
that genotype does not predict the 60-fold variation in phenotype and that previously reported nutritional factors may
account for a large portion of this variation. GSTA1, which detoxifies the DNA-reactive N-acetoxy-HAAs, exhibits a haplotype that abolishes an SP1-binding site, resulting in lower expression and higher risk for colon cancer. SULT1A1, which
activates N-hydroxy-HCAs and detoxifies phenolic PAHs, shows 5 functional SNPs but these only account for about 20%
of the observed phenotype, although both genotype and phenotype are associated with prostate cancer risk. Finally, we
have recently re-sequenced the promoter region of GSTM1, which detoxifies PAH diol epoxides, and discovered an allele
that creates a retinoid-responsive element. This results in overexpression of the enzyme, which may lead to cancer
resistance or increased response to retinoid chemotherapy.
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ORAL PRESENTATIONS
KL-3
NEW INSIGHTS FROM AN OLD ASSAY: THE SALMONELLA ASSAY REVISITED
David M. DeMarini
US EPA, RTP, NC, USA
Although the Salmonella (Ames) mutagenicity assay is 36 years old, it is still the primary assay for routine mutagenicity testing and uniquely suited for some basic research questions. Using genomic hybridization, we showed that the
Ames strains are missing 15-119 genes due to the ΔuvrB mutation. We now show that the ΔuvrB mutation makes the
Ames strains more sensitive to most mutagens compared to strains that are mutated in only the uvrB gene. Based on
molecular analysis of mutants induced by various mutagens, combined with analysis of both stable DNA adducts (by
32
P-postlabeling) and unstable DNA adducts (by an aldehydic site assay), we have found that the mutation spectrum
of dibenz[a,l]pyrene is similar to its spectrum of stable DNA adducts, whereas this is not the case for benzo[a]pyrene,
which induced ~600X more unstable than stable adducts. We found that a sulfone-phenanthrene, which was the most
mutagenic form of sulfur-containing phenanthrenes examined, produced only unstable DNA adducts. The drinking-water
mutagen MX also produced only unstable DNA adducts. Using specially designed microarray chips, we found that MX
altered expression of many genes, including those involved in porphyrin metabolism. This may be due to the structural
similarity of MX, which is a furanone, to pyrrole. In human studies, we have analyzed both conjugated and unconjugated urinary mutagenicity by acid-hydrolyzing the urine. In a clinical case-control study, we found that unconjugated
urinary mutagenicity is a biomarker and risk factor for colon cancer. In a feeding study, we now show that cruciferous
vegetables and yogurt reduced this category of urinary mutagenicity but enhanced conjugated urinary mutagenicity.
These studies show the continued value of the Ames assay for basic and applied research. [Abstract does not necessarily reflect the views of the US EPA.]
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ORAL PRESENTATIONS
O-001
VALIDATION OF MICRONUCLEI ASSAY IN PERIPHERAL BLOOD LYMPHOCYTES AS EARLY
CANCER RISK BIOMARKERS BY A NESTED CASE-CONTROL STUDY IN ITALY
Roberto Barale
Murgia E, Maggini V,Rossi AM
Department of Biology, Pisa University, Pisa, Italy
AIM: Aim of this work was to assess the predictive value of micronuclei (MN) in peripheral blood lymphocytes (PBL) for
the risk of developing cancer diseases.
METHODS: Blood samples from 1,650 healthy subjects selected from the general population were collected between
June 1991 and November 1993, and slides were immediately prepared for MN counting. The vital status, or the cause
of death, was monitored for all subjects until January 2005. At the end of the follow-up,14 years later, 111 deaths
and 52 cancer cases were recorded. For the present nested case-control study, 49 cancer cases were eligible and 101
healthy controls, matched for age and gender, were selected. Two thousands binucleated (BN) cells/subject were scored
for the MN assay.
RESULTS: MN frequency was significantly higher (Kruskall-Wallis test p=1.4 x 10-8) in cases (4.7 ± 3.4 MN/1000 BN
cells) than in the control group (1.5 ± 1.7 MN/1000 BN cells). No influence of other factors, such as age, gender, smoking and drinking habits, was observed in a logistic regression analysis. CONCLUSIONS: MN confirmed to be a good
cytogenetic biomarker that can be potentially used as predictor of cancer susceptibility with a very high sensitivity
(0.80; 95%C.I. 0.69-0.91). This study will contribute to the current European studies on the assessment of the predictive value of micronuclei for the risk of cancer onset.
O-002
COMPARISON OF AUTOMATED ANALYSIS METHODS FOR MICRONUCLEUS ASSAYS IN HUMAN
AND RODENT CELLS
Marlies De Boeck
Johnson & Johnson Pharmaceutical Research & Development, , Beerse, Belgium
Primary cells and cell lines are widely used for the assessment of micronucleus frequencies in biomonitoring studies,
genetic toxicology testing and fundamental research. To overcome the labour-intensive microscopic scoring of these
relatively rare events, over the years, different automated analysis methods have been developed. Image analysis,
flow cytometric and laser scanning cytometric methods are the most common. Increased throughput, objectivity and
accuracy are the main reasons to switch from manual to (semi)-automated analysis. Some of the early versions of
automation suffered from drawbacks such as artefact scoring. Nowadays, the systems undergo thorough biological and
technical validation and adequate quality control measures are incorporated. In addition, automation favours multiple
endpoint analysis and has its application in determining mechanisms of action (e.g. threshold assessment). The choice
of the automated system depends on the intended use (e.g. high throughput screening versus human biomonitoring).
A brief introduction on some of the methods that are not presented in this HUMN workshop will be given.
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ORAL PRESENTATIONS
O-003
FLOW CYTOMETRIC METHODS FOR SCORING MICRONUCLEI IN ERYTHROCYTES AND
NUCLEATED CELLS
Stephen D. Dertinger1
Svetlana L. Avlasevich1, Steven M. Bryce1, Siva Sugunan1, Dorothea K. Torous1, Yuhchyau Chen2, Russell
E. Ware3, Jack Bishop4, Kristine Witt4, Ruth Wilkins5 James McNamee5
Litron Laboratories, Rochester, NY; Department of Radiation Oncology, University of Rochester Medical Center, Rochester, NY2; St.
Jude Children’s Research Hospital, Memphis, TN3; NIEHS, Environmental Toxicology Program, Research Triangle Park, NC4;
Consumer and Clinical Radiation Protection Bureau, Health Canada, Ottawa, Canada5
The frequency of human blood micronucleated reticulocytes is a chromosomal damage endpoint that does not require
cell culture, and can be rapidly scored via flow cytometry (FCM). The endpoint is likely to find wide use after more
complete characterization of 1) its ability to assess acute and chronic genotoxicant exposures, 2) its utility for studying clinically and environmentally relevant exposures, and 3) the degree that host factors influence micronucleated
reticulocyte frequencies. This laboratory and others continue to address these issues by collecting data based on FCM
analyses using a 3-color labeling procedure and a biostandard for consistent instrument setup (commercially known
as MicroFlow® kits). Using this technique, the following populations have been studied, and will be discussed here:
healthy adults; patients undergoing cancer therapy; pediatric sickle cell disease patients (with and without hydroxyurea
exposure); pregnant women and newborn infants treated with zidovudine.
Aside from reticulocyte-based analyses, this laboratory also continues to develop FCM approaches for scoring micronucleus frequencies in nucleated cells. A staining procedure has been developed whereby necrotic and mid/late-stage apoptotic cells are labeled with
ethidium monoazide. Cells are subsequently stripped of their cytoplasmic membranes and incubated with a pan-nucleic acid dye. This
process provides a suspension of free nuclei and micronuclei, differentially stained relative to chromatin associated with dead/dying
cells. Representative in vitro data will be presented. Health Canada investigators have extended these studies by treating human blood
lymphocytes with ionizing radiation ex vivo. For these analyses, a third fluorescent reagent (DiA as a membrane dye) was incorporated
into the basic staining protocol. Data will be presented which demonstrate a highly reproducible dose-response relationship in the 0 - 4
Gy range, with a sensitivity limit of at least 1 Gy. The assay may provide for rapid analysis of radiation exposures, and therefore be particularly beneficial in emergency triage situations.
O-004
THE MICRONUCLEUS ASSAY IN HUMAN ERYTHROCYTES
Lilianne Abramsson-Zetterberg
National Food Administration, Box 622, 751 26 Uppsala, Sweden
The micronucleus (MN) assay in vivo has developed a lot since it first was described. The use of a flow cytometer instead
of a microscope when analysing the cells has increased the capacity and also the sensitivity. There are in principal two
flow cytometer-based MN methods described, one using a single-lazer and the other using a double-lazer flow cytometer. From the double-lazer flow cytometer-based MN assay data from > 20 different agents are so far published.
Furthermore, the automation of the analysis has been extended to cover also human erythrocytes. Using erythrocytes the variation in genotoxic exposure can be studied over short periods, which is different from e.g. assays using
peripheral lymphocytes. The use of flow cytometer allows a great number of cells (about 500 000 per sample) to be
analysed, giving the high sensitivity, which is a prerequisite in monitoring studies. The frequency of MN in very young
erythrocytes in human peripheral blood (fMN-Trf-Ret) can be determined. In a blood sample, a separation of these very
young cells (Trf-Ret) from older erythrocytes makes this micronucleus determination possible (L.Abramsson-Zetterberg
et al. (2000), Environ. Mol.Mutagen 36: 22-31). This separation is made with an immuno-magnetic method. The mean
background variation in the fMN-Trf-Ret from the same person with one week between sampling occasions is 0.2‰.
Recently published results show a correlation between low folate status and a high fMN-Trf-Ret, (L.Abramsson-Zetterberg
et al. (2006), Mut. Res. 603, 33-40.). This correlation was determined in blood from persons, both men and women,
with a normal serum folate status. Others have observed a correlation between folate status and chromosome stability
but then using the MN assay in human lymphocytes.
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ORAL PRESENTATIONS
O-005
IMMUNOMAGNETIC SEPARATION TECHNIQUE WITH SINGLE-LASER FLOW CYTOMETRY TO
ISOLATE AND ANALYZE MICRONUCLEI IN RETICULOCYTES
Ashutosh Lal1
Wafa Atamna1, Tal Offer1, Bruce Ames1; Frans Kuypers2, Amrita Bhagat2
Nutrition & Metabolism Center1; Red Cell Biology Laboratory, Children’s Hospital Oakland Research Institute, Oakland CA94609,
USA2
Background: DNA damage from environmental stress, such as radiation, poor nutrition and free radicals, induces formation of micronuclei in erythroid precursors. Micronuclei persist transiently in the circulating immature reticulocytes
where they can be measured using high throughput assays.
Aim: To develop an automated human erythrocyte micronucleus assay for single-laser flow cytometer.
Methods: Blood was collected in EDTA from healthy volunteers. For comparison, blood was collected from patients with
thalassemia who have marked increase in oxidative stress from elevated body iron. Washed blood (250 μL) was depleted
of white blood cells by incubation with a combination of mouse anti-human antibodies against CD14 and CD45 antigens.
Immunomagnetic beads (Dynal Cellection) coated with anti-mouse IgG were added. The supernatant was collected after
magnetic separation, and mixed with immunomagnetic beads attached to mouse anti-human CD71 antibody. Beads
were collected after magnetic separation, cells were stained with mouse anti-human CD71–FITC conjugated antibody,
and released from beads with DNase. After overnight fixation in methanol at -80C, the cells were rehydrated in PBS
and treated with RNase for 1 hour. After incubation in PBS and 7-AAD for 30 minutes, 100,000 cells were analyzed with
FACSCalibur. Excitation source was 488 nm laser, CD71-FITC emission was collected in FL1 channel, and 7-AAD emission
in FL3. The gate for micronucleated reticulocytes was set between the main group of 7-AAD negative reticulocytes and
the brightly stained white blood cells.
Results: In healthy volunteers the mean MN frequency by this method is approximately 2%. Patients with thalassemia
had up to 5 fold increase in MN frequency. Significant increase in reactive oxygen species in RBC’s was demonstrated
with dichlorofluoroscein diacetate and C11-BODIPY.
Conclusion: Combination of 7-AAD and FITC-conjugated anti-CD71 antibody allows use of single-laser flow cytometer
to measure frequency of micronuclei in immunomagnetically enriched population of reticulocytes.
O-006
COMPARISON OF THE PERIPHERAL BLOOD MICRONUCLEUS TEST IN RAT AND MOUSE
EXPOSED TO ANEUGENS: INVOLVEMENT OF THE SPLEEN IN THE SELECTIVE REMOVAL OF
THE MICRONUCLEATED ERYTHROCYTES
Zoryana Cammerer1
Azeddine Elhajouji 1, Willi Suter 1; Micheline Kirsch-Volders 2
Novartis Pharma AG, Basel, Switzerland 1; Vrije Universiteit of Brussels,Laboratory of Cell Genetics, Brussels, Belgium 2
Detection of clastogenic compounds in the peripheral blood MN test in rats is well established. However, based on the
available data in the literature the assessment of aneugen induced MN in rat peripheral blood shows contradictory data.
In our study a comparative evaluation of the peripheral blood flow cytometry micronucleus test in Wistar Han rat and
CD1 mouse exposed to two aneugens (vinblastine and vincristine) was performed. In addition both compounds were
tested in the rat bone marrow MN test. The treatment with vinblastine (0.25, 0.5, 1, mg/kg) or vincristine (0.025, 0.05,
0.1 mg/kg) induced no statistically significant increase in micronucleated polychromatic erythrocytes (MN-PCE) in rat
peripheral blood. In rat bone marrow, however, a clear, statistically significant increase in MN-PCE was found. The positive effect in the bone marrow MN test shows that the target organ was exposed to the appropriate concentration levels
of the respective aneugens. In mouse the peripheral blood flow cytometry analysis after the treatment with vinblastine,
vincristine and colchicine showed clear statistically significant increase in MN-PCE with all three compounds. These data
suggest that micronucleated cells induced by aneugens are removed from blood circulation by spleen due to the large
size of micronuclei. To investigate this hypothesis experiments with splenectomized rats treated with vincristine (0.05,
0.1, 0.15 mg/kg) and colchicine (0.7, 1, 1.3 mg/kg) were performed and statistically significant increases in MN-PCE
were found with all three doses of both compounds. Our results demonstrate that spleen is playing an important role
in the elimination of the MN-PCE induced by aneugens in Wistar Han rats. Based on our data it is concluded that the
flow cytometry peripheral blood micronucleus test is an appropriate test system for evaluating the genotoxic effects of
aneugens in mouse but probably not in rats.
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ORAL PRESENTATIONS
O-007
AUTOMATED IN VITRO AND IN VIVO MICRONUCLEUS SCORING USING THE METAFER
MICRONUCLEI SLIDE SCANNING SYSTEM
Christian Schunck
Tilman Johannes, Peter Haub
MetaSystems, Robert-Bosch-Str. 6, D-68804 Altlussheim
Analyses of micronuclei (MN) in vitro and in vivo are well established methods to assess DNA damage induced by chemical agents or ionizing radiation. However, often MN analyses tend to involve large sample numbers, and reproducibility
appears to be crucial for the comparison of results between different experiments or studies.
It is obvious that analysis automation of both the in vitro and the in vivo MN assay would help to streamline the tests,
and would also provide a higher degree of reproducibility. It is already known that the slide scanning platform Metafer
with the software module MicroNuclei generates highly precise results for the in vitro MN assay (Varga et al., 2004;
Patino-Garcia, B. et al., 2006). Recently we developed a set of system parameters (classifier) for Metafer with the
software module MetaCyte to assess column purified in vivo MN assay slides. With this classifier Metafer does a proper
selection of PCE and mature erythrocytes (NCE) based on color values, and automatically counts MN in both populations.
We could show that that the time needed to scan for 2,000 cells is about 5 – 10 min. Search and analysis is done unattended by the system, so that the operator time per slide is less than 1 min. Results obtained with the system could be
shown to be highly reproducible, and comparable with manual results from the same slides.
References
Varga, D. et al. (2004) An automated scoring procedure for the micronucleus test by image analysis. Mutagenesis 19,
391-397.
Patino-Garcia, B. et al. (2006) Scoring variability of micronuclei in binucleated human lymphocytes in a case-control
study. Mutagenesis (electronically published ahead of print).
O-008
A NEW INTEGRATED QUANTITATIVE IMAGING METHOD FOR AUTOMATED MICRONUCLEI
DETECTION AND SCORING USING PATHFINDER™ IMAGE CYTOMETER
Françoise Soussaline1
Homsy C. 1; Sallette J. 1; Tatarinova E. 1 ; Decordier I. 2; Plas G. 2; Roesems S. 2; Kirsch-Volders M. 2
IMSTAR SA, 60 rue Notre Dame des Champs, 75006 Paris, France www.imstar.fr1, Laboratory of Cell Genetics, Vrije Universi teit
Brussel, Pleinlaan 2, 1050 Brussels, Belgium2
The ex vivo/in vitro Micronucleus (MN) cytokinesis block assay is a well-validated methodology for biomonitoring, providing a measure of both chromosome breakage and chromosome loss. Moreover, the MN frequency and the characteristics of micronucleated cells within a population may be a valid biomarker of carcinogenic risks and phenotypic effects
of genotoxic agents. Our study is carried out in the frame of NEWGENERIS, an Integrated Project of the EC 6th FP (Food
quality and safety), where WP7 objectives are: 1) to implement a high throughput automated facility scoring of the in
vitro MN cytokinesis-block assay, 2) to assess the genotoxicity of a selected number of potential food carcinogens and
3) to study the influence of diet and environment on mother versus child genotoxic responses.
Using HUMN scoring criteria, IMSTAR and the VUB laboratory determined the most robust protocol of slide preparation
adapted to automated scoring. A standardized protocol was established using peripheral blood lymphocytes samples
from different European partner laboratories and standard slides.
Fine tuning of PATHFINDERTM Cellscan µN (IMSTAR), a fully automated image cytometer is expected to allow more objective and less time-consuming scanning and MN scoring than human scoring.
The system was tested on Giemsa stained slides, including negative and positive control samples, and samples collected
after treatment with clastogenic and aneugenic agents. Working steps for automated detection include images capture,
cell detection based on the cytoplasm and nuclei detection in each cell, as well as morphology characterisation of each
cell, eventually leading to recognition of cells undergoing mitosis or apoptosis.
For each individual cell, Pathfinder™ system was within cell populations the total number of cells, the % of mono-, biand poly (more than three) -nucleated cells, relative nuclei/cytoplasm area, number of nuclei per cell, and the number
of micronuclei per cell, size of micronuclei. Total time for capture and detection per slide (i.e. more than 1000 cells)
did not exceed 30 minutes at the present time. In this validation study, we compare the results obtained with various
compounds from manual scoring and from automated detection. Possible improvements in fluorescent dyes and identification of micronuclei (in particular small micronuclei induced by clastogenic agents) are discussed with regard to
complete automation for analysis using a slide feeder (IMSTAR ACCELL SFD).
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O-009
AUTOMATED MICRONUCLEUS SCORING USING IMAGE ANALYSIS AT NOVARTIS
Wilfried Frieauff 1
Joseph Gabriels 2, Claudia McGinnis 2
Novartis Pharma AG, Genetic Toxicology and Safety Pharmacology, 4002 Basel, Switzerland 1; Novartis Institutes for BioMedical
Research, Cambridge, MA, USA 2
Novartis has increasingly been using automated image analysis in genotoxicity testing (micronucleus test, comet assay,
metaphase finding) since 1988. To obtain higher throughput and less compound consumption, extensive automation
and miniaturization are preferred options. For the micronucleus screening test (MNT) to predict the chromosome aberration test (CA) in human peripheral lymphocytes, we changed from Mouse Lymphoma (L5178Y) to TK6 cells. These are
immortalized cells of human lymphoblastoid origin, p53 proficient, and are comparable with L5178Y cells regarding ease
of cultivation, growth in suspension, miniaturization of the assay, and amenability to automatic analysis. For a comparison of the predictivity of the CA result by the MNT, several Novartis compounds from different chemical classes were
processed in both assays. The Mouse Lymphoma MNT showed an overall predictivity of 77% (31 compounds tested),
while the TK6 MNT yielded a predictivity of 86% for a total of 43 tested compounds.
The image analysis application was developed to run on a PC connected to a fully automated microscope and a robotic
slide feeder. This system (ROBIAS) is capable of scoring 130 slides consecutively without any user interaction, leading
to a 3-fold increase in throughput. ROBIAS also serves as platform for other genotoxicity tests, making image analysis
a powerful analysis tool.
In addition, in early drug discovery (lead finding and optimization phase), the Cellomics ArrayScan® micronucleus
screening system has been established, using a 20-hour (-S9) treatment protocol of CHO-K1 cells with very low compound consumption. This 96-well, high-content imaging based on automated fluorescence microscopy allows for much
higher throughput and reduced costs in micronucleus assessment. The assay validation was based on more than 50
Novartis compounds, showing a very good correlation (predictivity ~90%) with the slide-based TK6 MNT, making the
system a valuable tool for rapid first tier screening of > 1200 compounds per year.
O-010
TOWARDS AUTOMATIC SCORING OF THE CYTOKINESIS-BLOCK MICRONUCLEUS CYTOME
ASSAY
Ida-Maria Sintorn1
Paul Jackway1, Pascal Vallotton1, Michael Fenech2
CSIRO Mathematical and Information Sciences1; CSIRO Human Nutrition, Adelaide, Australia2
The CBMN Cytome assay is an extended version of the established CBMN assay which is restricted to measurement of
micronuclei (MN) in binucleated (BN) cells resulting from chromosome breakage and loss. Based on the number of nuclei
and morphological appearance, cells can be classified into five main classes: mono-nucleated; bi-nucleated; multinucleated; apoptotic; and necrotic. The bi-nucleated class can be further divided into subclasses based on presence and
number of MN, nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs). Mono nucleated cells can be further divided
into subclasses based on presence and number of MN and NBUDs. All this information provides important insights into
the mechanisms of genome damage. For example, the presence of NPB in BN cells is indicative of DNA damage misrepair and/or telomere end fusion, while NBUDs are a biomarker of gene amplification. The origin of MN, NPB and NBUDs
can be further investigated by using fluorescent stains for certain markers such as centromeres and telomeres.
Towards developing an automated image based scoring method for all nuclear phenotypes in the CBMN Cytome assay,
we are focusing on robustly identifying and classifying cells as mono-, bi- or multi-nucleated, as well as detecting MN
and NBUDs in mono and BN cells, and NPBs in the BN cells. We are currently doing this in colour brightfield microscopy images of Diff-quick stained isolated lymphocyte cells. We are simultaneously investigating a number of different
options for slide preparation, staining, and image acquisition to establish a suitable protocol for automated image analysis that will remain as simple as possible. The two main criteria that need to be met are well separated cells and good
contrast between nuclei and cytoplasms. We are investigating whether fluorescence staining is necessary or if brightfield
staining with/without RNAase treatment is sufficient for these purposes.
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O-011
ADVANTAGES AND DISADVANTAGES OF DIFFERENT AUTOMATED SYSTEMS AND CURRENT
KNOWLEDGE GAPS
Ilse Decordier
Laboratory of Cell Genetics, Vrije Universiteit Brussel, Belgium
The scoring of micronuclei is per definition a cell by cell analysis which assesses the formation of micronuclei during
the metaphase-anaphase transition. Therefore it is essential to take into account cell division. Both flow cytometry and
image analysis can fulfil these requirements, as far as adequate preparation methods, stainings and selection criteria
are applied. The recommended methodology will be dependent on the question (cumulative low level versus recent
acute exposure), cell type (erythrocytes or lymphocytes), application (biomonitoring or hazard assessment) and the
sensitivity, specificity and reproducibility of the results obtained with a given methodology. Interlaboratory variation
would therefore be recommended
Validation of a methodology should take into account these three parameters, and in particular the capacity to discriminate accurately between small micronuclei induced by clastogens and large micronuclei induced by aneugens.
Practically, this consists in evaluating for a selected number of clastogens and aneugens which are the proportions of
false negatives and false positives with a given method. Without this validation step no data can be accepted. These
reference molecules would therefore serve as controls in later studies.
O-012
DEVELOPING A COLLABORATIVE NETWORK FOCUSSED ON AUTOMATED MICRONUCLEUS
ASSAY SYSTEMS AND THEIR VALIDATION AS PART OF THE HUMN PROJECT
Michael Fenech
CSIRO Mathematical and Information Sciences, Adelaide, Australia, CSIRO Human Nutrition, Adelaide, Australia
In this presentation I shall provide a brief overview of the objectives and achievements of the HUMN project (www.
humn.org) an international collaborative project on the use of micronucleus assays to study genome damage in human
individuals and populations.
This presentation will also be an introduction to the Automated Micronucleus Workshop organised by the HUMN project
with the aim of:
Reviewing current progress in development of automated systems for measuring micronuclei in human and mammalian
cells using image and flow cytometry.
Discussing current limitations with visual and automated scoring and how consistency can be improved in these systems.
Establishing criteria for comparing results between visual scoring and automated scoring in order to facilitate the validation process of automated systems.
Exploring the possibility of developing a collaborative network focussed on automated systems and their validation as
part of the HUMN project
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O-013
TOXICOGENOMIC ANALYSIS OF GENE EXPRESSION AN EMERGING APPROACH FOR
DIFFERENTIATING GENOTOXIC MECHANISMS
Jiri Aubrecht
Safety Sciences, Pfizer Inc, Groton, USA
The response to toxic stress leads to activation of complex molecular pathways involved in cell survival and/or cell
death. The development of genomic approaches at transcriptional or functional levels for studying molecular processes
elicited by treatment with chemicals, system toxicology, has a great potential for investigating mechanisms of toxicity.
Although standard in vitro genetic toxicity testing provides relatively simple and accurate hazard detection, interpretation of positive findings in terms of relevant risk, oncogenic potential in animals and humans, is difficult due to the
limited insight into underlying genotoxic mechanisms. The potential of chemicals to induce tumors in vivo, carcinogenicity, is evaluated using a 2-year bioassay in rats and mice that is very costly and time consuming. Thus development
of mechanism-based approaches capable of differentiating a wide range of genotoxic and oncogenic mechanisms is
expected to significantly improve risk assessment. The goal of this presentation is to summarize current developments
in toxicogenomic analysis of genotoxic mechanisms and provide a perspective on application of genomic approaches in
risk assessment.
O-014
DISCRIMINATION OF GENOTOXIC FROM NON-GENOTOXIC CARCINOGENS BY EXPRESSION
PROFILING IN VITRO
Jos Kleinjans
Dept. of Health Risk Analysis & Toxicology, Maastricht University, The Netherlands
Demands for better tests for genotoxicity and chemical carcinogenesis are increasing: For pharmaceuticals, the current
test battery on genotoxicity has been assessed to predict rodent carcinogenicity correctly by not more than 38 % while
simultaneously producing high percentages of false positives. A recent survey of over 700 chemicals demonstrated that
even 75–95% of non-carcinogens gave positive (i.e. false positive) results in at least one test in the in vitro test battery.
The current rodent cancer bioassays provide inadequate data to estimate human cancer risk at low dose; accuracies of
approximately 60 % are achieved. In improving current test models for genotoxicity and carcinogenicity, toxicogenomics
assays are considered to offer tremendous promise. In this respect, we investigated whether gene expression profiling
can discriminate carcinogens with major differences in their mode of actions, namely genotoxins versus non-genotoxins, by analysing modulation of gene expression profiles induced by 20 model carcinogens in HepG2 cells in vitro by
microarray technology. Using all data, the correctness for classification appeared 81%. Exclusion of the treatments
that had only marginal effects on the expression profiles, improved the discrimination for the training and validation
sets to 92 and 100% correctness, respectively. Exclusion of the gene expression signals that were hardly altered, also
improved classification, namely to 94 and 80%. We furthermore applied this model to investigate whether chemical
carcinogens belonging to the same chemical class, e.g. polycyclic aromatic hydrocarbons, induce discriminative gene
expression profiles according to their genotoxic/carcinogenic potency. Results demonstrate that genotoxic class-specific
gene expression profiles are promising as a tool in predictive toxicology and chemical risk assessment.
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O-015
CAN WE INTEGRATE TRANSCRIPTOMICS IN REGULATORY TOXICOLOGY?
Raffaella Corvi
Thomas Hartung
European Centre for the Validation of Alternative Methods (ECVAM), IHCP, Joint Research Centre of the European Commission,
Ispra, Italy
The new EU Chemicals Policy will require the assessment of some 30 000 chemicals, which will have serious animal
welfare implications unless alternative methods are developed, validated, and implemented in the context of the REACH
system. The 7th Amendment to the Cosmetics Directive foresees to phase out animal experiments within 10 years for
all human health effects by introducing bans on the animal testing of cosmetics and on the marketing in the EU of
cosmetics tested on animals.
In the context of REACH based on a Weight of Evidence approach, an Intelligent Testing Strategy should be developed
with the aim of generating sufficient data for a chemical to support its classification or exclusion from classification and
its risk assessment. The strategy should seek to ensure that the data requirements are met in the most efficient and
humane manner so that animal usage and costs are minimised. The careful evaluation of existing data should enable
the identification of the data gaps that need to be filled in to meet the requirements of REACH.
Transcriptomics-based tests are now widely applied in toxicology and biomedical research and have the potential to be
integrated in conjunction with other tests in the current testing strategies. Whether or not these tests or simply the use
of molecular markers may contribute in filling in the data gaps needs to be defined. Several EC research coordinated
efforts are currently addressing this issue. They cover in particular the challenging areas of toxicology as the ones of
chronic toxicity and carcinogenicity where there is a need for new approaches for a better safety assessment, which
also reduces the number of animals used.
In addition international organizations are already drafting programs related to the possible use of transcriptomicsbased methods for the use of hazard and risk assessment.
O-016
TOXICOGENOMICS OF CARCINOGENS: IDENTIFICATION OF CHARACTERISTIC EXPRESSION
SIGNALS IN LIVER AND KIDNEY
Hans J. Ahr
Heidrun Ellinger-Ziegelbauer, Christina Weiland
Bayer Healthcare AG, Molecular and Special Toxicology, Wuppertal, Germany
Application of gene expression techniques, using microarrays in toxicological studies (toxicogenomics), allows an unprecedented, hypothesis-free insight into mechanisms underlying toxicological effects and thus facilitates the interpretation
of a toxic compound’s mode of action. This would be especially valuable, if gene expression changes after short term
treatment would allow predictions of the outcome of a long term bioassay. We therefore investigated whether genotoxic
and non-genotoxic carcinogens at doses known to induce tumors in liver or kidney in the two year bioassay deregulate
characteristic sets of genes in a short term in vivo study in rats for up to 14 days. By global expression analysis, the
characteristic early events in the two target organs following treatment with carcinogens were identified and mechanistically characterized. Distinct cellular pathways were affected by the non-genotoxic and genotoxic carcinogens. Genotoxic
carcinogens primarily induced DNA damage response and activated proliferative and survival signaling. Non-genotoxic
carcinogens were characterized by responses to oxidative DNA or protein damage as well as cell cycle alterations and
signs of regeneration. A remarkable similarity of the characteristic gene expression profiles was observed for liver and
kidney. This provides an opportunity to use such combinations of pathway – associated gene expression profiles from
short term studies to predict a genotoxic or non genotoxic potential of a compound in various target organs.
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O-017
LEVERAGING COMPLEMENTARY DATA: TOXICOGENOMICS BASED ON COMBINED PROTEOMICS
AND TRANSCRIPTOMICS REFERENCE COMPENDIA
Jochen König
Hans Gmuender, Maria Wendt
Genedata AG, Basel, Switzerland
Toxicogenomics is a novel approach for predicting a compound's toxicity using gene, protein and metabolite expression
information. Changes to the molecular expression profiles following exposure to a drug together with conventional clinical endpoints are used to build reference libraries of known compounds against which novel compounds can be tested.
Biomarker candidates are then deduced from such libraries and characterized further to determine the causal relationship between the biomarker and toxicity in animal models and humans, respectively.
The basic steps in generating a toxicogenomics knowledge base for the evaluation of novel compounds include the collection of data from conventional toxicology, establishing quality-checked and normalized expression data, constructing
a reference database with a number of well-known and well-characterized compounds, and selection of toxicological
marker genes based on statistical methods. After validating and refining the reference compendium it is used to classify novel, uncharacterized compounds. Inclusion of related functional genomics data enables the characterization of
gene function and regulatory mechanisms to determine the compounds' mechanisms-of-action at various toxicological
endpoints.
Genedata together with 13 pharma and three academic partners will apply this approach to a series of proprietary
compounds that were dropped from development after they failed conventional toxicology tests. The efforts by the
PredTox consortium are actively supported by the European Commission as part of the ’Innovative Medicines for Europe’
(InnoMed) programme. During the project high throughput molecular techniques (transcriptomics, proteomics and
metabolomics) will be combined with conventional toxicology methods to build a unique toxicity profile of each compound. These profiles will be used to gain additional mechanistic understanding and eventually to evaluate the likelihood
that a drug candidate will fail for lack of safety.
O-018
NEW DATA SUPPORTING INDIRECT GENOTOXICITY DAMAGE AS A POSSIBLE MECHANISM OF
OCHRATOXIN A CARCINOGENICITY BY ANALYSING MODULATION OF GENE EXPRESSION IN
HK-2 CELLS
Leire Arbillaga1
Amaia Azqueta 1; Adela Lopez de Cerain 1; Joost HM van Delft 2
Department of Food Sciences and Toxicology, Faculty of Pharmacy, University of Navarra, Spain, Pamplona, Spain 1; Department of
Health Risk Analysis and Toxicology, Faculty of Health Sciences, University of Maastricht, The Netherlands, Maastricht,
Netherlands 2
Ochratoxin A (OTA) is a nephrotoxic mycotoxin often found in cereals. There is a great evidence of renal carcinogenicity
in male rats although its mechanism of action is unknown.
In general there are two mechanisms implicated in chemical carcinogenicity, the first by a direct DNA damage (genotoxic carcinogens), and the second, where a variety of cellular processes are involved (nongenotoxic carcinogens). One
hypothesis for OTA-mediated carcinogenicity is its genotoxicity, based on the formation of DNA adducts and oxidative
DNA damage, but data obtained using the alkaline comet assay in vitro at 3 and 6 hours with different concentrations
of OTA in HK-2 cells revealed that OTA does not induce DNA damage at concentrations and times where the viability is
above 80%. There is, however, a significant increase in oxidative DNA damage at cytotoxic concentrations, suggesting
an indirect DNA damage.
Therefore, the aim of the present work was to investigate the mechanisms implicated in the nephrotoxicity and carcinogenicity of OTA by analysing modulation of gene expression in HK-2 cells using Affymetrix Human Genome U133 A
2.0 Gene Chips.
For this purpose we chose a concentration (50 µM) at two time points, 6 and 24 hours. At 6 hours OTA induced 20%
cytotoxicity and oxidative DNA damage, and at 24 hours, 50% cytotoxicity and DNA strand breaks and oxidative damage. Many of the modulated genes were involved in several biological pathways: electron transport chain was the
predominant pathway induced at the two time points, suggesting an induction of mitochondrial reactive oxygen species (ROS) production; oxidative stress and inflammatory response were also induced at 24h, and apoptosis, cell cycle
arrest, MAPK signalling pathway among others, were inhibited. These data support the hypothesis that OTA may cause
DNA damage by an indirect mechanism which may be mediated by reactive oxygen species or OTA-mediated cytotoxicity
products and not through direct covalent binding to DNA.
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O-019
THE EFFECT OF CELLULAR TYPE AND STATE IN THE RESPONSE TO DNA DAMAGE
Eugenia Dogliotti
Department of Environment and Primary Prevention, Istituto Superiore di Sanitá, Rome, Italy
The epidermis has evolved to provide a barrier against the environment, which is essential for survival. This barrier
is constituted and continuously regenerated by terminally differentiating keratinocytes. We have investigated the role
played by the cell type in DNA damage response by using as model cell system keratinocytes and fibroblasts derived
from the same skin biopsy. The effects of different DNA-damaging agents, namely UVB radiation and oxidative stress,
have been analysed. Keratinocytes present a more sophisticated network of defence mechanisms as compared with
fibroblasts: 1) they are protected from UVB damage and are provided of a strong anti-oxidant capacity to prevent ROS
attack; 2) they present a more efficient nucleotide excision repair (global genome repair) than fibroblasts and preserve
this mechanism well into terminal differentiation using mechanisms other than p53; 3) they present an attenuated G1
checkpoint following DNA damage induction and, 4) upon damage, they facilitate cell death by apoptosis. These features
support the notion that keratinocytes, as first line of defence of the organism, require very sensitive mechanisms to
maintain genomic integrity. The cell type-specific effects of environmental agents should be included in any future study
aimed to understand the mechanisms of environmental carcinogenesis.
O-020
DNA REPAIR PROTEINS AND ACQUIRED IMMUNITY
Hans E. Krokan
Bodil Kavli, Sonja Andersen, Geir Slupphaug
Department of Cancer Research and Molecular Medicine, Faculty of Medicine,Norwegian University of Science and Technology,
N-7489 Trondheim, Norway
Repair of small base lesions in DNA takes place by base excision repair (BER) or by direct base repair by methyltransferases or oxidative demethylases, such as hABH2 and hABH3. BER involves removal of the damaged base by a DNA
glycosylase and gap-filling in a multi-step pathway. Uracil in DNA generally results from spontaneous deamination of
cytosine, giving rise to mutagenic U:G mispairs, or misincorporated dUMP-residues, resulting in U:A pairs. Repair of
uracil in DNA is initiated by a uracil-DNA glycosylase, the major ones being nuclear UNG2, mitochondrial UNG1 and
SMUG1. However, in the Ig locus of B-cells, DNA-uracil is an important physiological intermediate both in somatic
hypermutation (SHM) and class switch recombination (CSR). SHM is responsible for the remarkable diversity and high
affinity of antibodies. CSR results in new effector functions, by switching from production of IgM to IgG, IgA and IgE.
SHM and CSR are triggered by activation-induced cytosine deaminase (AID) that is induced by antigens. AID generates
U:G mispairs that cause CG to TA transitions in the Ig locus after replication. When UNG2 removes uracil, the resulting
abasic site is bypassed by one or more error-prone polymerases. This increases the mutational spectrum in SHM. UNG2
is also required for generation of strand breaks that triggers CSR. Not only UNG2, but also mismatch repair proteins
are involved in SHM, while double strand break repair proteins are essential for CSR. Humans deficient in AID or UNG2
display the hyperIgM syndrome (HIGM) characterised by recurrent infections, lymphoid hyperplasia, elevated IgM and
profoundly decreased IgG, IgA and IgE. SMUG1 can not complement the UNG-deficiency. Furthermore, Ung-deficient
mice have lymphatic hyperplasia and ~20-fold increased risk of developing B-cell lymphomas, mainly follicular lymphomas and diffuse-large B-cell type. In conclusion, there are extensive interactions between the immune system and
DNA repair.
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ORAL PRESENTATIONS
O-021
CONTROL OF MULTIPLE DNA POLYMERASES DEALING WITH LESIONS INDUCED BY
ENVIRONMENTAL MUTAGENS
Takehiko Nohmi
Kiyoko Kokubo, Masami Yamada, Petr Gruz
National Institute of Health Sciences (Tokyo), 1-18-1 Kamiyoga, Setagaya-ku, Tokyo, Japan
Genomic DNA is continuously exposed to a variety of endogenous and exogenous genotoxic agents. To deal with the
damage induced by the genotoxic agents, cells possess multiple DNA polymerases, i.e., 16 and 5 DNA polymerases in
humans and Escherichia coli, respectively. These include replicative and non-replicative DNA polymerases responsible
for the chromosome replication and the repair or bypass of damage, respectively. To understand better the roles of
multiple DNA polymerases in lesion-bypass leading to mutations, we have systematically disrupted the genes encoding
three SOS-inducible DNA polymerases, i.e., Pol II, Pol IV and Pol V, in Salmonella typhimurium TA1538. This strain bears
a mutational hotspot of CGCGCGCG sequence where -2 frameshift mutations are induced. Interestingly, 26 chemicals
examined in this study were classified into four categories. Class I is a group of compounds, e.g., benzo[a]pyrene,
whose mutagenicity was reduced by the deletion of dinB encoding Pol IV. Class II is a group of compounds, e.g., 7,12dimethylbenzo[a]anthracene, whose mutagenicity was reduced by the deletion of either dinB or umuDC encoding Pol V.
Class III is a group of compounds, e.g., 1,8-dinitropyrene, whose mutagenicity was reduced by the deletion of umuDC.
The mutagenicity was also partially reduced by the deletion of polB encoding Pol II. Class IV is a group of compounds,
e.g., Glu-P-1, whose mutagenicity was not reduced by the deletion of any of the genes encoding the SOS-inducible
DNA polymerases. From the results, we conclude that different sets of DNA polymerases are involved in lesion bypass
leading to -2 frameshift and also that the replicative DNA polymerase, i.e., Pol III holoenzyme, is involved in the bypass
reactions. The roles of DNA polymerases may be different depending on the lesion structures, types of mutations (base
substitutions versus frameshift) and sequence contexts where the lesions are located.
O-022
RADIATION INDUCES MICROHOMOLOGY MEDIATED RECOMBINATION IN TRANS
AT UNDAMAGED SITES
Robert H Schiestl1
Cecilia Chan 2, Zorica Scuric 3, Markus Kiechle 4
University of California, Los Angeles, Los Angeles, USA 1, University of California, Los Angeles, Los Angeles, USA 2, University
of California, Los Angeles, Los Angeles, USA 3, GSF, Munich, Germany 4
Ionizing radiation causes DNA double-strand breaks and genome rearrangements that can lead to cancer. Illegitimate
recombination repairs a DNA double-strand break in the absence of extended sequence homology. Some illegitimate
recombination events, also called microhomology-mediated recombination, occur between several basepairs of homology in the genome that generate about 50% of large deletions causing human genetic diseases. Ionizing radiationinduced genome rearrangements often show such microhomologies at their junctions, implying that radiation-induced
double-strand breaks are preferentially repaired by such recombination events. However, events at undamaged sites
have so far not been documented. Here, we report that both ionizing radiation and restriction enzymes induce microhomology-mediated integration of the DNA substrate in trans at sites that are not damaged. Irradiated yeast cells
displayed 82% microhomology-mediated nonhomologous integration, compared to only 27% in unirradiated cells.
Restriction enzymes enhanced both integration events at genomic restriction sites and random non-restriction sites via
microhomology-mediated recombination. Furthermore, exposure of yeast and mammalian cells to ionizing radiation
before transformation of an end-joining substrate showed increased utilization of microhomology of end joining events
in a transfected plasmid that has not been exposed to radiation. These results suggest that genomic double-strand
breaks caused by ionizing radiation or restriction enzymes induce a genome-wide microhomology-mediated illegitimate
recombination pathway that facilitates integration in trans at non-targeted sites and might be involved in the generation
of large deletions and rearrangements.
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O-023
INHIBITION OF NUCLEOTIDE EXCISION REPAIR BY NEUTROPHILS:
ROLE OF MYELOPEROXIDASE
Nejla Gungor
Roger WL Godschalk, Frederik J Van Schooten, Ad M Knaapen
Department of Health Risk Analysis and Toxicology, Maastricht University, Maastricht, Netherlands
Inflammation has been recognized as an important factor in cancer development. However, the biological processes
underlying this relationship have not yet been fully defined. In the lung, the influx of polymorphonuclear neutrophils
(PMN) is considered as a specific etiological factor linking inflammation with carcinogenesis. More specifically, neutrophils are implicated in pulmonary carcinogenesis through their capacity to accelerate the metabolism of inhaled
chemical carcinogens, causing enhanced formation of pro-mutagenic bulky DNA adducts. We aimed to investigate
the effect of neutrophils on the DNA repair processes that are involved in the removal of such adducts. Specifically,
we tested the hypothesis that neutrophils are potent inhibitors of nucleotide excision repair (NER). Alveolar epithelial
cells (A549) were co-incubated with PMA-activated human neutrophils. Phenotypical assessment of NER capacity in
the exposed A549 cells was performed using a modified comet-assay. The co-incubated epithelial cells showed a PMN
dose-dependent reduction of NER capacity. Moreover, we found that addition of the specific myeloperoxidase (MPO)
inhibitor 4-aminobenzoic acid hydrazide abrogates NER inhibition by PMN. In further experiments we showed that the
MPO product HOCl also dose-dependently reduced DNA repair capacity in the A549 cells, indicating that NER inhibition
by PMN is mediated by MPO-dependent formation of HOCl. Importantly, these effects were observed in the absence of
cytotoxicity or loss of cellular ATP. We finally showed by using 32P-postlabeling that HOCl-induced inhibition of NER was
associated with a delayed removal of DNA adducts in benzo[a]pyrene-exposed A549 cells. Our data show that neutrophils are potent inhibitors of nucleotide excision repair through MPO-mediated formation of HOCl. Generally, these
observations may provide a further biological explanation for the observed association between neutrophilic inflammation and lung cancer risk in smokers.
O-024
DNA LESIONS INDUCED BY N-NITROSODIETHYLAMINE: DOES METHYLTRANSFERASES PLAY
A ROLE?
Claudia Aiub1,2,3
Luiza Stankevicins 2, Lia Jascone 2, José Luis Mazzei 2, Luis Felipe Ribeiro Pinto 2, Israel Felzenszwalb 2
Salzburg University, Salzburg, Austria 1, Rio de Janeiro State University, Rio de Janeiro, Brazil 2, Gama Filho University,
Rio de Janeiro, Brazil 3
Mutagenicity (Ames) and survival assays were used to evaluate the participation of methyltransferases in the repair
of DNA lesions induced at low N-nitosodiethylamine (NDEA) concentrations. Positive NDEA mutagenicity response was
detected for YG7104 and YG7108 strains. Our results suggest that Ogt and Ada methyltransferases may be efficient
in the repair of nitrosamine-induced DNA damage and thereby able to prevent the incorporation of heritable mutations. We also assayed a fixed concentration of NDEA (36.5 µg/mL) varying the length of pre-incubation period (20 and
90 min) in the presence of different concentrations of S9 metabolic activation mixture (2.4, 4.8, 9.6 and 19.1 mg total
protein/mL). Results of this experiment suggest that NDEA reactive aldehydes can be originated either by exogenous or
endogenous metabolization and have direct genotoxic effects causing an impairment to cellular defense mechanisms,
such as DNA repair.
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O-025
ACCUMULATION AND PERSISTENCE OF MUTATIONS INDUCED IN SOMATIC STEM CELLS
OF MICE DURING IRRADIATION WITH LOW DOSE-RATE GAMMA RAYS FOR 483 DAYS,
A PRELIMINARY REPORT
Nao Kagawa 1
Tetsuya Ono 2, Isamu Hayata 3, Tsuneya Matsumoto 4, Yoichi Oghiso 4, Kimio Tanaka 4, Kazuaki Ichinohe 4,
Shingo Nakamura 4, Satoshi Tanaka 4
Kinki University, Higashiosaka, Japan 1; Tohoku University, Sendai, Japan 2; Natinal Institute of Radiological Sciences, Chiba,
Japan 3; Institute for Environmental Sciences, Rokkashomura, Japan 4
To determine whether mutations continually induced by low-level radiation persist and accumulate in somatic stem cells
in vivo, mice heterozygous at the Dlb-1 locus (Dlb-1b/Dlb-1a) were irradiated at a dose rate of 0.04, 0.86 or 15.56 mGy/
day for 483 days from 8 to 78 weeks of age to attain total doses of 0.02, 0.42 or 8.0 Gy, respectively. Small intestines
were sampled from control and irradiated mice 2 weeks after the completion of irradiation and subjected to the Dlb-1
mutation assay in which evidence of mutations of Dlb-1b gene in the stem cells was detected as mutant clones on the
surface of villi. About 10000 villi were observed for each of mice. Frequency of mutant clones (F) was defined as the
number of clones detected per 10000 villi observed. F values in the control and the low, middle and high dose groups
were 25.60±0.8, 25.62±0.8, 26.1±0.8, 35.2±1.0, respectively (N=10 for all groups). From linear regression analysis of
the dose-response data we obtain F = (23.1±1.3) +(1.38±0.33) D, where D is dose in Gy. Within experimental errors,
the induced rate of mutant clones estimated as regression coefficient is equal to 1.20 ±0.08 that had been estimated
based on F values determined for young heterozygous mice at 8 to 13 weeks of age after gamma irradiation up to 8 Gy
at a dose-rate of 0.3, 1 or 3 mGy/min. These results support the conclusions that (1) mutations continually induced by
low-level radiation accumulate in somatic stem cells and persist for prolonged time, most probably, by the end of life
span; (2) susceptibility of stem cells to radiation mutagenesis does not change with age.
O-026
SUPF MUTATIONS INDUCED BY URBAN PARTICULATE MATTER IN A METABOLISM FREE
SYSTEM
Michael N Routledge
Katherine Healey, Elizabeth C Smith, Christopher P Wild
Molecular Epidemiology Unit, University of Leeds, Leeds, UK
Urban particulate matter (UPM) is known to contribute to lung cancer incidence. UPM has been shown to be genotoxic to mammalian cells and to induce mutations in the Ames assay. Here, we have studied the induction of mutations
generated by direct acting mutagenic components of UPM, using the supF forward mutation assay, in the absence of
metabolism. Plasmid pSP189 was exposed to UPM in aqueous solution in the presence of sucrose buffer, to reduce strand
breaks. The mutation frequency induced by 1 μg/μl UPM was 4.99 mutants per 104 colonies, compared to a background
mutation frequency of 0.35 mutants per 104 colonies. The UPM induced mutation frequency was reduced to 0.84 and
1.48 mutants per 104colonies by addition of 1 mM mannitol or 1mM EDTA, respectively. A large percentage of mutant
plasmids contained frame shift mutations (57%), and 31% of mutant plasmids contained multiple mutations. Of the
base substitution mutations, 88% where at GC pairs, with twice as many transversions as transitions. The types of
mutations induced, the reduction of mutagenicity by the inclusion of the free radical scavenger, mannitol, or the metal
chelator, EDTA, and the sequence context of the induced mutations all support the conclusion that the majority of mutations were induced by reactive oxygen species generated by metal ions present in the UPM. Most mutation studies with
UPM have focused on organic carcinogens present on UPM. Our results highlight the potential contribution of metal ions
to the mutagenicity of UPM.
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O-027
HIGH EFFICENT DNA REPAIR PATHWAYS CAN BE DETRIMENTAL TO HUMAN HEALTH
Alain Sarasin 1
P. Dessen 1, A. Kauffmann 1, F. Rosselli 1, S. Michiels 2, A. Spatz 2, V. Winnepenninckx 3, J.J. Van den
Oord 3
Genomes and Cancers, FRE 2939, CNRS, Institut Gustave Roussy, Villejuif, France 1, Institut Gustave Roussy, Villejuif, France 2,
University Hospitals, Katholieke Universiteit Leuven, Leuven , Belgium 3
Melanoma is the most life-threatening human neoplasm of the skin and shows a world-wide dramatic increase in incidence and mortality. Whereas the clinical and histological features of melanoma progression are well known, the underlying molecular events leading to metastasis have not been clearly elucidated. Using a collection of frozen primary and
metastatic melanomas we determined their genome-wide gene expression profiles.
Thanks to a new multiple random validation strategy we identify a minimal signature of 60 genes allowing us to predict
with a high probability distant metastasis free-survival as well as overall survival at 4 years.
Because melanoma development is partly due to ultraviolet exposure, we analyzed the role of genes involved in DNA
replication, DNA repair and recombination in primary melanomas that are going to metastasize. Using a new strategy
of classification of genes by functional pathways, we could determine that the expression of a series of replication and
repair genes are associated with a probability around 10-12.-10-14 with a bad prognosis.
Most of the genes involved in the regulation of replication origin firings are overexpressed in primary melanomas that
will lead to metastasis as well as numerous genes involved in the maintenance of genetic stability, such a homologous
recombination and replication fork maintenance. Indeed few genes are directly involved in nucleotide excision repair,
base excision repair, mismatch repair and translesion synthesis. However, a vast majority of genes regulating the
pathways leading to recovery of replication fork stalling, probably by homologous recombination, are overexpressed in
primary tumours that are going to metastasize. It looks if genome stability is necessary for a primary tumour cell to
invade and succeed inducing distant metastasis.
O-028
LUNG CARCINOGENESIS MEDIATED THROUGH HNRNP B1: A CROSS TALK BETWEEN RNA
SPLICING REGULATORS AND DNA REPAIR SYSTEM
Eisaburo Sueoka
Department of Internal Medicine, Faculty of Medicine, Saga University, Saga, Japan
Lung cancer is the most common cause of cancer death among males in Japan. Its overall prognosis is poor, and is
strongly correlated with the stage of disease at diagnosis. Heterogeneous nuclear ribonucleoprotein B1 (hnRNP B1) was
first identified as an RNA binding protein regulating RNA splicing and telomerase activity. hnRNP B1 is overexpressed
in an early stage of lung cancers and premalignant lesions, such as bronchial dysplasia, suggesting that hnRNP B1 is
involved in lung carcinogenesis. We recently found that hnRNP B1 interacted with DNA-dependent protein kinase (DNAPK) complex, and the proteins were co-localized in the nucleus of lung cancer cell lines after irradiation. Furthermore,
recombinant hnRNP B1 protein dose-dependently inhibited DNA-PK activity both in vitro and in intra cellular comet assay
system. Using deletion mutants of hnRNP B1 and Ku proteins, regulatory subunits of DNA-PK, the responsible regions of
hnRNP B1 for the interaction with DNA-PK complex were identified. In addition, recombinant hnRNP B1 protein inhibited
the formation of holoenzyme of DNA-PK, dose dependently. Considering these results, we assume that the overexpression of hnRNP B1 occurring in the early stage of carcinogenesis inhibits DNA-PK activity, resulting in subsequent accumulation of erroneous rejoining of DNA double-strand breaks, causing tumor progression. To confirm the hypothesis,
transgenic mice carrying a chimera gene of hnRNP B1 and surfactant protein C promoter, which overexpress hnRNP B1
in the lung, were established. In this paper, we discuss possible new mechanisms of carcinogenesis by hnRNP B1 mediated through inhibition of DNA repair system.
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O-029
MAMMALIAN CHROMOSOMAL SINGLE-STRAND BREAK REPAIR AND NEURODEGENERATIVE
DISEASE
Keith Caldecott 1
Natasha Illes 1, Satchin Katyal 2, Peter McKinnon 2; Mustafa Saifi 3, Jim Lupski 3
Genome Damage and Stability Centre / University of Sussex, Sussex, UK 1; St Jude Children’s Research Hospital Memphis,
Memphis, USA 2; Baylor College of Medicine, Houston, USA 3
Thousands of DNA single-strand breaks (SSBs) arise in cells each day posing a continuous threat to genetic integrity,
and recent data has implicated defects in SSBR in hereditary neurodegenerative disease. Although lacking any known
enzymatic activity of its own, XRCC1 protein plays a major role in facilitating the repair of SSBs in mammalian cells, via
an ability to interact with multiple enzymatic components of the single-strand break repair (SSBR) process. Two of the
proteins with which XRCC1 interacts, polynucleotide kinase and aprataxin, do so via a divergent type of FHA domain.
Both aprataxin and PNK appear to be involved in the end processing step of SSBR, during which damaged termini are
converted into the 3’-hydroxyl and 5’-phosphate moieties needed for DNA gap filling and DNA ligation. Recently, we
have identified a third protein member of this divergent FHA domain family, encoded by a previously uncharacterised
human open reading frame. The relationship of this protein to DNA strand break repair processes, and the relationship
of SSBR to neurodegenerative disease, will be discussed.
O-030
UPREGULATION OF ADAPTIVE DNA POLYMERASES BETA AND KAPPA IMPEDES FORK
PROGRESSION WITHOUT ACTIVATING THE REPLICATION CHECKPOINT
Jean-Sébastien Hoffmann 1
Marie-Jeanne Pillaire 1, Christophe Cazaux 1; Rémy Bétous 1, Chiara Conti 2, Aaron Bensimon 3, Jerzy
Czaplicki 3, Philippe Pasero 4
Group “Instabilité génétique et cancer” équipe labellisée par La ligue contre le cancer,Institut de Pharmacologie et de Biologie
Structurale, CNRS UMR 5089, 205 route de Narbonne, 31077 Toulouse cedex 4, France 1 ; Unite de Stabilité des Génomes, Institut
Pasteur, 75724 Paris, France 2 ; Equipe Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089, 205 route de
Narbonne, 31077 Toulouse cedex 4, France 3 ; Institute of Human Genetics, CNRS UPR 1142, 34396 Montpellier Cedex 5, France 4
There is rising evidence that cancer development is associated from its earliest stages with DNA replication stress, a
major source of spontaneous genomic instability. However, the origin of these replication defects has remained unclear.
We have investigated the consequences of upregulating error-prone adaptive DNA polymerases (pol) beta and kappa
on chromosomal DNA replication. These enzymes are misregulated in different types of cancers and induce major chromosomal instabilities when overexpressed at low levels. Here, we have used DNA combing to show that a moderate
overexpression of pol beta or pol kappa is sufficient to impede replication fork progression and to promote the activation
of additional replication origins. Interestingly, alterations of the normal replication program induced by excess adaptive
polymerases were not detected by the replication checkpoint. We therefore propose that upregulation of adaptive DNA
polymerases induces a checkpoint-blind replication stress that contributes to genomic instability and to cancer development.
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O-031
8-NITROGUANINE AS A BIOMARKER FOR INFLAMMATION-RELATED CARCINOGENESIS
Yusuke Hiraku
Shosuke Kawanishi
Mie University Graduate School of Medicine, Tsu, Mie, Japan
Infection and chronic inflammation are important risk factors of human cancer. Infection with hepatitis C virus,
Helicobacter pylori and the liver fluke Opisthorchis viverrini are risk factors of hepatocellular carcinoma, gastric cancer
and cholangiocarcinoma, respectively. Inflammatory bowel diseases and oral diseases, such as oral lichen planus and
leukoplakia, are associated with colon and oral carcinogenesis. During chronic inflammation, reactive nitrogen and oxygen species generated from inflammatory and epithelial cells are considered to play the key role in carcinogenesis and
tumor progression. 8-Nitroguanine is a nitrative DNA lesion formed during chronic inflammation and considered to be
potentially mutagenic. By using immunohistochemical technique, we firstly demonstrated that 8-nitroguanine is formed
in association with inflammation-related carcinogenesis. 8-Nitroguanine formation was observed specifically in epithelial cells at the sites of carcinogenesis in humans (BBRC 2004, J. Hepatol. 2005, Cancer Sci. 2005, Nitric Oxide 2006)
and animal models (Carcinogenesis 2004, Cancer Sci. 2005). Antibacterial, antiviral and antiparasitic drugs dramatically diminished 8-nitroguanine formation and iNOS expression (J. Hepatol. 2005, Int. J. Cancer 2006). Moreover, we
have recently demonstrated that in cholangiocarcinoma patients, 8-nitroguanine formation occurred to a much greater
extent in cancerous tissues than in the adjacent non-cancerous tissues and that the formation of this DNA lesion is
closely associated with tumor invasion (World J. Gartorenterol. 2005). On the basis of our findings, we propose that 8nitroguanine can be a promising biomarker to evaluate the potential risk of inflammation-mediated carcinogenesis and
efficacy of the treatment of inflammatory diseases. Moreover, this DNA lesion may be used to predict the prognosis of
patients with inflammation-related cancer.
O-032
CHARACTERISATION OF A NATURAL MUTATOR VARIANT OF HUMAN DNA POLYMERASE
LAMBDA, AND ITS IMPACT IN DNA REPAIR AND MUTAGENESIS
Luis Blanco1
Gloria Terrados 1, Arancha Sanchez 1, Tomas Kirchhoff 1, Miguel García-Díaz 1,2, Katarzyna Bebenek 2,
Thomas Kunkel 2, Jean-Sébastien Hoffmann 3, Yvan Canitrot 3, Christophe Cazaux 3, Jean-Pascal Capp 3,
Santiago Ramón Cajal 4
Centro de Biología Molecular Severo Ochoa (CSIC-UAM). Universidad Autónoma, Madrid, Spain 1, Laboratory of Molecular Genetics,
National Institute of Environmental Health Sciences, Research Triangle Park, USA 2, Institute of Pharmacology and Structural
Biology. Centre National de la Recherche Scientifique, Toulouse, France 3; Department of Pathology, Hospital Vall d'Hebron,
Barcelona, Spain 4
Human DNA polymerase λ (Poll) is a recently identified DNA repair polymerase whose biochemical properties are partly
similar to those of Polb, suggesting a role of Poll in DNA repair. Indeed, as Polb, Poll has an intrinsic dRP lyase, an activity that is critical for the BER pathway. Unlike Polß, Polλ contains an N-terminal BRCT domain, required for an stable
interaction with non-homologous end-joining (NHEJ) factors, which suggests the involvement of Polλ in double-strand
break (DSB) repair.
According to the mutator phenotype hypothesis, the genome of tumor cells must acquire increased mutability resulting
from a malfunction of a network of genome stability systems, e.g., cell cycle arrest, DNA repair, and high accuracy of
DNA synthesis during DNA replication. Together, these highly conserved functional pathways act to limit cancer risk. In
the course of the identification of the possible events that lead to a mutator phenotype, we have identified and characterized two novel allelic variants of human Polλ: a double nucleotide polymorphism identified at the 3´-non-translated
region of the gene, and a single nucleotide polymorphism (SNP) resulting in amino acid change Arg to Trp in codon
438 (R438W). RFLP based analysis has been developed to detect the frequency of both polymorphic variants in normal
versus cancer patient populations. In vitro enzyme activity assays of both Arg and Trp 438 allelic variants showed a
decrease in the fidelity of the W438 variant. Ectopic expression in cells of the Poll W438 variant induces high mutation
frequency, aneuploidy, as well as chromosome aberration. Thus, we find that expression of the error-prone R420W Poll
appears to induce major genetic changes associated with a malignant phenotype. Recent resolution of the 3D-structure
of human Poll allows to discuss the molecular basis of this mutator W438 variant.
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O-033
EVALUATION OF GENOTOXICITY AND ANTIGENOTOXICITY BY FOOD-DERIVED COMPOUNDS
IN HUMAN COLON, PROSTATE, BREAST AND BLOOD CELLS USING GENOMICS AND
TRANSCRIPTOMICS-BASED METHODS
Beatrice L. Pool-Zobel
Thomas Hofmann, Stefanie Klenow, Yvonne Knöbel, Marian Raschke, Julia Sauer, Claudia Steiner,
Selvaraju Veeriah
Institute for Nutrition, Friedrich-Schiller-University, Jena, Germany
Diet contributes to cancers of colon, breast and prostate. Food-derived carcinogens include contaminants and endogenous products of oxidative stress. Their genotoxic potentials are governed by metabolic conversions in the tumor
target cells. Several anti-cancer types of food ingredients enhance metabolic detoxification, a mechanism of chemoprotection. We have studied butyrate (produced during fermentation of dietary fibre in the gut lumen), complex mixtures
of polyphenols (from fruits) and individual compounds (genistein, gallic acid) for protective effects towards hydrogen
peroxide (H2O2), ferric iron and 4-hydroxynonenal (HNE), menadione and hemoglobin. The strategy is to assess how
protective ingredients modulate gene expression (c-DNA microarrays, real-time PCR, western blot, measurements of
enzyme activities) in human cells and how this affects the genotoxicity of selected risk factors (comet-challenge assay).
We have shown that butyrate reduces the genotoxicity of HNE in human colon cells, probably by enhancing expression
of glutathione S-transferases (GSTs). In MCF-10A breast cells and in LAPC-4 prostate cells genistein enhances deactivation of menadione and H2O2, respectively, by inducing appropriate detoxification enzymes. Apple polyphenols induce
GSTT2, thus explaining a decreased genotoxic activity of H2O2 in human colon adenoma cells. Ex vivo, GSTP1 protein
was induced in peripheral lymphocytes of human volunteers after ingestion of fruit juices containing different polyphenols. If these types of effects are shown in cells in which tumors arise or in cells from early stages of preneoplasia, they
reveal an effective mechanism of primary cancer prevention, by inhibiting the initiation of cell transformation or the
early progression of transformed cells. Employed to peripheral blood lymphocytes, the measured parameters also serve
as non-invasive biomarkers for clinical studies.
O-034
COOKED AND PRESERVED MEAT CARCINOGENS AND CANCER
Rashmi Sinha
Nutritional Epidemiology Branch, Division of Cancer Epidemiology and Genetics, NCI, Bethesda, MD 20892, USA
There are several plausible biological mechanisms to explain an association between meat consumption and cancer
such as content of fat, protein, iron, and/or products from meat preparation (e.g., cooking or preserving methods).
We found in case-control studies that heterocyclic amines (HCAs) were significantly associated with tumors of the
colorectum, breast, stomach, pancreas, and lung and that benzo[a]pyrene (B[a]P) was associated with an increased
risk of colorectal adenoma. In contrast, we did not find associations with either bladder or Non-Hodgkin's lymphoma. In
a prospective study, the NCI Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial, we found a modest
increased risk for colorectal adenomas with high intake of red meat, well-done red meat, two HCAs (MeIQx and PhIP),
and B[a]P in 3,696 cases of colon adenoma as compared to 34,817 controls. We also observed an association between
higher intake of processed meats such as bacon and sausage with increased risk of colorectal adenomas. There was
no association with intake of total meat, red meat, or white meat intake in 1,338 prostate cancer cases in the PLCO
trial. However, the highest category of very well-done meat consumption (>10 g/day) was associated with a significant
69-percent increased incidence of prostate cancer when compared to those who did not consume well-done meat.
Furthermore, intake of PhIP was associated with a significant 28-percent increased incidence of prostate cancer in men
in the highest quintile of intake. There may be certain genetically susceptible groups who may be at substantially higher
risk but many of the studies did not have sufficient power to investigate the interaction adequately. In summary, even
though the results from case-control studies lend support to an association between meat cooking-mutagens and cancer
larger prospective studies are not consistent. Other components of red meat, such as heme iron, fat, nitrite/nitrosamine
need to be explored further.
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O-035
MEAT COOKING, HETEROCYCLIC AMINES AND CANCER RISK
Jakob Linseisen
Technical University of Munich, Munich, Germany
O-036
ANTICARCINOGENIC ACTIVITY OF PROBIOTIC BACTERIA
Joseph Rafter
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O-037
FORMATION OF POLYCYCLIC AROMATIC HYDROCARBON-DNA ADDUCTS IN HUMAN
LEUKOCYTES
Miriam C Poirier 1
Rao L Divi 1, Roel Vermeulen 1, Nathaniel Rothman 1, Rashmi Sinha 1, Martin Kuldorff 2, Marc J Gunter 3
National Cancer Institute, NIH, Bethesda, USA1, Harvard Medical School, Boston, MA, USA2, Department of Epidemiology and
Population Health, Bronx, New York, USA 3
Ingestion of cooking-derived polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (B[a]P), in meat has
been associated with risk of colorectal adenoma in epidemiological studies. In experimental models PAH-DNA adduct
formation is considered necessary but not sufficient for PAH-induced tumorigenesis, and may therefore provide a
biomarker of human cancer risk. Here we investigated a potential link between human leukocyte PAH-DNA adduct
formation and human colorectal adenoma in a clinic-based case-control study. The study comprised individuals who
were non-smokers at the time of screening, and included 82 cases of colorectal adenoma and 111 polyp-free controls.
All individuals donated blood samples at the time of sigmoidoscopy or colonoscopy. Leukocyte PAH-DNA adducts were
measured by competitive chemiluminescence immunoassay (CIA) using an antiserum elicited against DNA modified with
(±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE) that recognizes DNA adducts of several carcinogenic PAHs. For human samples, assay values are read from a BPDE-DNA standard curve but considered to
reflect the presence of multiple PAH-DNA adducts because of the broad specificity of the antiserum used. In this study
PAH-DNA adduct levels were higher among colorectal adenoma cases, compared to polyp-free controls (1.4 adducts
per 108 nucleotides vs.1.2 adducts per 108 nucleotides, respectively, p = 0.02). Compared to individuals in the lowest
quartile for PAH-DNA adduct formation, those in the highest quartile had an odds ratio of 2.1 for colorectal adenoma
(95% CI, 0.9 - 4.9), after adjustment was made for age and gender. When examined as a continuous variable, colorectal adenoma risk increased by 50% for every additional adduct per 108 nucleotides (95% CI, 1.1-2.2). This study
demonstrates a direct link between a biomarker of PAH exposure and colorectal adenoma.
O-038
INHIBITION OF FRIED MEAT-INDUCED DNA DAMAGE: USE OF CRUCIFEROUS VEGETABLES,
YOGURT, AND CHLOROPHYLLIN IN A DIETARY INTERVENTION STUDY IN HUMANS
David M DeMarini 1
Daniel T Shaughnessy 2, Zong-Li Xu 2, David M Umbach 2, Lisa Gangarosa 3, Barbara Schliebe 3,
Robert S Sandler 3, Jack Taylor 4
US EPA, RTP, NC, USA 1, NIEHS, RTP, NC, USA 2, School of Medicine, University of North Carolina, Chapel Hill, NC, USA 3, NIEHS,
RTP, USA4
Dietary exposures may be risk factors in colorectal cancer, but their effects in either causing or reducing DNA damage have not been
assessed directly in colon tissue in humans in vivo. In this pilot study, 16 subjects were enrolled in a 4-week controlled feeding study. In
a crossover design, 8 subjects were fed diets for 2-weeks that contained meat cooked at either low or high temp.; the diet also included
non-cruciferous vegetables. In the second phase, the remaining 8 subjects were fed either the high-temp. meat diet or a diet containing
high-temp. meat plus three types of antimutagens: cruciferous vegetables, yogurt, and chlorophyllin tablets, also in a crossover design.
Urine, feces, and blood were collected, and rectal biopsies were obtained from subjects each week during study. DNA damage in colonic
epithelium and lymphocytes was assessed using the comet assay, and urine and fecal mutagenicity were evaluated in the Salmonella
plate-incorporation assay. The high-temp. meat was highly mutagenic and had high levels of heterocyclic amines (HCAs), whereas
the low-temp. meat was weakly mutagenic and had undetectable HCA levels. Tail Moment values were significantly lower in subjects
consuming diets with antimutagens plus high-temp. meat compared to diets containing the meat alone (p = 0.026). Urine mutagenicity
increased 1.9-fold in subjects consuming the high-temp. vs. the low-temp. meat diet. For subjects consuming the diet containing the
antimutagens, the unconjugated urine mutagenicity decreased in 4/8 subjects, whereas the conjugated mutagenicity increased 2.5-fold
in these subjects. This study provides the first evidence in humans that these dietary antimutagens (a) reduce DNA damage in the target
organ (colon) and (b) alter the systemic levels of genotoxicants associated with consumption of a mutagenic diet. [Abstract does not
necessarily reflect the policy of the US EPA.]
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O-039
DETECTION OF 8-OH-DG, RIBONUCLEOSIDE 8-OH-GUO AND FREE BASE 8-OH-GUA IN FISH
FOOD PRODUCTS
Kazuaki Kawai
Peter Svoboda, Hiroshi Kasai
Department of Environmental Oncology, Univ. of Occupational and Environmental Health, Kitakyushu, Japan
8-Hydroxydeoxyguanosine (8-OH-dG) is a major form of DNA damage induced by reactive oxygen species (ROS), and
is analyzed in cellular DNA and urine as a marker of oxidative stress. Recently, we succeeded in analyzing 8-OH-dG,
8-OH-Guo (ribonucleoside) and 8-OH-Gua (free base), by an HPLC column switching technology using anion exchangeand reverse phase- columns coupled with an electrochemical detector (HPLC-ECD). This method is also applicable
for analyzing various biological fluids, such as serum, saliva and cell culture media. In this presentation, we report
that these 8-OH-Gua compounds were detected in fish food products, such as salted dried sardine (Maruboshi) and
dried small fishes (Iriko), which are often consumed in Japan. Water extracts of these fish products were analyzed by
aforementioned HPLC-ECD method. 8-OH-Gua (25 ng-22mg/g), 8-OH-Guo (28-406 ng/g) and 8-OH-dG (5-60 ng/g)
were detected in various amounts in these foods. The amounts of these compounds increased upon heating or broiling
of these fish products. A rodent diet including fish powder, such a CE-2, also contained these compounds. After the
administration of 8-OH-Gua to mice via drinking water, increased levels of 8-OH-Gua were detected in their urine. These
results provide two warnings. First, because these 8-OH-Gua compounds may be incorporated into DNA by a salvage
pathway (1) and show genotoxicity (2), it is possible that they are food carcinogens. Second, when these compounds
are analyzed as oxidative stress markers, fish foods (in human) and the CE-2 (CLEA Japan, Inc.) diet (in rodents) should
be avoided. 1) Hah SS, et al. Bioorg. Med. Chem. Lett.15, 3627-3631(2005); 2) Arashidani K, et al., Mutat. Res. 403,
223-227 (1998).
O-040
IDENTIFICATION OF THE HUMAN CYTOCHROME P450 ENZYMES INVOLVED IN
THE BIOACTIVATION OF THE HERB-BASED CARCINOGENS ESTRAGOLE AND METHYLEUGENOL
Suzanne MF Jeurissen 1,2
Marelle G Boersma 1, Jan JP Bogaards 3, Teris A van Beek 2, Gerrit M Alink 1, Ernst JR Sudhölter 2, Ivonne
MCM Rietjens 1,4
Division of Toxicology, Wageningen University, Wageningen , Netherlands1, Laboratory of Organic Chemistry, Wageningen
University, Wageningen , Netherlands2, TNO Quality of Life, Zeist, Netherlands 3, WU/TNO Centre for Food Toxicology, Wageningen,
Netherlands4
Estragole and methyleugenol are important constituents of herbs such as basil, anise and tarragon. These alkenylbenzenes are genotoxic
and carcinogenic and restrictions in use are indicated. The question remains whether the use of such herbs, herbal supplements, and
foodstuffs containing such herbs should be restricted as well. Effects of other herbal ingredients on the bioactivation enzymes involved,
may influence the ultimate risk upon exposure to low doses of alkenylbenzenes in complex herbal food matrices. The aim of this study
was to identify the human cytochrome P450 enzymes responsible for bioactivation of estragole and methyleugenol to their proximate
carcinogenic 1′-hydroxymetabolites. Different assays using recombinant enzymes and human liver microsomes were performed. For the
enzymes showing intrinsic activity for estragole or methyleugenol 1′-hydroxylation, the kinetic parameters kcat, Km and enzyme efficiency
(kcat/Km) were determined. For estragole both CYP2A6 (kcat/Km = 341 min−1 mM−1) and CYP1A2 (kcat/Km = 59 min−1 mM−1) are involved
and for methyleugenol CYP1A2 is the most important enzyme (kcat/Km = 167 min−1 mM−1) at physiologically relevant concentrations.The
activities of CYP1A2 and CYP2A6 may vary in humans due to genotype- and lifestyle-based influences and interindividual differences
in sensitivity towards 1′-hydroxymetabolite mediated adverse effects may occur. People bearing polymorphisms in CYP2A6 that lead
to poor metabolizer phenotypes might be at lower risk. For CYP1A2, lifestyle factors such as smoking and consumption of charbroiled
food can increase the activity of CYP1A2 and might increase the bioactivation. The bioactivation of estragole and methyleugenol after
consumption in an herbal matrix might be reduced by the presence of other CYP1A2 or CYP2A6 substrates or inhibitors. This issue is
presently under investigation.
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O-041
INTERACTION OF NUTRITIONAL AND GENETIC FACTORS IN COLORECTAL CANCER
Gunter Kuhnle
Diet and Cancer Group, MRC Dunn Human Nutrition Unit, Cambridge, UK
Background: The European Prospective Investigation of Cancer (EPIC) of 519,978 individuals aged 25 to 70 years is
the largest prospective study of diet, gene variants and cancer to date worldwide. Results have shown strong protective effects of dietary fibre against colorectal cancer, particularly left sided colon cancer. In addition, further data from
the EPIC study have shown that red and processed but not white meat is associated with increased risk of colorectal
cancer, particularly in low fibre consumers, whereas fish consumption is protective. Studies of the interaction with gene
variants and dietary factors are ongoing.
Methods: To investigate the underlying interaction between diet and DNA damage which would explain the association
between red meat and colorectal cancer risk, we have fed increased levels of red meat and measured ATNC (Apparent
Total N-nitroso Compounds) and measured the effects on DNA damage in faecal samples in a series of studies of volunteers maintained under controlled conditions.
Results: A consistent dose response in ATNC excretion to red meat consumption, but no significant effects of white
meat has been shown. Whilst red meat diets increase faecal ATNC, the equivalent amount of protein from eggs, milk,
cheese and vegetable protein has no effect. Furthermore, there appears to be a specific effect of haem whereas inorganic iron has no effect. Under certain conditions, haems are known to be nitrosated, and act as nitrosating agents.
Faecal water contains little haem compared with faecal homogenates which may explain why studies with the Comet
assay on faecal water have not shown effects of diet. However, in colonic exfoliated cells, the percentage staining positive for the NOC-specific DNA adduct, O6-carboxymethyl guanine (O6CMG) was significantly (p < 0.001) higher on the
high red meat diet. Faecal ATNC were positively correlated with the percentage of cells staining positive for O6CMG (r2
= 0.56, p = 0.011).
Conclusions: As these O6CMG adducts are not repaired, and if other related adducts are formed and not repaired, this
may explain the association of red meat with colorectal cancer.
O-042
THE EFFECTS OF CEREAL COMPONENTS ON THE PROPERTIES AND PROLIFERATION OF COLON
CANCER CELLS
Bjorn Akesson
Gunilla Önning, Per Mercke
Biomedical Nutrition, Lund University, Lund, Sweden
Background: The constituents of cereals can provide several disease prevention effects. For oats much focus has
been directed to the effects of the soluble fiber beta-glucan as studied in the EU project Beta-glucan (Design of foods
with improved functionality and superior health effects using cereal beta-glucans). To study in more detail the effects
of cereal components on processes related to cancer and blood lipid metabolism, experiments in model systems are
necessary.
Methods: Ca, 2 cells were incubated with hydroxycinnamic acids and the metabolic activity and cell proliferation were
followed. For more detailed studies cells were processed for the isolation of RNA which was subjected to labelling and
cDNA generation. Spotted cDNA microarrays with duplicate reporters and a total of 55488 spots representing 26819
sequence-verified IMAGE clones from the Research Genetics IMAGE clone library were used. The slides were produced
and processed at the Swegene DNA Microarray Resource Center (www.swegene.org/dna_microarrays).
Results: Initial experiments showed that ferulic acid and p-coumaric acid could inhibit cell proliferation. The gene
expression profiles induced by the two compounds differed significantly. Ferulic acid changed mainly the expression of
genes belonging to categories associated with DNA/chromosome organization while p-coumaric acid changed mainly
those belonging to energy/RNA/protein metabolism. Ferulic acid up-regulated 214 genes and down-regulated 266
genes. p-Coumaric acid up-regulated 258 genes and down-regulated 581 genes. Both compounds affected the expression of genes regulating cell cycle and proliferation, although different pathways seemed to be affected.
Conclusions: In summary, hydroxycinnamic acids had antiproliferative effects on Ca, 2 human cancer cells. Ferulic acid
and p-coumaric acid had differential effects on global gene expression.
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ORAL PRESENTATIONS
O-043
NUTRIGENOMICS AND INFLAMMATORY BOWEL DISEASE
Lynnette R. Ferguson
Ivonne Petermann, Claudia Hübner, Martin Philpott, Brian Browning, Andrew Shelling, Karen Munday,
LeeAnn Lewis
Faculty of Medical and Health Sciences / The University of Auckland, Auckland, New Zealand
Inflammatory bowel disease (IBD) is a complex disorder characterised by chronic inflammation of the gastrointestinal
tract. There are two main clinical subtypes, Crohn's disease and ulcerative colitis. Crohn’s disease can affect any part of
the intestine, and is associated with discontinuous, transmural lesions of the gut wall. In Ulcerative colitis, inflammation
is confined to the colon and rectum, and lesions are continuous and superficial. Most studies support a polygenic model
of inheritance, and environmental and dietary factors contribute significantly to both types of IBD. Data from two different New Zealand populations strongly implicate CARD15/NOD2, DLG5, MDR1, SLC22A4 and SLC22A5 genes in the
development of IBD, although other susceptibility genes must exist. The major aim of Nutrigenomics NZ is to develop
foods or identify food components that may benefit individuals carrying variant genetic polymorphisms that lead to IBD
susceptibility. Foods likely to enhance or reduce disease symptoms are being identified from dietary questionnaires,
done in parallel with genetic studies in humans. Candidate foods are fractionated, and the ability to overcome relevant
genetic variants tested in reporter gene assays. Finally, prime candidate foods are tested in animal models, before being
considered as candidates for biomarker studies in humans.
O-044
HOST-MICROBE CROSSTALK
Sven Pettersson (presented by Joseph Rafter)
Karolinska Institutet, Novum, Stockholm
Nuclear receptors (NRs), around fifty in mammals, are described as ligand induced transcription factors which regulate
the expression of a large number of genes involved in a variety of cellular processes. NRs are implicated in nearly every
aspect of vertebrate development and adult physiology, and alterations in structure, function and expression of NRs can
manifest in metabolic and reproductive diseases as well as in the progression of cancer. The GI tract and allied organs,
such as the liver, pituitary gland and spleen express a number of NRs. It is our hypothesis that NRs might be master
regulators of many of the known effects of microorganisms in the gut.
The indications of a correlation between inflammatory responses and metabolism are intriguing, and the cross-reactivity of these signaling pathways, possibly through NRs, merits investigation. We have already initiated a project for the
descriptive identification of genes or gene products in the NMRI mouse strain that are affected by intestinal colonization
with bacteria, locally (colon) and systemically (liver, pituitary gland and spleen). Global gene expression on DNA-microarrays containing >20 000 unique gene fragments is being exploited. The present project focuses on NR signaling. Thus,
the starting point of our studies on the relationship between metabolism and microorganisms will be analysis of NRs.
We will compare NR signaling in gut epithelial cells from germfree (GF) and conventional rodents. In the global gene
expression analyses, we will specifically put focus on the NRs and the genes they regulate (when known). When the
expression profiles of the relevant genes have been identified, the cellular localization of the proteins will be verified
using immunohistochemistry, following in situ hybridization assays. The expression of selected proteins can be further
investigated on tissue-arrays, which include most human tissues. We will also monitor, using RT-PCR, NR signalling in
the alimentary tract of humans suffering from obesity, allergy, inflammatory bowel disease and colon cancer.
Thus, we plan for the first time to carry out a thorough examination of the crosstalk between our microbes and all host
NRs. If this crosstalk can be identified, it would open possibilities to counteract potentially negative, disease-causing
effects arising in the GI tract, through the use of synthetic/diet derived NR ligands or via modulation of the composition of the intestinal microflora by novel foodstuffs/probiotic bacteria. NRs are already an important target for novel
drug development by the drug companies and the results of the present project may open up an entirely new field of
molecular targets for the food industry to exploit in their search for new and better functional foods.
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ORAL PRESENTATIONS
0-045
PROFILING OF GENE EXPRESSION MODULATION IN HUMAN LYMPHOCYTES IS NOT
A SENSITIVE MARKER FOR EXPOSURE TO AH-RECEPTOR AGONISTS.
Pim de Waard 1
Theo M de Kok1, Frederik-Jan van Schooten 1, Ron Hoogenboom 2, Ad Peijnenburg 2, Jac Aarts 3
University Maastricht; Dept. Health Risk Analysis and Toxicology, Maastricht, Netherlands 1, RIKILT, Institute for Food Safety,
Wageningen, Netherlands 2, Wageningen University; Dept. Toxicology, Wageningen, Netherlands3
Background: Cruciferous vegetables and citrus fruits have been shown to contain significant amounts of natural aryl
hydrocarbon receptor (AhR) agonists (NAhRAs) or their precursors. Binding to the AhR is known to activate the main
pathway by which dioxins, like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exert their toxicity. Despite this, NAhRAs
are reported to possess beneficial health properties and are generally presumed to be non-toxic. By using microarray
technologies, we found a remarkable similarity between the changes in gene expression profiles induced by NAhRAs
and TCDD in human Caco-2 cells.
Aim: To establish whether the expression of the same genes is also affected in human lymphocytes, both after ex vivo
exposure and in vivo after a dietary intervention,
Methods: Using RT-PCR analysis, we examined the gene expression profiles in both freshly isolated human lymphocytes
exposed to NAhRAs and TCDD and after a dietary intervention study with NAhRA containing foods.
Results and conclusion: After the ex vivo exposure, only a marginal differential regulation by TCDD and one NAhRA could
be detected in lymphocytes. RT-PCR analysis of the well-known dioxin and NAhRA responsive genes for CYP1A1, CYP1B1
and NQO1 showed no effect in the dietary intervention study. We therefore conclude that gene expression profiling in
human lymphocytes can probably not serve as a sensitive biomarker for exposure to (natural) AhR agonists, because
of a very weak responsive AhR in these cells.
O-046
PREDICTIVITY OF MICRONUCLEI IN CANCER RISK AND CHROMOSOMAL ABERRATIONS FOR
QUANTITATIVE ANALYSIS FOR CANCER RISK
Stefano Bonassi
Unit of Molecular Epidemiology, National Cancer Research Institute, Genoa, Italy
In the last 15 years a sizeable number of articles have been published addressing the issue of validating biomarkers
of chromosome damage as predictors of cancer risk. Cohorts from 11 European countries were followed-up showing in
most cases a significant association of cancer incidence with the frequency of chromosomal aberrations (CA) in healthy
subjects. In the last years, in the framework of the European project CancerRiskBiomarkers, an effort has started to
pool all available data from these cohorts in a unique database, and perform an overall estimate of the link between CA
frequency and cancer risk. Preliminary results, based on 22358 subjects and 774 cancer cases confirmed the increased
risk for subjects with medium (RR=1.33 95% CI 1.09-1.64) and high (RR=1.46 95% CI 1.20-1.78) frequency of CA.
Among specific cancer sites, a stronger association was found for the stomach cancer (RR= 3.46, 95% CI 1.15-10.40,
in the High CA frequency). More recently an international cohort was assembled in the framework of the HUMN collaborative project collecting data on the micronucleus assay (MN). The analysis of this new cohort has been just completed
and – in analogy with CA - showed a significantly increased risk of cancer in subjects with medium (RR= 1.84; 95%
CI 1.28-2.66) and High (RR=1.53; 95% CI 1.04-2.25) frequency of MN. These results open the perspective for using
biomarkers of chromosome damage for regulatory purposes in occupational and environmental health. The perspectives
and drawbacks of using these biomarkers at individual level will be discussed in parallel with the case of cholesterol level
and the risk of coronary hearth diseases.
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ORAL PRESENTATIONS
O-047
THE INTERPRETATION OF CYTOGENETIC ANALYSIS BY FISH
Radim J. Sram
Olena Beskid, Andrea Rossnerova, Pavel Rossner, Ivo Solansky
Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Prague, Czech Republic
FISH (fluorescence in situ hybridization) is a new technique, one method using DNA probes for the whole chromosome
painting. This method is widely used to evaluate a radiation exposure, as translocations frequencies do not essentially
change with time. But FISH painting has been applied rarely to evaluate human exposure to chemical carcinogens. In
our study we used conventional cytogenetic analysis (CCA) and FISH painting for chromosomes #1 and #4 (Cambio,
UK) to compare the effectiveness of both methods on the same occupationally exposed groups. 100 metaphases per
subject were scored for CCA and 1000 for FISH. We followed the impact of the occupational exposure to acrylonitrile,
ethyl benzene, 1,3-butadiene, carcinogenic polycyclic aromatic hydrocarbons (c-PAHs), and radiation at the nuclear
power plants. Our results of cytogenetic analyses consist of data on 859 occupationally exposed subjects and 417
matched controls. FISH cytogenetic analysis indicated the increase of tranclocations in groups exposed to acrylonitrile,
1,3-butadiene, and c-PAHs, no chromosomal damage was detected in these groups by CCA. In the group exposed to
c-PAHs (environmental exposure in city policemen) we observed for the first time the relationship between DNA adducts
and chromosomal aberrations by FISH. All our results suggest that the FISH technique is more sensitive than the CCA.
We may put forward an idea that for the risk assessment for human health translocations should be evaluated as more
significant chromosomal aberrations than breaks. The increase of genomic frequency of translocations represents a
significant health risk for their carriers, especially for the process of carcinogenesis. It will be pertinent to use this knew
knowledge about an increased genotoxic risk in the preventive medicine as well as in the legislation.
Supported by the Czech Ministry of Environment VaV-SL/5/160/05 of and by the AS CR 1QS500390506.
O-048
USE OF METABOLOMICS TO FIND NEW BIOMARKERS OF EFFECT
Bennard van Ravenzwaay
BASF AG, Ludwigshafen, Germany
Will metabolomics have a greater chance of success in toxicology and biomarker assessment that genomics and proteomics? In the events following a toxic insult genomics analyses the earliest change, followed by proteomics and
metabolomics. Consequently, metabolomics is closer to the toxicological outcome than the other omics. A further advantage of metabolomics is that changes are detectable in blood. If the analysis of a great number of individual organs can
be replaced by one matrix then this will provide significant advantages (less invasive method, no need to kill animals,
time coarse analysis possible). Metabolomics can focus on the analyses of all possible changes but this will result in
both known and unknown analytes some of which will be real metabolites of yet unknown chemical structure while
others will be artefacts generated during the analytical procedures. We have chosen to perform the analysis of blood
metabolites in such a way as to minimize the risk of artefacts and to have a high number of known metabolites. We have
also reduced the extent of variation in the biological system as well as during analysis. To investigate the potential of
metabolomics to find biomarkers or specific patterns of change we have analysed the blood metabolome of rats treated
with HPPD inhibitors, a novel class of herbicides. The results demonstrated that a single metabolite, tyrosine, can be
used as a biomarker. In addition to tyrosine we also found a specific pattern of change that involved 9 metabolites.
Though the extent of change was less than for tyrosine the consistent changes of these metabolites is diagnostic for
this (toxicological) mode of action. As the identity of all of these metabolites was known we were able to relate nearly
all of these changes to the mode of action.
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26.6.2006 16:05:46
ORAL PRESENTATIONS
O-049
TELOMERIC LENGTH AS A BIOMARKER OF INDIVIDUAL SENSITIVITY TO GENOTOXICANS
Ricardo Marcos 1
Jenifer Moreno 2, Maria Castella 3, Jordi Surrallés 3
Universitat Autonoma de Barcelona, Cerdanyola del Valles, Spain 1, Universitat Autonoma de Barcelona, Cerdanyola del valles,
Spain 2, Universitat Autonoma de Barcelona, Cerdanyola del Valles, Spain 3
Genomic instability syndromes are characterized by high sensitivity to genotoxic compounds and high incidence of cancer. In addition, telomere dysfunction has also been pointed out as leading to chromosome instability.
In most somatic human tissues and primary cells telomeres shorten with each cell division and, as a result, telomere
lengths are reduced with every cell division until a critical short length is reached, eliciting a senescent response; thus,
telomeric length might modulate the cell response to genotoxic insults.
To detect whether individual telomeric length acts a modulating factor of the level of genetic damage, both basal and
induced, age is a critical factor due to the replicative shortening. Thus groups very homogeneous in age, or cells from
newborn (umbilical cord cells) must be used to evaluate the role of telomere length as an individual sensitivity factor.
When telomere length is measured using terminal restriction fragments (TRF), by a conventional enzymatic digestion
and Southern hybridization method using a telomeric probe, high interindividual variability in telomer length is observed,
both in newborn and in and homogenous group aged around 22 years. Genetic damage can be measured using the
micronucleus assay at both spontaneous and induced levels; the induced damage observed after both chemicals and
ionizing radiation treatment of blood lymphocytes. Results show association between telomere length and genetic damage, with more damage in individuals with short telomeres. This suggest that telomeric length can be considered as an
individual sensitive factor.
0-050
THE CYTOKINESIS-BLOCKED MICRONUCLEUS ASSAY AS A NOVEL BIOMARKER FOR LUNG
CANCER RISK
Randa A El-Zein
Matthew B Schabath , Carol J Etzel, Mirtha S Lopez, Jamey Franklin, Margaret R Spitz
The University of Texas MD Anderson Cancer Center, Houston, TX, USA
Background/aims: There are a variety of biomarkers evaluating susceptibility to the carcinogenic effects of benozo[a]pyrene, however,
there are no assays that specifically evaluate susceptibility to nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
(NNK), a potent inducer of lung adenocarcinoma, the most common histologic subtype of lung cancer. Methods: We therefore modified
the cytokinesis-block micronucleus (CBMN) assay, an established biomarker of DNA damage and genomic instability to evaluate susceptibility to NNK by measuring the frequency of NNK-induced chromosomal damage endpoints (micronuclei MN; nucleoplasmic bridges
NPBs and nuclear buds) per 1000 binucleated cells. Results: Both spontaneous and NNK-induced levels of chromosomal damage were
statistically significantly higher in lymphocytes from 139 lung cancer cases than in 130 matched controls. 47% of the cases (compared
with 12% of the controls) had ≥ 4 spontaneous MN, 66% of the cases (and no controls) had ≥ 4 spontaneous NPBs and 25% (versus
5%) had >1 spontaneous bud (P < .001). Similarly, 40% of the cases (versus 6% of the controls) had ≥ 5 NNK-induced MN, 89% of the
cases (and no controls) had ≥ 6 induced NPBs and 23% (versus 2%) had >2 induced buds (P < .001). As continuous variables, these
measures were associated with 2-, 29- and 6-fold increases in risk for spontaneous and 2.3-, 45.5-and 10-fold increase in cancer risk for
NNK-induced MN, NPBs and buds respectively. We also evaluated the use of the CBMN assay as a predictor of risk based on number of
MN, NPBs and buds defined by percentile cut points in controls. The probability of being a case was 0.94 and 0.99 for the 95th percentile
of spontaneous and NNK-induced MN and NPBs respectively. Conclusions: Our study indicates that the CBMN assay appears to be an
extremely sensitive test for detection of NNK-induced genetic damage and a good predictor of lung cancer risk.
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ORAL PRESENTATIONS
0-051
REPAIR COMPETENCE AND COMET ASSAY AS BIOMARKER OF EFFECT AND SUSCEPTIBILITY
(EXPAH STUDIES)
Antonina Cebulska Wasielwska 1,2
Blanka Binkova 3, Radim J. Sram 3, Ivan Kalina 4, Teodor Popov 5, Peter B. Farmer 6
Department of Radiation and Environmental Biology, The H.Niewodniczański Institute of Nuclear Physics PAN, Kraków, Poland 1,
Chair of the Epidemiology and Preventive Medicine, Collegium Medicum of Jagiellonian University, Kraków, Poland 2, Institute of
Experimental Medicine AS CR, Prague, Czech Republic3; Department of Molecular Biology of the P.J.Šafárik University, Kosice,
Slovakia 4 , Department of Toxicology, National Center of Hygiene, Sofia, Bulgaria5; Cancer Biomarkers and Prevention Group,
Leicester, UK6
Environmental exposures’ importance has been evident in cancer incidence and mortality. Elevated frequencies of
chromosome aberrations (CA) are accepted biomarkers of hazardous genotoxic effects and CA studies have shown
dependence on the exposure and the association with adverse health outcomes. However, faster method for monitoring
the individual susceptibility to the induction of critical damage is still needed. Previous studies reported good correlations between DNA damage detected after same treatment by the single cell gel electrophoresis (SCGE) assay and CA
observed in a subsequent mitosis. The aim of our study performed under the EXPAH project was to investigate whether
exposure to carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) affects cellular susceptibility to induction of DNA
damage and its repair capacities. A challenging dose of radiation was applied. The induced DNA damage was analyzed
with SCGE before and after the incubation allowing cells to complete the fast DNA repair. Repair competence was investigated in groups of policemen, or drivers exposed to the environmental c-PAHs and subjects occupationally unexposed
to c-PAHs as controls. Results show variability between donors in a response to treatment and in the efficiency of DNA
repair. The significant influence of the occupational exposure to c-PAHs on cellular capacities have been observed.
Results also show that the decrease of DNA repair efficiency is significantly affected by genetic polymorphism and other
life-style related factors (smoking habit, diet). The results confirm that the SCGE applied with repair competence assay
can be used as a fast and reliable, phenotype related biomarker. In molecular epidemiology the biomarker can indicate
a growing risk to human health and could effectively predict cellular susceptibility to environmental, occupational, or
therapeutic genotoxic exposures.
Acknowledgment Research supported by: BBN/CM-4100/523/2/2006/501/NKL/188/L, EC-EXPAH QLK4-CT-200000091.
O-052
BIOMARKERS DURING PRENATAL AND EARLY POSTNATAL LIFE: EXAMPLES FROM AMERICAN
STUDIES
Nina Holland1
K. Huen 1, M. Kwan 1, A. Bradman 1, K. Harley 1, P. Buffler 1, I. Tager 1, B. Eskenazi 1,C. Furlong 2
University of California, Berkeley, Berkeley, USA 1, University of Washington, Seattle, USA 2
A growing number of diseases in children, including asthma and childhood leukemia, have been linked to environmental exposures during prenatal and early postnatal development. The main risk factors include pesticides, air and
water pollution, lead, environmental tobacco smoke, infections, and inadequate diet. Twelve Centers for Children’s
Environmental Health and Disease Prevention were funded by the National Institute of Environmental Health Sciences
and Environmental Protection Agency since 1998. Each Center has a unique research focus ranging from the study of
respiratory diseases, childhood learning issues, and developmental disabilities. Several of these Centers have collected
biological and environmental samples, that have already been analysed for multiple biomarkers of exposure and effect,
including mercury, lead, cotinine, pesticides, phtalates, PAHs, allergens, endotoxin, cytokines, cholinesterase etc. Four
large studies have shown that two agricultural communities in California and Washington State and two multi-ethnic
birth cohorts in New York City exhibit high variation of pre- and postnatal exposure to organophosphate pesticides
(OP). OP exposure was associated with shorter gestational age, increased frequency of abnormal reflexes in neonates
and other developmental problems. The activity of specific genes such as paraoxonase (PON1), can modify the effects
of OP exposure and developmental health outcomes. Newborns are shown to have several fold lower PON1 levels than
their mothers, and their OP sensitivity can be further affected by their PON1 genotype. Examples from several studies
including the Northern California study of childhood leukemia, and the CHAMACOS birth cohort of Latino mothers and
children from agricultural community, will be used to illustrate the advantages of employing multiple biomarkers of
exposure as well as genetic and immunological biomarkers for pregnant mothers and children. These data will help to
identify susceptible subpopulations and inform future policy decisions regarding allowable exposures to young children
and pregnant women.
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ORAL PRESENTATIONS
O-053
MOTHER-CHILD COMPARISON IN GENOMIC RESPONSE TO AIR POLLUTION EXPOSURE
Jos C Kleinjans 1
Danitsja M van Leeuwen 1, Marcel H van Herwijnen 1, Joost H van Delft 1, Marie Pedersen 2, Edyta Bajak 3,
Radim J. Sram 4, Micheline Kirsch-Volders 5, Lisbeth E Knudsen 2
Maastricht University, Maastricht, Netherlands 1, University of Copenhagen, Copenhagen, Denmark 2, Karolinska Institute, Sweden 3,
Laboratory of Genetic Ecotoxicology, Prague, Czech Republic 4, Vrije Universiteit Brussel, Brussels, Belgium 5
Children’s health is of increasing concern as children may be more vulnerable to environmental genotoxic exposure.
Molecular epidemiological studies including mothers and their children may give better estimates of the biological differences between these subpopulations to be used in environmental health risk assessment. Transcriptomic analysis
represents a promising tool for monitoring environmental carcinogenesis as the analysis of genome wide altered gene
expressions provide more generic and more mechanistic information on adverse health effects in humans than the classical cytogenetic biomarkers. Differences in exposure and in cellular response to air pollution between adults and children have been reported. The aim of this study was to apply transcriptomic analysis for comparing peripheral blood gene
expression profiles in mothers and their children living in two areas in the Czech Republic and differentially exposed to
air pollution, by using Agilent oligonucleotide microarrays. To evaluate associations of individual gene expression profiles
with individual cellular responses to environmental exposure, correlations of gene expressions with micronuclei frequencies in peripheral blood, an established biomarker of genotoxic effect, were studied. Relatively increased micronuclei
frequencies were found in environmentally exposed mothers and children. Considerable numbers of significantly differentially expressed genes and biological processes were found between subjects from the contaminated area and the
control area, in mothers as well as in their children. Differences and similarities in genomic responses between mothers and children will be presented at the level of modulated biological processes. In addition, associations of individual
gene expressions with micronuclei frequencies will be reported. Results demonstrate gene expression profiling to be a
valuable tool in biological effect monitoring.
The study was financed by the ChildrenGenoNetwork (QLK4-CT-2002-02198).
O-054
BASELINE CHROMOSOME ABERRATIONS IN CHILDREN
Domenico Franco Merlo 1
Elena Stagi 1, Marcello Ceppi 1, Pavel Rossner 2, Radim J. Sram 2
National Cancer Research Institute, Genova, Italy 1, Institute of Experimental Medicine AS CR, Praha, Czech Republic 2
There is a body of scientific evidence supporting the usefulness of including biomarkers of genetic damage in epidemiologic studies of children exposed to environmental pollutants. Chromosome aberrations are consistently increased in
children exposed to ionizing radiation and industrial chemicals than in referent children. Exposure(s) occurring during
childhood (and in utero) may continue for several years, become chronic, and eventually play a relevant role in the
aetiology of childhood as well as adulthood cancers. Indeed the association between CA frequency in peripheral blood
lymphocytes and cancer risk detected in occupationally exposed subjects supports the hypothesis that CA is a predictor
of cancer.
Estimates of the baseline frequency of CA in pediatric population is a prerequisite in planning epidemiologic investigations of children exposed to low level of environmental genotoxic agents. As reported for other cytogenetic biomarkers,
CA frequency may vary with gender and age in children as well as in adults. Baseline CA values are therefore needed
in planning sound field studies on genomic damage among the youth. The levels were estimated from 16 published
epidemiologic studies and from a large sample of Czech children aged 7-16 years (n.=1,214) and 206 newborns. All
children served as referents. For the whole referent population (age range 0-19 years) the mean CA estimated from the
published findings was 1.24%, 95%CI=1.05-1.47. Baseline values for boys and girls were 1.22%, 95%CI=1.12–1.32,
1.21, 1.14-1.29, respectively. Among newborns CA mean level was 1.14%, 95%CI=0.96-1.32. Based on the reviewed
and reanalysed data, CA baseline levels were similar in boys and girls and failed to show any clear increase with age.
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ORAL PRESENTATIONS
O-055
INHERENT SUSCEPTIBILITY OF CHILDREN
Ilse Decordier 1
Micheline Kirsch-Volders 1, Kelly De Bont 1, Roberta Ciardelli 2, Dominique Haumont 2
Laboratory of Cell Genetics, Vrije Universiteit Brussel, Brussels, Belgium 1, Neonatal Unit, Saint-Pierre University Hospital, Brussels,
Brussels, Belgium 2
Specific research projects have been initiated to protect children against environmental toxicants and carcinogens. The
central question is whether children’s susceptibility is different from that of adults.
Aim: To determine whether genotype and/or the DNA strand break repair phenotype in combination with the MN assay
would allow to estimate the relative sensitivity of a new born child as compared to his mother for a given genotoxic
exposure. H2O2 was chosen as model mutagen, since oxidative stress is a major issue for newborns suddenly exposed
to a high oxygen concentration.
Methods: We compared the in vitro genetic susceptibility for H2O2 in PBMC of 10 mother-newborn daughter pairs taking into account genotypes for relevant repair (hOGG1, XRCC1, XRCC3, XPA, XPD) and folate metabolism (MTHFR)
polymorphisms. After in vitro challenge with H2O2 the repair capacity was assessed by the Comet assay and genotoxic
effects by the cytokinesis-block MN assay.
Results: Initial H2O2-induced DNA damage was similar for daughters and their mothers. After 2 h repair, DNA repair was
faster in the daughters than in the mothers. When correlating with the genotypes only the MTHFR222 variant genotype
had a significant increasing effect on the induction of DNA damage. The amount of fixed DNA damage was assessed
by the MN assay. No significant differences between mothers and daughters were observed. Correlation with the genotypes showed that mothers with the XRCC1399 variant allele or XRCC3241 Thr/Thr genotype accumulated more MN when
exposed to H2O2. Multivariate analysis revealed a significant protective effect of maternal antioxidant supplementation
during pregnancy. Conclusions: Newborn daughters show a better DNA repair capacity after oxidative stress compared
to their mothers. Moreover these results demonstrate the combined interaction between genotype and antioxidant supplementation for the protection of children in utero and at birth.
O-056
IMPACT OF GENETIC POLYMORPHISMS ON RESPIRATORY MORBIDITY IN CHILDREN
Jan Topinka1
Miroslav Dostal 1, Irena Chvatalova 1, , Radim J. Sram 1 , Irva Hertz-Picciotto 2, Jesse P. Joad 2, Poh-Sin
Yap 2, Teri Greenfield 2
Institute of Experimental Medicine AS CR, Prague, Czech Republic 1, University of California, Davis, USA 2
We examined genetic polymorphisms and gene-environment interactions in relation to early childhood bronchitis. A
birth cohort from two districts in the Czech Republic was followed up at 3 to 4.5 years of age. Pediatric records were
abstracted for all illnesses. Bronchitis was defined as a physician diagnosis of J20 (acute bronchitis). PCR-RFLP techniques were used for genotype analysis of xenobiotic metabolizing (GSTM1, GSTT1, GSTP1, CYP 1A1, EPHX1) and DNA
repair genes (XPD 6 and 23, XRCC1, hOGG1) in samples of fetal tissue from placentas of 800 births. Poisson regression
was used to model the number of episodes of bronchitis during the first two years of life in relation to child’s genotype,
with adjustment for ethnicity, maternal smoking, child’s sex, mother’s age and education, season of birth and use of
coal for home heating or cooking. Additionally, XPD-6 was examined for modification of the effects of acute air pollution
in a repeated measures logistic regression analysis. More than 60% of the children experienced 1 or more episodes of
bronchitis in the first two years of life. Xenobiotic metabolizing genes did not predict the number of bronchitis events,
but XPD-6 polymorphisms did. Those with the XPD-6 AA genotype (40%) experienced bronchitis more frequently than
the homozygous CC and heterozygous children combined (RR=1.45, 95% confidence interval (CI)=1.36, 1.55). Those
with the CC genotype had a similar risk to the heterozygotes. In our data, the C-allele of XPD-6 appears to confer some
protection against bronchitis in early childhood, after adjusting for ethnicity and other factors. Neither XPD-6 nor XPD23 renders children more susceptible to short-term respiratory effects of ambient particles or PAHs. Although studied
mainly for their involvement in cancer risk, XPD may play a role in immune competence. Supported by Czech Ministry
of Environment (VaV – IC/6/504) and by Academy of Sciences CR (AV0Y50390512).
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O-057
RESPONSES TO MUTAGENIC COMPLEX MIXTURES – AN OVERVIEW
Brinda Mahadevan
L.A. Courter, William M. Baird
Department of Environmental & Molecular Toxicology, Oregon State University, Corvallis, USA
Chemical exposure to complex mixtures such as coal tar, ambient air pollution, and automobile exhaust is a serious
concern because humans are exposed to more than one of these mixtures. Studies have shown that many of the components of these environmental mixtures, such as polycyclic aromatic hydrocarbons (PAH) are common and can be
genotoxic, mutagenic and/or carcinogenic. To understand the genotoxic/carcinogenic effects of complex PAH mixtures
on metabolic activation, tumor initiation and gene expression, we designed experiments using environmental mixtures.
Standard reference material (SRM) 1597 (coal tar), SRM 1649a (urban dust) and SRM 1650a (Diesel exhaust particulate) or SRM 1975 (Diesel exhaust extract) were obtained from the National Institute of Standards and Technology. ,
treatment of the SRM with two potent carcinogenic PAH benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) were also
used to better understand the influence of these complex mixtures on PAH. DNA adduct formation on exposure to
SRM alone or on , treatment with BP or DBP both in vivo and in vitro were monitored by 33P post labeling and HPLC.
Induction of cytochrome P450 enzymes was observed as measured by ethoxyresorufin-O-deethylase assay. Gene
expression upon exposure to SRM 1649a and SRM 1650a in vitro was measured by using Affymetrix oligonucleotide
microarrays (U133A). In addition, to relate the gene expression results obtained in vitro, mouse skin initiation-promotion tumorigenesis model was utilized. This was followed by validation of expression of specific genes in the mice
tumors. Overall, the results from our studies suggest that testing for DNA adduct formation is important as it is the first
step during the multistage process of chemical carcinogenesis. Furthermore, the data provides not only a transcriptional
signature to chemical carcinogen exposure but also suggest that understanding the relationship between tumor induction and gene expression may allow the prediction of early genotoxic effects of environmental mixtures.
O-058
DETERMINATION OF URINARY PAH METABOLITES AS BIOMARKERS TO ASSESS
THE EXPOSURE OF THE GENERAL POPULATION TO PAH MIXTURES
Albrecht Seidel
Gerhard Dettbarn, Juergen Jacob
Biochemical Institute for Environmental Carcinogens, Grosshansdorf, Germany
Polycyclic aromatic hydrocarbons (PAH) are widespread occurring environmental contaminants some of which are well
known human carcinogens. Apart from personal smoking habits, the general population is exposed to low level of PAH
in ambient air particulate matter and contaminated food such as cereals, oils, fats, and grilled meat. Biological monitoring of PAH metabolites in human urine is the method of choice to assess the endogenous burden of an individual
by PAH as it reflects all different uptake routes including ingestion, inhalation and dermal resorption. Urinary excreted
phenols of naphthalene, pyrene and phenanthrene are selected to be determined as biomarkers of an exposue to PAH
mixtures because metabolites of higher moleculat weight PAH, such as benzo[a]pyrene, are urinary excreted only in
very small amounts. Among the phenols 1-hydroxypyrene (1-OHP) is the most frequently determined PAH metabolite
and in Germany and the United States references values for this biomarker have been calculated for the general population from environmental surveys performed in these countries. During the last years several analytical procedures have
been developed based on HPLC and GC/MS methodologies allowing to determine not only 1-OHP but also an additional
battery of phenols from naphthalene, phenanthrene and other PAH. Recently, analytical procedure became available
to determine more polar PAH metabolites excreted in urine of the general population, e.g. dihydroxypyrenes formed
from 1-OHP and the highly polar phenanthrene tetrol. The latter biomarker reflects the metabolic activation pathway
of carcinogenic PAH to bay region diol epoxides. The various types of phenanthrene metabolites have been proposed
to be used in future studies for phenotyping of individuals to allow to determine their possible susceptibility to develop
cancer upon exposure to PAH mixtures.
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O-059
3-NITROBENZANTHRONE, A POTENTIAL HUMAN CANCER HAZARD IN DIESEL EXHAUST
AND URBAN AIR POLLUTION
Volker Manfred Arlt
Institute of Cancer Research, Section of Molecular Carcinogenesis, UK
Epidemiological studies have shown that exposure to diesel exhaust and urban air pollution is associated with an
increased risk of lung cancer.
3-Nitrobenzanthrone is an extremely potent mutagen and suspected human carcinogen identified in diesel exhaust
and ambient air particulate matter. The main metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), was found in the
urine of salt mine workers occupationally exposed to diesel emissions, indicating that human exposure to 3-NBA due to
diesel emissions can be significant and is detectable. There is clear evidence that 3-NBA is a genotoxic mutagen forming DNA adducts after metabolic activation through simple reduction of the nitro group. Several human enzymes have
been shown to activate 3-NBA and its metabolites in vitro and in cells leading to the formation of purine adducts at
the C8 and N2 position of guanine and at the C8 and N6 position of adenine. The predominant DNA adducts in vivo, 2(2´-deoxyguanosin-N2-yl)-3-aminobenzanthrone (dG-N2-ABA) and N-(2´-deoxyguanosin-8-yl)-3-aminobenzanthrone
(dG-C8-N-ABA) are also the most persistent adducts in target tissue in rodents, and are most probably responsible for
the induction of GCRTA transversion mutations observed in vivo. It is concluded that these adducts not only represent
premutagenic lesions in DNA but are of primary importance for the initiation of the carcinogenic process and subsequent
tumour formation in target tissue. Indeed, 3-NBA is carcinogenic in rats after intratracheal instillation, inducing mainly
squamous cell carcinoma in lung. Because of its widespread environmental presence, 3-NBA may represent not only an
occupational health hazard but also a hazard for larger sections of the general population. For an accurate risk assessment more epidemiological studies on 3-NBA-exposed individuals and a broader monitoring of environmental levels of
3-NBA are required.
[Arlt (2005) Mutagenesis 20, 399-410]
O-060
CYTOGENETIC BIOMARKERS FOR MONITORING HUMAN POPULATIONS EXPOSED TO PHYSICAL
OR CHEMICAL MUTAGENS
A.T. Natarajan
University of Tuscia, Viterbo, Italy 1, Leiden University Medical Center, Leiden, Netherlands 2
Most mutagens induce chromosomal aberrations in the target cells and their frequencies can be used to estimate the
extent of exposure. Ionizing radiation induced chromosome aberrations are formed very fast following exposure and
the frequencies of unstable aberrations (dicentrics) in lymphocytes of exposed individuals can be used for estimating absorbed radiation dose even after months after exposure. Stable aberrations, such as reciprocal translocations,
which persist for a long time can be detected by FISH and used for retrospective dosimetry. High LET radiation, such
as plutonium α – rays, leave fingerprints in lymphocytes of exposed populations in the form of increased frequencies of
intra-chromosomal aberrations (inversions and deletions) which can be detected by m-band FISH. On the other hand,
chemically induced DNA lesions are constantly repaired and only the persistent lesions that are present at the time
of replication will lead to chromatid type of aberrations, the frequencies of which can be used to estimate the extent
of exposure. Some carcinogens, such as BPDE, have been shown to induce aberrations in specific sites of some chromosomes. The frequencies of micronuclei in lymphocytes (using cytogenesis blocked cells) as well as micronuclei in
reticulocytes can also be employed as surrogate for chromosome aberrations have been employed in several studies.
The relative merits of these techniques will be discussed.
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O-061
COMPARISON OF GENOTOXIC AND NONGENOTOXIC POTENCIES OF COMPLEX
ENVIRONMENTAL MIXTURES IN AIRBORNE AND RIVER SEDIMENT EXTRACTS
Miroslav Machala 1
Katerina Pencikova 1, Sona Marvanova 1, Pavel Krcmar 1, Zdenek Andrysik1,2, Brinda Mahadevan 3, William
M. Baird 3, Miroslav Ciganek 1, Jiri Neca 1, Jan Vondracek1,2
Veterinary Research Institute, Brno, Czech Republic 1, Institute of Biophysics, Brno, Czech Republic 2, Oregon State University,
Corvallis, USA 3
A series of in vitro bioassays, assessing genotoxic and tumor-promoting activities of xenobiotics in liver cellular models,
was used for determination of toxic potencies of extracts of complex abiotic environmental samples and their fractions.
Polycyclic aromatic hydrocarbons (PAHs) were found to be predominant toxicants in the studied samples. Based on our
previous studies on toxic modes of action of individual PAHs and their methylated derivatives, we could classify PAHs
into three groups based on distinct patterns of effects: strong genotoxins forming high levels of DNA adducts, increasing phosphorylation of histone H2AX and p53 protein, and inducing high increase of S-phase cells and apoptosis; AhR
agonists inducing cell proliferation in contact-inhibited cells; low-molecular-weight PAHs inhibiting gap junctional intercellular communication (GJIC) in rat liver epithelial WB-F344 cells. In this study, the AhR-mediated activity, determined
both as the activation of the AhR-dependent luciferase reporter gene expression in hepatoma cells and as the induction
of CYP1A1/1B1 mRNA expression in WB-F344 cells, was the most sensitive endpoint reacting to exposure with complex
samples. A release of confluent WB-F344 cells from contact inhibition, a potential tumor-promoting effect associated
with AhR activation, was also observed at low concentrations. Contrary to the activation of AhR, the CYP1A1A/1B1dependent EROD activity, genotoxic effects and apoptosis were significantly affected by the presence of other PAHs and/
or other compounds in complex mixtures; DNA adduct formation was detectable only at high concentrations. Similarly,
complex PAH mixtures induced only a partial inhibition of GJIC, which did not correspond with inhibitory potencies of
individual PAHs. (This work is a part of the MODELKEY project supported by EC 6th Framework programme http://www.
cordis.lu/sustdev/environment/home.html Contract-No. 511237 (GOCE)).
O-062
INTERACTIONS OF AROMATIC HYDROCARBONS IN THEIR BINARY MIXTURES; AN IN VITRO
STUDY
Alena Gábelová 1
Veronika Poláková 1, Katarína Poloncová 1, Zuzana Valovičová 1, Gabriela Prochazka 2
Cancer Research Institute, Bratislava, Slovakia1, Karolinska Institute, Stockholm, Sweden2
Millions of people are chronically exposed to low doses of noxious chemicals, frequently present in complex mixtures.
Individual components of a mixture can undergo various interactions and in this way they might enhance/multiply the
adverse health effects of the mixture. The improvement of our knowledge about the mechanisms of mutual interactions of the chemical agents in the mixtures might contribute to better assess the impact of environmental pollution
on human health.
The objective of this study was to investigate the cell response to exposure to individual aromatic hydrocarbons and
their binary mixtures in dependence on the proportion of particular carcinogen in the mixture. Benzo[a]pyrene (BaP)
and 7H-dibenzo[c,g]carbazole (DBC), ubiquitous environmental pollutants, were used as model agents and various
mammalian cell lines with different drug-metabolizing capacity were utilized as model system. The ratio of BaP:DBC in
the mixture was 1:1, 1:0.1 and 1:0.01. Both BaP and DBC decreased the cell viability in a dose-dependent manner and
increased significantly the level of micronuclei (MNs) and frequency of gene mutations (6-TGr mutations). In general,
cell exposure to a binary mixture resulted in the same or rather lower level of MNs and 6-TGr mutations in comparison
to a single carcinogen and led to a slight reduction of cell cytotoxicity. An antagonism to additivity was determined in
particular binary mixture depending on the ratio of BaP:DBC. The strongest antagonism was detected in V79MZh1A1
cells stably expressing single human cytochrome P4501A1 while a sub-additive effect was found in human hepatoma
HepG2 cells with a broad activation capacity. Based on these data we supposed that a competition of individual carcinogens for biotransformation enzymes or saturation of activating enzymes might restrict the formation of the reactive
intermediate and thereafter decreased the genotoxicity of the binary mixtures.
This study was supported by the VEGA grants No. 2/3092 and No. 2/6063.
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O-063
CANCER RISK MODEL BASED ON IN VIVO DOSIMETRY: STUDIES ON ACRYLAMIDE
AND BUTADIENE
Margareta Littorin Törnqvist 1
C. Fred 1, F. Granath 2
Dept. of Environmental Chemistry, Stockholm University, Stockholm, Sweden 1, Dept. of Medical Epidemiology, Karolinska Institute,
Stockholm, Sweden 2
In the 1970-ies Lars Ehrenberg at Stockholm University initiated a research line aiming at improved methods for cancer
risk estimation of genotoxic chemicals, which was based on experience from radiation biology. As a basis for quantitative
work, a dose concept was defined for genotoxic compounds (electrophiles), which was comparable with dose of ionising
radiation. Since then methods for the measurement of in vivo doses of electrophiles through adducts to haemoglobin
(Hb) are developed. The developments now have advanced so far so it is possible to test the applicability to chemical
carcinogens of the multiplicative cancer risk model used for ionising radiation. Cancer studies of 1,3-butadiene have
shown large differences in the sensitivity between the mouse and the rat. This is believed to be related to the differences
in the metabolism to the bifunctional diepoxybutane (DEB). In comparison with the monofunctional epoxy-metabolites,
DEB is a highly effective mutagen. The applicability of the multiplicative cancer risk model to the cancer test data for
butadiene was evaluated. According to the multiplicative risk model the risk increment is proportional to the in vivo
dose of the genotoxic agent and its mutagenic potency, and the background cancer incidence. The evaluation showed
a good predictability by the risk model of the tumour incidence on the basis of in vivo doses of the epoxy-metabolites,
measured as Hb adducts, and mutagenic potency. Furthermore, according to this evaluation DEB was found to be the
major contributor to the observed tumour incidences in mice. In contrast, the monoepoxy-metabolites were found to
be the major cause to the observed incidences in rats. These results are promising with regard to the usefulness of the
approach for cancer risk estimation suggested by Ehrenberg. Ongoing evaluation concerns acrylamide, as the general
dietary exposure makes this compound to a challenge for cancer risk estimation procedures.
O-064
MECHANISMS OF INFLAMMATORY DISEASES
Curtis C. Harris
S. Perwez Hussain
Laboratory of Human Carcinogenesis, National Cancer Institute, NIH, Bethesda, MD 20892-4258, USA
Free radicals are ubiquitous in our body and are generated by normal physiological processes, including aerobic metabolism and inflammatory responses, to eliminate invading pathogenic microorganisms. Free radicals can also inflict cellular
damage. Several defense mechanisms have evolved both to protect our cells from radicals–such as the p53 and the
Rb tumor suppressor pathways, and antioxidant scavengers and enzymes–to repair DNA damage. The nitric oxide and
COX2 pathways are also linked in many inflammatory diseases. Nevertheless, many chronic inflammatory diseases, e.g.,
ulcerative colitis, are associated with increased cancer risk. Understanding the relationship between chronic inflammation and cancer provides insights into the molecular mechanisms involved. In particular, we highlight the interaction
among nitric oxide, microRNA expression and the p53 stress response pathway.
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O-065
GENE-ENVIRONMENT INTERACTIONS IN ATHEROSCLEROSIS AND GLAUCOMA
Alberto Izzotti
Cristina Cartiglia, Mariagrazia Longobardi, Barbara Di Marco, Tanya Melissa Pennisi, Silvio De Flora
Department of Health Sciences, University of Genoa, Italy, Genoa, Italy
DNA damage is a common pathogenetic determinant in several chronic degenerative diseases. DNA adducts in aorta
smooth muscle cells (SMC) from 85 atherosclerotic patients were significantly correlated with atherogenic risk factors, including age, number of cigarettes, hypertension, blood triglycerides and total/HDL cholesterol, fluorescent DNA
adducts, and 8-oxo-dG, whose levels were even higher in the aorta inner-layer. DNA adducts were higher in patients
having a null GSTM1 genotype, while GSTT1, NAT1 and NAT2 did neither affect DNA adducts nor 8-oxo-dG. The 4977 bp
mtDNA deletion was detected in aorta SMC. Plasma malondialdehyde correlated with the number of cigarettes. Plasma
homocysteine was higher in 84 atherosclerotic patients than in 74 controls and was positively correlated with blood
triglycerides and inversely correlated with total glutathione and fruit consumption. Plasma homocysteine was increased
by homozygosity for slow MTHFR. Trombophilic polymorphisms affected the 4% of the atherosclerotic patients. All
patients have been now followed for up to 14 years in order to assess the prognostic relevance of molecular epidemiology data.
Glaucoma is the most frequent cause of irreversible blindness worldwide. We demonstrated that a significant increase
of 8-oxo-dG occurs in trabecular meshwork cells (TMC) of open-angle glaucoma patients. Oxidative DNA damage was
correlated with the increase of intraocular pressure and visual field defects,. Both within controls and glaucoma patients,
8-oxo-dG levels were significantly higher in subjects having a GSTM1-null polymorphism. The analysis of a variety of
gene polymorphisms related to oxidative damage, including OGG1, GSTT1, FAS, MPO1 and SOD, is in progress. To shed
light on the source of ocular oxidative damage in glaucoma we are analyzing the occurrence of 4977 bp mtDNA deletion
in TMC of glaucoma patients.
O-066
WHAT WE KNOW ABOUT MOLECULE GENETIC SUBCLASSES OF ASTHMA AND ATOPY RELATED
PHENOTYPES SO FAR?
Tarja Laitinen
Geneos Ltd, Helsinki Finland
Asthma is a multifactorial disease in which both environmental triggers and individual’s genetic makeup play a role. So
far genome wide scans have been reported in 17 study populations. By means of genome wide linkage and hierarchical
association analysis six positional candidate genes (ADAM33, PHF11, DPP10, GPR154, HLA-G, and CYFIP2) for asthma
related traits have been cloned. The interactions of the proteins encoded by these genes and the biological relevance
of these signalling pathways in the development of asthma are still poorly understood. Also the disease mechanisms
due to the genetic variance in the genes identified, remain largely unknown. This information is, however, rapidly accumulating. In the meanwhile it is import to examine the statistical robustness of each genetic finding in combination
with the limited data available on the functional properties of the corresponding proteins to estimate the strengths and
weaknesses in the chains of evidence.
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O-067
ROLE OF EPHX1 GENOTYPES IN THE DEVELOPMENT OF ASBESTOS AND SMOKING INDUCED
NON-MALIGNANT PULMONARY DISEASES
Ari Hirvonen1
Mari Kukkonen 1, Simo Kaleva 1, Tapio Vehmas 1, Matti Huuskonen 1, Harri Vainio 1, Päivi Piirilä 2
Finnish Institute of Occupational Health, Helsinki, Finland 1, Helsinki University Hospital, Helsinki, Finland 2
Background. The lungs are subject to oxidative stress from, e.g., cigarette smoke. An excess of oxidants and free radicals in the lungs are anticipated to promote cellular and tissue damage and are the major initiators of lung diseases.
Tobacco smoke is the commonest identifiable risk factor for, e.g., lung cancer and emphysema. Asbestos exposure,
in turn, is the sole known initiator of malignant mesothelioma and several non-malignant asbestos related pulmonary
disorders. The individual susceptibility to asbestos and smoking induced pulmonary diseases have been suggested to
be modified by genetic polymorphism of metabolic enzymes like the microsomal epoxide hydrolase (EPHX1). EPHX1 is
an enzyme involved in the first-pass metabolism of highly reactive epoxide intermediates and is expressed at varying
levels in most tissue and cell types. Two common aberrant alleles of EPHX1 gene have been detected, which confer the
slow and fast enzyme activity.
Methods. The study population consisted of 575 asbestos-exposed workers and 2105 population controls. The workers
were diagnosed with pleural diseases (n=181), emphysema (n=147), marked adhesion (n=109), fibrosis (n=68), or
combined fibrosis and emphysema (n=70). The EPHX1 genotypes were determined by real-time PCR based methods
and the statistical evaluations were performed using logistic regression analyses.
Results. The EPHX1 low activity genotypes were associated with elevated risk of fibrosis (OR 2.9; 95% CI 1.0-8.7),
pleural disease (OR 1.8; 95% CI 1.0.-3.2), and emphysema (OR 1.8; 95% CI 1.0-3.6) compared with the high activity
genotypes.
Conclusions. Our findings provide further support for the hypothesis that the inherited EPHX1-associated detoxification
capacity is an important modifier of individual susceptibility to asbestos and smoking induced pulmonary diseases.
This study was supported by the Finnish Work Environment Fund.
O-068
TOBACCO SMOKE-ASSOCIATED LARYNGEAL CANCER. DIFFERENTIATION BETWEEN MULTIPLE
PRIMARY TUMOURS AND TUMOR RECCURRENCE
Krzysztof Szyfter1
M Poznan 1,2, M Giefing 1, K Szukala 1, M Rydzanicz 1, M Jarmuz 1
Institute of Human Genetics / Polish Academy of Sciences, Poznan, Poland 1, Otolaryngology Clinic / K.Marcinkowski Medical
University, Poznan, Poland 2
Laryngeal cancer is regarded as a result of tobacco smoking and strong alcohol abusing. A treatment initially recognized
as successful in a considerable fraction of subjects can be followed by disease progression connected with tumor relapse
(up to 20% within one year) or an incidence of second primary tumor (SPT, 5-12%). Molecular epidemiology offers some
techniques to estimate a genetic risk of cancer progression as well as to categorize the new tumor burden. The latter
is required to take a right decision concerning treatment.
Subjects with SPT (larynx as an initial tumor site) were found to be highly-mutagen-sensitive as shown by the bleomycin
test. Bleomycin-induced breaks were not randomly distributed on chromosome but clustered in loci known to harbor
genes involved in carcinogenesis as 22q11 (GSTT1) or 8p21 (NAT2). Further, by PCR-based genotyping it was established that the risk to develop SPT is associated with overrepresentation of CYP1A1*1/*4 and GSTM1null genotypes as
well as a coincidence of some combinations of CYP1A1, GSTM1, GSTT1, NAT2 and XPD gene variants.
A differentiation between SPT and recurrencies was based on estimation of clonality estimated studied by a set of 12
microsatellite markrs to compare a loss of heterozygosity (LOH). In the set of 22 pairs of tumors 6/22 were found to be
clonally unrelated ("true" SPT) and 3/22 carried clonal genetic changes (micrometastasis or reccurrency). The results
concerning remaining 14/22 cases were insufficient to determine the clonality status. An extention of a number of microsatellite markers should improve a diagnosis potential.
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O-069
DNA DAMAGE AND DNA REPAIR CAPACITY IN CHILDREN WITH TYPE 1 DIABETES MELLITUS
Rudolf Stetina1
Jana Varvařovská 2, Renata Pomahačová 2, Konrad Siala 2, František Stožický 2, Zdeněk Rušavý 3, Silvie
Lacigová 3, Jaroslav Racek 4, Ladislav Trefil 4
Faculty of Military Health Sciences, Hradec Kralove, Czech Republic, Hradec Kralove, Czech Republic 1, Pediatric Department ,
Charles University Hospital, Plzen, Czech Republic, Plzen, Czech Republic 2, 1st Clinic of Internal Diseases, Charles University
Hospital – Diabetology Center, Plzen, Czech Republic , Plzen, Czech Republic 3, Institute of Clinical Biochemistry and Haematology,
Charles University, Plzen, Czech Republic , Plzen, Czech Republic 4
Diabetes mellitus (DM) is connected with oxidative stress (OS), causing the damage to lipids, proteins as well as to
DNA. We followed markers of the oxidative stress (superoxid dismutase (SOD), glutathion peroxidase (GPx), plasma
antioxidant capacity (AOC), reduced glutathione (GSH) and malondialdehyde (MDA)), as well as DNA breaks (SSB) and
the individual capacity to repair the oxidative damage to DNA (DNArc) in group of children (8 girls, 12boys, age 13.26±
SD 2.98) with diabetes mellitus Type1 (T1DM). SSBs and DNArc were measured by modified comet assay in peripheral
lymphocytes and their extracts, respectively. Results obtained in paediatric patients were compared with those of a
group of 23 adult diabetic individuals without microvascular complications, and with 30 adult T1DM patients having any
of the following complications: retinopathy, nephropathy and/or neuropathy.
Levels of SOD, MDA and GSH in diabetic children were significantly lower, compared with healthy controls, however,
increased DNA breaks in peripheral lymphocytes of diabetic children were observed. DNArc was significantly increased in
diabetic children (p<0.05). In comparison with adults, children had lower numbers of DNA breaks. On the other hand,
DNArc of peripheral lymphocytes in children was significantly greater than in both adult groups (p<0.01 a p<0.001).
Our results seem to confirm, that children and adult patients with T1DM have increased parameters of OS and increased
levels of DNA breaks compared with the age-related healthy population. In all groups of diabetics, the capacity to repair
DNA containing oxidised bases was significantly higher than in healthy controls. The DNA repair capacity decreased with
the age, being significantly higher in children compared to adults.
Acknowledgements: This study was supported by the grant of Internal Grant Agency of the Czech Ministry of Health
no. NR7919-3
O-070
THE USE AND LIMITATIONS OF DNA ADDUCTS IN DETERMINING HUMAN EXPOSURE TO
CARCINOGENS AND THE AETIOLOGY OF HUMAN CANCERS
David Phillips
Institute of Cancer Research, Sutton, UK
The formation of DNA adducts by an agent is strong evidence of its genotoxic potential. Characterisation of DNA adducts
can reveal information about the nature of its reactive intermediates and pathways of activation. The influence of
enzyme cofactors, inducers and inhibitors on the levels of adducts can reveal the mechanism of metabolic activation. In
mammalian tissues adducts are useful markers of carcinogen exposure, providing an integrated measurement of intake,
activation and delivery to the target macromolecule in target tissues. Accessible surrogate tissues, such as blood cells,
provide the means to investigate occupational or environmental exposure in healthy individuals. Such exposure, e.g. to
polycyclic aromatic hydrocarbons, has been demonstrated in several industries and in defined populations, respectively,
by the detection of higher levels of adducts. Adducts detected in many tissues of smokers are at higher levels than in
non-smokers, although the magnitude of the elevation does not predict the magnitude of the risk. In target tissues there
is often a linear relationship between dose and adduct formation. Adduct formation itself does not predict target organ
specificity, although interventions that inhibit DNA adduct formation invariably inhibit carcinogenesis. Furthermore, the
relationship between mutation frequency and adduct formation in different organs is complex, and does not always
predict which are the target organs for carcinogenesis. Compelling evidence for the cause /effect relationship between
adduct formation and tumour formation has come from studies on sites of carcinogen-induced DNA damage in specific
genes (e.g. TP53) and correlations with sites of mutation of these genes in human tumours. Despite these indicators,
much remains to be determined about carcinogen organotropism, the origins of mutations in human tumours and the
relative significance of exogenous and endogenous sources of DNA damage. Identifying changes in gene expression that
are specific to target organs may shed light on this.
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O-071
SYSTEMIC GENOTOXIC EFFECTS OF FORMALDEHYDE DETECTED IN BIOMONITORING STUDIES
AND EVALUATION OF THEIR PLAUSIBILITY IN AN EX VIVO APPROACH
Guenter Speit
Petra Schuetz, Oliver Schmid
University of Ulm, Department of Human Genetics, Ulm, Germany
Formaldehyde (FA) is genotoxic in vitro in cultured mammalian cells. A variety of evidence suggests that the primary
DNA alterations after FA exposure are DNA-protein cross-links (DPX). DPX can arrest DNA replication and lead to the
induction of other genotoxic effects such as sister chromatid exchanges (SCE) in proliferating cells. Incomplete repair
of DPX can lead to the formation of mutations, predominantly chromosome aberrations (CA) and micronuclei (MN).
Conflicting results have been obtained regarding genotoxic effects in peripheral blood of FA-exposed subjects. Positive
effects were reported in some studies for DPX, SCE, CA and MN. We critically reviewed these papers with regard to the
quality of the test performance and the consistency and plausibility of the results. In order to imitate the biomonitoring
approach, we performed ex-vivo experiments with human blood samples (FA-treatment of blood immediately before
cultivation) to follow FA-induced DNA damage (DPX), its repair and its genetic consequences in form of SCE and MN.
Our results clearly indicate that DPX (determined by the comet assay) are induced at FA-concentrations higher than
25µM and are efficiently removed before lymphocytes start to replicate. SCE are induced at concentrations higher than
100µM whereas cytotoxicity (reduction of the replication index) was already observed at 100µM. MN were not induced
by FA-concentrations up to 350µM under these experimental conditions because FA-induced cytotoxicity (reduction of
the nuclear division index) obviously prevented division of damaged cells. MN were only significantly induced in human
blood when proliferating cells were exposed to FA. It can be concluded from our study that mutagenic effects of FA are
very unlikely to occur in peripheral blood of FA-exposed subjects. The positive findings reported in biomonitoring studies are probably not directly related to FA-exposure. The relevance of systemic genotoxic effects for FA carcinogenicity
will be discussed.
O-072
THE POTENTIAL OF MASS SPECTROMETRY FOR THE DETECTION OF DNA DAMAGE
Peter B. Farmer
Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester, Leicester, UK
The power of mass spectrometry (MS) to achieve qualitative and quantitative analyses of genotoxin-induced DNA damage has increased greatly in recent years with the development of improved liquid chromatographic interfaces and
ionisation sources. The advantages of MS are that it will unequivocally characterise the chemical nature of DNA adducts,
and that it provides increased chemical specificity, accuracy and reproducibility in their quantitative analysis. Our recent
work has demonstrated the potential of electrospray ionisation (ESI) liquid chromatography-tandem MS (LC-MS/MS)
for the determination of a wide range of DNA adducts. Many deoxynucleoside adducts undergo a common dissociation
[MH-116]+ involving the loss of deoxyribose from the protonated adducted deoxynucleoside molecule ion, resulting in
the adducted base being the major product ion. Monitoring this fragmentation allows selective analysis of these adducts.
We have applied this methodology to the determination of oxidative DNA damage (8-oxo-2’-deoxyguanosine) in humans
who had been exposed to environmental air pollution, and to an investigation of the mutagenic effects of exposure
to environmental carcinogenic polycyclic aromatic hydrocarbons, including the measurement of benzo[a]pyrene and
dibenzo[a,l]pyrene DNA adducts (collaboration with S Kyrtopoulos, Athens). When used to detect benzo[a]pyrene diol
epoxide-2’-deoxyguanosine adducts formed in vivo, the method showed comparable sensitivity to an existing 32P-postlabelling method.
MS may also be used for sensitive analysis of adducted bases liberated by hydrolysis of carcinogen-modified DNA. We
have observed using ESI LC-MS/MS that a generic method for monitoring N-7-alkylguanines is monitoring the transition
of the [MH]+ precursor ion to protonated guanine (m/z 152). This approach has wide applicability for the monitoring of
adduct formation following exposure to exogenous and endogenous low molecular weight alkylating agents.
Acknowledgements to the UK Medical Research Council and the European Commission.
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O-073
BIOMONITORING OF INHALATORY EXPOSURES; FROM INVASIVE TO NON-INVASIVE METHODS
Frederik-Jan Van Schooten
Dept. Health Risk Analysis and Toxicology, Universiteit Maastricht, Maastricht, Netherlands
The role of inhalatory mutagenic exposures in human carcinogenesis is indisputable. For instance epidemiological studies have shown that smoking is related to tumorigenesis in many tissues such as lung or bladder. Human biomonitoring to assess the mutagenic body load may help identifying those that are at particular genotoxic risk by measuring
carcinogens, their metabolites and their interaction products with macromolecules. To be successfully applicable in
molecular epidemiological studies biomonitoring should be performed in accessible surrogate tissues representing levels
in target-tissues, such as the lung. As an example monitoring of smoking related carcinogen-DNA adduct levels has been
performed in human lung tissues, bronchoalveolar lavage cells (BAL cells), cells derived from induced sputum, buccal
mucosa and white blood cells. In general, DNA adduct levels 1. were enhanced in lung tissue and BAL cells compared to
white blood cells, 2. varied according to changes in exposure, 3. varied over extended periods time (months), 4. varied
depending on individual characteristics as body mass index (BMI) and/or genetic variants in PAH-metabolism. Generally,
we are at the stage of effectively using DNA adduct measurements as biomarkers in molecular epidemiological studies.
At the same time, since studies are becoming larger and larger to reach sufficient statistical power, there is a need to
develop less invasive sampling strategies and more high through put methodologies.
O-074
DNA-PHOSPHATE ADDUCTS – A POTENTIAL BIOMARKER?
Johanna Haglund
Department of Environmental Chemistry, Stockholm University, Stockholm, Sweden
Although there is evidence that phosphate oxygens in DNA are modified by several electrophiles leading to phosphotriester (PTE) formation, the PTEs have not been paid much attention. Studies of their biological significance are few.
Little is also known about the repair of PTEs. The phosphate adducts are not expected to interfere directly with the base
pairing. However, there are studies showing other types of effects. Since it was observed that PTEs in DNA in vivo are
more stable than most base adducts their use as dose monitor was suggested. Early efforts to quantify PTEs in DNA
comprised cleavage of DNA by alkali-induced strand breaks. Later, the 32P-postlabelling method was further developed
for determination of PTEs. PTEs are chemically relatively stable under physiological conditions except for adducts containing oxygen, sulphur or nitrogen in the beta position. It was early shown that PTEs are weak electrophiles, a property
well studied in chemical systems but hardly in biological contexts. The possibility of utilising the weakly alkylating property of the PTE by transferring the alkyl group to a strong nucleophile was therefore explored. Standard nucleophiles,
e.g. aniline and thiosulfate, were shown to perform nucleophilic displacement on the alkyl group in the PTE with the
formation of an alkyl-nucleophile complex suitable for analysis. However, the reaction-kinetic studies demonstrated too
low rates of the transalkylation to be practically useful. Therefore, the supernucleophilic cob(I)alamin was tested and
was shown to perform the same transalkylation 4,000 times faster. The alkyl-cobalamins formed are analysed by miniaturised LC-MS/MS using column switching techniques. In addition, the PTEs formed in calf thymus DNA after treatment
with ethylnitrosourea have been structurally characterised by LC-MS/MS. The transalkylation method is being developed
and the question if DNA-phosphate adducts are useful as an alternative and complementary biomarker remains?
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0-075
GENE-EXPOSURE INTERACTION ON ANTI-BENZO[A]PYRENEDIOLEPOXIDE-(B[A]PDE)-DNA
ADDUCT FORMATION IN LYMPHOMONOCYTES OF HUMANS
Sofia Pavanello
Alessandra Pulliero, Bruno Onofrio Saia, Erminio Clonfero
Occupational Health Section, Department of Environmental Medicine and Public Health, University of Padova, Padova, Italy
We evaluated the influence of exposure and genetic determinants on anti-benzo[a]pyrenediolepoxide-(B[a]PDE)-DNA adduct formation
(an adduct induced by the ultimate carcinogen of B[a]P) in lymphomonocytes of humans occupationally and environmentally exposed to
polycyclic aromatic hydrocarbons (PAHs). We first demonstrated that an HPLC/fluorescence method could be applied to anti-B[a]PDEDNA measurements to assess high and chronic occupational exposure to PAHs (B[a]P) (n= 200 workers). Adduct levels were significantly
higher in coke oven workers and chimney sweeps than controls, but not in aluminium plant workers and psoriatic patients. No influence of
smoking or diet was detected due to overlap with the high occupational exposure. In coke oven workers, the greater risk of anti-B[a]PDEDNA adduct formation was also related to the lack of glutathione s-transferase activity M1 (GSTM1 *0/*0 genotype). Additionally in
more highly exposed coke oven workers (with urinary 1-pyrenol exceeding the proposed BEI (2.28 micromol/mol creatinine)) the increase in
adduct levels was significantly related to some low-activity NER (nucleotide excision repair) genotypes together with GSTM1*0/*0. These
genetic factors appear to be as important as occupational exposure to PAHs in modulating the levels of the biomarker. Recently antiB[a]PDE-DNA adduct formation in lymphomonocytes of general population exposed to low doses of PAHs (B[a]P) was also evaluated
(n=585). Our results indicate that anti-B[a]PDE-DNA adduct can be detected in the general population and its formation, associated not
only with tobacco smoke exposure, but also with indoor and diet exposures, is modulated by GSTM1 genotype. The information provide
here are important since DNA adduct formation in surrogate tissues is considered an index of genotoxic exposure also in target organs
(e.g., lung) and their increase, linked both to exposure and genetic characteristic, may also be predictive of higher risk of PAH-related
cancer.
0-076
CYTOGENETIC BIOMARKERS TO ASSESS THE GENETIC DAMAGE INDUCED IN CHRONIC
ALCOHOLICS WITH MALIGNANT, PREMALIGNANT, AND NON-MALIGNANT DISEASES
Sarolta Gundy 1
Gabor Szekely 1, Eva Remenar 1, Attila Pulay 2
National Institute of Oncology, Budapest, Hungary 1, National Institute of Psychiatry and Neurology, Budapest, Hungary 2
We wished to clarify whether frequency of chromosomal aberrations (CAs) and bleomycin (BLM) sensitivity might be
influenced by disturbed liver metabolism following chronic alcohol intoxication in malignant, premalignant and nonmalignant alcohol-related diseases. Untreated head and neck cancer patients (HNCP), alcoholics with fatty liver and
cirrhosis (ALCL) and alcoholics applying for admission to a withdrawal treatment (ALC) were examined.
Self-reported chronic exposures to alcohol and tobacco were nearly similar in the 3 groups, however, clinical markers
of the liver function showed the most unfavourable values in ALCs.
The aberrant cell frequencies for 150 HNCPs (2.85%), and 61 ALCLs (2.76%) were higher than those for 100 ALCs
(2.05%) and 150 healthy controls (2.00%), respectively. The HNCPs´ and ALCLs’ mutagen sensitivity expressed in BLMinduced chromatid breaks/cell (b/c) was significantly higher than the sensitivity of controls (1.16 and 1.30 b/c vs. 1.02
b/c), but the b/c values were the highest (1.73 b/c) in ALCs without manifestation of any known disease.
Elevated CAs characterised mainly genetic instability leading to malignant (HNC) or premalignant (ALCL) diseases.
(Fatty liver and cirrhosis of ALCLs often leads to hepatocellular cancer). BLM sensitivity was, however, expressed in a
smaller extent in these patients than in ALCs, indicating that greater elevation of b/c of ALCs occurs probably due to
faster ethanol metabolism in the liver than in patients suffering from a malignant or premalignant disease.
The same potential mutagens and carcinogens and their metabolic products activate different repair- and detoxification
processes in alcoholics with different diseases. CAs are probably more stable biomarkers of the overall cancer susceptibility, while the results of BLM-assay suggest rather an association with the effects of endogeneous genotoxicants of
hepatic ethanol metabolism.
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O-077
EPIGENETIC MECHANISMS IN REGULATION OF CELLULAR PROCESSES AND TUMORIGENESIS
Zdenko Herceg
International Agency for Research on Cancer (IARC), 150 Cours Albert Thomas, F-69008, Lyon, France
In recent years, epigenetic changes have emerged as key mechanisms in the development of human cancer (1). The
term 'epigenetic' defines all heritable changes in gene expression and chromatin organization that are not coded in the
DNA sequence itself. Epigenetic inheritance including DNA methylation and histone modifications are essential mechanisms that allow the stable propagation of gene activity states from one generation of cells to the next. Importantly,
deregulated epigenetic mechanisms are strongly implicated in cancer development and it has become apparent that
cancer is as much a disease of abnormal epigenetic as it is a disease of genetic mutations. However, although both scientific and medical communities now recognize the importance of epigenetic changes in cancer, the precise contribution
of epigenetic mechanisms and cellular targets epigenetic alterations to human cancers are largely unknown. We have
studied the role of epigenetic mechanisms including histone modifications in cellular processes and cancer development
(2-4). These studies have shed new light on the epigenetic mechanisms and revealed new concepts in this emerging
field. We have further investigated the significance of epigenetic mechanisms in normal cellular processes and abnormal
events that lead to oncogenic transformation and tumour development. These new results and concepts will be presented as well as their implication for risk assessment, molecular diagnostics and cancer prevention.
1. Jones, P.A. and Baylin, S.B. (2002) Nat Rev Genet, 3, 415-428.
2. Herceg Z., Hulla W., Gell D., Cuenin C., Lleonart M., Jackson S. & Wang Z.Q. (2001) Nature Genetics 29: 206-211.
3. Li H., Cuenin C., Murr M., Wang Z.-Q. & Herceg Z. (2004) EMBO J. 23: 4824-4835
4. Murr R., Loizou L., Yang Y.-G., Cuenin C., Li H., Wang Z.Q. and Herceg Z. (2006). Nature Cell Biology, 8: 91-99.
O-078
TARGETING OF GENES FOR SILENCING BY PROMOTER HYPERMETHYLATION: INFLUENCE OF
ENVIRONMENTAL EXPOSURE
Steven A. Belinsky
Lovelace Respiratory Research Institute, Albuquerque, NM 87112, USA
Gene promoter hypermethylation now rivals mutation as a critical factor in cancer initiation and progression. One focus
of our studies has been the influence of environmental exposures that result in different types of DNA damage on gene
promoter hypermethylation. Our initial approach to this issue was to ask whether carcinogen exposures that damage
DNA through different mechanisms such as single- versus double-strand breaks would induce lung tumors that arise
via inactivation of different genes or inactivate genes at a higher prevalence. We first examined the methylation of the
estrogen receptor
(ER) CpG island in lung tumors from humans, rats, and mice. ER methylation was seen at a similar
low prevalence (~20%) in lung tumors induced in humans by cigarette smoke or in rats and mice by NNK. Tumors in
unexposed mice or never smokers showed a much higher prevalence for methylation of the ER gene. The most striking differences were seen in rat lung tumors induced by exposure to X-ray or plutonium (Pu) where the ER gene was
methylated in 38% and 82% of tumors, respectively. A subsequent study examined the prevalence for methylation of
a panel of genes in adenocarcinomas from Pu-exposed workers at MAYAK, the first Russian nuclear enterprise established to manufacture weapons plutonium (Pu), compared to nonworker controls. The prevalence for methylation of
p16 and GATA5 was increased significantly in tumors from workers compared to nonworker controls. Stratification of
Pu exposure into tertiles also revealed a striking dose response for methylation of the p16 gene. The influence of Pu on
inducing methylation of the p16 gene was also seen in an animal model in which the prevalence for methylation of this
gene was increased in rat lung tumors induced by Pu or Pu plus cigarette smoke compare to cigarette smoke alone.
Together, these studies show that gene-specific promoter methylation could be modulated differentially depending on
carcinogen exposure.
These findings have now been extended to address several key questions. What role does DNA damage play in initiating
gene promoter methylation? What is the sequence of events that occur to cause gene silencing and finally, do defined
carcinogen exposures target different genes for silencing? Several in vitro model systems are being used to address
these questions. A transient transfection model has been developed using a firefly luciferase expression vector that
contains the first 162 bp immediately upstream of the translational start site of the rat p16 gene to study transcriptional regulation of this gene. Studies investigating the effect of exposure to bleomycin, overexpression of cytosine-DNA
methyltransferses (DNMTs) and a single methylation site on transcriptional activity will be described. (Supported by
ES008801).
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O-079
MUTATION, CELL CYCLE AND APOPTOSIS: PRESERVING GENOMIC INTEGRITY IN SOMATIC
AND EMBRYONIC STEM CELLS
Peter J. Stambrook
The development of somatic cell nuclear transfer is a procedure with promise for the production of embryonic (ES) stem
cells that obviates the use of early embryos. Among the problems that exist are extensive epigenetic modifications of
DNA and chromatin proteins that can affect gene expression. These are potentially reversible. In addition, there are
irreversible changes such as loss of heterozygosity (LOH) due to mitotic recombination. A key distinction between germ
cell and somatic cell function is the greater requirement for maintenance of genomic integrity in the former. This implies
that germ cells, and probably ES cells have more robust (or different) mechanisms to preserve the integrity of their
genomes. To test this proposition, we have used a mouse model in which the endogenous reporter gene for mutation
detection, Aprt, is heterozygous. Mutation frequencies in somatic cells approach 10-4, and the majority of these events
occur as a consequence of LOH due to mitotic recombination. Although somatic cells can sustain such high levels of
DNA damage, this level of DNA damage is untenable for the function of germ cells, and by extrapolation embryonic
stem (ES) cells. Damage to somatic cells can be tolerated by the organism, and such damage may accumulate with age
and manifest at a later time as somatic disease (e.g. cancer). Such a high level of damage to ES cell DNA would not
be tolerable since such damage will affect all of the cells derived from a mutant ES cell and will be passed on to future
generations. It is likely, therefore, the ES cells have mechanisms beyond those of somatic cells to protect the integrity
of their genomes. We have examined two such mechanisms. Firstly, we have shown that mutation frequencies and frequencies of mitotic recombination in ES cells are about 100-fold lower than in adult somatic cells or in isogenic mouse
embryonic fibroblasts (MEFs). Significantly, uniparental disomy (UPD), as a consequence of nondysjunction, emerges
as a major pathway of mutagenesis. It is unlikely, however, that a 100-fold reduction in mutation frequency would be
sufficient to protect the ES cell genome to the extent required. A second, complementary protective mechanism would
be to eliminate those ES cells that have acquired an undue mutational burden, thereby maintaining the population
pristine. We have investigated this possibility and produced data that are consistent with this hypothesis. ES cells lack
a G1 checkpoint and the two known signaling pathways that mediate the checkpoint are compromised. The checkpoint
kinase, Chk2, which participates in both pathways is sequestered at centrosomes in ES cells and does not phosphorylate
its substrates (i.e. P53 and Cdc25A) that must be modified to effect a G1 arrest. Ectopic expression of Chk2 does not
rescue the P53-mediated pathway, but does restore the pathway mediated by Cdc25A. After exposing wild type ES cells
to ionizing radiation, no cells accumulate in G1 but do so in S-phase and in G2. ES cells expressing ectopic Chk2 arrest
in G1 as well as G2, and appear to be protected from apoptosis.
O-080
EPIGENETIC EPIDEMIOLOGY OF GASTRIC CANCER
Yasuhito Yuasa 1
Hiromi Nagasaki 1, Yoshimitsu Akiyama 1, Kazue Imai 2, Kei Nakachi 2
Tokyo Medical and Dental University, Tokyo, Japan 1, Radiation Effects Research Foundation, Hiroshima, Japan 2
Epigenetic gene silencing through DNA methylation is one of the important steps in the mechanism underlying tumorigenesis, including of the stomach. Past lifestyle factors of cancer patients, such as intake of vegetables, are very
important to affect gastric carcinogenesis. However, the relationship between DNA methylation and past dietary habits
in cancer patients remains largely unknown except the high incidence of hypermethylation of genes, such as p16/INK4a,
in cigarette smokers. The CDX2 homeobox transcription factor plays a key role in intestinal development, but CDX2 is
also expressed in most of intestinal metaplasia and a part of carcinomas of the stomach. We analyzed the methylation
status of the CDX2 5' CpG island in gastric cancer cell lines by methylation-specific PCR (MSP), and then CDX2 mRNA
was found to be activated after 5-aza-2'-deoxycytidine treatment of the methylation-positive cells. We further examined
the methylation status of CDX2 in primary gastric carcinomas by MSP and compared it with the past lifestyle of the
patients, including dietary habits. Methylation of CDX2 was found in 20 (34.5%) of the 58 male patients and 1 (6.7%)
of the 15 female patients. Since the methylation frequency was low in the female patients, the analysis was performed
only on the male cases. CDX2 methylation was correlated with the decreased intake of green tea and cruciferous
vegetables, and also with full or overeating habits. These findings are consistent with epidemiological observations on
gastric cancer. Thus, diet could be an important factor determining the methylation status of genes such as CDX2 and
the resultant aberrant expression of genes involved in carcinogenesis. We are analyzing the methylation status of other
genes in gastric carcinomas, and their relationships with lifestyle factors will be discussed.
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O-081
THE DUAL ROLE OF AHR IN GENOTOXIC AND NONGENOTOXIC MECHANISMS OF TOXICITY
OF ENVIRONMENTAL PAHs IN LIVER EPITHELIAL CELLS
Jan Vondracek1
Zdenek Andrysik 1,2, Lenka Svihalkova-Sindlerova 1, Alois Kozubik 1 , Pavel Krcmar 2, Miroslav Machala 2,
Sona Marvanova 2, Dagmar Faust 3, Cornelia Dietrich 3, Jan Topinka 4, Oksana Sevastyanova 4
Institute of Biophysics, Brno, Czech Republic 1, Veterinary Research Institute, Brno, Czech Republic 2, Johannes Gutenberg
University, Mainz, Germany 3, Institute of Experimental Medicine, Prague, Czech Republic 4
Polycyclic aromatic hydrocarbons (PAHs) have been extensively studied as genotoxic, initiating agents. In contrast,
their nongenotoxic effects that might be linked to carcinogenesis are poorly characterized. We investigated role of the
aryl hydrocarbon receptor (AhR) in the effects of weakly mutagenic benz[a]anthracene (BaA), benzo[b]fluoranthene
(BbF) and of the strongly mutagenic benzo[a]pyrene (BaP) on induction of PAH-metabolizing enzymes, formation of
DNA adducts, and disruption of cell proliferation control in contact-inhibited rat liver epithelial WB-F344 ‘stem-like’ cells.
Using expression of a dominant-negative AhR mutant (dnAhR), transient transfection with siRNA targeted against AhR,
and chemical inhibitors of cell proliferation, we studied the mechanisms of cell proliferation control in WB-F344 cells.
We found that up-regulation of cyclin A/cdk2 complex activity plays a significant role in AhR-dependent, BaA- and BbFinduced disruption of contact inhibition. However, neither BaA nor BbF modulated expression of the principal cdk inhibitor involved in maintenance of contact inhibition, p27Kip1, or pRb phosphorylation. In contrast, BaP induced high levels of
DNA adducts, leading to apoptosis, a decrease in total cell numbers and significantly higher percentage of S-phase cells
than either BaA or BbF. Given the high levels of cyclin A/cdk2 activity, downregulation of p27Kip1 and hyperphosphorylation of pRb, the accumulation of cells in S-phase was probably due to a compensatory proliferation. Both apoptotic and
proliferative effects of BaP were dependent on AhR. Thus, activation of AhR is directly involved in disruption of contact
inhibition by weakly mutagenic PAHs and in genotoxic effects of BaP, both leading to enhanced cell proliferation and
increasing a likelihood of fixation of mutations. (Supported by grant No. B6004407 from the Grant Agency of ASCR and
No.00002716201 from the Ministry of Agriculture.)
0-082
EFFECT OF PHENOBARBITAL ON THE LEVEL METHYLATION OF PROMOTER REGION OF P16
GENE IN RAT LIVER
Katarzyna Urbanek
Grażyna Kostka , Katarzyna Urbanek, Bożena Wiadrowska, Robert Bańkowski, Jan Krzysztof Ludwicki
National Institute of Hygiene, Department of Environmental Toxicology, Warsaw, Poland
The changes in DNA methylation are being considered as one of the mechanisms of non-genotoxic carcinogens
activity.
The aim of this study was to determine the effect of phenobarbital (PB), nongenotoxic rodent liver carcinogen on the
methylation level of the promoter region of suppressor gene p16, hepatomegaly, DNA synthesis and DNA-methyltransferase activity (DNA-MTase) in rat liver. Male Wistar rats received PB in 1, 3 and 14 daily oral doses (at 24-h intervals)
equivalent to 1/10 LD50. The methylation level was determined by PCR-based methylation sensitive restriction enzyme
analysis (MSRA). Obtained PCR products were electrophoresed on a 6% polyacrylamide gel. The activity of DNA-MTase
was assayed by Adams et al. method and was expressed as cpm/µg protein. DNA synthesis (S-phase) was measured
by [3H] thymidine incorporation into nuclear fraction of the liver homogenates (dpm/mg DNA).
Short-term treatment of rats with PB resulted in hepatomegaly due to DNA synthesis. There was 2,5-fold and 2,7-fold
(p<0,01) increase in thymidine incorporation following PB administration for 1 and 3 days at a dose 92,8 mg/kg b.w.
per day. The effect of PB on DNA synthesis correlated with changes in methylation pattern of p16 promoter sequence.
The increase in methylation of p16 gene, 25 and 63% were present, as compared with control. The parallel biochemical
measurements showed an increase (37 and 53%) in nuclear DNA-MTase activity. After prolonged administration (14
days) DNA synthesis declined to control level. No changes in methylation of p16 gene and DNA-MTase activity were
found.
Thus, we can conclude that PB produced reversible changes of studied endpoints.
This work was supported by Polish Grant no. 2 P05D 028 26.
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O-083
THE THRESHOLD OF TOXICOLOGICAL CONCERN CONCEPT IN RISK ASSESSMENT
Robert Kroes
Institute for Risk Assessment Sciences / Utrecht University, Utrecht, Netherlands
The concept that “safe levels of exposure” for humans can be identified for individual chemicals is central to the risk
assessment of compounds with known toxicological profiles. The traditional approach to risk assessment is separated
into hazard identification, hazard characterisation, exposure assessment and risk characterisation, and data from toxicity studies on the specific chemical under evaluation are necessary for hazard identification and characterisation.
The Threshold of Toxicological Concern (TTC) is a concept that refers to the establishment of a level of exposure for
all chemicals, whether or not there are chemical-specific toxicity data, below which there would be no appreciable risk
to human health. The concept proposes that a low level of exposure with a negligible risk can be identified for many
chemicals, including those of unknown toxicity based on knowledge of their chemical structures.
The TTC principle was examined for general toxicity end points as well as for specific end points including carcinogenicity, teratogenicity, reproductive toxicity and immunotoxicity. In addition, consideration was given to structural alerts
for high potency carcinogens, endocrine disrupting chemicals, food allergens and to the potential for metabolism and
accumulation. A decision tree approach, which incorporates a tiered approach for applying the TTC principle, has been
proposed as a preliminary step in food safety evaluation (Kroes et al., 2000 and 2004).
The TTC principle, its use to date, its potential future applications and the incorporation of the TTC principle in the Risk
Assessment paradigm are described.
O-084
A RATIONALE FOR CONTROLLING GENOTOXIC IMPURITIES IN PHARMACEUTICALS
- THE STAGED TTC CONCEPT
Lutz Mueller
F. Hoffmann-La Roche Ltd, Basel, Switzerland
The synthesis of pharmaceutical products frequently involves the use of reactive reagents and the formation of intermediates and by-products. Low levels of some of these may be present in the final drug substance and drug product
as impurities. Such chemically reactive impurities may have at the same time the potential for unwanted toxicities
including genotoxicity and carcinogenicity and hence can have an impact on product risk assessment. This ppresentation outlines a procedure for testing, classification, qualification, toxicological risk assessment, and control of impurities
possessing genotoxic potential in pharmaceutical products. Referencing accepted principles of cancer risk assessment,
this document proposes a staged threshold of toxicological concern (TTC) approach for the intake of genotoxic impurities
over various periods of exposure. This staged TTC is based on knowledge about tumorigenic potency of a wide range
of genotoxic carcinogens and can be used for genotoxic compounds, for which cancer data are limited or not available.
The delineated acceptable daily intake values of between ~1.5 μg/day for ~lifetime intake and ~120 μg/day for ≤ 1
month are virtually safe doses. Based on sound scientific reasoning, these virtually safe intake values do not pose an
unacceptable risk to either human volunteers or patients at any stage of clinical development and marketing of a pharmaceutical product. The intake levels are estimated to give an excess cancer risk of 1 in 100,000 to 1 in a million over
a lifetime, and are extremely conservative given the current lifetime cancer risk in the population of over 1 in 4. While
the proposal applies to all clinical routes of administration and to compounds at all stages of clinical development it is
important to note that certain types of products, such as those for life-threatening indications for which there are no
safer alternatives, allow for special considerations using adaptations of the principles put forward.
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O-085
IWGT AND ILSI HESI INITIATIVES ON THE INTERPRETATION OF AND FOLLOW-UP TO
POSITIVE IN VITRO GENOTOXICITY RESULTS
Véronique Thybaud
Sanofi Aventis / Drug Safety Evaluation, VITRY SUR SEINE, France
The in vitro genotoxicity tests are key components of the regulatory test battery conducted for safety assessment.
Data accumulated during the past years demonstrate a high rate of positive in vitro results for non-carcinogenic compounds. Two initiatives, International Workshop on Genotoxicity Testing (IWGT), and Health and Environmental Sciences
Institute of International Life Sciences Institute (ILSI HESI) are currently on going to improve the interpretation of
in vitro positive results and to develop appropriate follow-up testing strategies. In both cases, tripartite committees
involving recognized experts from industry, academic laboratories and regulatory authorities are working together to
address the issue. At the IWGT meeting in September 2005, the Working Group defined circumstances where the in
vitro positive results lead to very low or no concern, and no follow-up testing would be needed. Parameters to consider
are for example the reproducibility, the level of cytotoxicity, the relationship of results to the control range of values,
and the total weight of evidence across assays. In case of clear positive results, when follow-up testing is needed, the
WG recommended selecting the additional tests based on the knowledge about the mode of action and the nature of the
responses (e.g. end-points). Genotoxic events might occur via mechanisms other than interaction with DNA, showing
non-linear dose-response and resulting in no concern at human exposure levels. More recently in June 2006, the newly
created ILSI HESI subcommittee met to define how a collaborative effort (e.g. database, study) could help to advance
the scientific basis for the interpretation of positive results and facilitate the development of follow-up testing strategies
and criteria for determining the relevance of findings to human health. The aim of the two complementary initiatives is
to facilitate the integration of genotoxicity data into a weight-of-evidence based risk assessment process.
O-086
THE BONE MARROW MICRONUCLEUS TEST: POSITIVE COMPOUNDS INACTIVE IN VITRO
AND ‘POSITIVE’ COMPOUNDS THAT DO NOT INDICATE GENOTOXIC HAZARDS: REPORT
OF THE IWGT WORKING GROUP”
David Tweats
Centre for Molecular Toxicology and Genetics, University of Wales Swansea, Singleton Park, UK
In vivo genotoxicity tests play a pivotal role in genotoxicity testing batteries. They are used both to determine if potential genotoxicity observed in vitro is realised in vivo and to detect any genotoxic carcinogens that are poorly detected
in vitro. It is recognised that individual in vivo genotoxicity tests have limited sensitivity but good specificity. Thus,
a positive result from the established in vivo assays is taken as strong evidence for genotoxic carcinogenicity of the
compound tested. However, there is a growing body of evidence that compound-related disturbances in the physiology of the rodents used in these assays can result in false positive results for genotoxicity. For rodent bone marrow or
peripheral blood micronucleus tests, these disturbances include changes in core body temperature (hypothermia and
hyperthermia) and increases in erythropoiesis following prior toxicity to erythroblasts or by direct stimulation of cell
division in these cells. There is existing data in the literature on these areas and also new data was collected on behalf
of the IWGT via a questionnaire. This has shown that there are numerous examples where increases in micronuclei
in bone marrow cells do not indicate direct genotoxicity by the test compound. However, simple changes in the test
protocol can alert the researcher and the regulator to those compounds where this is an issue and thus improve interpretation of these studies.
A second IWGT survey has identified a number of compounds that appear to be more readily detected in vivo than in
vitro. The reasons for this property varies from compound to compound and includes metabolic differences; the influence of gut flora; higher exposures in vivo compared to in vitro; and effects on pharmacology, in particular folate depletion. A number of receptor kinases fall into the latter category. It is possible that at least some of these compounds are
detectable in vitro if a specific in vitro test is chosen as part of the test battery, but the ‘correct’ choice of test may not
always be obvious when testing a compound of unknown genotoxicity. It is noted that many of the compounds identified
in this study interfere with cell cycle kinetics and this can result in either aneugenicity or chromosome breakage. These
results have regulatory implications on the design of test batteries. There is pressure for all in vitro test batteries, which
may be possible in the future if the specificity of testing in mammalian cells can be improved. If this occurs there will
be a need to identify the classes of genotoxin identified by this study so that further testing can be carried out.
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O-087
HOW TO REDUCE FALSE POSITIVE RESULTS WITH IN VITRO GENOTOXICITY TESTING
AND AVOID UNNECESSARY FOLLOW-UP ANIMAL TESTS: REPORT OF AN ECVAM WORKSHOP
David Kirkland
Covance Laboratories Ltd., Harrogate, UK
Genotoxicity tests in mammalian cells in vitro produce a remarkably high occurrence of false positive results when
compared with rodent carcinogenicity. This leads to additional in vivo genotoxicity testing. If there were more accurate,
predictive in vitro tests for genotoxicity there would be a significant reduction in the number of animals used. More than
20 invited genotoxicity experts from academia, government and industry gathered at ECVAM (April, 2006) to address
these problems. The following were agreed: Deficiencies in metabolism, p53 function and DNA repair capability in rodent
cell lines (e.g. CHO, CHL, V79, L5178Y) contribute to their high false positive rate. Human lymphocytes may have a
lower rate. Better guidance on understanding the likely mechanisms and interpretation of positive results irrelevant for
humans is urgently needed both for practitioners and regulatory reviewers.
High concentrations and high levels of cytotoxicity currently required in mammalian cell tests may not be justified. A thorough review of
published and industry data is needed to determine whether such levels are justified for the detection of in vivo genotoxins and DNAreactive, mutagenic carcinogens.
High levels of cytotoxicity may lead to false positives, and various measures are currently allowable under OECD guidelines. A detailed comparison of multiple measures of cytotoxicity is needed to see if different measures would select
different concentrations for test.
Human cell that are p53 and DNA-repair proficient, and have defined Phase 1 and Phase 2 metabolism, offer the best
hope for reduced false positives in the future. Human lymphocytes may have a future role but HepG2 and TK6 cell
systemss also show promise. Other human cell lines such as HepaRG should be evaluated for genotoxicity. A collaborative research programme is needed to identify and evaluate new cell systems with appropriate sensitivity but improved
specificity.
O-088
A HIGH SPECIFICITY GENOTOXICITY ASSAY MEASURING THE RESPONSE OF THE HUMAN
GADD45 ALPHA GENE
Richard M Walmsley1
Nick Billinton 2, Paul Hastwell 3
University of Manchester, Manchester, UK 1, Gentronix Ltd, Manchester, UK 2, GSK, Ware, UK 3
High sensitivity in the existing regulatory in vitro mammalian cell genotoxicity assays has been shown to come at the
cost of low specificity: there are too many false positive results (Kirkland et al, 2004). A new assay, exploiting fully p53
dependent expression of the GADD45 alpha gene in human cells, demonstates a very much higher specificity than the
existing tests without loss of sensitivity. A study of well-characterised direct acting genotoxic and non genotoxic compounds with diverse mechanisms of DNA damage (including aneugens) reveals that the assay responds positively to all
classes of genotoxic damage with both high specificity and high sensitivity. A successful proof-of-principle has also been
deomstrated for S9 metabolic activation with the model compound cyclophophamide. Data from over 100 assays with
toxic and non-toxic genotoxins, as well an non-genotoxins, and including marketed pharmaceuticals will be presented
and compared with published data from other genotoxicity and carcinogenicity assays. The utility of the assay in screening and analytical studies will be discussed.
D. Kirkland, M. Aardema, L. Henderson and L. Muller. Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens: I. Sensitivity, specificity and relative predictivity.
Mutation Research/Genetic Toxicology and Environmental Mutagenesis 584 (2005) 1-256.
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O-089
HUMAN AND RODENT ENZYME SYSTEMS FOR ACTIVATING AMINO- AND NITROARENES
IN GENOTOXICITY TESTS
Hansruedi Glatt
Eva Muckel, Ulrike Pabel, Walter Meinl
German Institute of Human Nutrition (DIfE), Potsdam-Rehbruecke, Germany
The chemical classes of amino- and nitroarenes encompass industrial intermediates, consumer products (such as hair
dyes), phytochemicals, pyrolysis products, pesticides and drugs. Some amino- and nitroarenes are established human
carcinogens. Further congeners demonstrated carcinogenicity in animals, whereas others were inactive. Many nitro- and
aminoarenes are potent mutagens in bacterial test systems, but inactive or weakly active in standard mutagenicity tests
in mammalian cells in culture. None of these results are closely associated with carcinogenic activity. Differences in
biotransformation may lead to false positive as well as false negative results. Thus, bacterial nitroreductases (converting nitroarenes to hydroxylamines) and acetyltransferase (converting hydroxylamines to reactive esters) differ in their
substrate specificity from corresponding mammalian enzymes. Mammalian acetyltransferases (NATs) are absent or low
in standard mammalian target cells. Sulfotransferases (converting hydroxylamines to even more reactive esters than
NATs) are absent in bacteria, and low or absent in standard mammalian target cells. We have expressed 50 individual
SULT and NAT forms (from different mammalian species and human genotypes) in bacterial and/or mammalian target
cells and explored their activation capacity. This genetic engineering strongly improved the detection of carcinogenic
congeners as mutagens; in particular, strong mutagenic effects now also occurred in mammalian cells. Among the
human phase-II enzymes, SULT1A1, SULT1A2 and NAT2 showed clearly the highest activation potential (often with high
substrate specificity). In the rat, Sult1c1, Nat1 and Nat2 were most active. In the mouse, Sult1d1 was the most active
SULT form (NATs have not been studied). Many SULTs and NATs are expressed with high tissue specificity and are subjected to genetic polymorphisms in humans – aspects that might be important for predicting/explaining organotropisms
and individual susceptibility.
O-090
BIOTRANSFORMATION OF CARCINOGENS: ENZYMES, DNA ADDUCTS, AND RELEVANCE
F. Peter Guengerich
Vanderbilt University School of Medicine, Nashville, TN, USA
Research over the past 60 years has shown the importance of the metabolism of chemical carcinogens in the formation of DNA adducts, mutations, and cancer in animal models. The cytochrome P450 (P450) enzymes are prominent in
bioactivation and detoxication, and the roles of human P450 enzymes in metabolism of many major carcinogens have
been established. More recent studies in this laboratory have shown the involvement of human P450 1A2 in the activation of aminophenyl norharman, a procarcinogen formed by the P450-mediated fusion of norharman with aniline. Other
work has involved the abilities of newly characterized human P450s in carcinogen metabolism. P450 2W1 is expressed
mainly only in tumors and was found to activate a wide variety of procarcinogens. Interestingly, P450 2S1 is induced
via the AhR system but has not been found to be involved in carcinogen metabolism. Bis-electrophiles, e.g. ethylene
dibromide, are able to crosslink glutathione and the DNA repair protein O6-alkylguanine DNA alkyltransferase to DNA
and produce mutations. This effect is rather general for all bis-electrophiles, although the mechanism of mutation is
still not established. Proteomic searches have shown that other nuclear proteins with highly nucleophilic groups can
also be crosslinked to DNA by bis-electrophiles; studies are in progress to determine if these crosslinks are cytotoxic
or mutagenic. Both vinyl monomers and lipid peroxidation lead to the formation of etheno (e) DNA adducts. Recent
work with the bacterial DNA polymerase Dpo4 has revealed structural details of how 1,N2-e-G adducts are processed.
Several differences in the process are seen with human translesion DNA polymerases. Collectively, we know much about
the details of DNA adduct chemistry. Analytical systems now provide detection and analysis of DNA adducts at very low
levels. The issue of how many adducts are dangerous to humans is still an open issue.
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O-091
CARCINOGEN-ACTIVATED TRANSCRIPTION FACTORS CONTROLLING BOTH, CARCINOGEN
METABOLIZING ENZYMES AND CELL CYCLE
Franz Oesch
Carsten Weiss, Cornelia Dietrich and Barbara Oesch
Institute of Toxicology, University of Mainz, Obere Zahlbacherstr. 67, D-55131 Mainz, Germany
In most cases the difference in risk posed by a given carcinogen to diverse species, including human as the species of
special interest, critically depends on the provision of the given species (and organ and cell type) with individual carcinogen biotransformation enzymes controlling the metabolic activation of the (pre)carcinogen and the detoxication of
the ultimately carcinogenic metabolite(s). This may in definable cases introduce practical metabolic thresholds of carcinogenic action even for DNA-damaging genotoxic carcinogens. A matter of special concern in this regard is the fact that
various carcinogens can induce biotransformation enzymes leading to quantitative and in some cases even qualitative
differences of the pattern of carcinogen biotransformation enzymes before and after induction. The individual transcription factors responsible for such inductions typically interact with the enhancer region not only of one but of several
carcinogen biotransformation enzymes, a given transcription factor leading to changes in an entire group of carcinogen
biotransformation enzymes (a “gene battery”). Results will be presented which show that such transcription factors in
addition interact with the enhancer region of genes coding for proteins not involved in biotransformation. When the
expression of genes is affected which code for proliferation controlling proteins the resulting inductions can not only lead
to a greater accumulation of carcinogen activating enzymes thereby increasing tumor initiation, but at the same time
also to tumor promotion. This combination can render the respective compounds in the respective species to especially
powerful carcinogens. In addition, both of these activities may have their own thresholds. The more we learn about
these basic mechanisms the more rational predictions and improved risk assessment will become feasible.
O-092
ACTIVATION AND DETOXICATION OF ANTICANCER DRUG ELLIPTICINE AS A MODEL
GENOTOXICANT: ENZYMES, DNA, ADDUCTS, EFFECTS
Marie Stiborova1
Eva Frei 2
Charles University, Prague, Czech Republic 1, German Cancer Reserach Center, Heidelberg, Germany 2
Introduction: Ellipticine is a drug exhibiting the multimodal mechanism of action. While the prevalent mechanisms
of ellipticine antitumor and mutagenic activities were suggested to be intercalation into DNA and inhibition of DNA
topoisomerase II, based on our data, ellipticine should be considered a drug, whose efficiency or genotoxic effects are
dependent on its activation by CYP and peroxidase.
Results and discussion: We found a new mode of ellipticine action, formation of covalent DNA adducts mediated by its
oxidation with CYP and peroxidase. Such adducts were found in vitro, in V79 cells with CYP1A1, 1A2 and 3A4, adenocarcinoma MCF-7 cells, leukemia HL-60 and CCRF-CEM and neuroblastoma cells and in vivo in rats exposed to ellipticine.
We report the molecular mechanism of ellipticine oxidation by CYPs and identify human and rat CYPs responsible for
ellipticine metabolic activation and detoxication. We also present a role of peroxidases (myeloperoxidase, cyclooxygenase, lactoperoxidase) in ellipticine oxidation leading to ellipticine-DNA adducts. 9-Hydroxy- and 7-hydroxyellipticine
formed by CYPs and the major product of ellipticine oxidation by peroxidases, the dimer, in which the two ellipticine
skeletons are connected via N6 of the pyrrole ring of one ellipticine molecule and C9 in the second one, are the detoxication metabolites. 13-Hydroxy- and 12-hydroxyellipticine, produced by ellipticine oxidation with CYPs, the latter one also
spontaneously from another metabolite, ellipticine N2-oxide, are metabolites responsible for formation of two deoxyguanosine adducts in DNA. The results shown here allow us to propose two carbenium ions, ellipticine-13-ylium and
ellipticine-12-ylium, as reactive species generating two major DNA adducts seen in vivo in rats treated with ellipticine.
The study forms the basis to further predict the susceptibility of human cancers to ellipticine.
GACR (203/06/0329), ME CR (1M4635608802, MSM0021620808)
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O-093
LIPID PEROXIDATION INDUCED DNA DAMAGE AND REPAIR IN CANCER-PRONE
INFLAMMATORY DISEASES
Jagadeesan Nair
Helmut Bartsch
Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany
In response to chronic oxidative stress several enzymes such as cyclooxygenase-2 (COX-2), lipoxygenases (LOX) and
inducible nitric oxide synthase (iNOS) are over expressed in cancer prone inflammatory diseases. Common pathways
of chronic degenerative diseases also involve over production biologically relevant reactive oxygen species (ROS) and
reactive nitrogen species (RNS). Oxidative stress and lipid peroxidation are implicated in the development of human
cancers where chronic inflammation and persistent infections are involved. We have studied DNA damage (etheno-DNA
adducts) induced by 4-hydroxynonenal -6 PUFAs) such as arachidonic acid (AA)ω-6 polyunsaturated fatty acids (ω(HNE)
primarily formed by lipid peroxidation of and linoleic acid. -6 PUFAs also metabolized by enzymes overexpressed during chronic inflammationω such as COX-2, LOX and iNOS to form reactive aldehydes. We have reported increased DNA
damage caused by reactive aldehydes to occur in colonic polyps of familial adenomatous polyposis patients where
COX-2 is overexpressed. In the DMBA-TPA multistage mouse skin carcinogenesis model, a strong positive correlation
was observed between the formation of HNE-derived DNA adducts and the LOX-catalyzed AA metabolites, hydroxyeicosatetraenoic acids (HETE). In the SJL mouse model and in p53 knock-out mice, HNE-derived DNA adducts in affected
tissues were elevated due to an increased iNOS and nitric oxide overproduction. Our studies provided evidence for
increased LPO induced etheno-DNA adducts in several cancer prone diseases such as inflammatory bowel disease disease (IBD), chronic pancreatitis (CP), and alcoholic liver diseases. In IBD and CP ethenodeoxycytidine levels were found
to be several fold higher than that of ethenodeoxyadenosine. Literature reports suggests a role of differential DNA repair
for this difference. Taken together our results clearly demonstrate an increased DNA damage to occur during chronic
inflammation as a result of overexpressed stress response enzymes, oxidative stress and lipid peroxidation; the ensuing
mutations and genetic instability may drive cells towards malignancy.
O-094
OXIDATIVE STRESS AND BREAST CANCER
Regina M Santella1
Sybil Eng 1,Pavel Rossner 1, Mary Beth Terry 1, Meenakshi Agrawal 1, FangFang Zhang 1, Alfred I Neugut 1,
Sharon Sagiv 2, Marilie D Gammon 2, Mia Gaudet 2, Julie A Britton 3, Susan L Teitelbaum 3
Columbia University, New York, USA 1, Univ. North Carolina, Chapel Hill, USA 2 , Mt Sinai School of Medicine, New York, USA 3
Reactive oxygen species (ROS) are generated during normal cellular metabolism, as a result of the influence of various environmental factors, as well as during pathological processes. ROS are responsible for DNA, lipid and protein
damage and play an important role in the development and progression of many human diseases, including cancer. To
investigate the role of ROS in breast cancer risk, we determined levels of several oxidative stress biomarkers (oxidized
plasma proteins and urinary isoprostanes and 8-oxodeoxyguanosine (8-oxodG)) in breast cancer cases and controls
in the Long Island Breast Cancer Study Project. In this population-based case-controls study, women in Nassau and
Suffolk Counties, NY over 20 years of age with newly diagnosed, primary in situ or invasive breast cancer were identified
between August 1, 1996, and July 31, 1997. In addition, single nucleotide polymorphisms in hOGG1, involved in the
base excision repair of oxidative DNA damage, were determined. Elevated levels of urinary isoprostanes and oxidized
plasma proteins were significantly associated with increased breast cancer risk. In contrast, urinary 8-oxodG levels,
suggested to be a measure of DNA repair capacity, were inversely related to risk (P trend = 0.04). Among controls, age
and cigarette smoking were found to impact on biomarker levels. No associations were observed between individual
OGG1 polymorphisms, haplotypes or diplotypes and breast cancer risk. The association between having at least one
variant allele and breast cancer risk was stronger among moderate alcohol drinkers and those with higher body mass
index but only one interaction was statistically significant assuming a multiplicative model. These results suggest that
biomarkers of oxidative stress may be associated with breast cancer risk.
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O-095
OXIDATIVE DAMAGE TO DNA AND RISK OF CANCER
Steffen Loft
Peter Moller
Oxidative damage to DNA is well established in experimental carcinogenesis. Biomarkers of such damage, including
oxidised guanine in leukocyte DNA or excreted repair products, have also been extensively studied in biomonitoring
and dietary interventions. Recently, assays of the related repair activity, especially of OGG1, have been added. Many
carcinogenic exposures, including air pollution, increase biomarkers of oxidative damage to DNA. A survey of 132 intervention and cross-sectional studies concluded that ingestion of antioxidants may be associated with reduced level of
DNA damage biomarkers in white blood cells and urine of humans. Recent data suggest that decreased levels of damage may be due to enhanced repair.
A meta-analysis indicates that the common variant Ser326Cys in OGG1 can be a risk factor for lung cancer, whereas
a rare variant in OGG1 and germ line mutations in the corresponding mismatch repair gene MYH are risk factors for
hereditary colon cancer. The level of oxidative DNA damage and repair activity can be quite different between tumor
and normal tissues and case-control studies have shown increased level of oxidised DNA bases and decreased repair
capacity in leukocytes from cases. Similarly, urinary biomarkers of DNA damage may be elevated in patients with cancer.
However, such studies are likely to be associated with reverse causality.
Cohort studies are required to provide evidence that a high level of DNA oxidation implies a high risk of cancer. However,
this represents a real challenge considering the large number of subjects and long follow-up time required with likely
spurious oxidation of DNA during collection, assay and/or storage of samples. At present, easily storable biomarkers
such as urinary excretion of 8-oxodG and 8-oxogua or measures of the related repair capacity can be applied to existing
biobanks. In that context associations between urinary excretion of 8-oxodG and later cancer risk have been found.
O-096
OXIDANTS AND ANTIOXIDANTS IN THE PREVENTION OF MUTATION – RELATED DISEASES
Silvio De Flora1
Francesco D'Agostini 1, Carlo Bennicelli 1, Maria Bagnasco 1, Anna Camoirano 1, Alberto Izzotti 1, Roumen
Balansky 1,2
Department of Health Sciences, University of Genoa, Italy, Genoa, Italy 1, National Centre of Oncology, Sofia, Bulgaria 2
Oxidative mechanisms play an important role in the pathogenesis of chronic degenerative conditions, such as cancer,
atherosclerosis, heart diseases, chronic obstructive pulmonary diseases, glaucoma, etc. Reduced glutathione (GSH)
provides a major defense system against oxidants and electrophiles. GSH analogues and precursors, such as N-acetylcysteine (NAC), are among the most promising chemopreventive agents. The potential ability of NAC to prevent mutation and cancer is supported by a number of studies in vitro, in animal models and in phase II clinical trials. Among in
vitro studies, we recently demonstrated that human mammary stem-derived cells have an increased susceptibility to
DNA damage and oxidative stress induced by 7,12-dimethylbenz(a)anthracene. These effects were inhibited by NAC.
Several studies showed that NAC exerts protective effects in rodents towards (a) DNA alterations that spontaneously
occur in lung during the perinatal period, (b) smoke-induced damage of mitochondrial DNA that is typically involved
in aging, and (c) functional genomic changes induced in both adults and transplacentally exposed foetuses. These
alterations included the formation of 8-hydroxy-2’-deoxyguanosine and of bulky DNA adducts, overexpression of genes,
detected by cDNA microarrays, and formation of proteins, detected by antibody microarrays, which protect against
oxidative stress. NAC was also found to inhibit apoptotic mechanisms due to redox unbalances and to attenuate the
smoke-related loss of Fhit protein. In vitro and in vivo studies provided evidence for the antiangiogenetic properties of
this antioxidant, which suppressed VEGF expression in Kaposi’s sarcoma cells and inhibited the growth of this human
tumor in nude mice, often resulting in total regression. Unfortunately, under some circumstances antioxidants can
behave as prooxidants. As an example of combined chemoprevention, the adverse effects of ascorbic acid and selenium
were counteracted in the presence of NAC, which maintains a reducing environment.
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O-097
REPAIR OF DNA LESIONS INDUCED BY OXIDATIVE AND NITROSATIVE STRESS
Hiroshi Ide
Hiroshima University, Graduate School of Science, Department of Mathematical and Life Sciences, Higashi-Hiroshima, Japan
Reactive oxygen species generate structurally diverse DNA base lesions. SMUG1, originally identified as a single-strand
selective monofunctional uracil-DNA glycosylase, is a relatively new member of human DNA glycosylases involved in
the repair of oxidative base damage. Unlike other human DNA glycosylases such as NTH1 and NEIL1, SMUG1 exhibits
a stringent damage specificity and excises uracil derivatives with an oxidized group attached to the C5 position (5hydroxymethyluracil, 5-formyluracil, 5-hydroxyuracil). The damage-excising activities for these lesions in HeLa cell-free
extracts are neutralized almost completely by an antiserum to SMUG1, indicating that SMUG1 plays a pivotal role in the
repair of a certain class of oxidative base lesions. A series of active site mutants of SMUG1 are constructed, and the
catalytic and elaborate damage recognition mechanisms are elucidated. Reactive nitrogen species such as nitric oxide
and nitrous acid induce nitrosative damage to DNA bases. Nitrosative deamination of guanine gives rise to xanthine and
oxanine (Oxa). Oxa reacts further with polyamines (spermine and spermidine) and DNA binding proteins, yielding bulky
cross-link adducts. The role of base excision repair (BER) and nucleotide excision repair (NER) systems in the repair of
Oxa and an Oxa-spermine cross-link adduct (Oxa-Sp) has been investigated. The results with purified DNA glycosylases
and cell-free extracts suggest that Oxa is processed poorly by BER and NER in E. coli and human cells. Conversely, OxaSp is excised efficiently by prokaryotic UvrABC. Cell-free extracts from XPA or XPF cells exhibits no incision activity for
DNA containing Oxa-Sp, but activity is restored when extracts from the two cells are combined. These results indicate
Oxa-Sp adducts are repaired by the NER pathway.
O-098
SPERM DNA FRAGMENTATION AS A MARKER IN REPRODUCTIVE TOXICOLOGY
Donald P. Evenson
HCLD. South Dakota State University, Brookings, USA
Background: Volumes of literature have concluded that sperm with damaged DNA produce negative reproductive outcomes. Since the classical semen parameters of sperm count, morphology and motility are relatively poor predictors of
pregnancy outcomes and sometimes not correlated with genotoxic exposure, sperm DNA damage may be an excellent
independent marker for exposure to toxicants.
Aims: To evaluate sperm DNA damage by the Sperm Chromatin Structure Assay (SCSA), terminal Deoxynucleotidyl
transferase-mediated dUTP nick end labeling technique (Tunel), Single-cell gel electrophoresis assay (Comet), Halo
Sperm Assay and Chromomycin A3 (CMA).
Methods: Sperm DNA fragmentation/chromatin techniques with emphasis on Sperm Chromatin Structure Assay (SCSA)
as well as SCSA flow cytometry sorted sperm populations.
Results: The extent of sperm DNA damage was often highly dose responsive with the exposure level as seen for air
pollution, radiation, genotoxic chemicals, certain medications, heat and fever. CMA staining was highly correlated with
the SCSA defined High DNA Stainable (HDS) values. FCM sorted moderate-DNA fragmentation index (DFI) sperm were
normal for morphology and morphometry but one-half were Comet positive. High DFI sperm had abnormal morphology
and morphometry and all were Comet positive.
Conclusions: The low pH treatment of sperm followed by acridine orange staining is an extremely precise and repeatable
protocol with CV’s in the range of 1 – 2%. Furthermore, the SCSA is highly sensitive and dose responsive. Although not
fully understood, the test likely measures the sites of ss and ds DNA breaks. The high correlation between HDS and CMA
strongly suggests that these sperm have retained histones. The SCSA test is currently cited as being the only sperm
DNA fragmentation test that is sufficiently rigorous for critical diagnosis and prognosis in the human andrology clinic.
Likewise, the SCSA has been cited as the most accurate and sensitive test for sperm toxicology studies.
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O-099
TRANSMISSIBLE GERM CELL MUTAGENESIS AND CARCINOGENESIS IN MICE AND HUMANS
Taisei Nomura
Germ cell exposure to radiation and chemicals induces mutations in higher plants and animals, and other adverse effects
(e.g., tumor, malformation, etc) in mice, though these findings have not been supported in the children of atomic bomb
survivors. It will be important to follow the human subjects at molecular level and clinical level (especially adult type
cancer) throughout their lives to determine whether the mouse studies can predict human response. Germ-line exposure induced persistent hypersensitivity to tumor induction in the progeny, producing clusters of tumors by the postnatal
treatment with tumor promoters, suggesting that transmissible alterations must be imprinted in germ cells for the future
development of cancer by the postnatal environment.
Cytogenetic analyses provided evidence for the presence of chromosomal anomalies in the F1 progeny with malformations, but no evidence for visible chromosomal changes in tumor-bearing progeny. Mutations of oncogenes and tumor
suppressor genes such as p53 were also detected in specific tumors observed in cancer prone descendants, but rarely
in commonly observed ones in the strain used. Cumulative changes in many normal gene loci concerning immunological,
biochemical and physiological function may slightly elevate or enhance tumor incidences. In fact, Gene Chip analyses
showed suppression and/or over-expression of many functional genes in the progeny of mice exposed to radiation.
Alternatively, genetic instability can modify tumor occurrence in transgenerational manner. Radiation induces mini-satellite and micro-satellite mutations in mice and also some in humans. Links of such molecular changes to transmissible
genetic risks will be discussed. (Supported by JSPS, MEXT. Heiwa Nakajima Fund and Space Forum of Japan)
O-100
SEMEN QUALITY OF YOUNG FERTILE MEN IN THE CZECH REPUBLIC
Jiri Rubes
Roman Rybar, Ludmila Faldikova, Drahomira Svecova, Atanaska Zajicova, Zdenek Veznik
Veterinary Research Institute, Brno, Czech Republic
Semen quality was evaluated in young men aged 20-30 years (n=100) coming from two localities of the Czech Republic
(CR): Prague (the capital of CR; a high level of air pollution) and Brno (the second largest city of CR; a markedly lower
level of air pollution).
Sperm analysis (volume, concentration, motility, morphology, viability etc.) was performed and sperm chromatin integrity was measured by the SCSA. Chromatin integrity was quantified as DFI (DNA fragmentation index) and immature
cell forms as HDS (high DNA stainability) parameters.
Sperm chromatin quality assessed by the level of DFI and HDS parameters was significantly lower in men coming
from Prague in comparison with the group of men from Brno (P=0.026, P<0.0001, respectively). Lower ejaculate volumes (P=0.04), percentages of motile spermatozoa (P=0.001), percentages of spermatozoa with normal morphology
(P=0.009) and lower speed of movement (P=0.003) were also recorded in men from Prague. Even in case normospermic men were evaluated only, the men from Prague had significantly higher DFI and HDS levels than the group of
normospermic men from Brno (P=0.006, P<0.021, respectively). SCSA parameters of non-smokers did not differ from
SCSA parameters of smokers.
Negative correlations between DFI and motility (r=-0.270; P<0.01), % of live sperm (r=-0.363; P<0.01), and also
negative correlations between HDS and sperm concentration (r= -0.295; P<0.01), motility (r=-0.467; P<0.01), speed
of movement (r=-0.365; P<0.01) and % of live sperm (r=-0.445; P<0.01) were detected. Positive correlations between
both DFI and HDS and percent of sperm morphology abnormalities were recorded (r=0.412; P<0.01, r=0.373; P<0.01,
respectively).
The results of the study suggest that air pollution may adversely affect male reproduction. The air pollution may be
sensitively reflected by sperm chromatin integrity level, even though conventional parameters of ejaculates remain
unchanged.
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O-101
INTEGRATING NEW TECHNOLOGIES INTO THE ASSESSMENT OF HERITABLE GENETIC EFFECTS
Rosalie K. Elespuru
FDA/CDRH, Rockville, USA
Since the pioneering experiments on the induction of heritable mutations in mice by radiation and chemicals, evidence
of similar effects in humans has been sought, but not realized. The challenge of estimating human health effects resulting from damage to the germ line may be met with new technologies. New systems for germ cell monitoring are being
developed, including analysis of microsatellite mutations (multiple systems), or gene mutations in transgenic (Big Blue)
fish. In addition, genetic testing in populations as part of personalized medicine could be integrated into a strategy for
assessing germ cell effects in humans. This would include the selection of new sentinel genes among those likely to
be monitored broadly. Candidates could include CYP genes (related to metabolism of xenobiotics) that are important in
optimizing drug doses, the high frequency LDLR mutations (related to familial high cholesterol) that predict myocardial
infarction in 50% of individuals in their lifetime, as well as genetic diseases that are commonly monitored (CF and PKU,
and an increasing number of genes planned for newborn screening programs). Third, new epidemiological approaches
may now be feasible. These could take advantage of birth defects monitoring systems already established in many
states in the US, if information on the exposure of parents to common genotoxins such as tobacco smoke and certain
drugs were taken. Recent advances in genetic analysis should make differentiation of new germ cell mutations possible
in a larger background of developmental (non-genetic) and inherited factors. Conclusion: Multiple new sources of monitoring, health outcome-based or exposure-based, could be developed to examine the relationship between genotoxins
and heritable alterations in the genome. These new approaches could be integrated into strategies for pre-market safety
analysis or post-market monitoring of regulated products.
O-102
CHROMOSOME ABERRATIONS IN LYMPHOCYTES FROM GREY SEAL (HALICHOERUS GRYPUS)
PUPS FROM ESTONIA AND NORWAY
Aase Krokje
Chris Bingham
Department of Biology, Norwegian University of Science and Technology, 7491 Trondheim, Norway
Persistant organic pollutants (POP) are highly lipophilic and thus bioconcentrated in nutritional food chains. Since
marine organisms often have high lipid contents, predators in marine food webs are subjected to the highest levels of
accumulated POP. In this project grey seal (Halichoerus grypus) pups from two free-living populations, in Froan Nature
Reserve, Central Norway and near Saaremaa, Estonia, were studied. Previous studies have shown that POP levels are
more than five times higher in the pups from Saaremaa than from Froan. The aim of this project was to compare the
genotoxic effects in a heavily exposed seal population in Saaremaa with a low exposed seal population in Froan. Blood
samples were taken from weaning pups. Lymphocytes, isolated from whole blood, were cultured on site. From metaphase enriched cultures slides for cytogenetic analysis were made from 38 individuals from Froan and 40 individuals
from Saaremaa. The percent damaged metaphases is higher in the Saaremaa samples than in the Froan samples.
There was a difference between the sexes in the frequency of damaged metaphases. The results are consistant with
a correlation of measured overall genetic damage and 21 polychlorinated biphenyls (PCBs). On the other hand, the
differences between the two populations were clearer, when the chromosomal aberration data were treated as group
totals rather than at the individual level.
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O-103
BIOMONITORING STUDIES OF DNA DAMAGE IN MUSSELS EXPOSED TO POLLUTED COASTAL
WATERS IN DENMARK
Jette Rank
Department of Environment, Technology and Social Studies, Roskilde University, Denmark
Genotoxic biomarkers may be used as indicators of effects on organisms and may be also as predictors of long term consequences on populations. However, in the present study, DNA strand breaks in mussel gill cells detected in the comet
assay were used only as a biomarker of exposure to genotoxic pollutants. DNA damage were studied in indigenous and
transplanted blue mussels (Mytilus edulis) from different coastal areas in Denmark and among these sites some were
potentially affected by anthropogenic pollution originating from chemical dumping sites or wastewater outlets. The
results indicate response to pollution from some of the suspected areas. Caged mussels exposed to leaking chemicals
from coastal dumping sites showed statistically significant increase in tail moments compared to mussels from reference sites. DNA damage in native mussels from other polluted sites also showed significant DNA damage two to three
times higher than the back ground levels. The concentrations of some heavy metals and organic chemicals (PAHs and
PCBs) in biota and sediment were compared with the levels of DNA damage and correlations were found for some of the
chemicals. The results show that genotoxic biomonitoring can be useful as an indicator of genotoxic pollution, and it can
be recommended to use such methods before and after cleaning of polluted sites to demonstrate the effect of the management. However, genotoxic biomonitoring in coastal areas also have some challenging problems regarding influence
of different factors as weather conditions, seasonal variations and adaptation which need to be investigated further.
0-104
INDUCTION OF DNA DOUBLE STRAND BREAKS IN THE H4IIE CELL LINE CAUSED BY CO
EXPOSURE TO COPPER, CADMIUM AND ZINC SINGLY AND IN COMBINATIONS
Renate Haldsrud
Department of Biology, Norwegian University of Science and Technology (NTNU), N-7491Trondheim, Norway
Xenobiotics such as heavy metals exist in nature as a mixture of compounds. Knowledge about how these xenobiotics
interact in the context of effect will help to determine the risk facing organisms inhabiting polluted areas. DNA double
strand breaks (dsbs) are a serious form of genetic damage. In addition to cancer development, DNA damage is also
associated with toxic endpoints such as enzymatic changes, metabolic disturbances, accelerated ageing, development
of diseases, and a diminished ability for reproduction, adaptation and survival. In addition, induction of damage in germ
cells may increase the mutational load of the population. The aim of this study was to quantitatively evaluate DNA-dsbs
as a biomarker for metals. The induction of DNA-dsbs was assessed in the H4IIE rat hepatoma cell line following exposure to the metals cadmium, copper and zinc both singly and in combinations. A technique involving in situ lysis within
agarose plugs of cellular material prior to agarose gel electrophoresis was used to separate DNA fragments according
to size whilst minimizing handling damage. Fragment size distributions were acquired from gel image data, and median
molecular length values were calculated and used as a quantitative measure of induced DNA-dsbs. Exposure to high
concentrations of copper and cadmium in combination caused a significant increase (p < 0,05) in the number of strand
breaks in the H4IIE cells. This effect was cancelled when the cells were exposed to high concentrations of copper and
cadmium in combination with zinc. In this exposure, the number of DNA-dsbs was significantly higher (p < 0,001) than
the control values. Environmentally relevant concentrations of the metals cadmium, copper and zinc did not appear to
cause an elevated level of DNA strand breaks in the in vitro exposed H4IIE cell line.
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0-105
THE USE OF EMBRYOS OF THE LAMBDA CII TRANSGENIC MEDAKA FOR ENVIRONMENTAL RISK
ASSESSMENT. APPLICATION TO SEDIMENTS OF THE SEINE ESTUARY (FRANCE)
Jérôme Cachot1
Mac Law 2, Michelle Norris 3, Richard N Winn 3, Karine Le Menach 4, Hélene Budzinski 4
University of Le Havre, Le Havre, France1, North Carolina State University, Raleigh, USA 2 , University of Georgia, Athens, USA3 ,
University Bordeaux I, Bordeaux, France4
The Lambda cII is a transgenic Japanese medaka which has been specifically developed for sensitive detection of mutagens in the aquatic environment. Lambda cII embryos were exposed for ten days to sediments amended with three different concentrations of an organic extract of sediment from the Seine estuary. This sediment contained 8348 ng.g-1 of
PAHs and 206 ng.g-1 of PCBs. Mortality, skeletal deformities and cII mutations were recorded at different developmental
stages. Striking results were obtained with the 6X organic extract. The success of hatching declined significantly (5.7
times) and a two-day delay of hatching was reported. Fry survival decreased significantly to only 21% and about 15%
of adults exhibited spinal deformities. Levels of cII mutations in fry liver were significantly enhanced (2.2 times above
background) and corresponded mainly to G:C to T:A transversions. The study confirmed that sediments of the Seine
estuary contain numerous organic toxicants including mutagens which can induce a large panel of effects including
embryotoxicity, teratogenesis and mutagenesis. It also demonstrated the suitability and sensitivity of embryos of the
Lambda cII medaka for genotoxic risk assessment in the aquatic environment. The study was supported by the SeineAval program and the Agence de l’Eau Seine-Normandie (France).
0-106
DISCOVERY AND FUNCTIONAL ANALYSIS OF VARIATION IN HUMAN DAMAGE RESPONSE
PATHWAYS
Douglas A. Bell
National Institute of Environmental Health Sciences, RTP, NC, 27709, USA
Dissecting gene-environment interaction in disease requires the ability to identify genetic susceptibility loci regulating
exposure-related phenotypic traits. Genome databases (dbSNP) and gene expression were incorporated into bioinformatics and functional genomics methods to identify sequence variants (e.g., SNPs) in gene regulatory elements (Tomso
et al. PNAS, 2005) and we have applied these to p53 and antioxidant response pathways. The antioxidant response
element (ARE) is a cis-acting enhancer sequence found in the promoter region of genes encoding anti-oxidative and
Phase II detoxification enzymes. In response to oxidative stress, the transcription factor NRF2 binds to ARE elements,
activating transcription and modulating in vivo defense mechanisms against oxidative damage. We have developed
computational methods to (1) statistically model a position-weight matrix (PWM) (2) identify a set of SNPs whose
sequences fit the ARE motif; (3) map the selected polymorphic AREs to within 5Kb of gene transcription start sites;
and (4) prioritize candidate polymorphic ARE-containing genes by phylogenetic footprinting and gene expression profiling. We have identified 2839 ARE SNPs that map to upstream regions of 2306 human genes. Phylogenetic footprinting of human, mouse, and rat genomic sequences identified 161 genes containing 321 conserved AREs. By mining of
microarray profiles from oxidant exposure studies in NRF2 knockout mice, 278 genes were reported as oxidative stress
(NRF2)-regulated genes. The intersection of these criteria (high-impact SNPs, conserved AREs, NRF2-regulated expression) produced our top candidates for further analysis. Among the 28 highly conserved ARE SNPs mapped to 21 NRF2
regulated genes are several recognized oxidative stress pathway genes. We are evaluating the impact of these SNPs
on NRF2 binding, gene expression phenotype, and response to oxidative stress in model systems. SNPs with functional
impact genes will be tested in oxidant related disease.
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0-107
ROLE OF N-ACETYLTRANSFERASE GENETIC POLYMORPHISMS IN CANCER RISK
David Hein
University of Louisville School of Medicine, Louisville, KY, USA
0-108
GENE – GENE ENVIRONMENT INTERACTION AND CANCER RISK
Emanuela Taioli1
Michael Okobia 1, Seymour Garte 1, Paolo Vineis 2
University of Pittsburgh Cancer Institute and Graduate School of Public Health, Pittsburgh, USA 1, Imperial College, London, UK 2
The interaction between genetic and environmental factors is complex, and includes the activity of several genes,
some of which interact with each other, some of them with different tissue distribution and tissue specific inducibility.
Therefore, association studies of metabolic gene polymorphisms and cancer require large numbers in order to have
adequate power. This can be obtained through a meta analytic approach, or a pooled analysis.
We analyzed data pooled through the GSEC study, a collaborative study on genetic susceptibility to environmental
carcinogens (http://www.upci.upmc.edu/research/ccps/ ccontrol/index.html) to study the interaction between multiple
genes on a pathway of metabolism of known environmental carcinogens, and their effect on cancer risk. Breast and
lung cancer are presented as examples.
In a pooled sample of 611 lung cancer cases and 870 controls where individual genetic information on CYP1A1, GSTM1
and GSTT1, and environmental exposure to tobacco smoking was available, the risk of lung cancer with the combination
of CYP1A1*4 or *2B alleles (exon 7), GSTM1 and GSTT1 homozygous deletion was 8 times higher than that observed
in subjects carrying the wild type form of the CYP1A1 gene, and who had both GSTM1 and GSTT1 functional genes.
However, since less than 5% of the overall population carried this high risk genotype, the population attributable risk
for this combination is low.Similar data were obtained with a large sample of breast cancer cases and controls (3,391
cases and 3,451 controls), where both CYP1B1 and CYP1A1 were available, as genetic markers of estrogen metabolism.
In this analysis, an interaction between CYP1B1 genotype and age around menopause was observed in populations of
European descent, but not in Asians or in populations of African descent.Pooled analysis is a useful approach to analyze
multiple gene interactions and scientifically relevant subgroups of subjects, which cannot be evaluated in individual
studies because of small numbers.
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0-109
GENETIC SUSCEPTIBILITY, GENOMIC INSTABILITY AND USEFULNESS IN CANCER THERAPY
William W. Au
Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, Galveston, Texas 77555-1110,
USA
Inheritance of certain susceptibility genes for chemical metabolism (CYP 1A1, GSTM1) and for DNA repair (XRCC1, XPD)
is significantly associated with the development of cancer from exposure to environmental mutagens. The development
of cancer is most likely initiated by the induction of gene mutations and chromosome aberrations (CA). The presence
of CA in lymphocytes has been shown to be an excellent predictor for cancer, indicating that such abnormalities have
also been induced in target cells. Indeed, CA and more specifically genomic instability are hallmarks of cancer cells.
Additionally, specific genes such as p53 are often inactivated. The latter may be instrumental in allowing cells with
genomic instability to survive and to transform into cancer cells. Scientists can make use of such information to develop
molecular procedures to intervene the cancer process. We have used cervical cancer (CC) cells to develop molecular
intervention/gene therapy protocols which include interference RNA (siRNA) and dominant negative estrogen receptor
blocker. Transfection of the blocker gene via adenovirus into CC cells caused significantly reduced expression of HPV E6
and E7 mRNA and cyclin D1 protein, based on Western blot analyses. These changes are substantiated independently
by the Flow analysis which indicates immediate arrest of cells in S phase and the subsequent expression of apoptosis.
These observations suggest that blockage of estrogen receptors led to the suppression of viral oncogene expression and
reactivation of p53. The latter recognized the existence of genomic instability in the cancer cells as severe stress signal
and initiated a cascade of cellular responses that included cell cycle arrest and self-destruction. Precise knowledge of
environmental carcinogenesis is therefore essential to the development of effective cancer prevention and intervention
protocols.
0-110
MOLECULAR EPIDEMIOLOGY OF IMMUNE DEFENSE AGAINST CANCER THERAPY
Kei Nakachi
Tomonori Hayashi, Yoichiro Kusunoki, Kazue Imai
We have previously shown that natural cytotoxic (NK) activity of peripheral-blood lymphocytes was inversely related to
cancer development, based on a prospective cohort study. A wide variation of NK activity among individuals cannot be
explained by environmental factors alone, and the genetic fraction of NK activity needs to be clarified. A case-control
study within this cohort was designed: 102 cancer cases with lymphocyte DNA available and three control groups, each
of which consisted of 204 subjects selected from non-cancer cohort members for each tertile level of NK activity. We
first carried out a phenotype-genotype association analysis between two control groups with high and low NK activity, in
terms of the SNPs, scattered over 270 kb, in the natural killer complex gene region on chromosome 12p, identifying the
major haplotype alleles that were closely associated with NK activity. Next, cancer risks of individuals were assessed for
these haplotypes by this case-control study. The phenotype-genotype association analysis showed that there were two
haplotype blocks, NKG2D hb-1 and NKG2D hb-2, localized in the NKG2D gene region, and that each of these generated
two major haplotype alleles: For NKG2D hb-1, low-activity-related LNK1 (P<0.00008) and high-activity-related HNK1
(P<0.0001); for NKG2D hb-2, LNK2 (P<0.0002) and HNK2 (P<0.0008). Furthermore, the haplotypes LNK1/HNK1 and
HNK1/HNK1 revealed a decreased risk of cancer development, with odd ratios of 0.754 (95%CI 0.473-1.20) and 0.471
(0.233-0.952), respectively, taking the risk of LNK1/LNK1 as reference. Individuals who are genetically predisposed to
have low or high natural cytotoxic activity can in part be determined by NKG2D haplotyping, which in turn reveals an
increased or decreased risk of cancer development. These results suggest that the immunogenetical factor plays a role
in immunosurveillance mechanisms against cancer.
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0-111
BIOTRANSFORMATION ENZYMES AS MODIFIERS IN BREAST CANCER
Pavel Soucek1
Ivan Gut 1 , Radka Vaclavikova 1, Miluse Hubackova 1, Roman Kodet 2, Marcela Mrhalova 2, Jan Novotny 3
Group for Biotransformations, Center of Occupational Medicine, National Institute of Public Health, Prague, Czech Republic 1,
Institute of Pathology and Molecular Medicine, Teaching Hospital Motol and 2nd Medical Faculty, Charles University , Prague, Czech
Republic 2, Institute of Oncology, General Teaching Hospital and 1st Medical Faculty, Charles University, Prague, Czech Republic 3
The genetically variable biotransformation enzymes: cytochrome P450 (CYP) epoxide hydrolase (EPHX1), NAD(P)H:
quinone oxidoreductase (NQO1), and glutathione S-transferases (GST) metabolize and conjugate drugs, carcinogens,
and natural products. High number of human cancers results from exposure to environmental carcinogens suggesting
that individual effectiveness in the detoxification of these chemicals may influence susceptibility to malignant disease.
Our study aimed at determining: 1/ whether any association exists between genetic polymorphisms in CYP1B1, EPHX1,
NQO1, GSTM1, GSTP1, GSTT1 and individual susceptibility to breast cancer and 2/ the expression of four important CYPs
(CYP1B1, 2C9, 2E1 and 3A4) in human carcinomas of the mammary gland, breast tissue of the same patients without
morphological evidence of presence of tumor cells and peripheral blood lymphocytes.
The distribution of genotypes in NQO1 (Pro187Ser) was significantly different between the control group and breast
cancer cases. Individuals simultaneously lacking GSTM1 and carrying at least one GSTP1-105Val allele were at significantly higher risk of breast cancer. Combinations of either GSTM1null or GSTP1-105Val with low activity of EPHX1 also
presented a significant risk of breast cancer.
The expression of CYP1B1 in tissues and lymphocytes was highest followed by CYP2E1. Expression levels of CYP2C9
and CYP3A4 were under limit of quantification in majority of individuals. The level of CYP1B1 expression in mammary
gland correlated with expression in lymphocytes.
Taken together, our findings seem to suggest an influence of genetic polymorphisms in biotransformation enzymes,
particularly NQO1 and some high frequency two-gene combinations on the susceptibility to breast cancer. The high
expression of CYP1B1 in breast tissue may evoke formation of carcinogens, e.g. the formation of 4-hydroxyestradiol
during estradiol metabolism or metabolic activation of polycyclic aromatic hydrocarbons.
0-112
MOLECULAR EPIDEMIOLOGY AS A TOOL FOR UNDERSTANDING SPORADIC COLORECTAL
CANCER
Pavel Vodicka1
Monika Hanova 1, Elena Tulupova 1, Veronika Polakova 1,Alessio Naccarati 1, Barbara Pardini 1,2, Ludmila
Vodickova 1,3, Pavel Soucek 3, Jan Novotny 4, Kari Hemminki 5
Inst. Exper. Med., Acad. Sci. Czech Rep., Prague, Czech Republic1, Dept. Biology, University of Pisa, Pisa, Italy2, Natl. Inst. Publ.
Health, Prague, Czech Republic3, First Medical Faculty, Charles University, Prague, Czech Republic4, German Cancer Res. Center ,
Heidelberg, Germany5
The majority of cancer cases occurs sporadically (70%) and an important involvement by genetic polymorphisms in
low penetrance genes (as biotransformation and DNA repair genes) may be expected. Colorectal cancer (CRC) poses
a heavy health problem in the Czech Republic (Czech population is ranking third worldwide in the incidence of colon
cancer, and is a world leader in the incidence of the rectal cancer), the prospects of molecular epidemiological approach
in assessing individual susceptibility towards CRC is being discussed. Genetic polymorphisms in coding and regulatory
sequences may result in structural alterations in enzymes, leading to increased cancer susceptibility, as polymorphisms
in EPHX1, NQO1, XPD, exon 10 and XRCC3, exon 7 genes are associated with increased risk of breast cancer, and individuals with repair capacity below the population mean can be at an increased risk of developing various kinds of cancer. In our study 525 patients with sporadic CRC and 525 healthy controls, matched for sex and age, were assayed for
polymorphisms in XME (CYP1B1, EPHX1 exons 3 and 4, GSTM1, GSTP1, GSTT1, NQO1 and ABCB, exon 26), DNA repair
genes (XPD, exon 23, XPG, exon 15, XPC, exon 15, codons 499 and 939, XRCC1, exons 6, 9 and 10, APE1, hOGG1,
XRCC3 and NBS1), MMR genes (MSH2, intron 12 and MSH6, codon 39) and genes controlling cell cycle (CCND1, exon
4, TP53, intron 3 and codon 72, and P21, codon 31). Single genotypes as above and genotypes in particular binary
combinations were analysed for their modulating risk of sporadic CRC patients and the data were evaluated in relation
to the age, sex, life style and the data on diagnosis and tumor localization and staging. Well designed molecular epidemiological study may contribute to the understanding susceptibility to sporadic CRC.
The work was supported by GACR 310/05/2626 and by IGA NR 8563-5/2005
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O-113
THE CONTRIBUTION OF THE ECNIS NETWORK TO THE VALIDATION OF MARKERS FOR RISK
ASSESSMENT
Paolo Vineis
Fondazione ISI, Turin, Italy, Imperial College, London, UK
The purpose of the presentation is to summarize the state-of-the art concerning the validation of biomarkers for
inclusion into epidemiological studies on environment and cancer, and to identify criteria for the inclusion of validated
biomarkers into risk assessment practices. ECNIS is a Network of Excellence that is energetically addressing several
issues, both technological and methodological, for the improvement of “molecular epidemiology”. The purpose is to
understand whether the last 20 years of research in the field of molecular epidemiology have produced usable biomarkers, and in particular to know where we are with the development of biomarkers of exposure and biomarkers of early
effect. Also, much emphasis has been put recently on high-throughput technologies for genetic analyses, and similar
efforts are now put into the development and validation of high-throughput markers of exposure (DNA adducts, oxidative damage) and intermediate markers (proteomics). A new generation of studies in the field of environmental cancer
will soon become possible, and the results of these studies will impact also on risk assessment procedures.
O-114
EVALUATION OF IN VIVO MUTAGENICITY OF ENVIRONMENTAL CHEMICALS USING
TRANSGENIC MODEL ANIMALS
Yasunobu Aoki
National Institute for Environmental Studies, Tsukuba, Japan
Gene mutation is an essential process in the initiation of cancer. Diesel engine emissions are a major source of mutagens
in urban air, and are suspected of causing lung cancer. We evaluated the in vivo mutagenicity of diesel exhaust (DE),
DE particles (DEP), and potent mutagens in DEP, including benzo(a)pyrene (B(a)P) and 1,6-dinitropyrene (1,6-DNP),
as model environmental mutagens for assessing the risk of lung cancer. A single intratracheal instillation of B(a)P or
1,6-DNP into the lung of gpt delta mice, which carry the gpt (guanine phosphoribosyl transferase) gene as a mutation
target, resulted in a dose-dependent increase of mutant frequency (MF). The relative mutagenic potency of 1,6-DNP
was 32 × 10–5 (MF/mg), about 13 times that of B(a)P. The major mutations induced by B(a)P and 1,6-DNP were a G:
C to T:A transversion and a G:C to A:T transition, respectively. The in vivo mutagenicity of DE was estimated from
mice that inhaled DE at a concentration of 3 mg/m3 or that received a single intratracheal instillation of DEP or DEP
extract. MF was elevated with duration of DE inhalation for 12 weeks. Instillation of DEP and DEP extract increased MF
linearly with dose; relative mutagenic potencies were 2.7 × 10–5 and 5.6 × 10–5 (MF/mg), respectively, indicating that
the mutagenicity of DEP is derived mainly from compounds in DEP extract, as 50% by weight of DEP is DEP extract. A
major mutation induced by DE inhalation was a G:C to A:T transition, and positions of mutation hotspots in gpt induced
by DE inhalation and DEP instillation were almost identical to those induced by 1,6-DNP instillation, suggesting that the
predominant mutagen in DE is 1,6-DNP or a related compound. These results indicate that transgenic animals designed
for detecting mutations potentially make a good system for the evaluation of in vivo mutagenicity of environmental
chemicals and the identification of compounds inducing in vivo mutations.
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O-115
PROGRESS WITH INTEGRATED RISK ASSESSMENT?
Hans Kromhout
University of Utrecht, Utrecht, The Netherlands
O-016
TOXICOKINETIC PATTERN OF PHTHALIC ACID FOR RISK ASSESSMENT APPROACH
Byung-Mu Lee
Duck S Lim
Sungkyunkwan University, Suwon, Korea
The toxicokinetic patterns of phthalic acid (PA), one of the metabolites of phthalic acid esters (PAEs), were studied
in rats after oral administration. After oral administration of dose 20, 100, and 500 mg/kg PA/kg to rats, urinary or
serum concentrations of PA were determined by a high performance liguid chromatography (HPLC) system. After oral
administration of 500 mg/kg, dose-normalized (20 mg/kg) area under the curve (AUC) (146.9 ug*hr/ml) was significantly greater than at 20 mg/kg (44.7 ug*hr/ml). The plasma concentrations of PA in rats showed a biexponential
increase following oral administration of doses ranging from 20 to 500 mg/kg in rats. The terminal elimination half-lives
(t1/2) of PA at the 20, 100, and 500 mg/kg dose levels were 6.5±1.1, 5.1±3.6, and 5.1±1.2, h, respectively. The total
clearances (CL) of PA at the 20, 100, and 500 mg/kg dose levels were 97.4±4.2, 215.0±55.4 and 721.1±51.8 ml/h, ,
respectively. The apparent volume of distributions of PA at the 20, 100, and 500 mg/kg dose levels were 903.3±125.3,
1419.9±527.5 and 5264.9±993.7 ml, respectively. PA was absorbed rapidly after an oral dose of 500 mg/kg with peak
concentration (Cmax) in blood (20.5±3.6 ug/ml) reached after 30 min of drug administration. Urine analysis indicates
that 25.5±0.45 % of the administered dose of PA (500 mg/kg, p.o) is recovered unchanged in urine within 0-24 h.This
would be the first attempt to show the toxicokinetic properties of PA in animal models and may be useful for the development of risk assessment.
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O-117
HUMAN EXPOSURE ASSESSMENT OF HETEROCYCLIC AMINES IN FOODS
Tai-Kyung Ryeom 1
Kyung-Jin Min 1, Seung-Woon Myung 2, Cheon-Ho Jo 2, Hyo-Min Lee 1
Korea Food and Drug Administration, Seoul, Korea 1, University of Kyonggi, Kyonggi-do, Korea 2 , University of Kyonggi, Kyonggido, Korea
In this study the average daily exposure level of heterocyclic amines (HCAs) was estimated to characterized the cancer
risk by food intake of general Korean people, and to identify the relationship between the internal and external dose of
dietary intake using analysis of uninary PhIP. For the exposure assessment of HCAs by foods ingestion, the average body
weight for adult groups (20-64 years) and consumed values form report on national health and nutrition survey and
our laboratory's nutrition survey were used. The estimated lifetime daily intake of PhIP and MeIQx were 3.44 and 3.69
ng/kg bw/day. And, cancer potency of PhIP and MeIQx were 3(mg/kg/day)-1, 11(mg/kg/day)-1, respectively. The dietary
excess cancer risk estimated using cancer potency of PhIP and MeIQx were from 1.0×10-5 to 4.1×10-5, respectively.
O-118
CRISES IN THE CHEMICAL GENOTOXICITY PARADIGM: STEM CELLS, CELL-CELL
COMMUNICATION AND SYSTEMS BIOLOGY AS IGNORED CONCEPTS
James E.Trosko
Michigan State University, East Lansing, USA
BACKGROUND: Mutagenesis, cytotoxicity and altered gene expression contribute to diseases. The current paradigm in
toxicology is that if a chemical causes any disease, it involves DNA damage/ mutations. It stems from “genotoxicity”
assays, with positive results for chemicals linked to diseases; with mutations in oncogenes and tumor suppressor genes;
with chemicals inducing oxidative stress; with DNA adducts in exposed tissues; and with heredity diseases. The lack of
data on what are the “target” cells in exposed tissues contributes to the genotoxic paradigm.
METHODS: Many toxicants do not induce nuclear DNA damage, even though they induce oxidative stress, and are teratogens, tumor promoters, immunotoxicants, reproductive-and neuro-toxicants, indicating a need to assess the epigenetic risk of chemicals. Gene expression is a consequence of a network of extra-intra-, and gap junctional inter-cellular
signaling pathways, which causes a proper balance of proliferation, differentiation and apoptosis. Tissue homeostasis
requires signaling between all cell types.
Results: Chemicals, such as PAH’s, DDT, TCDD, PCB’s, PBB’s, peroxisome proliferators, etc., that can induce birth
defects, cancer, immunological, reproductive and neurological effects, are non-genotoxic. Inhibition of gap junctional
communication causes the proliferation of cells and blockage apoptosis and differentiation. With the ability to isolate
human adult stem cells, differential responses of the stem cells, their committed progenitor daughter cells and the terminally differentiated daughters to toxicants will demonstrate how toxicants alter tissue homeostasis.
CONCLUSIONS: Using the multi-stage model of carcinogenesis, and ignored “hallmarks” of cancer (e.g., stem cells as
target cells for carcinogenic initiation; role of gap junctional intercellular communication during the promotion phase),
a new paradigm will be offered to help explain the paradoxes of the “crises in the genotoxicity” paradigm.
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O-119
LOOKING TO THE FUTURE: WHERE WILL "OMICS" AND SYSTEMS BIOLOGY LEAD RISK
ASSESSMENT
Christopher Portier
National Toxicology Program, Research Triangle Park, USA
The interactions of cellular components, cross-talk between signaling pathways and non-linear kinetics of biochemical
processes have always made it difficult to fully characterize the interplay between changes to regulatory circuits in a
cell leading to changes in tissue response leading to host morbidity and mortality. For the last 5 years, the explosion
in "omics" technologies and the development of systems biology has created promise that these mysteries can be
unraveled and used to drive risk assessments. But what is hypothetical and what is real? In this presentation, I will
discuss these issues in the context of risk assessment discussing what is possible today and what may be possible in
the next 5 to 10 years. The focus of the talk will be on three basic issues: identifying hazards and defining "modes-ofaction", defining doe-response relationships, and measuring exposures.
O-120
MOLECULAR EPIDEMIOLOGICAL STUDIES IN 1,3-BUTADIENE EXPOSED CZECH WORKERS
Richard J. Albertini1,3
Radim J. Sram2, Pamela M. Vacek3, Janice A. Nicklas3, Jake McDonald4, James A. Swenberg5
BioMosaics, Inc. Burlington, VT, USA1, Institute of Experimental Medicine AS CR, Prague, CZ2, University of Vermont, Burlington,
VT, USA 3, Lovelace Respiratory Research Institute, Albuquerque, MN, USA 4, University of North Carolina, Chapel Hill, NC, USA5
A continuum of biomarkers was measured in 1,3-butadiene (BD) exposed workers in the Czech Republic following
extensive exposure assessments over several months. Mean 8-hour TWA BD exposure levels for the 25 controls, 24
monomer production workers and 34 polymerization workers (all male) in an initial study were 0.010, 0.290 and 0.810
ppm. Biomarkers measured included urinary metabolites, haemoglobin adducts, SCEs, HPRT gene mutations, chromosome aberrations and metabolic genotypes at several loci. Urinary metabolite and haemoglobin adduct concentrations
were significantly elevated, correlating with BD exposure levels. Detoxification metabolism as reflected by urinary
metabolite ratios showed the dominance of hydrolysis. No significant relationships were seen between BD exposures and
gene mutations or cytogenetic endpoints. A second study compared females and males. In addition to the haemoglobin
adducts measured in the first study, analysis of a diepoxide specific adduct was added. BD levels had fallen since the
first study, TWAs are now 0.180 and 0.0035 ppm for 23 female exposed and 26 female control workers, respectively and
0.370 and 0.007 ppm for 30 male exposed and 25 male control workers, respectively. Metabolite ratios again reflected
the dominance of hydrolytic detoxification and were not different between the sexes. Diepoxide specific adducts were
non-detectible in any subjects. Again, neither HPRT mutations nor cytogenetic changes were related to BD exposures
in either sex. BD’s genotoxicity and therefore its carcinogenicity depend on electrophilic metabolites, particularily the
diepoxide, that result from oxidative metabolism. Humans form much less of this metabolite per unit dose of BD than
do rodents. This is reflected in its diminished genotoxicity in humans, with the weight of evidence (including these
Czech studies) indicating that it has yet to be demonstrated under modern exposure conditions. This has implications
for assessing its cancer risk. Supported from Health Effects Institute and American Chemistry Council.
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O-121
BURDEN OF CANCER DUE TO OCCUPATIONAL EXPOSURES
Paolo Boffeta
Mathieu Boniol, Philippe Autier
International Agency for Research on Cancer, Lyon, France
In their seminal in the early 1980s, Doll and Peto estimated the burden of occupational cancer in the United States to
6.8% of all cancer deaths in men. These estimates have been applied to other countries without a critical appraisal.
However, authors who have adopted an approach based on estimates of exposure and relative risks have resulted in
lower estimates.
We performed an evidence-based estimate of the burden of occupational cancer in France in 2000. We considered occupational exposures for which a causal association with human cancer has been definitely established. Relative risks were
extracted from meta-analyses or pooled analyses. For most agents, the number of exposed workers was obtained from
a nationwide survey that provided an estimate of the number of workers employed in each industry.
The total number of cancer deaths among men attributable to occupational agents in France in 2000 was 2437, or 1.8%
of all cancer deaths. The corresponding figure in women was 257 deaths, (0.5%). Asbestos contributed to about half
of the burden, and the other major contributors were polycyclic aromatic hydrocarbons, chromium and employment as
painter. Traditional occupational carcinogens, such as aromatic amines and radon had a limited impact.
Although our results might be underestimated because of our assumptions (e.g., exclusion of few carcinogens for which
no exposure data were available and of suspected carcinogens), they suggest that the role of occupational exposures
in determining the global cancer burden is lower than previously reported. This may be due to methodological limitations of previous estimates, but it also likely reflects the effect of the successful implementation of measures aimed to
reduce exposure to recognized carcinogens. Asbestos remains the most important occupational carcinogen. The burden
of occupational cancer in women is very low. Estimates of the burden of occupational cancer should be time- and placespecific.
O-122
INDIVIDUALS AT HIGH RISK OF STOMACH CANCER AMONG ATOMIC-BOMB SURVIVORS
IDENTIFIED IN RERF IMMUNOGENOME STUDY
Tomonori Hayashi
Yukari Morishita, Kazue Imai, Yoichiro Kusunoki, Kei Nakachi
Results of epidemiological studies conducted since the establishment of the Atomic Bomb Casualty Commission-Radiation
Effects Research Foundation (ABCC-RERF) in 1947 have clearly demonstrated several important long-term effects of
atomic-bomb (A-bomb) radiation in humans: radiation dose-dependent increases in the incidence of, and mortality due
to, malignant tumors. On the other hand, exposure to radiation greatly affects the immune system of the host, and
immunological host defense is considered to play an important role in the development of various cancers. Therefore,
we recently commenced an immunogenome study in a sub-cohort of the RERF Adult Health Study and investigated
stomach cancer development in atomic-bomb survivors in relation to immunogenetic factors and radiation exposure.
We conducted case-control studies within this cohort and assessed individual risk of stomach cancer on the basis of IL10 haplotypes. We found that, although overall risk of stomach cancer significantly increased with increased radiation
dose, this risk in both radiation-exposed and non-exposed survivors was greatly modulated by IL-10 haplotypes, making
possible the identification of people at high risk of radiation-associated stomach cancer. In addition, plasma IL-10 levels
were found in controls to vary with the IL-10 haplotypes as well as radiation dose. These results suggested that plasma
IL-10 levels may be a promising surrogate marker for prevention of stomach cancer, indicating that individual risk of
stomach cancer is in part determined by both radiation exposure and IL-10 haplotypes.
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0-123
OXIDATIVE STRESS MARKERS IN BUS DRIVERS
Pavel Roessner, Jr.
Vlasta Svecova, Alena Milcova, Zdena Lnenickova, Radim J. Sram
Institute of Experimental Medicine, Prague, Czech Republic
Exposure to ambient air pollution is associated with many diseases including cardiovascular disorders and cancer.
Oxidative stress, caused by excessive production of reactive oxygen species, is believed to be one of the major sources
of particulate matter (PM)-mediated adverse health effects. PM in ambient air arises from industry, local heating, as
well as vehicle emissions and poses a serious problem mainly in large cities. Oxidative stress causes damage to lipids,
DNA, and proteins. To measure the oxidative damage in humans various markers are available. 15-F2t-isoprostane (15F2t-IsoP) became widely used marker of lipid peroxidation. To analyze DNA damage, levels of 8-oxodeoxyguanosine
(8-oxodG) are studied. Levels of protein oxidation can be assessed by means of detection of carbonyl groups formed
on amino acid side chains.
To analyze the effect of air pollution on markers of oxidative stress we studied 50 bus drivers from Prague, Czech
Republic, and 50 matching controls. Levels of oxidative stress markers were significantly increased in the exposed
group. In bus drivers the median levels were: 0.81 nmol/mmol creatinine for urinary 15-F2t-Iso and 7.79 nmol/mmol
creatinine for urinary 8-oxodG, while in controls the corresponding values were 0.68 nmol/mmol creatinine for 15-F2tIso (p<0.01), and 6.12 nmol/mmol creatinine for 8-oxodG (p<0.05). The median carbonyl levels in the exposed group
reached 14.1 nmol/ml plasma, while in controls 12.9 nmol/ml plasma (p=0.001). Among controls 15-F2t-IsoP levels correlated with age (R=0.33, P<0.05), vitamin C (R=-0.31, P<0.05) and cotinine (R=0.47, p=0.001) levels. In conclusion,
our study indicates that exposure to PM in ambient air results in increased oxidative stress as measured by markers of
lipid peroxidation, DNA and protein oxidation.
Acknowledgements: The study was supported by the grant VaV-SL/5/160/05 of the Czech Ministry of Environment and
by the grant 1QS500390506 of the Academy of Sciences of the Czech Republic.
0-124
4-OXO-2-HEXENAL, A VOLATILE MUTAGEN FORMED BY OMEGA-3 FAT PEROXIDATION
Hiroshi Kasai1
K. Kawai 1, M. Maekawa 1, Y. Takahashi 2, H. Nakamura 2, R. Sawa 2, S. Matsui 3, T. Matsuda 3, K. Savela 4
Univ. Occupational & Environmental Health, Kitakyushu, Japan 1, Microbial Chemistry Res. Center, Tokyo, Japan 2, Kyoto Univ.,
Kyoto, Japan 3, National Veterinary & Food Res. Institute, Helsinki, Finland 4
To identify mutagens formed in a model reaction of lipid peroxidation, linolenic acid methyl ester and hemin were reacted with dG. As a result, a 4-oxo-2-hexenal-dG adduct (dG*) was identified in the model reaction mixture. The 4-oxo-2hexenal (4-OHE) showed mutagenic activity in the Salmonella typhimurium strains TA100 and TA104. The 4-OHE reacts
not only with dG but also with dC and 5-methyl-dC to yield adducts (dG*, dC*, 5-methyl-dC*). The structures of dC*and
5-methyl-dC* were determined mainly by mass-, 1H- and 13C-NMR- spectra. The structure of dC* was confirmed by
X-ray crystallography. After 4-OHE was orally administered to mice, 4-OHE-dC- and 4-OHE-5-methyl-dC adducts were
detected in esophageal, stomach and intestinal DNA. It is important to confirm the formation of 4-OHE during the
heat processing of edible vegetable oil, or during cooking. From ethylacetate traps (extracts) of the smoke released
during the broiling of fish, 4-OHE was detected by GC-MS. Commercial perilla oil, which is rich in linolenic acid (40-50
micro-g/g), the edible part of broiled fish (5-170 micro-g/g), and various fried foods (5-50 micro-g/g) also contained
high levels of 4-OHE. It was present at an especially high concentration (70-170 micro-g/g) in broiled saury. Workers
involved in the manufacturing of vegetable oils, such as colza oil and soybean oil, which contain linolenic acid, will be
exposed to 4-OHE during the crude oil extraction and purification. The same is true for workers manufacturing instant
noodles and snack foods, and manufacturing of EPA and DHA from fish oil. Kitchen workers involved in frying foods or
broiling fish in restaurants may also be exposed to 4-OHE. Since 4-OHE induces DNA adduct formation in experimental
animal organs, it is possible that 4-OHE is an important human carcinogen. Further studies on the carcinogenicity of
4-OHE and its detection in working environments will be required.
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0-125
CHROMOSOMAL ABERRATIONS IN WORKERS EXPOSED TO NITROTOLUENES: EFFECT OF
GENETIC POLYMORPHISM IN XENOBIOTIC-METABOLIZING ENZYMES
Hannu Norppa 1
Ari Hirvonen 1, Hilkka Järventaus 1, Hansruedi Glatt 2, Ovnair Sepai 3, Huifang Yan 4, Yu-Ying Liu 4,
Gabriele Sabbioni 5
Finnish Institute of Occupational Health, Helsinki, Finland 1, German Institute of Human Nutrition, Potsdam-Rehbrücke, Germany 2,
University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, UK 3, Institute of Occupational Medicine, Chinese Academy of Preventive
Medicine, Beijing, P. R. China 4, Institute of Environmental and Occupational Toxicology, Airolo, Switzerland 5
Nitrotoluenes, such as 2-nitrotoluene, 2,4-dinitrotoluene, and 2,6-dinitrotoluene, are carcinogens to which humans are
exposed in the workplace and in the general environment. In the present study, we studied the level of chromosomal
aberrations in workers exposed to high levels of nitrotoluenes during the production of dinitrotoluene and trinitrotoluene. We also determined genotypes for polymorphic phase II enzymes assumed to function in nitrotoluene metabolism,
namely glutathione S-transferases (GSTM1, GSTT1, GSTP1), N-acetyltransferases (NAT1, NAT2), and sulfotransferases
(SULT1A1, SULT1A2). The frequency of total chromosomal aberrations was increased in the exposed workers (n=91)
in comparison with a group of factory controls (n=60). The effect of nitrotoluene exposure on chromosomal aberrations
was primarily seen (1) in SULT1A1 and SULT1A2 wild-type homozygotes, but not in carriers of SULT1A1 and SULT1A2
variant alleles, and (2) in carriers of NAT1*10 and *11 alleles (“rapid acetylators”) but not in subjects without these
alleles (“normal acetylators”). In addition, GSTT1 (together with smoking) and GSTP1 genotypes were observed to
modify the level of chromosome damage in the nitrotoluene-exposed workers. Among all subjects, an effect of smoking
on the level of chromosomal aberrations was seen in GSTM1 null but not in GSTM1 positive subjects. In conclusion,
our results indicate that the effect of nitrotoluene exposure on the level of chromosomal aberrations depends on the
SULT1A1, SULT1A2, NAT1, GSTT1, and GSTP1 genotypes of the subjects, suggesting that polymorphisms of enzymes
probably involved in nitrotoluene metabolism affect the extent of genotoxic damage resulting from exposure to nitrotoluenes. We could also confirm earlier findings on an elevated sensitivity of GSTM1 null subjects to the genotoxic effects
of smoking. [Supported by European Commission Contract ERB-IC-CT97-0221].
0-126
GENOTOXIC EFFECTS AND SECONDARY OXIDATIVE STRESS IN WORKERS OCCUPATIONALLY
EXPOSED TO STYRENE
Alessio Naccarati 1
Ludmila Vodickova 1,2, Miroslava Kuricova 3, Rudolf Stetina 4, Pavel Soucek 2, Paola Manini 5, Giuseppe De
Palma 5, Bolek Marczynski 6, Barbara Pardini 1, Monika Hanova 1, Pavel Vodicka 1
Inst. Experimental Medicine, Acad. Science Czech Rep., Prague, Czech Republic 1, National Institute of Public Health, Prague, Czech
Republic 2, Slovak University of Health, Bratislava, Slovakia 3, Purkynje Military Medical Academy, Hradec Kralove, Czech Republic 4,
Laboratory of Industrial Toxicology, University of Parma, Parma, Italy 5, Research Institute of Occupational Medicine (BGFA), RuhrUniversity of Bochum, Bochum, Germany 6
Styrene is widely used in the production of plastics, synthetic rubber, and polyester resins. Occupational exposure in
the reinforced plastic industry is the heaviest and may entail a daily intake of gram quantities of styrene via inhalation.
Simultaneous analyses of exposure, early effect and susceptibility biomarkers were performed in a cohort of 79 styreneworkers and 49 unexposed controls from Czech Republic. Secondary DNA damage via oxidative stress was investigated
analyzing levels of 8-OH-deoxyguanosine in lymphocytes, and 8-OH-guanine and deoxyguanosine in urine. In addition, individual DNA repair capacity to remove γ-irradiation and oxidative induced-DNA damage was investigated in the
whole cohort. All parameters of exposure correlated with one another. Interestingly, 4-vinylphenol conjugate levels were
several-fold higher in exposed subjects, suggesting formation of intermediary 3,4-arene oxide in exposed individuals.
MN frequency was moderately higher in exposed (P=0.034), but MN were also modulated by age and sex. SSB levels
were significantly lower in exposed workers, while DNA repair capacity was two-fold higher in exposed than in controls,
suggesting a potential induction of DNA repair machinery in relation to occupational exposure. 8-OH-deoxyguanosine
levels in lymphocytes were lower in exposed than in control individuals and lower in subjects with higher repair capacity to remove oxidative DNA damage. 8-OH-deoxyguanosine levels in lymphocytes were the highest in individuals with
hOGG1 homozygous variant genotype (P=0.001), while repair capacity to remove oxidative DNA damage was the highest in those with hOGG1 wild-type genotype (P=0.037). The potential genotoxicity/carcinogenicity of styrene needs to
be extensively evaluated in relation to the individual susceptibility factors, as the variability in styrene detoxification and
in the individual repair capacity to remove styrene-induced DNA damage.
The work was supported by Diephy FOOD-CT-2003-505609.
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ORAL PRESENTATIONS
0-127
DNA DAMAGE IN POLICEMEN FROM PRAGUE
Bozena Novotna
Jan Topinka, Radim J. Sram
Aim of study: Analysis of DNA damage in a group of city policemen at various periods of the year in order to assess the
impact of different levels of air pollution on DNA integrity.
Methods: Blood samples were taken during January and September 2004 in a group of 57 city policemen and 11 controls (policemen working in the office) from different Prague districts. All persons were non-smokers. Information about
inhalation exposure was obtained by (1) stationary monitoring of basic air pollutants during sampling periods and (2)
personal exposure monitoring of carcinogenic polycyclic aromatic hydrocarbons (cPAHs) 48 h before blood sampling.
The data were completed by detailed lifestyle questionnaire. Alkaline version of comet assay combined with enzymes
of excision repair (endonuclease III and formamidopyrimidin-DNA-glycosylase) was used for analysis of DNA damage in
peripheral lymphocytes. Statistical analysis of the results was performed using non-parametric Mann-Whitney test.
Results: Regardless the year period, policemen exposed to the city air pollution exhibited a higher level of DNA damage than their colleagues from office (p < 0.05). Within exposed group, unspecific as well as oxidative DNA damage in
samples from January exceeded significantly the values detected in September. Positive correlation between the levels
of oxidative DNA damage and cPAH exposure was observed only during winter sampling period. This finding probably
reflected an increased oxidative stress as a result of high concentrations of respirable dust particles. Control persons
did not show significant seasonal variability in the degree of DNA damage.
Conclusion: Our preliminary results confirmed that the work of city policemen in Prague is associated with increased risk
of DNA damage. The role of particular factors contributing to this risk remains to be identified.
Acknowledgements: Supported by the Czech Ministry of Environment VaV/740/5/03 and VaV-IC/6/5/04.
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NOTES:
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POSTER
PRESENTATIONS
Biomarkers and Molecular Epidemiology
P-001 - P-037
DNA Damage and Repair, Genomic Instability
P-039 - P-088
Epigenetic Mechanisms in Carcinogenesis
P-089 - P-092
Food Mutagens Impact on Health (ECNIS)
P-093 - P-106
Germ-cell Mutagenesis/Epidemiology
P-107 - P-108
Impact of Genetic Variation on Individual Susceptibility
P-109 - P-125
Invited Speakers - Nutrigenomics (ECNIS)
P-126
Mutagenicity in Higher Plants
P-127
Nutrigenomics
P-128 - P-131
Occuppational Exposure to Mutagens and Carcinogens
P-132 - P-152
Oxidative stress response
P-153 - P-165
Regulatory Genetic Toxicology
P-166 - P-178
Risk Assessment
P-179 - P-188
Threshold for Genotoxic Compounds
P-189 - P-191
Toxicogenomics
P-192 - P-197
Transgenic Models in Genetic Toxicology
P-198 - P-199
Transplacental Exposure and Health Consequences
P-200 - P-202
26.6.2006 16:05:52
P-001
BIOMARKERS OF SUSCEPTIBILITY AND EFFECTS IN HUMANS OCCUPATIONALLY EXPOSED
TO URBAN AIR POLLUTION
Sabrina Angelini 1,2
Patrizia Hrelia 1; Giorgio Cantelli-Forti 1;Fabio Carbone 1; Francesca Maffei 1;Rajiv Kumar 2;
Kari Hemminki 2;
Department of Pharmacology, University of Bologna, Bologna, Italy1, Division of Molecular Epidemiology, German Cancer Center,
Purpose. Among the potentially toxic chemicals present in the air of urban centers, benzene raises particular concern,
due to its haematoxicity and leukaemogenic risks. The aim of the present study was to investigate the relationship
between benzene exposure, biomarkers of early biological effect and biomarkers of susceptibility [NQO1, GSTT1 and
MPO genotypes].
Material & methods. Peripheral blood lymphocytes were collected from 52 policemen [mean age 39.5 ± 7.1] carrying on
different outdoor activities in high traffic area of Bologna city, and 45 indoor workers [mean age 40.1 ± 7.2] enrolled
as controls. Early biological effect were assessed by the micronucleus assays (MN).
Results. The average benzene exposure during the work-shift was significantly higher in the traffic police as compared
with controls [24.3 ± 14.4 vs 4.39 ± 0.99; P = 0.001]. MN frequency was significantly increased in policemen as compared with controls [7,13 ± 2,29 vs 5,76 ± 2,15; P = 0,018]. All the policemen were genotyped. Preliminary results
showed higher frequency of MN in subjects with the GSTT1 and GSTM1 null genotypes and the NQO1 and MPO variant
allele compared with those wild-type.
Conclusion. Results support the idea that long term exposure to urban air pollution increases DNA damage and that
polymorphisms analysis should be taken into account in understanding benzene-induced adverse health effects.
P-002
TP53 MUTATIONS AND ARG72PRO GENE POLYMORPHISM IN A HUNGARIAN PRIMARY LUNG
CANCER STUDY POPULATION
Livia Anna 1
Bernadette Schoket 1; Erika Gyorffy 1; Zoltan Gyori 2; Judit Segesdi 2; Janos Minarovits 2; Ibolya Soltesz 3;
Szilard Kostic 3; Attila Csekeo 3; Reetta Holmila 4; Kirsti Husgafvel-Pursiainen 4
Fodor Jozsef National Center for Public Health, Budapest, Hungary1; Johan Bela National Center for Epidemiology, Budapest,
Hungary2; Koranyi National Institute for Pulmonology, Budapest, Hungary3; Finnish Institute of Occupational Health, Helsinki,
Finland4
TP53 tumour suppressor gene has a regulatory role in various cellular mechanisms including response to genotoxic
stress, cell-cycle control, DNA repair and apoptosis. The aim of the study was to reveal possible associations among
TP53 mutations, TP53 Arg72Pro gene polymorphism, smoking status, and histological type of lung cancer. The study
population comprised 104 patients with primary squamous cell carcinoma or adenocarcinoma who underwent lung
resection. After screening the tumour samples for the presence of mutations in exons 5, 6, 7, 8, 9, and 11 of the TP53
gene with denaturant gradient gel electrophoresis and/or automated capillary electrophoresis single strand conformation
polymorphism, the mutations were identified by direct sequencing. Arg72Pro polymorphism was detected by PCR-RFLP
method from non-tumorous tissue. The results indicate that 46% of the tumour samples carried mutations. The highest
mutation frequency was in exon 5 (22%). In the ever smokers group, there was a significant difference in mutation
frequency between the squamous cell carcinoma and adenocarcinoma histological types (P=0.024). The most frequent
mutation types observed were G->A, G->C and G->T nucleotide changes. The predominant mutation type for never
smokers and patients who gave up smoking more than one year before the surgery was G->A. G->T mutations were
found exclusively in current smokers and in patients who gave up smoking within one year before the surgery. Regarding
the Arg72Pro polymorphism, the Pro/Pro homozygous rare variant genotype was found in the adenocarcinoma group at
10% frequency, whereas none was observed in the squamous cell carcinoma group. In conclusion the results suggest
that smoking and TP53 Arg72Pro genotype may have differential influences in non-small cell lung cancer.
Acknowledgment: The research was supported by OTKA T034616, Finnish-Hungarian S&T SF-02/01 and SF-14/03, EU
FP6 NoE ECNIS 513943.
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P-003
THE POSSIBILITY OF APPLICATION OF COMET ASSAY IN ASSESSMENT OF IN VIVO
CARDIOTOXICITY OF OXIDATIVE STRESS INDUCERS
Anna Cieślak 1
Agnieszka Bartoszek 1; Anita Piasek 1; Jolanta Łukowicz 1,2; Grażyna Peszyńska-Sularz 1,2; Stefan
Popadiuk 2; Włodzimierz Grajek 3
Dept. of Pharmaceutical Technology and Biochemistry, Gdańsk University of Technology, Gdańsk, Poland1; Medical University of
Gdańsk, Gdańsk, Poland2; Dept. of Biotechnology and Food Microbiology, The August Cieszkowski Agricultural University of Poznań,
Poznań, Poland3
This still ongoing research emerged during realisation of larger project on dietary intervention supporting cancer chemotherapy with doxorubicin. This anthracycline antibiotic belongs to the best antitumor drugs, however cardiotoxicity limits
its clinical usefulness. Therefore, it was of interest to gain some information, in a simple and quantitative way, whether
such undesirable side effects can be modulated by dietary means. Doxorubicin is known to induce oxidative stress and
also apoptosis in heart tissue. Both these events are detectable by means of comet assay, a well-elaborated and quick
test. Comet assay has been used so far for the assessment of genotoxic damage only in isolated myocytes treated ex
vivo with doxorubicin. So, in the first stage we had to develop a mild procedure of isolation of nuclei from heart tissue.
For the study, healthy or leukaemia L1210 bearing mice were treated with doxorubicin at toxic (20 mg/kg up to 20 hr
before tissue collection) or therapeutic dose (7.5 mg/kg 7 days before tissue collection) or with tert-buthyl peroxide
(1.35 mg/kg, 2 hr), a model compound inducing oxidative stress. The collected hearts were frozen in a PBS solution
containing 10% [v/v] DMSO and kept at -85°C until used. Still slightly frozen tissue was minced with Polytron and spun
down. After rinsing, the pellet was suspended in hypotonic buffer and centrifuged through concentrated Ficoll solution
to remove tissue debris. The collected nuclei were counted in cell counter and the appropriate amount was taken for
typical comet assay procedure. Since for the isolation whole hearts were taken, even at the stage of counting of nuclei,
the differences in recovery of nuclei indicative of heart damage between animals groups could be noticed. This study is
still in progress and the final detailed results will be presented, however even the initial observations suggest that comet
assay may become an easy and quick means of cardiotoxicity as well as its reversal assessment.
P-004
CYTOGENETIC BIODOSIMETRY: THE ROLE OF THE MICRONUCLEUS TEST IN MONITORING LOW
LEVEL EXPOSURE TO IONIZING RADIATION
Claudia Bolognesi 1
Cristina Balia 1; Elena Giordano 1; Paola Roggieri 1; Maria Concetta Nucci2; Vittorio Lodi 2; Francesco
Saverio Violante3
Carcinogenesis Unit, National Institute for Research on Cancer, Genova, Italy1; Environmental Servizio di Sicurezza Igiene e
Medicina del Lavoro, Universitŕ, Bologna, Italy2; Unitŕ Operativa Medicina del Lavoro, Universitŕ, Bologna, Italy3
Biological dosimetry has a very important role to contribute in the early period after an accident where many persons
may have been exposed and to monitor long term risk of low level exposure to ionizing radiation. Four cytogenetic endpoints, associated to a chromosomal damage, have been considered for biodosimetry: dicentrics, fluorescence in situ
hybridization (FISH) based translocations, prematurely condensed chromosomal breaks and micronuclei. Biodosimetry,
based on the analysis of dicentric chromosomes, has been considered the most sensitive method, however this technique is time-consuming and demands expertise. The ex vivo lymphocyte micronucleus test (MN) combined with the
evaluation of the nucleoplasmic bridges, as an index of dicentric chromosomes, allows to evaluate the complex pattern
of chromosomal alterations produced by the ionizing radiation. In recent years this technique has been proposed as a
valid and less laborious alternative for large scale studies. To further validate the use of MN test in biodosimetry, calibration curves were defined in healthy non irradiated subjects after in vitro irradiation, showing a satisfactory dose relationship for a range 0,15- 2 Gy. A biomonitoring study was carried out in in 30 hospital workers exposed to low levels of
ionizing radiation compared with a gender-, age- and smoking habits-matched control group of 30 subjects. Comparable
MN frequencies in binucleated lymphocytes was observed in control and exposed subjects. An increase, although not
statistically significant was detected in a subgroup of the mostly exposed subjects (MNBNcells/1000 cells 4,5 vs 3,7).
A statistically significant increase of MN frequency in mononucleated lymphocytes was observed in exposed subjects,
revealing the effect of a chronic exposure. Our results suggest that MN test might be considered a valid biomarker for
assessing genetic damage in populations exposed to low level of ionizing radiation.
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P-005
SEPARATION AND DETECTION OF FLUORESCENCE LABELED RNA MODIFICATIONS
BY CAPILLARY ELECTROPHORESIS WITH LASER-INDUCED FLUORESCENCE DETECTION
Michael G. Cornelius 1,2
Manfred Wiessler 1; Heinz H. Schmeiser 1
German Cancer Research Center, Division of Molecular Toxicology, Heidelberg, Germany1; Karolinska Institute, Novum, Department
of Biosciences and Nutrition, Huddinge, Sweden2
We investigated the separation and detection of the 5’-monophosphates of ribonucleosides selectively conjugated with
BODIPY-FL-EDA (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride)
at the 5’-phosphate group using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). BODIPY
conjugates of the four common nucleoside-5’-monophosphates (guanosine-5’-monophosphate, adenosine-5’-monophosphate, cytidine-5’-monophosphate and uracil-5’-monophosphate) were prepared and subjected to CE-LIF to serve
as standard compounds for peak assignment and to develop separation conditions for the analysis of RNA. Also, two
products arising from the deamination of the purinic nucleosides (inosine-5’-monophosphate and xanthine-5’-monophosphate) were included in the above procedure. All BODIPY conjugates were detected and resolved by CE-LIF after
digestion of RNA or oligonucleotides to 5’-monophosphates by nuclease P1 and fluorescence labeling without further
purification step. Comparison with two previously developed assays with BODIPY-FL-EDA postlabeling for analysis of
2’-deoxynucleoside-3’- and -5’-monophosphates showed that all three versions were equally efficient and sensitive.
Moreover, using our new assay we were able to analyze and quantify pseudouracil in samples from different types of
RNA and RNA from different organisms. This indicates that fluorescence postlabeling of nucleoside-5’-monophosphates
after digestion of RNA with nuclease P1 has the potential to measure naturally occurring modified bases and modifications from exogenous sources in RNA.
P-006
DEGREE RELATIVES
Erdem Coskun1
Gonca Demirgcigil Cakmak1; Neslihan Aygun Kocabas1; Sema Burgaz1; Faik Cetindag2; Osman Sunter 2;
Hayriye Edinsel 2
Gazi University, Faculty of Pharmacy, Dept. of Toxicology, Ankara, Turkey1, Abdurrahman Yurtaslan Oncology Hospital, Dept.of
Radiation Oncology, Head and Neck Group, Ankara, Turkey2
Studies have suggested that not only reduced DNA repair capacity (phenotype) but also genetic susceptibility (genotype) may play an important role in risk of head and neck cancer (HNC). Further support for genetic susceptibility is
evidenced by aggregation of HNC patients with their first degree relatives (FDRs). For this purpose, in our study, we
have investigated the relationship between the basal and radiation-induced micronucleus (MN) frequencies with XRCC1
gene polymorphism (Arg399Gln) both for HNC patients (n=38) and their FDRs (n=21), as well as healthy controls
(n=27). XRCC1 (X-ray repair cross-complementing group 1) is a base excision repair protein that plays a central role
in the repair of DNA base damage and strand breaks. The XRCC1 alleles were detected using PCR-RFLP technique. For
the micronucleus assay, blood samples were exposed in vitro to 2 Gy γ rays (60Co) at a dose rate of 0.62 Gy/min. HNC
patients, FDRs and controls variants had elevated but not significant basal MN levels (p>0.05). For the induced MN frequencies, variants had decreased frequencies in all groups but not different significantly (p>0.05). Smoking didn’t affect
on MN frequncies and also we did not find an interaction between Arg399Gln polymorphism and smoking (p>0.05). Our
data seemed to be supported by other studies that found a decreased risk for the genetic variation in XRCC1 codon 399
in HNC patients. However, further work is needed to resolve the importance of other polymorphisms of XRCC1 including
codon 194, as well as other DNA repair systems e.g. XRCC3 and OGG1.
This study was supported by Gazi University Scientific Research Fund (Project No. 02/2004-17)
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P-007
ROLE OF CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL LYMPHOCYTES IN POPULATIONBASED BIOMONITORING OF THE CZECH GENERAL POPULATION
Milena Cerna 1,2
Hana Bavorova 1; Jiri Smid 1; Pavel Rossner 1; Dana Ocadlikova 1
National Institute of Public Health, Prague, Czech Republic1; Charles Univ., 3rd Fac. Med., Prague, Czech Republic2
Background: In the Czech Republic, the frequency of chromosomal aberrations (CA) in human peripheral blood lymphocytes has served since 1975 as a tool to monitor selected occupational exposures. In 1994, the Human Biological
Monitoring Project was launched within a nation-wide Environmental Health Monitoring System aiming to assess the
exposure of the Czech general population to environmental contaminants. The frequency of CA was included in the
spectrum of biomarkers with the aim to monitor control levels of aberrant cells in children and adults.
Methods: Blood from blood donors (N=2801) and children aged 8-10 y (N=2353), residents for a period of not less than
2 years, was sampled repeatedly at 2-3 year intervals in four selected urban areas, following the SOP. After explanation
and signing an informed consent, each person completed a short questionnaire. Furthermore, CA were analyzed in 420
cord blood samples obtained in 1994-1995 and in the blood of 88 children aged 5-6 years. Cytogenetic analysis was
performed in short-term (50 h) cultures. In each subject, one hundred metaphases were analyzed in coded slides.
Results: In adults, a declining trend in CA frequency in the period 1993 – 1999 changed to an upward trend beginning
the year 2001. In 2003, the mean level of CA, 1.65%, was again comparable to the historical control value of 1.77 %
before 1990. In children, the levels of CA vary between 1.23% in 1993 and 0.99% in 2001. The lowest value (0.59%)
was obtained in a group of pre-school children. In cord blood, the mean level was 1.08%. Neither differences between
smokers and nonsmokers nor any significant correlation of CA with the levels of toxic or benefit elements monitored in
the same blood samples were obtained.
Conclusion: Monitoring CA in the general population represents a useful tool reflecting population exposure to environmental pollution.
P-008
POLYMORPHISMS IN DNA REPAIR GENES, BIOMARKERS OF DNA DAMAGE AND PROCESS
OF AGING
Zuzana Džupinková
Ladislava Wsólová; Mária Dušinská
Background/Aims: Aging is an inevitable consequence of being a multicellular organism, associated with a decline in
physiological function and resulting in death. Polymorphisms have been identified in a number of critical genes that may
influence the processes of aging, risk of many diseases, including cancer and also susceptibility to many environmental
risk factors. Aim of our project “THE ROLE OF GENETIC PREDISPOSITION, IMMUNE SYSTEM AND NUTRITION IN AGING”
is to show if and how the polymorphisms in DNA repair genes can influence repair processes in human and if there is
any relation to human aging.
Methods: Blood samples were collected from 151 young people (20–25 years) and 140 seniors (65–70 years). PCRRFLP assays were used to determine eleven polymorphisms in genes of base excision repair (XRCC1, APE1, hOGG1);
nucleotide excision repair (XPD, XPA, XPC); homologous rejoining (NBS1), and direct damage reversal (MGMT). DNA
damage (strand breaks base oxidation and alkylation) and individual DNA repair capacity were measured in lymphocyte
using the comet assay.
Results: Polymorphism frequencies did not differ between young and old subjects. FPG-sensitive sites were higher in
young group compared with seniors (p=0.012). Based on multifactorial analysis of variance we found an interesting
correlation between polymorphisms in genes involved in NER and markers of oxidative DNA damage; the highest level
of FPG-sites was observed in the subjects with wild type of XPC gene in intron 9 and simultaneously with wild type of
NBS1 gene.
For the other NER genes, subjects homozygous to wild type alleles Asp312 and Lys751 in the XPD gene were more
sensitive to H2O2 treatment than variant type (p=0.023 and p=0.034).
Conclusions: These associations suggest a surprising and still unexplained involvement of nucleotide excision proteins
in repair of oxidative base damage.
Supported by SSTA Agency; contract no. APVT-21-013202.
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P-009
ASSESSMENT OF BIOMARKERS IN CERVICAL CANCER PATIENTS
Timea Farkasová 1
Soňa Gurská 1; Alena Gábelová 1; Zuzana Macháčková 2; Pavol Lukačko 2
Cancer Research Institute, Bratislava, Slovakia1; National Cancer Institute, Bratislava, Slovakia2
The present study is designed to find a correlation between a variety of biomarkers associated with individual radiosensitivity/radioresistence response among cervical cancer patients. The biomarkers presented are: the level of initial and
residual DNA damage, the kinetics of DNA strand break rejoining, radiation dose response, genetic variation of DNA
repair genes and their contribution to the repair phenotype.
The in vitro alkaline comet assay has been used to analyze radiation-induced DNA damage, DNA repair and radiation
dose response effect in peripheral blood lymphocytes. Genetic polymorphism of various DNA repair genes (XRRC1, PARP,
hOGG1) was analyzed by RFLP- PCR assay in the group of patients and controls. Until now we analyzed 18 cervical
cancer patients and controls as well.
Even though the number of subjects involved in the study was very small, we tried to determine which biomarker has
the strongest correlation to the clinical outcome of these patients. The initial DNA damage, kinetics of DNA strand
break rejoining, residual DNA damage, radiation dose response itself have not shown positive association to radiation
response.
However, we observed some weak correlations between combination of variant genotype and in vitro radiation response
in case of combining genotypes of repair genes with biomarkers outcomes. The variants of XRCC1 and OGG1 indicate
lower slopes of the dose response curves, but no correlation occurred for PARP. We found find a slower DNA rejoining
only in the variants of XRCC1. There was no association between polymorphisms of these genes and initial DNA damage.
To verify these findings it is necessary to enlarge the group of subjects.
This study was supported by the project 2003 SP 51 028 08 00/028 08 01 from the national program Use the Cancer
Genomics to Improve the Human Population Health.
P-010
RELATIONSHIP BETWEEN DNA REPAIR GENETIC POLYMORPHISMS AND SMOKING-RELATED
DNA ADDUCT LEVELS IN HUMAN BRONCHIAL TISSUES
Erika Gyorffy 1
Livia Anna 1; Bernadette Schoket 1;Zoltan Gyori 2; Judit Segesdi 2; Janos Minarovits 2; Ibolya Soltesz 3;
Szilard Kostic 3; Attila Csekeo 3
Jozsef Fodor National Center for Public Health, Budapest, Hungary1; Bela Johan National Center for Epidemiology, Budapest,
Hungary2; Koranyi National Institute of Pulmonology, Budapest, Hungary3
The formation of tobacco smoke-derived DNA adducts is considered a key event in the initiation of lung cancer that
may be influenced by genetic susceptibility factors. The aim of the study was to investigate the modulation of smoking-related aromatic DNA adduct levels in human bronchial tissue by the XPG His1104Asp, XPD Lys751Gln and XRCC1
Arg399Gln DNA repair genetic polymorphisms. The study population consisted of 223 Hungarian patients who underwent lung resection. Levels of aromatic DNA adducts in macroscopically normal bronchial tissue were determined by
the 32P-postlabelling method with nuclease P1 enrichment. Genotypes were detected by PCR-RFLP methods. DNA adduct
level of smokers (current smokers and those who had given up smoking within one year before surgery, n=146) was
9.46±5.3, of non-smokers (life-time non-smokers and those who had given up smoking more than one year, n=77) was
5.25±3.2 respectively (P<0.0001). Genotype frequencies in the study population were the followings: for XPG 32.9%
His1104His, 50.5% His1104Asp, 16.6% Asp1104Asp; for XPD 61.1% Lys751Lys, 33.0% Lys751Gln, 5.9% Gln751Gln;
for XRCC1 34.5% Arg399Arg, 55.2% Arg399Gln, 10.3% Gln399Gln. Neither XPG nor XPD nucleotide excision repair
polymorphisms influenced the bronchial adduct levels in smokers and non-smokers. No apparent influence of the
adduct levels was observed for XRCC1 base excision repair genetic polymorphism. There was no detectable influence
of the three genetic polymorphisms in various combinations on DNA adduct levels. There was no significant influence
of these gene polymorphisms on daily cigarette dose-related adduct levels and on estimated adduct elimination rate in
ex-smokers.
The research project has been supported by the Hungarian OTKA T 034616 research grant; Hungarian-Finnish InterGovernmental S&T Cooperations SF-02/01 and SF-14/03; EU FP6 ECNIS 513943 NoE.
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P-011
PAPILLARY THYROID CARCINOGENESIS AMONG ATOMIC-BOMB SURVIVORS
Kiyohiro Hamatani
Hidetaka Eguchi; Masataka Taga; Kei Nakachi
RERF (Radiation Effects Research Foundation) epidemiological studies of atomic-bomb survivors (A-bomb survivors)
have indicated that excess relative risk of papillary thyroid carcinoma (PTC) increased in radiation-exposed survivors
than in the non-exposed depending with radiation dose. However, molecular mechanisms of radiation-associated thyroid carcinogenesis have not yet been elucidated. Two findings suggest that RET oncogene rearrangements may be
important in the development of radiation-associated thyroid cancer: RET rearrangements (RET/PTC) directly induced
by X-irradiation in vitro and in vivo, and RET/PTC observed in 60%-80% of PTC in post-Chernobyl children and in
patients with history of radiation therapy. On the other hand, BRAF point mutation is detected at high frequency in PTC
from adult patients without history of radiation therapy, and is thought to also be involved in the early stages of PTC.
To clarify the mechanisms of PTC occurred in A-bomb survivors, we studied the association of RET/PTC and BRAFV600E
mutation with radiation dose and latency period (time period from A-bomb exposure to diagnosis) in 86 PTC A-bomb
survivors. First, we found that median radiation dose in PTCs with RET/PTC was significantly higher than that without
RET/PTC (P = 0.001). Second, median radiation dose in PTCs with BRAFV600E mutation was significantly lower than that
without the mutation (P < 0.001). A significant difference was found median latency period between exposed patients
with and without BRAFV600E mutation (31 vs. 20 yrs, P < 0.001). In addition, RET/PTC and BRAFV600E mutation were mutually exclusive (P < 0.001). These results suggested that RET/PTC or BRAF mutation might be involved more or less in
thyroid carcinogenesis in A-bomb survivors exposed to high radiation dose, respectively.
P-012
PROTEIN ADDUCTS FORMED IN VITRO BETWEEN HUMAN HAEMOGLOBIN AND DEUTERIUM
LABELLED PHTHALIC ACID ANHYDRIDE, CHARACTERIZED WITH MASS SPECTROMETRY
Marina C Jeppsson
Bo A.G Jönsson; Christian H Lindh
Department of occupational and environmental medicine, Lund, Sweden
Organic acid anhydrides (OAAs) are low molecular weight molecules that are highly reactive and widely used in the
industry. Exposure of OAAs can cause asthma and/or rhinitis, conjuctivities and cancer. In this study we characterized
adducts between haemoglobin and the OAA phthalic acid anhydride (PA). The primary use of PA is as a chemical intermediate in the production of phthalate esters, which function as plasticizers. Using protein adducts as a biomarker for
exposure assessment of carcinogenic and genotoxic compounds was suggested 1974 by Ehrenberg et al. In vitro conjugates were made with human haemoglobin (Hb) and deuterium labelled PA in order to identify the amino acids forming
adducts with PA. Three conjugstes with different molar ratio were made; 1:10, 1:1 and 1:0.1 (Hb:PA). Nanospray-quadropole-time of flight mass spectrometer and liquid chromatogapy- triple quadropole mass spectrometer was used for
analysis. The conjugate with molar ratio 1:0.1 (Hb:PA) is the most relevant conjugate since it is the one closest one to
real exposure levels. It was found that Hb-PA conjugate with molar ratio 1:0.1 are adducted to four amino acids in the
α-chain (val1, lys16, lys40 and lys61) and to three amino acids to the β-chain (val1, lys66 and lys144). We also identified that
PA can bind to the amino acids in two forms, as phthalamide and as phthalimide. The phthalimide adduct was found on
all the lysine residues mentioned above but not on the valine residues. The conclusion is that valine and lysine are the
main amino acids adducted. The phthalimide protein adduct has not been described before. This knowledge could be
used in the development of biomarkers of PA-exposed workers.
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P-013
THE ABILITY OF Γ-TOCOPHEROL TO DECREASE OXIDATIVE DNA DAMAGE
Clara Johansson 1
Lennart Möller 1; Jonas Nygren 2; Bengt Vessby 3
Unit for Analytical Toxicology, Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden1; Laboratory of
Molecular and Cellular Toxicology, Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health,
Helsinki, Finland2; Department of Public Health and Caring Sciences, Clinical Nutrition and Metabolism, Uppsala University, Uppsala,
Sweden3
Background: Vitamin E consists of related substances such as α-tocopherol (αT) and γ-tocopherol (γT). Vitamin E supplements often contain only αT which has been questioned since αT has been shown to decrease the level of γT. Recent
studies indicate that γT is important in preventing oxidative stress and has properties not shared by αT. Aim: The aim
of this study was to examine if oxidative stress can be decreased by consumption of supplements designed to imitate
a natural healthy diet, containing more than 20 different antioxidants including αT and γT. Method: For six weeks, middle-aged, over-weight men consumed antioxidant supplements. The participants were randomly divided into a control
group consuming placebo, a low dose group consuming roughly half the recommended intake and a high dose group
consuming approximately the recommended daily intake of these 20 antioxidants. Oxidative DNA damage was measured with the Comet assay before and after antioxidant administration. An in vitro study was also performed in order
to investigate the ability of γT to protect cells against DNA damage. Result: There was a significant increase in the level
of γT, after antioxidant administration in both dose groups. Furthermore, the average level of oxidative DNA damage in
the high dose group after administration of antioxidants was significantly lower than in the control group. In the in vitro
study there was a significantly lower level of oxidative DNA damage, induced by peroxynitrite in cells pre-incubated
with γT compared to controls. Conclusions: The low doses of standardized fruit and vegetable extracts protected middle-aged men against oxidative DNA damage, at doses similar to normal dietary intake. Further, the protection against
DNA damage was correlated to an increase in the plasma level of γT but not αT. This suggests that γT might be a key
substance to reduce oxidative stress.
P-014
THE EFFECTS OF GSTM1 AND GSTT1 POLYMORPHISMS ON MICRONUCLEUS FREQUENCIES
IN HUMAN LYMPHOCYTES IN VIVO
Raluca Mateuca 1
Mathieu Roelants 2; Michael Fenech -leader of the HUMN project 3; Micheline Kirsch-Volders 4
Vrije Universiteit Brussel (VUB), Brussels, Belgium1; Vrije Universiteit Brussel (VUB), Brussels, Belgium2; CSIRO Health Sciences
and Nutrition, Adelaide, Australia3; Vrije Universiteit Brussel (VUB), Brussels, Belgium4
The influence of genetic polymorphisms in GSTM1 and GSTT1 genes on micronucleus (MN) frequencies in human
peripheral blood lymphocytes was assessed through a pooled analysis of data from seven laboratories which performed
biomonitoring studies using the in vivo cytokinesis-block MN assay. A total of 301 non-occupationally exposed individuals (207 males, 94 females) and 343 workers (237 males, 106 females) occupationally exposed to known or suspected
genotoxic substances were analysed by Poisson regression.
The results of the pooled analysis indicate that the GSTT1 null subjects had lower MN frequencies than their positive counterparts in the total population (FR = 0.55; 95%CI. = 0.33-0.89). The protective effect of this genotype is
reversed with increasing age, with a FR of 1.33 (95%CI. = 1.06-1.68) in subjects aged 60. A significant overall increase
in MN frequency with age and gender (p <.001 and p = .024, respectively) was observed, females having higher MN
frequencies than males, when occupationally exposed (p = .002). Non-occupationally exposed smokers had lower MN
frequencies than non-smokers (p = .001), while no significant difference in MN level was observed between smokers
and non-smokers in the occupationally exposed group (p = .79).
This study confirms that pooled analyses, by increasing the statistical power, are adequate for assessing the involvement
of genetic variants on genome stability and for resolving discrepancies among individual studies.
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P-015
LONG-TERM ELEVATION OF INFLAMMATORY MARKERS AMONG ATOMIC-BOMB SURVIVORS:
ACCELERATED IMMUNOLOGICAL AGING
Yukari Morishita
Tomonori Hayashi; Hiroko Nagamura; Mayumi Maki; Yoshiko Kubo; Yoichiro Kusunoki; Kei Nakachi
Even after more than half a century has passed, exposure to atomic-bomb radiation continues to have long-term health
effects on survivors. It is known through numerous reports that among patients exposed to high-dose radiation in accidents or medical treatment acute inflammatory reactions occur several days following the exposure, but the long-term
late effects of radiation on inflammation are yet to be clarified. In this study, we measured proportions of peripheral
lymphocyte subsets and plasma levels of inflammatory markers and immunoglobulins to comprehensively determine
their relationship with radiation dose. Excluding patients with history of cancer, rheumatoid arthritis, chronic bronchitis,
or myocardial infarction, we selected 442 study subjects randomly from the Adult Health Study population consisting
of atomic-bomb survivors and unexposed people. We observed statistically significant elevation of the following markers with increased radiation dose: (1) Inflammatory cytokines (IL-6, TNF-α, IFN-γ, and IL-10), (2) CRP, total ROS, and
erythrocyte sedimentation rate (ESR), and (3) Immunoglobulin levels. Also observed was a decreased percentage of
CD4T cells with increased radiation dose. Since both radiation exposure and aging were related to the elevation of most
of the inflammatory markers examined in this study, we conducted our review by converting radiation effects to an
acceleration of aging. Namely, judging from IL-6, TNF-α, IL-10, CRP, total ROS ESR, and total Ig, exposure to 1 Gy of
radiation was found to correspond to about 9 years of aging. These results suggest that atomic-bomb radiation might
have accelerated inflammation status, which is usually promoted by aging.
P-016
DNA-ADDUCT COMPARISON BETWEEN 3- AND 2-NITROBENZANTRONES IN RATS
Nagy Eszter 1
Möller Lennart 1; Duan Jianxin 1; Zeisig Magnus 1;Adachi Shuichi 2;Takamura-Enya Takeji 3;
Karolinska Institutet, Huddinge, Sweden1; Sagami Women's University, Kanagawa, Japan2,National Cancer Center Research
Institute, Tokyo, Japan3
Content: Background: 3-nitrobenzanthrone (3-NBA) is a nitro-PAH, formed during the combustion of diesel fuel, which
is genotoxic and carcinogenic in in vitro and in vivo models, respectively. 3-NBA exists in high concentrations near the
emission source but rearranges to its more abundant isomer 2-nitrobenzanthrone (2-NBA), which in in vitro studies is
1/3 as genotoxic as 3-NBA, and also causes oxidative stress.
Aim: In this study, rats were intratracheally administered both 3-NBA and 2-NBA, in order to evaluate the genotoxic
potential of the substances. Two different human cell lines (A549 and HepG2) were also exposed to 3-NBA and 2-NBA
in order to compare the DNA adduct patterns in vitro and in vivo. 3D models of characterized 3-NBA DNA-adducts were
also generated using Molecular Modeling to examine the binding of the adducted moiety to DNA.
Results: 2-NBA is somewhat less potent in cells lines and mammals compared to 3-NBA, but the DNA adduct pattern
is similar in both models. While DNA adduct formation after 3-NBA exposure in vivo first rapidly increases and then
decreases with time, the formation after 2-NBA exposure display a slow, but continuous increase.
Molecular Modeling of a tredecamer model of standard B-DNA, with the introduced 3-NBA-derived DNA adducts in the
sequence, showed that these 3-NBA-derived DNA adducts are either positioned orthogonally to the base pair in the
major groove or clammed in the minor groove.
Conclusion: Different model systems showed that the same type of DNA adducts were formed in cells as in tissues. The
less soluble and more stable 2-NBA isomer seemed to be stored longer in the body compared to 3-NBA, which raises
the question if it has the potential to bioaccumulate and thereby pose a greater and more long-term threat to health
compared to 3-NBA.
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P-017
THE USE OF BIOMARKERS OF EXPOSURE TO POLYCYCLIC AROMATIC HYDROCARBONS (PAHs)
IN PSORIATIC PATIENTS UNDERGOING COAL-TAR THERAPY
Anna Pastorková 1
Marek Brabec 1; Milena Černá 1,2; Lenka Borská 3; Zdeněk Fiala 3
National Institute of Public Health, Prague, Czech Republic1; Charles Univ., 3rd. Fac. Med., Prague, Czech Republic2; Charles Univ.,
Fac. Med., Hradec Králové, Czech Republic3
Background: Psoriasis is a chronic proliferative skin disease affecting 1-2% of the world’s population. Goeckerman’s
method which combines dermal application of coal tar ointment and UV radiation, is often used as the first option of
treatment. PAHs in coal tar may represent a health risk for patients due to their genotoxicity. Therefore, several biomarkers of exposure were chosen to confirm exposure to PAHs in treated patients. Methods: Urine and blood samples of
23 treated patients (age16-78, 14 male, 9 female; 9 smokers) hospitalized during treatment (8-30 days), were collected before, in the middle and after final application of 3-5% therapeutic coal tar ointment. The range of ointment
applications varied from 32-77% of body surface. The biomarkers used were: (a) urinary mutagenicity using Salmonella
typhimurium TA98 and YG1041 strains in the plate incorporation assay after incubation with β-glucuronidase, (b) determination of 1-OH pyrene by GC/MS, and (c) cytogenetic analysis of human peripheral lymphocytes. Urinary levels of
creatinine were analyzed spectrophotometrically. Results: Before therapy, mutagenicity was observed in 7 urine samples
(TA98+S9, mean 2.8 rev/mg creatinine), in 14 samples (YG1041-S9, mean 5.8 rev/mg creatinine) and in 22 samples
(YG1041+S9, mean 13.5 rev/mg creatinine) respectively. The maximum increase of mutagenicity was observed in the
middle of therapy using YG1041+S9 strain (23 samples, mean 86.6 rev/mg creatinine). Levels of 1-hydroxypyrene
varied from 0.3 to 97.4 with mean values of 1.2 µg/mmol/creatinine before therapy and 2.8 and 2.7 µg/mmol creatinine during and after therapy, respectively. No correlations between 1-OH pyrene levels, chromosomal aberrations
and urinary mutagenicity values have been found. Conclusion: The urinary mutagenicity test remains a suitable tool to
monitor population exposure to genotoxic carcinogens. Supported by Research Project No. 00179906 and Grant IGA NR
8154-3 of the Czech Ministry of Health
P-018
Beatriz Pérez-Cadahía 1,2
Josefina Méndez 1; Blanca Laffon 1,2; Eduardo Pásaro 2; Joao Paulo Teixeira 3; Susana Silva 3; Joana
Roma-Torres 3; Olga Mayan 3
Department of Cell and Molecular Biology, University of A Coruńa, La Coruńa, Spain1; Toxicology Unit, University of A Coruńa, La
Coruńa, Spain2; Environmental Health and Toxicology Department, National Institute of Health, Porto, Portugal3
In the environment of tyre producing plants presence of several organic solvent vapours and airborne particulate matter
has been characterized. In addition, elevated mutagenicity and genotoxicity have been observed in air and particulate
samples. The objective of this study was to determine the genotoxic effects in a group of individuals engaged in the
production of rubber tyres from a Portuguese factory. Urinary thioethers were measured as biomarker of exposure.
Peripheral blood samples were collected from 32 exposed workers and 32 controls, and micronucleus (MN) test and
sister chromatid exchanges (SCE) were performed to evaluate the genotoxicity. Microsomal epoxide-hydrolase codons
113 and 139 polymorphisms were determined to estimate the expected activity of the enzyme. Thioethers excretion was
found significantly higher in rubber workers. No significant difference was observed in MN or SCE frequencies between
exposed and controls. Higher levels of cytogenetic rates were obtained in epoxide-hydrolase expected low activity
donors with regard to medium and high activity individuals.
This work was partially supported by the Comissão de Fomento ŕ Investigação em Cuidados de Saúde and by a grant
from the Xunta de Galicia (PGIDT04PXIB10602PR).
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P-019
MOLECULAR AND GENETIC CHARACTERISTICS OF SPORADIC COLORECTAL CANCER
IN THE CZECH REPUBLIC
Veronika Polakova 1
Monika Hanova 1; Elena Tulupova 1; Jana Slyskova 1; Alessio Naccarati 1; Barbara Pardini 1; Pavel Erik
Vodicka 1; Ludmila Vodickova 1,2; Jan Novotny 3, Kari Hemminki 4
Institute of Experimental Medicine, Academy of Sciences of Czech Republic, Praha, Czech Republic1; National Institute of Public
Health, PRAHA, Czech Republic2; First Medical Faculty, Charles University, Praha, Czech Republic3; German Cancer Research Centre,
Colorectal cancer (CRC) is a common neoplasia in both men and women with estimated risk of 5% worldwide. The
Czech Republic ranks first in the incidence of rectal cancer and third in the incidence of colon cancer. Inherited deficiency in DNA repair and cell cycle control have been associated with an individual´s susceptibility to sporadic CRC.
Polymorphisms in DNA repair genes may modulate DNA repair capacity and, together with polymorphisms in genes
involved in cell cycle control may affect the risk of CRC.
We examined the association between polymorphisms in BER and NER DNA repair genes: XPD codon 751, XPC codon
949, XPG codon 1104, XRCC1 codons 194, 399, XRRC3 codon 241, hOGG1 codon 326, APE1 codon 148 and CRC, in a
population-based case-control study (525 cases and 525 controls, age- and sex-matched) using PCR-RLFP as well as
realtime-PCR allelic discrimination with TaqMan probes. The methods for the determination of polymorphism in genes
involved in cell cycle, such as CCND1, and cell cycle control genes, such as p53, p21 are currently under progress.
The results of our study demonstrate that XPG 1104Asp/His (OR 1,31 , 95% CI 1,03-1,68) and APE1 148Glu (OR 1,43,
95% CI 95% 1,12-1,82) alleles are related to the higher risk of CRC.
We also found that the 194Arg/Trp genotype of XRCC1 as well as 399Gln genotype of XRCC1 are associated with lower
risk of CRC. Our results are in agreement with recent findings in Taiwanians, though an increased risk of some types of
cancer has been mostly reported for XRCC1 399Gln.
Polymorphisms in other tested repair genes (XPD, XPC, XRCC3, hOGG1) did not show any association with the risk of
sporadic CRC.
Grant GACR No. 430/371 is hereby acknowledged.
P-020
DETECTION OF A METHYLGLYOXAL DNA ADDUCT BY HPLC COUPLED TO TANDEM MASS
SPECTROMETRY
Jean-Luc Ravanat
Lucie Mollard; Carine Badouard; Alain Favier
CEA Grenoble, Grenoble, France
Diabetes mellitus is the most common metabolic disorder and has late complications, including inflammation and oxidative stress, due to chronic hyperglycaemia. Methylglyoxal (MG), a known carcinogenic alpha-keto-aldehyde has been
shown to be highly produced in diabetic patients. Attempts were made in the present work to detect and quantify DNA
lesions induced by MG.
Our work was focused on the adduct generated by reaction of MG with 2'-deoxyguanosine (dGuo), one of the four DNA
nucleosides. We have shown that the generated adduct, named dGuo-MG that exists as two diastereomers, is not stable
and that its decomposition restores a mixture of dGuo and MG. Therefore, in order to be able to measure the MG adduct
in DNA, our strategy consists in converting the unstable adduct to a more stable one by a chemical reduction using
sodium borohydride. Then a sensitive HPLC-MS/MS method was developed to detect the corresponding stable nucleoside
dGuo-MGred, using an isotopically labelled internal standard. To measure the lesion in DNA incubated with increasing
concentration of MG, the MG adduct is first converted to stable dGuo-MGred and then DNA is digested enzymatically.
HPLC-MS/MS analysis of the resulting DNA hydrolysate shows that the amount of dGuo-MGred detected increases
linearly with the concentration of MG. Such an approach has been also used to detect the adduct in cells treated with
MG. For this purpose, reduction step is performed just after DNA isolation and prior to digestion. We have shown that
incubation of THP1 (human lymphocytes cell line) with MG is able to induce the formation of a MG-DNA adduct detected
as its reduction product dGuo-MGred. Work is in progress in order to measure the level of the dGuo-MG adduct, subsequently to reduction, in the DNA of lymphocytes isolated from diabetic patients.
151
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P-021
CHROMOSOMAL ABERRATIONS MEASURED BY FISH PAINTING AS BIOMARKER OF AIR
POLLUTION EXPOSURE – PROSPECTIVE STUDY OF POLICEMEN IN PRAGUE
Andrea Rössnerová
Olena Beskid; Pavel Rössner; Ivo Solanský; Radim J. Sram
Institute of Experimental Medicine, Prague, Czech Republic1
The effect of exposure to carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) adsorbed onto respirable air particles
(PM2.5, <2.5 mm) on chromosomal aberrations analyzed by fluorescence in situ hybridization (FISH, whole chromosome painting of #1 and #4) and conventional cytogenetic analysis (CCA) was studied in a group of 115 policemen.
Personal exposure was evaluated using personal samplers during work shift. Sampling periods were January, March,
June, and September in 2004. Personal monitoring data indicated that highest concentrations of c-PAHs were during
January – 1.58.ng/m3 for B[a]P and 9.07 ng/m3 for c-PAHs. In those periods, the mean frequency of translocations
measured by FISH (FG/100) was 1.32±1.07 vs.0.87±0.81 (p<0.05), by CCA 2.07±1.48 vs. 1.84±1.42 %AB C (not
statistically significant).
Translocations measured by FISH are supposed to be stable for the long period of time. In our study the frequency of
translocations decreased within the period of few months, and correlated with c-PAHs and DNA adducts.
Our previous results indicated that substantial decrease in the exposure to ethyl benzene and benzene in the course of
more than 6 months can also decrease the level of stable translocations. We can hypothesize that cells carrying several
translocations do not circulate for years, but may by also eliminated as cells carrying chromosomal breaks. It seems
to be pertinent to use FISH analysis repeatedly on the same subjects, trying to specify the relationship between the
exposure and effect.
Supported by the Czech Ministry of Environment VaV-SL/5/160/05 and by the AS CR 1QS500390506.
P-022
32P-POSTLABELLING ANALYSIS OF THYMINE DIMER IN URINE FROM CHILDREN
OF DIFFERENT SKIN TYPES
T I Sandberg 1
D.Segerbäck 1; N.Kotova 2; P.C.Turner 3; C.P.Wild 3; A.Sylla 4; M.S.Diallo 4
Karolinska Institute, Huddinge, Sweden1; Stockholm University, Stockholm, Sweden2; University of Leeds, Leeds, United Kingdom3;
Institut Pasteur de Guinée, Kindia, Republic of Guinea4
Formation of UV induced DNA lesions is associated with the development of different forms of skin cancer. The predominant lesion induced is a thymine-thymine (T=T) dimer which following DNA repair can be released intact into the urine.
A highly sensitive 32P-postlabelling assay has been developed, and it was shown that the amount of excreted T=T in
urine was directly correlated to the UV dose received.
Epidemiological studies show that dark skin is highly protective against skin cancer. In this study we compared urinary
T=T levels in children of different skin types after exposure to natural sunlight. Urine samples were collected from
Swedish children of Caucasian origin and in black children from Guinea, West Africa.
The T=T levels in the Guinean children did not differ dramatically from that of Swedish children in spite of the higher
UV exposure in Africa. A preliminary assessment of the difference after correction for the different UV doses received
by the two populations indicate that the amount of lesions in the African children were about one order of magnitude
less than in the Swedish children. This protection factor of black skin is in line with what has been found when analysing DNA damage in skin earlier, and shows that other factors than just DNA damage are important to determine the
risk of skin cancer.
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26.6.2006 16:05:55
P-023
DEVELOPMENT OF A MICRONUCLEUS ASSAY FOR MOUSE ALVEOLAR TYPE II CELLS
Hanna Sandvik 1
Ghita Falck 1; Tiina Santonen 1; Hannu Norppa 1; Julia Catalán 1,2
Finnish Institute of Occupational Health, Helsinki, Finland1; University of Zaragoza, Zaragoza, Spain2
Alveolar epithelium is composed of type I and II pneumocytes. Alveolar type II cells occupy only about 5 % of the lung
surface area, but have an important role in producing, secreting, and recycling lung surfactant. Unlike type I cells, type
II cells are able to divide, e.g., when damaged epithelium is repaired after lung injury. The aim of the present study
was to develop a micronucleus assay to detect genotoxic damage after inhalation exposure in mouse alveolar type
II cells, potential target cells for lung carcinogens. As a model carcinogen, we used ethylene oxide, a highly reactive
alkylating agent which has been shown to induce micronuclei in various in vivo and in vitro assays. Ethylene oxide is
used as a sterilising agent, e.g., in hospitals and as an intermediate in the chemical industry. Five male C57BL/6J mice
were exposed to ethylene oxide via inhalation at a concentration of 600 mg/m3 for 4 h. Five unexposed control mice
were also included. 72 h after the end of the exposure, the lungs of the mice were lavaged with a trypsin solution
and chopped with scissors. The cell suspension was filtered, and the isolated cells were plated on chamber slides and
allowed to attach for 48 h at 37 °C. To enhance the specificity of the assay, bronchiolar Clara cells, which are isolated
together with type II cells, were discriminated by blue tetrazolium staining. The frequency of micronuclei was scored,
using DAPI staining, in 2000 blue tetrazolium -negative cells (defined as type II cells) per mouse. A very clear (about
14-fold) increase in the frequency of micronucleated cells was seen in the ethylene oxide -exposed mice as compared
with the controls. Thus, mouse alveolar type II cells seem to be suitable for micronucleus analysis in studies aimed at
identifying genotoxic lung carcinogens.
P-024
DEVELOPMENT OF A LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY METHOD
FOR THE DETECTION AND QUANTITATION OF BENZO[A]PYRENE DERIVED DNA ADDUCTS
Rajinder Singh 1
Peter B Farmer 1; Margaret Gaskell 1; Rachel C Le Pla 1; Balvinder Kaur 1; Ali Azim-Araghi 1; Jonathan
Roach 1; George Koukouves 2; Vassilis L Souliotis 2; Soterios A Kyrtopoulos 2
Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester, University Road, Leicester, LE1 7RH, United Kingdom1,
Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece2
The polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P) is a proven animal carcinogen that is potentially carcinogenic to humans. B[a]P is an ubiquitous environmental pollutant as well as being present in tobacco smoke, coal
tar, automobile exhaust emissions and charred food. A liquid chromatography-tandem mass spectrometry (LC-MS/MS)
method using electrospray ionization and selected reaction monitoring has been developed for the detection of 10(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE-N2dG) adducts formed in DNA
following the metabolic activation of B[a]P to benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (B[a]PDE). The method
involves enzymatic digestion of the DNA sample to 2′-deoxynucleosides following the addition of a stable isotope internal
standard, [15N5]B[a]PDE-N2dG and then solid phase extraction to remove unmodified 2′-deoxynucleosides prior to analysis by LC-MS/MS. The limit of detection of the method was 10 fmol (~ 3 B[a]PDE-N2dG adducts per 108 2′-deoxynucleosides) using 100 µg of calf thymus DNA as the matrix. Three stereoisomers of the B[a]PDE-N2dG adduct were detected
following the reaction of calf thymus DNA with B[a]PDE in vitro. The levels of B[a]PDE-N2dG DNA adducts in mice livers
following intraperitoneal dosing with 50, 100 and 200 mg/kg B[a]P were found to increase in a dose dependent manner,
with adducts reaching maximal levels at 1 to 3 d and then gradually decreasing over time but still detectable after 28
d. A very good correlation (r = 0.962, p < 0.001) was observed between the results obtained for the mouse liver DNA
samples using LC-MS/MS compared to those obtained using a 32P postlabeling method. However, the levels of adducts
observed following 32P postlabeling using butanol enrichment where ~3.7 fold lower.
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P-025
INTER-STRAND DNA CROSS-LINKS IN THE PIG SKIN TREATED WITH SULPHUR MUSTARD
IN VIVO
Rudolf Stetina 1
Frantisek Oplustil 2
Faculty of Military Health Sciences, Hradec Kralove, Czech Republic, Hradec Kralove, Czech Republic1, Military Technical Institute for
Protection, Brno, Czech Republic, Brno, zech Republic2
The modiffied comet assay (single cell gel electrophoresis) was used for the meassurement of cross- links in DNA
induced with sulphur mustard (SM). The method was calibrated first using mouse lymphocytes or He La cells cells
treated with SM in vitro and with styrene 7,8- oxide to introduce DNA breaks. While in control cells untreated with SM
the alkaline DNA unwinding (measured as the % DNA in comet tail) was almost complete, in cells treated with SM a
dose – dependent decrease of DNA unwinding was found. This efect of SM on the DNA unwinding was linear within the
broad range of concentrations 0,16 – 5μM. In cells treated with concentrations higher then 5 μM the number of crosslinks is high and no DNA unwinding is observed.
SM was applied to the pig skin using silicone rubber matrix contaminated with 3 μl SM, which were left in contact with
the skin for different period of time ( 4-60 min.). The amount of the resorbed SM was time dependent. Samples of
exposed skin were excised and cells were isolated using the colagenase treatment. The relative amount of cross-links
was measured in these cells by the modified comet assay described above. Results showed approximately linear dosedependence of induced cross-links within the range of absorbed doses of SM (6-110 μg). Cross-links in the skin could
be valuable biomarker of exposition to SM.
P-026
CARCINOGENICITY TESTING BY A FLUORESCENCE-BASED RECOMBINATION ASSAY IN HUMAN
CELLS.
Lisa Wiesmueller
Nuray Akyuz; Cindy Baumann
Department of Obstetrics and Gynaecology at the University of Ulm, Ulm, Germany
Genotoxicity tests available today have several shortcomings. Thus, the most widely applied Ames Assay measures
point mutations in bacteria, thereby disregarding larger genome alterations and the physiological particularities of
human cells. Cytogenetic assays in mammalian cells miss small genome rearrangements and require additional drug
treatment, fixing, staining and microscopic analysis. Since there is evidence that deregulated recombination processes
are associated with cancer and with a broad spectrum of carcinogenic treatments (Schiestl, 1989), we exploited DNA
recombination as the endpoint for genotoxicity testing. We designed a fluorescence based assay for probing recombination in living cells (Akyüz et al., 2002). The test utilizes primary and immortalized cells from different mammalian
species and organs, is characterized by a fast response and fulfills the requirements of automated performance. We
detected genotoxicities in human cells after gamma-irradiation as well as after treatment with the antineoplastic drug
etoposide, which causes DNA breaks and is negative in the reverse mutation assay in Salmonella strains (Akyüz and
Wiesmüller, 2003). Recombination inducibility was similarly detected after treatment with chemotherapeutic drugs from
different classes, i.e. with the topoisomerase I inhibitor camptothecin, the alkylating agent MNNG, replication inhibitors,
and potentially carcinogenic drugs that do not directly damage DNA or affect metabolization. These and further data
provide proof of principle that the assay detects Ames Assay positive and negative carcinogenic compounds. References:
Akyuz N, Wiesmuller L (2003) ALTEX 20, 77-84. Akyuz N, Boehden GS, Susse S, Rimek A, Preuss U, Scheidtmann KH,
Wiesmuller L (2002) Mol Cell Biol 22, 6306-6317. Schiestl RH (1989) Nature 337, 285-288.
154
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P-027
THE FORMATION OF HYDROXYETHYL VALINE IN HOSPITAL STERILIZATION WORKERS:
EFFECTS OF GENETIC POLYMORPHISMS OF GLUTATHIONE S-TRANSFERASE THETA
AND MICROSOMAL EPOXIDE HYDROLYSE
Kuen-Yuh1
Kuen-Yuh Wu 1; Chia-Fang Wu 1; Ming-Fong Chen 1; Lan-Tyi Duann 2; Tsung Jen Cheng 3
Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan Town, Miaoli County,
Taiwan1; National Taichung Nursing College, Taichung, Taiwan2, Institute of Occupational Medicine and Hygiene, School of Public
Health, Taiwan University, Taipei, Taiwan3
The formation of N-(2-hydroxyethyl) valine (HEV) in hemoglobin has been considered as a biologically effective dose
for ethylene oxide (EO) exposures. Analysis of HEV in humans also represents the net effects of interactions between
genetic polymorphisms of glutathione S-transferase theta and microsomal expoxide hydrolase and EO which serve as
a model compound for other epoxides. In this study, 148 volunteers with no EO exposure history and 76 EO-exposed
hospital sterilization workers in Taiwan were recruited, 10 ml of blood was collected, and background information was
gathered using questionnaires from each study subject. HEV was processed by following the modified Edman degradation method and quantitated using a gas chrography coupled with mass spectrometry. Genetic polymorphisms of GSTT1
and mEH were also analyzed. Statistical analysis shows that the formation of HEV was significantly associated with
smoking status, EO exposure and genetic polymorphism of GSTT1. Further analysis shows that the contents of HEV in
study subjects were also associated with the combination of GSTT1 genotypes with the predicted activity of mEH in the
sterilization workers. These results are consistent with the observations in animal studies that EO can be detoxified by
glutathione transferase and epoxide hydrolse and suggest that EO detoxification mainly by glutathione transferase in
human.
P-028
ACCELERATED 32P-HPLC FOR LIPOPHILIC DNA ADDUCTS
Magnus Zeisig
Eszter Nagy; Lennart Möller
Karolinska Institutet, Dept. of Biosciences and Nutrition, Huddinge, Sweden
Background: DNA adducts are DNA addition products from various DNA-damaging agents and act both as biomarkers
and a measurement of the actual damage to the genome. Therefore they are of value to determine both exposed or
sensitive individuals or populations as well as agents causing DNA damage. Method: A common method to measure
DNA adducts is through DNA hydrolysis, adduct enrichment, 32P-postlabeling and chromatographic separation. HPLC
with inline radioactivity detection, 32P-HPLC, gives both high sensitivity and good resolution of even complex mixtures
of DNA adducts. One drawback with this method is limited capacity when dealing with high number of samples because
of time required for each analysis and inability of paralell analyses. The aim of this study was therefore to increase the
analytical capacity by reducing analysis time. Result: Change of HPLC columns to low backpressure ones and adaption of
elution to the new possibilities this offers, enables a reduction in time per analysis from 100 min to 20 min. This does not
affect sensitivity but lowers chromatographic resolution, even if lipophilic DNA adducts are still well resolved from other
DNA components. This is useful when the total amount of DNA adducts is the primary interest. When high resolution is
required this can be achieved by gradient modifications which increases the time required per analysis to 30 minutes.
Conclusions: Development of the chromatographic system in 32P-HPLC increases the capacity in number of samples per
HPLC system 3- to 5-fold, depending on resolution requirements without any negative effect on sensitivity.
155
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P-029
COMPARISON BETWEEN THE FREQUENCIES OF STABLE AND UNSTABLE CHROMOSOMAL
ABERRATIONS IN GENERAL POPULATION OF THE REPUBLIC CROATIA
Aleksandra Fucic 1
Davor Zeljezic 1; Nevenka Kopjar 1; Vilena Kasuba 1; Ruzica Rozgaj 1; Snjezana Ramic 1; Joe Nathan
Lucas2
Institute for Medical Research and Occupational Health, Zagreb, Croatia1; ChromoTrax, Inc., Frederick Innovative Technology
Center, Federick, United States2
Fluorescence in-situ hybridization (FISH) using whole chromosome paints is a potent tool in retrospective biological
dosimetry. Generally regarded as a ‘gold standard’, three-color FISH is used in studies for comparison of translocation
frequency with chromosome aberrations detectible by conventional cytogenetics. The aim of our work was to determine
the baseline translocation yield in the peripheral blood lymphocytes using whole-chromosome paint probes for chromosomes 1, 2 and 4, and to compare it with the results of the standard chromosomal aberration analysis. The study
comprised 20 individuals from the general population of the Republic Croatia (10 males and 10 females). Both gender
groups were of the similar age. The mean age of the male group was 47 years (range 37 - 58), and the female group 41
(range 31 – 50). For each subject more than 1000 genome equivalents were analysed. Translocation frequencies were
calculated according to Lucas et al. (1992). The translocation and chromosomal aberrations per 100 cells for male and
female subjects were not significantly different, 0.41 for males and 0.3 for females. In lymphocytes of all subjects, chromatid and chromosome breaks and acentric fragments detected were in concordance with historical background values
for this age group. The percentage of chromatid breaks found in males was 0.14, and in females 0.10. In females 0.1 %
of chromosome breaks (0.1 % in males) and 0.07 % of acentrics (0.02 % in males) was found. Detected translocation
frequency is in agreement with historical values for the analysed age group. Due to the limited number of European
biodosimetry centres, the launching of common databank and standardization of selection of chromosomes for genome
equivalent calculations is of great significance.
P-031
FACTORS AFFECTING BACKGROUND AND INDUCED LEVELS OF SCE AND MICRONUCLEI
IN FROZEN HUMAN LYMPHOCYTES
Andrea Zijno
Francesca Saini; Ester Siniscalchi; Riccardo Crebelli
Istituto Superiore di Sanitŕ, Rome, Italy
The use of frozen biological samples in human population studies with cytogenetic biomarkers is recommended in order
to optimise resources and to allow best experimental planning and repeated analyses. In addition to practical advantages, the use of frozen specimens can allow the a priori selection of study subjects, greatly improving the analytical power
of the study. However, the maintenance of the individual characteristics of fresh lymphocytes after freezing and thawing
is a prerequisite for their successfully use. This aspect has not been systematically investigated so far. In this pilot study
performed on 57 subjects, lymphocytes were frozen in aliquots of 500-700 μl at 107 lymphocytes/ml. Twenty subjects,
selected on the basis of their XRCC3 Thr241Met genotype, were tested in a challenge assay where micronuclei (Mn) and
nucleoplasmic bridges in binucleated cells and SCE were analyzed after mytomicin C and γ radiation treatments. Results
indicate that frozen lymphocytes maintain most of individual features observed in fresh samples. Age was confirmed
to be a strong modulator of basal Mn in the 20 frozen samples (p = 0.007). Also gender exerted a distinct influence
on basal Mn, with a higher incidence in the 4 females compared to males (16.0‰ vs 12.75). The comparison of Mn
observed in this study in frozen blood samples with those observed in the same subjects 5 years before analysing fresh
whole blood cultures showed a significant correlation (p = 0.001), even after correction for age and sex. After treatment of frozen cells with γ rays, a significant correlation of induced Mn with age was observed (p=0.046), confirming
similar findings obtained with fresh lymphocyte. Finally smoking habit, even if not significantly, influenced SCE. These
preliminary observation confirm the suitability of frozen lymphocytes for subsequent cytogenetic analyses, and their
potential for application in prospective studies on individual susceptibility to DNA damage and cancer.
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P-032
AN IN VITRO INVESTIGATION INTO LEVELS OF DNA DAMAGE IN LYMPHOCYTES FROM
MOTHERS AND BABIES’ CORD BLOOD
Diana Anderson 1
Natalie P Wyatt 1; Cesar Falque-Gonzalez 1; Diane Farrar 2; Derek Tuffnell 2; Donald Whitelaw 2
Dept. of Biomedical Sciences / The University of Bradford, Bradford, United Kingdom1; NHS Trust / Bradford Royal Infirmary,
Bradford, United Kingdom2
A report by the National Academy of Sciences (1993) ascertained that children may experience different levels of chemical exposures than adults, and that their sensitivities to chemical toxins may be increased or decreased by comparison
with adults [1]. Ethical approval was obtained from both the University of Bradford and the Research Ethics Committee
of the Bradford NHS Hospitals Trust. A blood sample from the mother was taken as close to the delivery time as possible
and venous cord blood was obtained at an appropriate time post-delivery. Lymphocytes were examined in vitro in the
Comet assay, from 24 mother and baby pairs using ethyl methanesulfonate (EMS) which is primarily a point mutagen,
and, from 16 mother and baby pairs using sodium nitrite (NaNO2) and hydrogen peroxide (H2O2). Lymphocytes from 20
mother and baby pairs were examined in vitro using EMS for occurrence of micronuclei, and, metaphase spreads from
4 mother and baby pairs were also examined for incidence of SCEs. There were overall no biologically significant differences in levels of DNA damage when lymphocytes from mothers and babies were examined in these various assays.
Therefore these studies do not support the above statement [1] with this particular compound that the sensitivities of
children to chemical toxins may be increased or decreased by comparison with adults.
P-033
NEWGENERIS: A NEW FP6 INTEGRATED PROJECT ON NEWBORNS AND GENOTOXIC EXPOSURE
RISKS
Maria Botsivali
NewGeneris Communication and Dissemination Office, National Hellenic Research Foundation, Institute of Biological Research and
Biotechnology, Athens, Greece
NewGeneris (“Newborns and Genotoxic Exposure risks: Development and application of biomarkers of dietary exposure to genotoxic and immunotoxic chemicals and of biomarkers of early effects, using mother-child birth cohorts and
biobanks”) is a FP6 Integrated Project (started 1/2/2006, duration 5 years), co-ordinated by Prof. J. Kleinjans, University
of Maastricht, with the participation of 25 partners. Its objective is to assess the role of maternal exposure, during
pregnancy, to dietary carcinogenic and immunotoxic compounds in the induction of increased risk of childhood cancer
and immune disorders. Biomarkers of exposure, carcinogenic and immunotoxic risks and susceptibility will be developed
and applied using high-throughput techniques. Existing mother-child birth cohorts and biobanks (Norway, Denmark, UK,
Spain) will be employed, and new ones will be set up (UK, Spain, Greece), representing a virtual European birth cohort
of approx. 300,000 mother-child pairs.
Dietary exposure of pregnant women to selected carcinogens and immunotoxins will be assessed using questionnaires,
while epidemiological surveys will be used to study associations between exposure and disease risks. Paternal exposures
will also be considered. Biomarkers in maternal and umbilical cord blood will be used to compare fetal and maternal
exposures, while genetic pathways indicative of risks of cancer and immune disorders will be studied by analysis of
gene expression and proteomics profiles. Inter-individual variability in responses will be evaluated by genotyping and by
phenotyping for DNA-repair activities. In vitro transplacental perfusion will be used to further study in utero exposure
to the selected toxins.
Overall public health implications and ethical issues will be addressed, and results will be disseminated to key stakeholders.
Contract no. FOOD-CT-2005-016320
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P-034
THE INFLUENCE OF DNA REPAIR GENES POLYMORPHISMS ON DNA DAMAGE AND ITS REPAIR
IN CHILDREN ENVIRONMENTALLY EXPOSED TO LEAD
Lucyna Kapka 1
Danuta Mielzynska 1; Marcin Kruszewski 2
Institute of Occupational Medicine and Environmental Health, Sosnowiec, Poland1; Institute of Nuclear Chemistry and Technology,
Warsaw, Poland2
A study on 88 children environmentally exposed to lead and 49 controls was performed in order to examine the influence
of lead exposition and genetic polymorphisms on the level of DNA damage and DNA damage repair capacity.
Exposure to lead was assessed by lead in blood (PbB) determination by AAS. The frequency of micronuclei (MN)
was determined by cytokinesis-block micronucleus assay. The steady-state level of SSB and FPG-sensitive sites was
determined using comet assay. The capacity of DNA damage repair was estimated by alkaline comet assay in a timecourse experiment after irradiation of cells with 2 Gy (X-rays). Polymorphism of DNA repair genes (XPA[(-4)G A],
XPDAsp312Asn, XRCC1Arg399Gln, XRCC3Thr241Met, TDGVal367Met) and gene of hem biosynthesis (ALADLys59Asn)
was determined by RFLP-PCR analysis.
Frequencies of MN (p<0.001), the level of FPG-sensitive sites (p<0.001) and repair capacity (the level of DNA damage
half hour after irradiation; (p=0.011) differed significantly between groups. Both, in the whole studied population and in
the exposed group, children possessing the AA genotype for XPA gene had higher frequencies of MN (p=0.044;p=0.017)
and the level of FPG-sensitive sites (p=0.025; p=0.033,respectively). The mean PbB level in exposed children, who
had at least one ALADAsn59 allele was higher than in those with homozygous ALADLys59 (5.76±1.60vs5.09±2.01;p=0.
143). Lower levels MN (p=0.009) and SSB (p=0.022) were also observed in environmentally exposed population with
homozygous XRCC3Met241 and XPDAsn312 genotypes as compared with children with “wild type” genotypes. The rate of
radiation-induced DNA damage repair was affected by XRCC1 and XRCC3 genes polymorphism, the genes involved in
the repair of SSB and DSB of DNA, respectively.
The results suggest the importance of evaluating markers of individual susceptibility, since they may modulate genotoxic
effects induced by environmental lead exposure.
P-035
EUROPEAN NETWORK ON CHILDREN’S SUSCEPTIBILITY AND EXPOSURE TO ENVIRONMENTAL
GENOTOXICANTS CHILDRENGENONETWORK (QLK4-CT-2002-02198)
Lisbeth E. Knudsen
Marie Pedersen
The Concerted Action aimed at collecting, validating and reviewing the available, though few, European and other
studies and study materials on children’s exposure and susceptibility to genotoxicants, with focus on air pollution. The
ChildrenGenoNetwork demonstrated availability and usefulness of existing data relating biomarkers to children's environmental exposure.
The network also initiated a number of national studies which contribute excellent platforms for intervention and adherence to the upcoming pilot project on human biomonitoring in Europe. The pilot project demonstrated usefulness of one
of the new omic techniques, namely transcriptomics to which relevant expectations as new monitoring tool is stated.
All such new techniques need validation and comparisons to traditional biomarkers are recommended.
Mother-child cohorts or more relevant triad studies including the fathers are useful constructions for sampling and
biobanking with the aim of surveillance of the exposure of the general population and specific research projects.
Risk assessment related to children’s exposure to environmental toxicants must take age specificity into account and
more data about exposures and effects are needed for proper assessment of differential susceptibility.
Ethical issues and social and legal impacts of studies with children needs more exploration as geographical and cultural
differences are seen.
Sixteen partners from 12 countries contributed. A number of papers can be found at www.pubhealth.ku.dk/cgn
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P-036
ETHICAL ISSUES RELATED TO INDIVIDUALISED BIOMONITORING STUDIES OF CHILDREN
Lisbeth E. Knudsen
Marie Pedersen
Major ethical problems related to individualised biomonitoring will be identified and analysed in order to develop a
coherent approach to human biomonitoring in Europe by ESBIO (Expert team to Support Biomonitoring). Specific attention will be devoted to the use of biobanks and information obtained in common databases and adhering regulations
in force and foreseen.
The first step consists of analysing existing information gathered from the inventory European biomonitoring studies by
SCALE and during the ChildrenGenoNetwork. There are no uniformity and agreement in Europe about the procedures
and contents of the process of informed consent. Measures are taken to ensure confidentiality and security of the data.
Ethical advice has been taken to determine the maximum volume of blood samples that may be taken in order to
minimize distress to the participant. Ethical questions on communication and access to own data (right to know, right
not to know) are therefore raised: e.g. is it acceptable not to report on the individual results? Also communication with
the public was reported to raise difficulties and to need careful consideration and preparation. An active involvement of
professionals in the field of communication and sociology may contribute to resolving these constraints.
The second step for ESBIO is to interview a number of different stakeholders (e.g., study persons, nurses, technicians,
researchers, regulators, etc.) in order to further investigate the processes of information of study persons as diverse
views and understanding of the advantages and limits of human biomonitoring may cause ethical conflicts. March 11th
to 14th 2007 a workshop with major stakeholders identifying major items for guidelines for ethical issues in Copenhagen
will take place.
www.eu-humanbiomonitoring.org and www.pubhealth.ku.dk/cgn
P-037
INVESTIGATION OF ANTIOXIDANT STATUS AND GST POLYMORPHISM ACCORDING TO DEGREE
OF AUTISM IN CHILDREN WITH DEVELOPMENTAL DELAY
Su-Yeon Kim 1
Eunju Park 1; Kyung-Im Jeon 1; Bo-Young Seo 1; Hyun-Jin Lee 1; Han-Woo Lee 2; Jung-Hwa Choi 3
Dept. of Food and Nutrition, Kyungnam Univeristy, Masan, Korea1; Div. of Social Welfare, Jinju International University, Jinju,
Korea2; Div. of Food Science, Jinju International University, Jinju, Korea3
Autism is a complex developmental disability that typically appears during the first 3 years of life. There is an evidence
that reactive oxygen species (ROS) play an important role in autism as well as other psychiatric disorders. GlutathioneS-transferases (GSTs) conjugate GSH to ROS or xenobiotics. GST activity depletes GSH levels and may either detoxify
or enhance the toxicity of a compound. Glutathione-S-transferase mu 1 (GSTM1) and glutathione-S-transferase theta 1
(GSTT1) have polymorphic homozygous deletion (null) genotypes resulting in complete absence of enzyme activity. The
purpose of this study was to investigate plasma antioxidant status and erythrocyte antioxidant enzyme activities according to autistic scores by Autism Behavior Checklist (ABC) and to explore whether the GST polymorphisms confer a risk
to an individual to develop autism in children with developmental delay. We divided 39 developmental delayed children
aged 3-9 into two groups using Autism Behavior Checklist (ABC); autistic (n=22) and non-autistic (n=17). The autistic
children showed significantly lower levels of plasma total radical trapping antioxidant potential (TRAP) and erythrocytes
antioxidant enzyme activities such as catalase and superoxide dismutase compared with the non-autistic children group.
However, neither GSTM1 nor GSTT1 genotypes in the autistic children was significantly different from the non-autistic
children. Our results suggest a possible role of increased oxidative stress and altered enzymatic antioxidants, both of
which may be relevant to the pathophysiology of autism in children with developmental delay. Further and large scale
study would be needed to confirm the relationship between GST polymorphism and etiopathogenesis of autism.
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P-038
GENOTOXIC EFFECTS IN THE EASTERN MUDMINNOW (UMBRA PYGMAEA L.) AFTER EXPOSURE
TO RHINE WATER USING THE SCE AND COMET ASSAY; A COMPARISON BETWEEN 1978
AND 2005
Gerrit M. Alink 1
Bert Spenkelink 1; Joris T.K. Quik 1,2; Serge G.P. Rotteveel 2; Hannie L. Maas 2; Eric J.M. Penders 3; Wim
Hoogenboezem 3
Chair Toxicology, Wageningen University, Wageningen, Netherlands1; RIZA Institute for Inland Water Management and Waste
Water Treatment, Lelystad, Netherlands2; Het Waterlaboratorium, RIWA Rhine Water Works, Nieuwegein, Netherlands3
For surface water used for drinking water preparation continuous monitoring of the presence of toxic compounds is warranted. For monitoring genotoxic compounds fish models have been developed such as the Eastern mudminnow (Umbra
pygmaea L.) because of its clearly visible 22 metacentric chromosomes. It was demonstrated in the late seventies that
Rhine water was able to induce chromosome aberrations and sister chromatid exchanges in this fish species (Alink,
1980). Although in vitro mutagenicity studies of the Dutch Rhine water work association (RIWA) have shown that the
genotoxicity of the river Rhine steadily decreased during the last decennia, there is still concern because of the presence
of some rest mutagenicity and because in most studies fractions of the water have been tested in in vitro test systems
such as the Salmonella-microsome test. For that reason and in order to be able to make a comparison with the water
quality 25 years ago, a study was performed with the same experimental design as previously in order to measure the
effect of Rhine water on the induction of SCE’s in the Eastern mudminnow. As a new test system the Comet assay was
performed. Fish were exposed to Rhine water or to groundwater for 3 and 11 days in flow through aquaria. Fish exposed
for 11 days to Rhine water had a significant higher number of SCE’s and an increased tail moment compared to control
fish exposed to ground water. After exposure for 3 days to Rhine water there was no difference in SCE’s and a slightly
increased tail moment compared to the control. It was concluded that still genotoxins are present in the river Rhine but
that the genotoxic potential has markedly decreased compared to 25 years ago.
P-039
EVALUATION OF DNA DAMAGE IN MURINE FIBROBLASTS TREATED WITH CIGARETTE SMOKE
CONDENSATE
Cristina Andreoli 1
Floriana Flamma 1; Francesco Mercati 1; Angela Martino 1; Fabio Caradonna 2; Maurizio Mauro 2; Giulia
Sciandrello 2
Research Department, BAT-Italia SpA, Naples, Italy1;Cellular and Developmental Biology Department, University of Palermo,
Palermo, Italy2
Cigarette Smoke Condensate (CSC) is a complex chemical mixture containing about 4800 compounds, many of them
have cytotoxic and mutagenic activities on mammalian cells. Most of these compounds are able to interact with DNA at
different levels. Cells may respond to DNA damage by following different pathways, such as the DNA repair processes
and the cell cycle and DNA damage checkpoint activation.
To the aim to evaluate the biological effects of CSC on cells, alkaline comet assay and flow cytometry were used to
examine DNA damage/repair and cell cycle progression. All experiments were performed by using CSC from standard
cigarettes in the range of doses 30-180mg/ml and Swiss 3T3 murine fibroblasts.
Results obtained by comet assay showed that CSC induces DNA strand breaks, significantly higher after 90 min of treatment than 3hrs. This difference, particularly evident at 100 and 150mg/ml, is probably due to a fast repair that can be
explained by the oxidative component of the DNA damage CSC-induced. To clarify these results, further investigations
on the evaluation of the oxidative damage are in progress by applying two different methods: the detection of the oxidised bases on the DNA by using the modified protocol of Comet assay with FPG and Endo III, and the evaluation of the
intracellular levels of ROS, based on the ability of treated cells to oxidise a fluorogenic substrate.
Previous results of long-term survival showed that cells lose their ability to form colonies in dose-dependent manner,
after 24hrs of CSC treatment and 168 hrs of culture. However, the cytofluorimetric analysis showed that a fraction of
cells, blocked in G2/M immediately after 24hrs of treatment, are gradually granted to continue the cell cycle, after incubation for further 6hrs in medium CSC-free. Further investigations on the cell cycle alteration are on going.
160
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P-040
INCREASED GENOMIC INSTABILITY IN PERIPHERAL LYMPHOCYTES OF DOWN SYNDROME
PATIENTS DUE TO INCREASED RATE OF CHROMOSOME NON-DISJUNCTION
Georgia Stephanou
Konstadinos Andrianopoulos; Michael Tyrakis; Asimina Kouloumenta; Constantinos Andrianopoulos;
Nikos A. Demopoulos
Division of Genetics, Cell and Developmental Biology, Department of Biology, University of Patras, Patras, Greece
Down syndrome (DS) is a chromosomal disorder with a general population frequency once in 700 live births and is
related with trisomy of chromosome 21. DS patients are characterized of mental retardation, premature aging, reduced
life expectancy, high risk for malignant diseases and increased sensitivity to various mutagens such as radiation, chemicals and viruses. Non-disjunction of chromosome 21 occurs more often in cells of trisomy 21 patients than in cells of
normal controls. Additionally mothers of DS patients show higher non-disjunction frequency of chromosomes 13 and
21 in their peripheral lymphocytes. The aim of this study is to compare genomic instability in DS patients in relation to
control individuals and especially to evaluate possible chromosome malsegregation rate of other chromosomes than 21.
The sex chromosome X and the autosome 8 were chosen and their segregation rate was analyzed in peripheral blood
lymphocyte cultures by means of CBMN assay combined with Dual-FISH using chromosome specific alphoid probes.
Micronucleus frequency was also estimated. In addition SCE frequency was studied in blood lymphocytes of the same
individuals. Lymphocyte cultures were established from 4 DS patients aged 15-34 (mean 25.5) and 4 healthy donors
of similar age. DS patients were determined to be karyotypically as 47,XY+21 or 47,XX+21. Our results showed that
there is no difference in SCE frequency of DS individuals in comparison with controls. Micronucleus frequency was higher
in DS patients but no statistically significant difference was determined. On the other hand a significant decrease of
Cytokinesis Blocked Proliferation Index (CBPI) was observed in lymphocytes from DS patients in comparison with cells
from karyotypically normal donors. Additionally increased malsegregation rate was identified in DS individuals for chromosomes X and 8 due to increased non-disjunction frequency. In conclusion non-disjunction of chromosome X and 8
takes place more frequently in cultured lymphocytes of DS patients.
P-041
POLY(ADP-RIBOSE) POLYMERASE-1 (PARP-1) ANTAGONIZES TOPOISOMERASE I-DEPENDENT
RECOMBINATION STIMULATION BY P53
Cindy Baumann 1
Lisa Wiesmüller 1; Gisa S Boehden 2; Alexander Bürkle 3
Universitätsfrauenklinik, Ulm, Germany1; Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie, Hamburg,
Germany2; Molecular Toxicology Group, Department of Biology, Konstanz, Germany3
P53 and topoisomeraseI, which participate in DNA recombination, interact with and are poly(ADP-ribosyl)ated by
PARP-1. Previously, we showed that both p53 and PARP-1 downregulate homology-directed double strand (DSB) repair
independently of the DNA sequence. Surprisingly, however, we also discovered that p53 enhances homologous recombination (HR) at the RARα breakpoint cluster region (bcr) comprising topoisomerase I recognition sites, when using an
SV40 based test system. In this system expression of PARP-1 counteracted HR enhancement by p53 at the RARα bcr,
although DNA replication was largely unaffected. However, both p53 and PARP-1 exerted anti-recombinogenic rather
than stimulatory activities, when the same DNA element (RARα bcr) was integrated in an episomal recombination plasmid. Strikingly, with DNA substrates integrated into cellular chromosomes, enhancement of HR by p53 and antagonistic
PARP-1 action was seen, very similar to the HR of viral minichromosomes. The essential role of topoisomerase I in this
regulatory mechanism was revealed by siRNA-mediated knockdown. The topoisomerase I-dependent effects by p53 and
PARP-1 were lost after I-SceI-meganuclease-mediated cleavage of the chromosomally integrated substrate, suggesting
a role in increasing the recombinogenicity of the DNA substrate. Further results with truncated PARP-1 indicate that
PARP-1 prevents p53 from stimulating spontaneous HR on chromosomes via topoisomerase I activity, probably through
competitive topoisomerase I interactions rather than poly(ADP-ribosyl)ation.
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P-042
ASSESSMENT OF ANTIDIABETIC AGENT PIOGLITAZONE GENOTOXICITY IN RATS BY COMET
ASSAY
Abdulkerim Bedir 1
Yuksel Aliyazicioglu 1; Muhlise Alvur 1; Zafer Yurdakul 1; Mehmet Uysal 1; Duygu Erol Suvaci 1; Ali
Okuyucu 1; Zeliha Cansel Özmen 1; Birşen Bilgici 1; Hakki Kahraman 2; Murat Hökelek 3
Ondokuz Mayis University, Faculty of Medicine, Department of Biochemistry, Samsun, Turkey1; Ondokuz Mayis University, Faculty of
Medicine, Department of Internal medicine, Samsun, Turkey2; Ondokuz Mayis University, Faculty of Medicine, Department of
Microbiology, Samsun, Turkey3
Objective: Pioglitazone, a member of the thiazolidinedione class of antidiabetic agents, improves glycemic control by
improving insulin sensitivity. Pioglitazone is a highly selective and potent agonist for peroxisome proliferator-activated
receptor-gamma (PPARγ). However, there have been no data in the literature concerning pioglitazone genotoxicity in
both animals and human beings. Thus, the aim of this study was to investigate the genotoxicity of pioglitazone in rats
by single cell gel (comet) assay.
Methods: Twenty male Sprague-Dawley rats were randomly distributed into four groups. Rats were dosed for 12 days
by oral gavage at doses of 10 (group III), 20 (group II), and 40 mg/kg/day (group I, highest dose equivalent to
approximately 10 times the maximum recommended human daily dose for rats); rats of group IV (negative controls)
were given on the same days an equal volume of the vehicle. Under short ether anesthesia, rats were euthanized by
exsanguinations, and peripheral blood and livers were collected and processed for comet assay.
Results: A statistically significant increase of DNA damage was observed in the hepatocytes of the pioglitazone treated
groups I and II compared with controls (p<0.001) as assessed by % tail DNA and Olive Tail Moment. As for the lymphocytes, the marked DNA damage was first detected at the group III (p<0.001), representing systemic effects of
pioglitazone.
Conclusion: Taken together, the data indicate that pioglitazone is able to induce primary DNA damage in both liver cells
and in lymphocytes of rats.
P-043
THE EUKARYOTIC PSO2P/SNM1P FAMILY REVISITED: IN SILICO ANALYSES OF PSO2P A, B
AND PLASMODIUM GROUPS
Diego Bonatto 1
Martin Brendel 2; Joao Antonio Pagas Henriques 3
Universidade de Caxias do Sul, Caxias do Sul, Brazil1; Universidade Estadual de Santa Cruz, Ilheus, Brazil2; Universidade Federal
do Rio Grande do Sul, Porto Alegre, Brazil3
The eukaryotic family of Pso2/Snm1 exo/endonuclease proteins has important functions in repair of DNA damages
induced by chemical interstrand cross-linking agents and ionizing radiation. These exo/endonucleases are also necessary for V(D)J recombination and genomic caretaking. However, despite the growing biochemical data about this family,
little is known about the number of orthologous/paralogous Pso2p/Snm1p sequences in eukaryotes and how they are
phylogenetically organized. In this work we have characterized new Pso2p/Snm1p sequences from the finished and
unfinished eukaryotic genomes and performed an in-depth phylogenetic analysis. The results indicate that four phylogenetically related groups compose the Pso2p/Snm1p family: (i) the Artemis/Artemis-like group, (ii) the Pso2p A group,
(iii) the Pso2p B group and (iv), the Pso2p Plasmodium group. Using the available biochemical and genomic information about Pso2p/Snm1p family, we concentrate our research in the study of Pso2p A, B and Plasmodium groups. The
phylogenetic results showed that A and B groups can be organized in specific subgroups, with different functions in
DNA metabolism. Moreover, we subjected selected Pso2p A, B and Plasmodium proteins to hydrophobic cluster analysis
(HCA) in order to map and to compare conserved regions within these sequences. Four conserved regions could be
detected by HCA, which are distributed along the metallo-β-lactamase and β-CASP motifs. Interestingly, both Pso2p A
and B proteins are structurally similar, while Pso2p Plasmodium proteins have an unique domain organization. The possible functions of A, B and Plasmodium groups are discussed.
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P-044
DEGREE RELATIVES
Erdem Coskun 1
Sema Burgaz 1; Neslihan Aygun Kocabas 1; Gonca Demircigil Cakmak 1; Faik Cetindag 2; Osman Sunter 2
Hayriye Edinsel 2
Gazi University Faculty of Pharmacy Department of Toxicology, Ankara, Turkey1; Abdurrahman Yurtaslan Oncology Hospital
Department of Radiation Oncology Head and Neck Group, Ankara, Turkey2
Studies have suggested that not only reduced DNA repair capacity (phenotype) but also genetic susceptibility (genotype) may play an important role in risk of head and neck cancer (HNC). Further support for genetic susceptibility is
evidenced by aggregation of HNC patients with their first degree relatives (FDRs). For this purpose, in our study, we
have investigated the relationship between the basal and radiation-induced micronucleus (MN) frequencies with XRCC1
gene polymorphism (Arg399Gln) both for HNC patients (n=38) and their FDRs (n=21), as well as healthy controls
(n=27). XRCC1 (X-ray repair cross-complementing group 1) is a base excision repair protein that plays a central role
in the repair of DNA base damage and strand breaks. The XRCC1 alleles were detected using PCR-RFLP technique.
For the micronucleus assay, blood samples were exposed in vitro to 2 Gy γ rays (60Co) at a dose rate of 0.62 Gy/min.
Mutant allel frequencies were 34.2% (n=26), 38.1% (n=16) and 40.7% (n=22) for patients, relatives and controls,
respectively. HNC patients, FDRs and controls variants had elevated but not significant basal MN levels (p>0.05). For
the induced MN frequencies, variants had decreased frequencies in all groups but not different significantly (p>0.05).
Smoking didn’t affect on MN frequncies and also we did not find an interaction between Arg399Gln polymorphism and
smoking (p>0.05). Our data seemed to be supported by other studies that found a decreased risk for the genetic variation in XRCC1 codon 399 in HNC patients. However, further work is needed to resolve the importance of other polymorphisms of XRCC1 including codon 194, as well as other DNA repair systems e.g. XRCC3 and OGG1.
This study was supported by Gazi University Scientific Research Fund (Project No. 02/2004-17)
P-045
THE HIGH LEVEL OF SPONTANEOUS ENDOREDUPLICATION IN EM9 MUTANT CELL LINE
CORRELATES WITH A LOW LEVEL OF DNA METHYLATION
Inmaculada Domínguez
Gloria Cantero; Santiago Mateos; Nuria Pastor; Manuel Luis Orta; Felipe Cortés
Department of Cell Biology, Faculty of Biology, University of Sevilla, Sevilla, Spain
We have recently shown that DNA demethylation caused by substitution of cytidine by 5-azacytidine (5-azaC ) into DNA
produced a failure in topoisomerase II function in chromosome segregation that can be detected as endoreduplication
(Mateos et al., Mutation Research 578: 33-42, 2005)
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P-046
OXIDATIVE DAMAGE TO DNA AND OXIDATIVE STRESS IS INVOLVED IN EARLY STAGES
OF COLON CANCER DEVELOPMENT
Daniel Gackowski 1
Ryszard Olinski 1; Rafal Rozalski 1; Agnieszka Siomek 1; Anna Szpila 1; Tomasz Dziaman 1; Jolanta Guz 1;
Karol Bialkowski 1; Marek Foksinski 1; Zbigniew Banaszkiewicz 2; Arkadiusz Jawien 2
Department of Clinical Biochemistry, Collegium Medicum Nicolaus Copernicus University, Bydgoszcz, Poland1; Clinic of Surgery,
Collegium Medicum Nicolaus Copernicus University, Bydgoszcz, Poland2
Oxidative damage to DNA often has been blame as a possible basis for the physiological changes associated with cancer
development. To have an insight into this important issue in the present study the parameters reflecting oxidative DNA
damage (determination of 8-oxo-2’-deoxyguanosine (8-oxodG) in DNA isolated from leukocytes, urinary excretion of
8-oxoguanine (8-oxoGua) and 8-oxodG and low-molecular weight antioxidants were analyzed in the control group, the
adenoma patients and the colon cancer patients.
Our results demonstrated that 8-oxodG level in leukocytes DNA and urinary excretion rate of 8-oxodG was significantly
higher in patients with adenomas in comparison with control group, while no further change was observed in carcinoma
patients. Moreover, we found strong support for a role of oxidative DNA damage in development of adenoma comparing
subjects with the high level (HL) of 8-oxodG level in cellular DNA vs the low lewel (LL), where odds ratio (OR) and 95%
confidence interval (CI) were 7.60 (CI:2.57-22.49), while OR value for a risk of carcinoma was 5.75 (CI:2.26-14.26).
Similar, OR values of urinary excretion rates of 8-oxodG were respectively 1.64 (CI:1.32-13.34) for the adenoma and
1.57 (CI:1.04-7.01) for the carcinoma.
The level of analyzed antioxidants gradually decreased in the sequence: control group – adenoma patients – cancer
patients. Significant reduction in the concentration of alpha-tocopherol and retinol was found in adenoma patients
in comparison with the control group what suggest they role in development of early stage of cancer development.
We found further support for a protective effect of antioxidants on the risk of colon cancer development; the ORs for
adenoma, comparing subjects in the HL of alpha-tocopherol and retinol vs the LL were 0.03 (CI:0.01-0.14) and 0.18
(CI:0.06-0.49) respectively. Summing up, it appears likely that oxidative DNA damage and low levels of antioxidant
vitamins are a hallmark of “high responders” to develop colon cancer.
P-047
CORRELATION BETWEEN DNA ADDUCT FORMATION AND CYTOGENETIC DAMAGE INDUCTION
IN MAMMALIAN CELLS EXPOSED TO ACRYLAMIDE AND GLYCIDAMIDE
Gonçalo Gamboa da Costa 1
Nuno G. Oliveira 2; Célia Martins 3; Marta Pingarilho 3; José Rueff 3; Jorge F. Gaspar 3; Vanda Martins 3;
M. Matilde Marques 4; Frederick A. Beland 5; Mona I. Churchwell 5; Daniel R. Doerge 5
The Institute of Cancer Research, Sutton, Surrey, United Kingdom1; Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal1;
Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal2; Department of Genetics, Faculty of Medical Sciences, UNL, Lisbon,
Portugal3; Centro de Química Estrutural, Technical University of Lisbon, Lisbon, Portugal4; National Center for Toxicological
Research, Jefferson, Arkansas, United States5
Acrylamide (AA) is an important chemical intermediate in industry, has widespread use in grouting and PAGE applications, and is present in tobacco smoke. AA is a well known human neurotoxic agent and has been classified as a
suspected carcinogen to humans. Recently, high levels of AA have been detected in starchy foods prepared at high
temperatures, raising interest on the effects of exposure of the general population to this compound. AA is metabolised by Cyp2E1 to glycidamide (GA), an electrophilic epoxide that is known to react with DNA, generating a major
depurinating DNA adduct, N7-(2-carbamoyl-2-hydroxyethyl) guanine (N7-GA-Gua). In order to determine the biological
significance of this DNA adduct, it is important to assess the correlation between its levels and the induction of cytogenetic damage in cells. In this work, we investigated the effects of exposing cultures of V79 cells, a rodent cell system
devoid of cytochrome P450 2E1, and peripheral blood human lymphocytes from healthy volunteers to AA and GA. A
clear dose-response was observed between the GA exposure levels and the formation of N7-GA-Gua, determined by
HPLC-ES-MS/MS, in both cell types. In contrast, exposure to AA did not induce the formation of DNA adducts, except
in V79 cells subjected to high levels of acrylamide. DNA adducts were not detectable in any of the control samples. In
V79 cells a strong correlation was found between the N7-GA-Gua adduct levels and the induction of sister-chromatid
exchanges (SCEs). GA also increased the frequency of chromossomal aberrations (CAs) excluding gaps. AA induction
of DNA adducts, SCEs and CAs excluding gaps was observed only at the 2.0 mM concentration and the effects were
moderate. These results indicate that in these model systems, the genotoxic potential of AA is dependent on metabolism
to GA, which is a potent inducer of DNA adducts, SCEs and CAs.
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P-048
DEVELOPMENT OF A HIGH-THROUGHPUT IMMUNOCHEMICAL ASSAY FOR THE ASSESSMENT
OF FUNCTIONAL METHYLGUANINE-DNA-METHYLTRANSFERASE (MGMT) LEVELS
Panagiotis Georgiadis
Vassiliki Pletsa; Stella Kaila; Soterios A Kyrtopoulos
National Hellenic Research Foundation, athens, Greece
The major cellular defense against the genotoxic alkylating agents is methylguanine-DNA-methyltransferase (MGMT),
a suicide enzyme that transfers the alkyl group from the O6- position guanine to a cysteine residue on its molecule.
Measurement of functional MGMT is essential for clinical research and molecular epidemiology studies. Inactivation of
MGMT by promoter hypermethylation is commonly seen in tumours and measurement of functional MGMT could be a
valuable prognostic tool for the prediction of the overall patient survival. The last decade, clinical trails were conducted
attempting to increase the responsiveness of tumours to alkylating agents by the concomitant administration of MGMT
inhibitors. In addition, the role of the exposure to alkylating agents, and subsequently of MGMT, in human cancer is
still unclear. So far, the only quantitative method for the detection of MGMT is based on the reaction of the MGMT with
[3H]-methyl-DNA. However, this method is tedious,very expensive and cannot be automated.
We have developed a sandwich ELISA method for the quantization of MGMT which is based on its property to react not
only with O6-belzylguanine (BG) but with O6-belzylguanine tagged with biotin as well. The final MGMT-BG-Biotin stable
complex is subsequently immobilized in anti-MGMT-coated microtiter plates. Streptavidine conjugated with hor?eradish
peroxidase is then added and chemiluminescence is produced by the addition of the appropriate substrate. The method
is highly sensitive (0.5 fmol MGMT protein relative to 3 fmol of the old method) has an extremely high dynamic range
(0.5-500 fmol relative to 3-20 fmol of the old method) is inexpensive and can be easily automated. This method will be
a valuable tool in biobank studies and clinical research.
P-049
CHROMOSOMAL INSTABILITY IN SALIVARY GLAND TUMORS – PRELIMINARY RESULTS
Maciej Giefing 1
Malgorzata Rydzanicz 1; Roksana Cegla 1;Maciej Kujawski 1; Malgorzata Wierzbicka 1,2; Krzysztof Szyfter 1,2
Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland1; Department of Otolaryngology, Medical University of
Poznan, Poznan, Poland2
In the last two decades an alarming increase of salivary gland tumors incidence was observed in Poland. This fact
together with scarce cytogenetic data available on salivary gland tumors encouraged us to undertake this study. A
cohort of salivary gland tumors was studied with CGH technique aiming for identification of common minimal regions
of gain and loss. Such regions potentially harbor oncogenes and tumor suppressor genes that may be involved in the
pathogenesis of this disease.
30 salivary gland tumor paraffin embedded tissue samples (mainly Whartin and mixed type tumors) were obtained from
the K. Marcinkowski Medical University in Poznan. DNA was isolated using standard phenol/chloroform method. DNA
from 16 salivary gland tumors (next 14 are planned) were studied by CGH.
The preliminary results indicate a non random distribution of DNA gain or loss across the tumor genomes. Several
recurrent regions including: del 1p35-pter (14/16); del 16p12.3-13.11 (11/16); del 19p13.2-q13.3 (10/16); amp 4q27q28.1 (9/16) were identified. Frequent changes were observed in regions harboring known tumor suppressor genes or
oncogenes like CCND1 loss (4/16); tp53 loss (3/16).
The most frequent observed change is del 1p35-pter. Our results indicate that this region known to be the most frequent human chromosomal deletion syndrome region plays an important role in tumor formation. Other example is the
amplified region 5q21 harboring the CHD1 gene. CHD1 may alter the chromatin structure by its chromo-domain and
thus regulate access of transcription factors and gene expression. Our results indicate that the salivary gland tumors
present a different chromosomal aberration pattern than head and neck tumors in general and thus should be treated
as a distinctive entity.
It should be noted, however, that some of the presented results may differ having completed the study.
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P-050
NEW SYSTEM FOR THE PRODUCTION OF MAMMALIAN DNA POLYMERASE KAPPA IN E. COLI:
PURIFICATION, CHARACTERIZATION AND IMMUNOCHEMISTRY OF THE HUMAN AND MOUSE
POLK PROTEINS
Petr Grúz
Naoko Niimi; Akira Sassa; Takehiko Nohmi
DNA polymerase k (POLK) is a member of the new Y-family of DNA polymerizing enzymes with orthologues spanning
all 3 kingdoms of life including prokarya, eukarya and archaea. These enzymes substitute for normal high fidelity DNA
replicases during the bypass of DNA adducts which would otherwise block replication and cause toxicity. In mammals,
POLK seems to have evolved to assist the replication machinery to cope with minor groove N2-guanine adducts such as
those inflicted by steroid hormone metabolism or induced by xenobiotics such as polycyclic aromatic hydrocarbons in an
error free manner. However, when acting on undamaged template POLK has low fidelity and is a promiscuous mispaired
primer termini extender suggesting that its deregulation would have deleterious consequences for genome stability. In
order to get deeper understanding of their biochemical properties in vitro as well as study their activities on cellular
and organ levels in vivo we have isolated both human and mouse POLK proteins overexpressed in E. coli and raised
polyclonal antiserum for their detection by immunological methods.
To maximize translation efficiency in bacteria, the new overexpression vector pYG8582 has been constructed by incorporating a cis acting translational enhancer into a standard T7-promoter based plasmid. This vector was used for the
overexpression of total 8 proteins counting catalytic domains as well as full size versions of the human and mouse POLK
and their catalytically dead variants. The catalytic domain of human POLK has been purified to homogeneity by FPLC
and used for raising polyclonal antiserum in rabbits. The antiserum detected both human and mouse POLK with high
efficiency and confirmed elevated expression in reproductive tissues corresponding to increased steroid metabolism. In
vitro POLK bypassed both (–)-trans-[BP]-N2-dG and with lower efficiency also (+)-trans-[BP]-N2-dG DNA adducts
derived from the potent environmental carcinogen benzo[a]pyrene.
P-051
ROLE OF DNA POLYMERASE DELTA IN THE REPAIR OF PSORALEN-PHOTOINDUCED DNA
INTERSTRAND CROSS-LINKS IN SACCHAROMYCES CEREVISIAE
Jaqueline M Cardone 1
Joao Antonio Pegas Henriques 1,4; Diego Bonatto 2; Martin Brendel 3
Federal University of Rio Grande do Sul/Biotechnology Center, Porto Alegre, Brazil1; University of Caxias do Sul/
Biotechnology Institute, Caxias do Sul, Brazil2; State University of Santa Cruz, Ilhéus, Brazil3 ; University of Caxias do
Sul/Biotechnology Institute, Caxias do Sul, Brazil4
Characterization of DNA interstrand cross-link (ICL) repair in Saccharomyces cerevisiae has allowed us to determine that
genes from all three epistasis groups of DNA repair [nucleotide excision repair, homologous recombination and postreplication/translesion repair] seem to encode proteins involved in this complex task. Although several of these genes
are known, there has been little progress in clarifying the final steps of ICL removal from DNA. A yeast strain lacking
subunit Pol32 of Pol δ, is about 3-fold more sensitive to 8-MOP+UVA whereas deletion of neither CTF4 nor of DPB3
genes resulted in increase of sensitivity over that of the isogenic wild type. Using a plasmid transformation assay, we
found that pol32∆ mutant is extremely deficient in joining non-cohesive DNA ends, particularly in cases of mismatched
5’ overhangs. Sequencing of the recovered plasmids showed that both WT and pol32 mutant strains are able to generate NHEJ products, but the structures of junctions from the pol32 strain were diverse with some of the joining events
without obvious filling. Based on these findings we propose a model for removal of ICL from yeast DNA where the DSB,
resulting from early steps in ICL processing, is subsequently repaired by DNA polymerase δ-mediated NHEJ. Supported
by CNPq, Capes and FAPERGS.
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P-052
GENETIC INSTABILITY IN MIGRANT/SEASONAL FARMWORKER (MSF) CHILDREN OF MEXICAN
ORIGIN RESIDING IN TWO TEXAS REGIONS.
María A. Hernández-Valero 1
Lovell A. Jones 2; Kevin J. Wolfe 3; Adele Guerin 3; Sherif Z. Abdel-Rahman 3
Department of Epidemiology & Center for Research on MInority Health, UT M.D. Anderson Cancer Center, Houston, United States1;
Department of Health Disparities Research & Center for Research on Minority Health, UT M.D. Anderson Cancer Center, Houston,
United States2; Department of Preventive Medicine & Community Health, UT Medical Branch, Galveston, United States3
Background: MSF children of Mexican origin from Texas have not been studied extensively even though they are chronically exposed to pesticides when they accompany their parents to the fields or work in the fields themselves. Levels of
several pesticides have been detected in these children, some of which are carcinogenic in laboratory animals. Since
higher frequency of chromosome aberrations (CA) in circulating lymphocytes can indicate an increased risk for cancer,
chromosomal damage in MSF children would indicate the need to implement stricter laws prohibiting children from entering or working in the fields. Methods: we are currently using the fluorescence in situ hybridization (FISH) cytogenetic
technique to detect CA in the lymphocytes of a random sample of 50 MSF children ages 5- 18 years who are enrolled
in the on-going study “Biomarkers of genetic susceptibility in environmentally exposed MSF children” Hernández-Valero,
PI (NCMHD P60MD000503) because (1) CA assays are widely employed biomarkers of genotoxic effects in exposed
populations, (2) studies with human lymphocytes have shown an elevated frequency of breakage in the heterochromatin
region 1q12 after exposure to a variety of environmental contaminants, & (3) our goal is to increase the sensitivity for
detecting genetic damage, as well as enhancing the ability to delineate the influences of polymorphisms in susceptibility
genes on the levels of the observed damage. Results: will be presented at the conference: Mean & standard deviation of
CA per 1000 cells will be compared between two groups of MSF children residing in a Texas (1) urban setting: Houston
metropolitan area (n=25) & in (2) an agricultural setting: Texas-Mexico border area (n=25) using two-sample t-tests, &
logistic regression & general linear models to determine the relationship between CA & predictors of exposure (gender,
age, field work, length of exposure, residencial area, etc.). Conclusion: will be presented at the conference.
P-053
INDUCTION OF COINCIDENT MITOTIC RECOMBINATION AT UNLINKED LOCI IN YEAST
AT FREQUENCIES HIGHER THAN PREDICTED FOR INDEPENDENT EVENTS
Kathryn M. Freeman
George R. Hoffmann
College of the Holy Cross, Worcester, MA, United States
Bleomycin (BLM) is a potent recombinagen and mutagen in Saccharomyces. Diploid strain D7, developed by F.K.
Zimmermann, is heteroallelic at trp5 (trp5-12/trp5-27) and homozygous for ilv1-92, permitting the detection of gene
conversion and point mutations by selecting for tryptophan- and isoleucine-prototrophy, respectively. Although heteroallelic at ade2, D7 is an adenine prototroph because of interallelic complementation. Mitotic recombinants that are
homozygous for ade2-40 or ade2-119 produce red or pink colonies, respectively, and red/pink sectored colonies arise
by mitotic crossing over. Given that the trp5, ade2, and ilv1 loci are on different chromosomes, we hypothesized that
frequencies of coincident (simultaneous) genetic events affecting more than one of them would be compatible with
independent probabilities. Thus, ade2 altered colonies should be equally frequent among all cells, Trp+ recombinants,
and Ilv+ revertants. Contrary to this expectation, there is a highly significant enrichment for ade2 mitotic recombinants
among BLM-induced Trp+ recombinants compared to that among all surviving cells or among revertants. We eliminated
the possibility that this is an artifact attributable to low frequencies of red or pink haploid cells arising in D7. The possibility that the excess represents overdispersion of damage caused by differential permeability to BLM, as reported in
mammalian cells, was eliminated by evidence that the phenomenon also occurs after mutagenesis with ß-propiolactone
or UV and that it applies to coincident recombination but not point mutations. We hypothesize that the excess of double recombinants is ascribable to a subpopulation in a recombination-prone state or hypersensitive to the induction of
mitotic recombination. The cause of altered recombination-proneness is unknown, but it is not a simple correlate of
growth phase or cell-cycle stage, and we have been unable to demonstrate its induction as an adaptive response to
DNA damage.
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P-054
ERROR-PRONE AND ERROR-FREE NONHOMOLOGOUS END-JOINING FOR REPAIRING DNA
DOUBLE STRAND BREAKS IN HUMAN CELLS
Masamitsu Honma
Mayumi Sakuraba; Tomoko Koizumi; Yoshio Takashima; Hiroko Sakamoto; Makoto Hayashi
Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double strand breaks (DSBs) in mammalian cells.
Because NHEJ is basically mutagenic pathway leading to deletion, however, it must be precisely regulated to maintain
genomic integrity. To investigate the genetic consequences of DSBs by NHEJ, we created a mammalian cell system to
trace the fate of chromosomal DSBs without selection bias. The human lymphoblastoid cell lines TSCE5 and TSCE105
are heterozygous (+/-) for the thymidine kinase gene (TK), and have single and double I-SceI endonuclease site in the
TK gene, respectively. DSBs at the I-SceI site were repaired by NHEJ resulting in TK-deficient deletion mutants (+/-).
NucleofectorTM (Amaxa) can efficiently introduce an I-SceI expression vector into cells and makes it possible to recover
mutants without the phenotypic TK-selection. We found mutations associated with the DSBs in 3% and 30% of randomly
isolated clones from TSCE5 and TSCE105, respectively. Most of the mutations in TSCE5 were small deletions ranging
from 1 to 30-bp, and others were over 60-bp deletions, an insertion, and a rearrangement. Mutants resulted by interallelic homologous recombination were also detected infrequently. TECE105 mutations, on the other hand, were mainly
large deletions encompassing the two I-SceI sites. The large deletion was generated by NHEJ for two ends of DSBs.
The joint sequence was similar to that of TSCE5 mutants. Interestingly, some mutants have a new I-SceI site by perfect
joining of the two I-SceI sites, implying that error-free NHEJ pathway work for that. These results support an idea that
majority of DSBs are repaired by NHEJ resulting small deletions or no deletions. NHEJ must strongly contribute to maintain genomic integrity by repairing DSBs as well as by preventing deleterious alterations in the genome as possible.
P-055
GENOTOXIC POTENTIAL OF ORGANOPHOSPHOROUS PESTICIDES PARATHION, PARAOXON
AND DIMEFOX
Irena Hreljac
Irena Zajc; Bojana Žegura; Tamara Lah Turnšek; Metka Filipič
National Institute of Biology, Department of Genetic Toxicology and Cancer Biology, Ljubljana, Slovenia
Organophosphorous substances (OPs) irreversibly inhibit acetylcholine esterase and this property is exploited for their
use as insecticides or warfare agents. Very little is known about the chronic effects of OPs on non-target tissues in
humans. Several reports have linked long-term exposure to OP pesticides to increased risk of Non-Hodgkin’s lymphoma
and different leukaemias, but the mechanism by which OPs affect cancer development is not yet clear. Our aim was to
investigate whether low concentrations of OP pesticides can cause DNA damage in non-target human cells and affect
expression of genes known to be involved in the DNA damage response.
Three model OP pesticides – parathion, paraoxon and dimefox were studied in human hepatoma HepG2 cell line. Using
the comet assay with or without inhibitors of DNA repair enzymes (arabinosylcytosine and hydroxyurea) we investigated
whether the sub-cytotoxic concentrations of OP pesticides could induce DNA damage. Dimefox did not increase the level
of strand brakes; parathion and paraoxon increased the level of strand breaks only at the highest concentration used.
Using RT-PCR, we found that exposure of HepG2 cells to parathion and paraoxon caused an increase in the expression
of genes that are involved in the response to genotoxic stress: P53, MDM2 and GADD45. Dimefox did not significantly
change the expression of MDM2 or GADD45, while at the higher concentrations it increased the expression of P53. These
data indicate that parathion and paraoxon may have some genotoxic potential, while dimefox probably does not.
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P-056
THE RELEVANCE AND SIGNIFICANCE OF N7-METHYLGUANINE AND N3-METHYLADENINE DNA
ADDUCTS ORIGINATING BY THE TOBACCO SMOKE METHYLATING CARCINOGENS
Jiří Chadt 1
Pavel Vodička 1; Robert Nilsson 2
Institute of Experimental Medicine, AS CR, Prague, Czech Republic1; Department of Genetics, Stockholm University, Stockholm,
Sweden2
DNA adducts induced by tobacco smoke may give a clue on the onset of cancer. Tobacco smoke represents a mixture
of approximately 4000 organic and inorganic compounds, already recognised in main stream, side stream, and secondhand tobacco smoke. More than 40 substances are known to be carcinogenic in animals (polycyclic aromatic hydrocarbons, aza-arenes, aromatic and heterocyclic amines, aldehydes, N-nitrosamines, inorganic compounds, benzene,
vinylchloride, styrene and 1,3-butadiene). Our major concern is devoted to tobacco specific nitrosamines (TSNAs) arising from metabolism of the nicotine, the potent human carcinogen (head and neck, lung cancer). Biotransformation of
TSNAs provides very reactive electrophilic intermediates, which readily interact with nucleophilic spots of DNA, such as
methyldiazohydroxide and others. Methyldiazohydroxide, a product of nitrosation of nicotine, is strongly methylating O6and N7- positions of guanine and N3- position of adenine most often. The most effective methylation occurs at the N7position of guanine and 5 – 7x lower at the N3-position of adenine. These exogenous methylations may be mutagenic
and carcinogenic, since N7-guanines and N3-adenines result in GC-TA transversion (N7-MeG) and AT-TA transversion
(N3-MeA). Less abundant O6-MeG results in GC-AT transition. Furthermore, under alkaline conditions 7-methylguanines
may be converted into corresponding formamidopyrimidines, resulting in mispairing. On the other hand, under acid
conditions 7-methylguanines can yield to the depurination and following single strand breakage. The biological consequences of the above methylations are related to the efficiency of DNA repair (N7-MeG and N3-MeA via BER, O6-MeG
via MGMT pathway). The aim of this work is to monitor specific methyl guanine and adenine adducts in vitro and after
in vivo by HPLC analytical methods in methylated DNA caused by products of TSNAs metabolism and consider possible
context to carcinogenity and mutagenicity.
P-057
EFFECT OF ALKB MUTATION ON MMS-INDUCED MUTAGENESIS IN E.COLI AB1157 STRAINS
Albert Jaworski
Jadwiga Nieminuszczy; Anna Sikora; Elzbieta Grzesiuk
It is widely accepted that MMS, the SN2 type alkylating agent, is a fairly strong inducer of SOS mutagenesis and adaptive response. In E. coli the mechanisms protecting cells against alkylating agents include: the tag and ogt genes,
expressed constitutively; ada, alkB (forming one operon) and alkA and aidB that are expressed after induction of the
adaptive response.
The role of AlkB protein, the product of alkB gene, in DNA repair has been established only recently. AlkB is a dioxygenase that oxidatively demethylates N1meA and N3meC in DNA in a reaction involving α-ketoglutarate, O2, and Fe2+,
resulting in recovery of the natural A and C bases. We have established that in E. coli AB1157 defective in alkB (strains
BS87 and HK82) the frequency of MMS-induced argE Arg+ reversion is 10 – 100-fold higher than in AB1157 alkB+.
Here we show that MMS-induced lesions leading to Arg+ reversions are repaired during transient starvation (conditions
for Mutation Frequency Decline, MFD repair). This repair is fully dependent on the umuDC encoded DNA polymerase V
since in E. coli AB1157alkB∆umuDC double mutant the frequency of argE Arg+ reversion is dramatically reduced. We
have also studied an effect of alkB mutation on MMS-induced mutagenesis and MFD repair in E.coli xth nth nfo triple
mutant deficient in base excision repair. In this strain we have observed poor survival and high level of MMS-induced
Arg+ revertants accompanied by the lack of MFD repair. Introduction of additional alkB mutation into this strain leads to
3-fold increase in the level of Arg+ revertants in comparison to E.coli xth nth nfo alkB+ strain.
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P-058
GENOTOXICITY OF IRINOTECAN IN HUMAN LYMPHOCYTES ASSESSED USING
THE CYTOKINESIS-BLOCK MICRONUCLEUS ASSAY IN VITRO
Nevenka Kopjar 1
Snježana Ramie 1; Mirta Milie1; Vesna Pavlica 2
Institute of Medical Research and Occupational Health, Zagreb, Croatia1; University Hospital for Tumors, Zagreb, Croatia2
Irinotecan is a promising antitumor agent, mostly used in the treatment of metastatic colorectal cancer. In spite of
widespread use, its cytogenetic effects were not extensively studied. The objective of present study was to evaluate
the level of genetic changes in human lymphocytes exposed to two therapeutic doses (180 mg/m2 and 350 mg/m2) of
irinotecan in vitro. The DNA damage was estimated using cytokinesis-block micronucleus (CBMN) assay. The frequencies of micronuclei (MN), nuclear buds, nucleoplasmic bridges as well as apoptotic morphological changes in fixed cells
were studied simultaneously. Moreover, nuclear division index both in treated and control cells was estimated. The
results show that irinotecan induced marked genetic changes, especially increased incidence of MN and nuclear buds as
compared to untreated control. Genotoxic effects of irinotecan on lymphocytes were dose-dependent. Both concentrations caused a significant increase in the fraction of apoptotic cells. Irinotecan also induced cytotoxicity as measured
by nuclear division index. Based on the results obtained we can conclude that both therapeutic concentrations of irinotecan are geno/cytotoxic to human non-target cells. Our results also point to the importance of biomarker studies in
non-target cells of cancer patients after successful chemotherapy since they could be a good predictive factor to detect
subpopulations of patients with genome instability.
P-059
EVALUATION OF TRYPTOPHOL TOXICITY IN HUMAN LYMPHOCYTES USING THE CYTOKINESISBLOCK MICRONUCLEUS ASSAY IN VITRO
Amalina Šafranie 1
Višnja Baeun-Družna1; Ivan Kosalec 2; Snježana Ramie3; Nevenka Kopjar 3
Faculty of Food Technology and Biotechnology, University of Zagreb, Croatia1; Faculty of Pharmacy and Biochemistry, University of
Zagreb, Zagreb, Croatia2; Institute of Medical Research and Occupational Health, Zagreb, Croatia3
Tryptophol (indole-3-ethanol) has been established as an endogenous plant constituent but is also formed in vitro by
biosynthesis of certain bacteria, yeasts and some seed plants. It is a compound that induces sleep, and is formed by the
parasite in trypanosomal sleeping sickness. Toxicity of tryptophol on human peripheral blood lymphocytes was investigated in vitro using the cytokinesis-block micronucleus (CBMN) assay. The concentrations tested were in range 0.25
mM, 0.50 mM, 1.0 mM and 2.0 mM. The frequencies of micronuclei (MN), nuclear buds, nucleoplasmic bridges as well as
apoptotic morphological changes in fixed cells were simultaneously evaluated. In addition, nuclear division index (NDI)
both in treated and control cells was estimated. Results indicate that tryptophol caused induction of MN, nuclear buds
and nucleoplasmic bridges, as well as the induction of apoptosis in exposed cells. Observed effects were concentrationdependent and their frequencies were the lowest in the sample treated with 0.25 M of tryptophol. We assume that the
majority of damage in tryptophol-treated lymphocytes was induced by aneugenic mechanisms. In concentrations tested,
tryptophol also affected cell membranes, which in turn led to the marked decrease of the cell volume. Tryptophol also
caused the delay in lymphocyte proliferation in vitro, and the values of NDI decreased in parallel with the increase of its
concentration. Present study elucidates only a part of tryptophol cytotoxicity to human cells, and leaves open area for
further investigations of potential effects on host organism mediated by its secondary metabolites.
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P-060
INFLUENCE OF THE DIETARY FACTORS ON THE RATE OF MODIFICATION OF OXIDATIVE DNA
DAMAGE REPAIR IN NEWBORN PIGS
Pawel Kowalczyk 1
Barbara Tudek 1; Aleksandra Bielen 1; Daniel Laubitz 2; Romuald Zabielski 3
Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw Institute of Biochemistry and Biophysics PAS,
Warsaw, Poland 1; The Kielanowski Institute of Animal Physiology and Nutrition, Jablonna, Poland2; Agricultural University, Warsaw,
Poland3
The diet exerts an important effect on the development and functioning of the digestive tract. We were investigating
the effect of supplementation of the diet of lactescent sows with grains rich in polyunsaturated fatty acids (PUFA: flaxseed, rape-seed 4 kg/100 kg of feed each) as well as L-carnitine (15 g/100 kg), taurine (100 g/100 kg) and vitamin E
(15 g/100 kg) on the rate of oxidative DNA damage excision in colons of their offspring.
Diet supplementation was performed from the 80th day of pregnancy till 28th day of lactation. Repair activities in colons
of the offspring from sows maintained on supplemented diet were higher than in unsupplemented group already on the
first day after delivery and stayed constant until the end of experiment (28th day of life). The repair activity for 8-oxoG
and ?A (measured by the nicking assay) increased about twice in pigs, whose mothers were fed with supplemented diet
rich in PUFA. However, diet supplementation had no effect on the repair activity of ethenocytosine (?C).
We also studied the effect of supplementation of pigs with different doses of iron. Iron was administered in single
injection at the dose 75mg or 200 mg after the 3rd day of birth. The iron dose 75 mg increased significantly the repair
activity for all three damages, but higher dose, 200mg increased only the activity of 8-oxoG and εA glycosylases but
not εC glycosylases.
Thus dietary factors can modulate repair capacity for 8-oxoG, and ?A, but have no effect on excision rate of ?C from
pigs intestines.
P-061
EFFECTS OF VITAMIN C ON OXIDATIVE DNA DAMAGE TO HUMAN LYMPHOCYTES AND HELA
CELLS
Amaya Azqueta 1
Adela López de Cerain 1; Andrew R Collins 2
Food Sciences & Toxicology, University of Navarra, Pamplona, Spain1; Department of Nutrition, University of Oslo, Oslo, Norway2
Vitamin C (vit. C) is a water-soluble antioxidant, which scavenges reactive oxygen and nitrogen species. It can promote the removal of oxidative DNA damage from the DNA and/or nucleotide pool, through the up-regulation of repair
enzymes. It has been accepted that a diet rich in vit. C from fruits and vegetables provides protection against cancer
and other diseases. Among numerous reports concerning positive effect of vit. C as an antioxidant there are also those
pointing to its potential pro-oxidant effect at high dose. The aim of this work is to study the protective effect of vit. C
against oxidative DNA damage caused by H2O2 and the photosensitizer Ro 19-8022 (Ro) in human lymphocytes and
HeLa cells using the comet assay. With this purpose cells were incubated with different concentrations of vit. C -0, 1,
5, 10, 50 y 100 µM- for 30 minutes at 37?C. After washing they were treated with 0.2 µM of Ro plus visible light for
9 minutes, or with 25 µM (lymphocytes) and 10 µM (HeLa cells) of H2O2 for 5 minutes on ice. Cells treated with different concentrations of vit. C were included as controls. After treatment cells were washed and prepared to carry out
the comet assays. Comets were visually scored and classified. The enzyme formamidopyrimidine glycosylase (FPG)
was used to detect oxidised purines in cells treated with only vit. C and with vit. C plus Ro. In both types of cells, vit.
C induced SSB at high doses, althought no FPG sensitive sites were detected. There was no protective effect of vit. C
against the oxidative DNA damage caused by H2O2. The same results were observed in cells treated with Ro but in those
pretreated with high concentrations of vit. C a decrease in FPG sensitive sites and an increase in single strand breaks
were observed. According to these results, high concentrations of vit. C have a pro-oxidant effect “in vitro” in both types
of cells. Further data are needed to understand the antioxidant/pro-oxidant role of vit. C.
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P-062
SPONTANEUS AND BLEOMYCIN-INDUCED MICRONUCLEI IN HUMAN LYMPHOCYTES:
CORRELATING POLYMORPHISMS IN DNA REPAIR GENES WITH MUTAGEN SENSITIVITY
Francesca Maffei 1
Sabrina Angelini 1; Fabio Carbone 1; Giorgio Cantelli-Forti 1; Patrizia Hrelia 1;
Rajiv Kumar 2; Kari Hemminki 2
Department of Pharmacology, University of Bologna, Bologna, Italy1; Division of Molecular Genetic Epidemiology, German Cancer
Research Center, Heidelberg, Germany2
Epidemiological studies have revealed a positive association between DNA repair capacity and risk of cancer. A decreased
repair capacity results in greater susceptibility to mutations and enhanced genetic instability. Consequently, the identification of individuals carrying genetic polymorphisms responsible for reduce DNA repair capacity has substantial
preventive implication.
We investigated the association between single nucleotide polymorphisms (SNP) in the XPD, XRCC1 and XRCC3 genes
and the levels of spontaneous and bleomycin (BLM)-induced chromosome damage in peripheral blood lymphocytes from
80 healthy individuals (45 women and 35 men, mean age 30.5±9.5) without history of cancer. Twenty-five percent
were mild smokers (less than 20 cigarettes smoked daily). BLM treatment was effective in the induction of chromosome
damages as we observed a statistically significant increase in micronuclei (MN) frequency (MN/ 1000 binucleated cells:
6.78±2.82 versus 14.75±4.50, P = 0.001). The results of Poisson regression analysis that included age, gender and
smoking status showed that smoking status exerted a marginal influence on the spontaneous MN frequency (P= 0.078),
whereas BLM sensitivity was not influence by any of them.
Genotype analysis did not reveal a clear association between the studied polymorphisms (XPD, XRCC1 and XRCC3) and
DNA damage however, a definitive conclusion can not be drawn, due to the relatively small sample size. When genegene interaction was considered, individuals bearing the lowest score for protective alleles were also the one with the
highest levels of spontaneous and BLM-induced DNA damage. These preliminary results have to be considered as a
starting point for the inherited basis of human mutagen sensitivity, and should encourage further investigation with a
large sample size and including more genetic polymorphisms that might lead to the identification of a genetic profile
responsible for the individual susceptibility to genotoxic compounds.
P-063
DNA DOUBLE-STRAND BREAKS REPAIR AS A FUNCTION OF THE SUBSTRATE ENDS IN NORMAL
AND CANCER HUMAN CELLS
Mariusz Malinowski 1,2
Janusz Blasiak 1; Tomasz Poplawski 1; Elzbieta Pastwa 3
Department of Molecular Genetics, University of Lodz, Lodz, Poland1; Department of Molecular and Medical Biophysics, Medical
University of Lodz, Lodz, Poland2; Molecular Genetics Department, Medical University of Lodz, Lodz, Poland 3
DNA double-strand breaks (DSBs) are the most serious damage. If they are not repaired or misrepaired, may lead to
cell death, mutations or cancer transformation. In human cells they can be repaired mainly via non-homologous DNA
end joining (NHEJ). The structure of the DSBs is an important feature of the mechanism underlying NHEJ. To investigate the dependence of the NHEJ repair reaction on the structure of DNA termini in normal and cancer human cells, we
employed an in vitro NHEJ assay. We used linearized plasmids containing several different configurations of DNA ends,
and fluorescent dye for rapid and direct visualization of rejoining products. In our studies we demonstrate that the efficiency of NHEJ is different for all examined DNA ends. Furthermore, the repair of complementary and blunt-ended DNA
was more efficient than the repair of more complex ends. We also observed that human cancer cells are able to repair
DSBs at different ratio compared to normal human cells. In conclusion, the present study showed that a distinct genetic
constitution of these cells could be responsible for various efficiency of non-homologous DNA end joining.
This work was supported by the State Committee for Scientific Research, Grant No. 3 PO4A 032 25.
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P-064
INDUCTION OF GENETICS VARIABILYTY IN OFFSPRING OF MICE, GAMMA-IRRADIATED
IN DOSE RANGE FROM 1 GY TO 3 GY
Andrey E. Myazin
Lylyvati K. Ramayia; Gadjiramazan O. Shaikhaev; Vladimir A. Shevchenko; Marina D. Pomerantseva
Institute of general genetics, Moscow, Russian Federation
It is known, that cells at various stages of spermatogenesis have various radiosensitivity. The aim of the present work
is study effects of a chronic and acute irradiation in various doses and also comparison genetic sensitivity of spermatogonia and spermatides to an irradiation in a range of doses 1-3 Gy. The analysis carried out with the use of the
RAPD-test (random amplified polymorphic DNA assay) which is based on amplification total genomic DNA with a series
of random primers and enables multi-locus scanning of hypervariable regions. Mice BALB/c were irradiated in doses 1-3
Gy on gamma-unit GYPOS (dose rate- 4,5 Gy/min, source- Cs-137). For comparison of sensitivity of different stages of
spermatogenesis males were crossed with females the same strain in two weeks and in three months after an irradiation. The offspring in both cases contained in standard conditions and killed in the age of 3-4 weeks. Purification and
amplification of DNA were performed, by using commercial kits of Izogen. For PCR amplificator PT-48 (TDL Company)
were used. Products of RAPD were separated into 1,5 % agaroze gel. Analysis of offspring patterns carried out on the
basis of comparison with parental patterns, with the purpose of registration of new, "not parental" bands. Results of the
analysis processed statistically. In case of an acute irradiation we received increasing of a level of the polymorphism
expressed as frequency of occurrence of new strips on one descendant in family, which significantly distinguished from
the control. Comparison of results of the analysis of sensitivity of spermatogonia and spermatids at all used doses, has
shown, that in pre-meiosis cells genetic effects of an irradiation are expressed in the greater degree, than in post-meiosis cells. Result of our work is is revealing dependence of levels of polymorphism in hypervariable sites of DNA in the
field of doses up to 3 Gy, and also detection of genetic effect at influence of low-levels chronic radiation.
P-065
THE FREQUENCY OF GC TO AT BASE SUBSTITUTIONS ARISING IN CAA - TREATED DNA
IS ALKB -DEPENDENT
Jadwiga Nieminuszczy
Agnieszka M Maciejewska; Jaroslaw T Kusmierek; Elzbieta Grzesiuk
The E. coli AlkB protein is a part of inducible Ada response. It is a DNA repair enzyme that belongs to the α-ketoglutarate-Fe2+-dependent-dioxygenase superfamily. AlkB removes alkyl lesions via an oxidative mechanism restoring native
DNA. Most recently, it was found that AlkB also repairs ethenoadducts (e-adducts). We generated e-adducts in plasmid
DNA by chloroacetaldehyde (CAA) treatment. The modified plasmid, which carried Miller’s mutation 102 identifying
GC?AT transitions, was transformed into bacterial cells of different genetic background and mutants were selected. The
102 mutation allows us to study mutations caused by eG and eC. In non adapted and adapted to alkylating agents
cells we observed a dose dependent increase of mutations in the alkB strain, which was correlated with a decrease in
transformation efficiency. The double alkB mug mutant (additionally lacking Mug glycosylase removing eC) showed an
enhanced mutagenic effect. The mutagenic effect of invalid alkA (lacking AlkA glycosylase removing eG) and alkA mug
was comparable to that observed in alkB and alkB mug, respectively. Interestingly, in alkB mutant the frequency of
mutations caused by initially formed 3,N4-a-hydroxyethanocytosine (HEC) has been higher than the one caused by the
product of its dehydration, eC. These in vivo findings were verified by in vitro reactions.
Our results confirm that AlkB repairs eC and suggest that the protein is also able to repair HEC, indicating that its substrate specificity may be broader than reported previously.
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P-066
INFLUENCE OF OCCUPATIONAL EXPOSURE TO ENVIRONMENTAL POLYCYCLIC AROMATIC
HYDROCARBONS ON DNA REPAIR
Antonina Cebulska-Wasilewska 1,2
Agnieszka Panek 1; Ivan Kalina 3; Jozef Zidzik 3; Peter B Farmer 4
Department of Radiation and Environmental Biology, The H.Niewodniczański Institute of Nuclear Physics PAN, Kraków, Poland1;
Chair of the Epidemiology and Preventive Medicine, CM UJ, Kraków, Poland2; Department of Molecular Biology of the P.J.Šafárik
University, Košice, Slovakia3; Cancer Biomarkers and Prevention Group, University of Leicester, Leicester, United Kingdom4
The aim of our study was to investigate whether occupational exposure to environmental polycyclic aromatic hydrocarbons (PAHs) can affect DNA repair efficiency. Monitoring was performed on lymphocytes from unexposed males (54
people aged 33.4) and exposed donors (52 policemen of average age 32) from Košice in Slovakia. A challenging dose
of X-rays and an alkaline version of single cell gel electrophoresis assay were applied to investigate cellular repair efficiency. The DNA damage was detected before and immediately after irradiation, and after incubation for periods allowing
cells to proceed with DNA repair processes. In all studies, samples from the same pool of lymphocytes of unexposed
healthy male donor were applied as the internal standards. No significant difference was observed between exposed
and unexposed groups in the mean values of the DNA damage detected in lymphocytes prior or immediately after challenging treatment. However, analysis of the amount of not repaired (residual) DNA damage detected after incubation of
irradiated cells, revealed a significantly lower efficiency of DNA repair process in lymphocytes of subjects occupationally
exposed to PAHs (residual DNA damage 60.7% for non exposed versus 69.8% for exposed, p<0.01). A significantly
negative influence of the smoking habit on the efficacy of the repair process was also observed (residual DNA damage
41.3% for non smokers versus 49.4% for smokers, p<0.037). Significantly lower efficiency (39.7% in mutant versus
60.3% in a wild type, p<0.009) of the repair process of the damage induced by the challenging dose was also observed
in lymphocytes of genetically polymorphic donors with mutation in exon 5 GSTP1 (GSTP1 Ile/Val).
Research was partially supported by grants: EC-EXPAH QLK4-CT-2000-00091/SPUB-M 620/E-77/SPUB-M/5.PRUE/
DZ74/2001.
P-067
ROLE OF O6 METHYLGUANINE DNA METHYLTRANSFERASE (MGMT) IN METHYLATING AGENT–
INDUCED APOPTOSIS
Vassiliki Pletsa 1
Apostolia Guialis 1;Soterios Kyrtopoulos 1; Meropi Patrinou-Georgoula 1; Wynand Roos 2; Filippo Rosselli 3
National Hellenic Research Foundation, Athens, Greece1; Division of Applied Toxicology-Institute of Toxicology, Mainz, Germany2;
Institut Gustave Roussy, Villejuif, France3
Methylating agents is a widely used class of anticancer drugs. The most biologically significant methylation adduct
is O6-methylguanine (O6-meG) repaired by the specific enzyme O6 methylguanine DNA methyltransferase (MGMT)
Prereplicative repair by MGMT and postreplicative Methyl-directed Mismatch Repair (MMR) detetermine the level of
O6-meG-induced genotoxicity and cell death. O6-meG accumulation due to inefficient repair can lead to cell death via
apoptosis although the mechanism seems to be cell type dependent.
RNA binding proteins are implicated in all aspects of RNA metabolism. Reports on their modifications during apoptosis
and interactions with apoptosis-associated elements indicate their potent implication in the control of apoptotic cascade.
Aim of this study is to investigate the role of DNA repair in methylating agent-induced apoptosis and examine alterations of RNA binding proteins to assess their possible use as markers of tumour chemoresistance/sensitivity.
An O6-methylguanine-DNA-methyltransferase inducible HeLa cell line was treated with different concentrations of Nmethyl-N-nitrosourea (MNU) under varying expression levels of the DNA repair enzyme MGMT. Cells lacking MGMT were
far more sensitive to MNU than those with even low levels of the enzyme. Flow cytometric analysis confirmed the protection provided by MGMT against MNU-induced apoptosis. Molecular analysis revealed caspase-7 instead of caspase-3
activation and PARP cleavage as late events prominent 72h after treatment. Poly(A) polymerase cleavage was detected
only in cells lacking MGMT.
Our results indicate that apoptosis is mainly induced in the absence of MGMT. We are currently investigating the molecular apoptotic pathway involved in HeLa and additional cell lines and possible modifications of RNA binding proteins in
order to assess their possible use as markers of chemosensitivity .
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P-068
INHIBITION OF HOMOLOGOUS RECOMBINATION (HR) BY P53 IS DEPENDENT
ON PHOSPHORYLATION ON SERINE 15
Anja Restle
Christine Janz; Lisa Wiesmüller
Universitätsfrauenklinik, Ulm, Germany
ATM/ATR mediated phosphorylation of p53 on serine 15 is a frequent modification and provoke a cascade of post-translational modifications. By use of a fluorescence based recombination assay and chemical ATM/ATR inhibition in cells
expressing wild-type p53 or p53 mutated on serine 15, we demonstrate, that suppression of error-prone homologous
recombination (HR) is dependent on p53 phosphorylated on serine 15 (p53pSer15). To further delineate the mechanisms that underly p53pSer15 functions in recombination control, we compared the nuclear localization of p53pSer15
and the key enzymes of HR after replication fork stalling. Our results show, that p53pSer15 colocalizes with 40-60 %
of Rad51 and Mre11 foci, whereas p53pSer15 subcompartimentalization was almost mutually exclusive with respect
to Rad52. Hence, possible sites of p53pSer15-dependent regulatory activities seem to be sites of Rad51- rather than
Rad52-dependent HR processes. Notably, after 6 h the association of Rad51 and Mre11 foci overlapping with p53pSer15
was less than 20 %, therefore, p53pSer15 interactions with repair complexes containing Rad51 or Mre11 exist only
during early HR processes. Next, we correspondingly investigated p53pSer15 and the RecQ helicase BLM, a tumor suppressor which, like p53, exhibits recombination control and pro-apoptotic functions. Our analysis revealed colocalization
within a fraction of approximately 70 % of the BLM foci and long-lived physical interactions until 6 h after replication
arrest. We propose that p53pSer15-BLM interactions play a dual role in suppressing error-prone HR at an early stage
as well as subsequent checkpoint signaling.
P-069
ANTICANCER PML PROTEIN DIFFERENTIALLY REGULATES GENE PROMOTERS AND PARTIALLY
COLOCALIZES WITH WRN HELICASE
Rasa Vaitiekunaite1,2
Rusin Marek1; Dorota Butkiewicz1; Agata Baginska1; Malgorzata Krzesniak1
Department of Tumor Biology, Center of Oncology, Maria Skłodowska-Curie Memorial Institute, Gliwice, Poland1;Institute of
Oncology, Vilnius University, Vilnius, Lithuania2
WRN and PML are anticancer proteins involved in cellular senescence. WRN is a DNA helicase, playing role in DNA replication, repair, transcription and apoptosis. The loss-of-function WRN mutations cause cancer-prone progeria known
as Werner syndrome. PML is a multifunctional protein involved in cell proliferation, apoptosis and regulation of transcription. It may exist in several isoforms. The disruption of PML normal function by its fusion to retinoic acid receptor
is associated with acute promyelocytic leukemia. Both WRN and PML proteins in their native, non-mutated state are
localized in cell nucleus. Some observations suggest possible functional interactions between PML and WRN, however,
this issue is poorly examined. The goal of this work was to determine whether WRN and splicing variants of PML colocalize in nucleoplasmic structures called nuclear “bodies” and to better understand involvement of PML in the process of
transcription regulation. For WRN-PML colocalization studies, U2-OS cells were cotransfected with WRN fused to mRFP,
and PML isoforms I-VI fused to EGFP. Five PML isoforms (I and III-VI) formed PML nuclear bodies, whereas isoform II
showed diffused nuclear localization. WRN and PML isoforms II, V and VI did not colocalize. For WRN and PML isoforms
I, III, and IV, partial colocalization was observed in a subset of cells – PML protein formed a conspicuous shell on some
WRN bodies. To determine the influence of PML isoforms on gene promoter activity, several cell lines were cotransfected
with PML and reporter vectors containing one of viral (CMV, HSV-TK and SV40) or human (BRCA1, BRCA2, p16, XPA
and WRN) gene promoters. The promoters were efficiently activated by PML isoform VI, by PML isoform IV, or by both
these isoforms. Moreover, we observed that the ability of PML to activate a promoter is cell-type dependent, indicating
a complex pattern of gene regulation by PML.
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P-070
INCREASED SPEED AND EFFICIENCY OF DNA STRAND BREAK REJOINING BY TEMPORARY
NON-GENOTOXIC STIMULATION OF POLY(ADP-RIBOSE) SYNTHESIS IN HUMAN CELLS
IN VITRO
Nadezhda I. Ryabokon 1,2
Joanna Rzeszowska-Wolny 1; Rose I. Goncharova 2; Gunars Duburs 3
Centre of Oncology, M. Sklodowska-Curie Memorial Institute, Gliwice, Poland1; Institute of Genetics and Cytology, National
Academy of Sciences of Belarus, Minsk, Belarus2; Latvian Institute of Organic Synthesis, Riga, Latvia3
Background. The poly(ADP-ribose) polymerases PARP-1 and PARP-2 play a crucial role in DNA base excision repair after
having been activated by a DNA strand interruption. The quantity of poly(ADP-ribose) rapidly synthesized from NAD+ is
proportional to the level of DNA damage, and can be decreased by NAD+ or PARP deficiency causing poor DNA repair
and genomic instability. The precise role of poly(ADP-ribose) in DNA repair is widely studied and not yet clear. Here we
studied the effect of transient non-genotoxic stimulation of poly(ADP-ribose) formation on repair of DNA single strand
breaks (SSBs). Methods. Poly(ADP-ribose) synthesis was induced by 100 µM H2O2 in lymphoblastoid Raji cells and further
stimulated by the 1,4-dihydropyridine derivative AV-153 at non-genotoxic concentrations of 1 nM –10 µM. Poly(ADPribose) formation was quantitated by in situ immunofluorescence in individual cells and confirmed by Western blotting
of whole-cell extracts. DNA damage was assessed by alkaline comet assays. Results. A ~100-fold increase in poly(ADPribose) was observed during the first 5 min of recovery from H2O2 treatment, and followed by a gradual decrease up to
15 min. This synthesis was completely inhibited by the PARP inhibitor 1,5-isoquinolinediol. In contrast, AV-153 showed
a concentration-dependent stimulation of poly(ADP-ribose) formation after the first minute of recovery. The extent of
transient overproduction of poly(ADP-ribose) in the presence of AV-153 reached 130% and was strongly correlated with
the speed and efficiency of DNA SSB rejoining. Conclusions. These results demonstrate (i) the importance of the level
of poly(ADP-ribose) synthesis for the speed and efficiency of DNA repair and (ii) the possibility of enhancing SSB repair
via stimulation of poly(ADP-ribose) synthesis. Acknowledgements. N.I.R.’s work in Centre of Oncology, Gliwice, Poland,
was supported by Fellowship funds from the NCI, OIA, Bethesda, MD, USA.
P-071
INFLUENCE OF POLYMORPHISMS IN DNA REPAIR GENES ON REPAIR KINETICS, MEASURED
BY THE COMET ASSAY
Charlotta M Ryk 1
Bo Lambert 1; Sai-Mei Hou 1; Rajiv Kumar 1,3; Michael N Routledge 2; Christopher P Wild 2
Department of Biosciences and Nutrition / Karolinska Institute, Stockholm, Sweden, Sweden1; Molecular Epidemiology Unit, The
LIGHT Laboratories / University of Leeds, Leeds, England, England2; Division of Molecular Genetic Epidemiology, German Cancer
Research Centre, Heidelberg, Germany, Germany3
Part of the inter-individual variation in DNA repair capacity that exists in a normal population may be attributable to
polymorphisms in DNA repair genes. We have used the single cell gel electrophoresis method (comet assay) to study
the functional impact of seven polymorphisms in six different DNA repair genes, involved in base excision repair (XRCC1
Arg194Trp, XRCC1 Arg399Gln, and APEX1 Asp148Glu), homologous recombination repair (XRCC3 Thr241Met and NBS1
Glu185Gln), mis-match repair (hMLH1 –93 A/G), and nucleotide excision repair (XPD Lys751Gln). Lymphocytes were
obtained from 52 healthy non-smoking volunteers. One portion of the cells was used for genotyping, using the PCR-RFLP
or Taq-Man allelic discrimination method. Another portion of the cells was stimulated with phytohemagglutinin (PHA) for
48 hours, treated with 0.5 mM of the direct acting alkylating agent methylmethane sulphonate (MMS) for 15 minutes,
and allowed to repair for 5, 20, 45 or 125 minutes. The DNA damage remaining at the different time points was evaluated both as pecent damaged cells and as a pseudo score (PS, comparable to percent tail DNA). The mean PS was 3.2
(+/-1.6) in untreated cells, 54.4 (+/-8.1) five minutes after exposure and 39.0 (+/-12.1) 125 minutes after exposure.
Statistically significant increased risk for high PS were seen in the APEX1 Asp148 allele carriers at all time points (5,
20 and 45 minutes) except the last one (125 minutes). Also the XRCC3 241Met allele was associated with an increased
risk for high PS, but only at the initial two time points. The NBS1 185Gln allele was associated with an increased risk at
the first, and a decreased risk at the following two time points. This indicates that the repair rates at early stages are
affected by these polymorphisms in the present experimental system.
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P-072
CELLULAR RESPONSE TO DNA LESIONS INDUCED BY DOXORUBICIN IN HUMAN FIBROBLASTS
PROFICIENT AND DEFICIENT IN NUCLEOTIDE EXCISION REPAIR
Mateus H Agnoletto 1
Temenouga N Guecheva 1; Jenifer Saffi 1,3; Antonio Pegas Henriques 1,3; Luis FZ Batista 2; Heleotonio
Carvalho 2; G P Amarante-Mendes 2; Joao Carlos F Menck 2
Federal University of Rio Grande do Sul,Porto Alegre,Brazil1; University of Sao Paulo, Sao Paulo, Brazil2,1; Lutheran University of
Brazil, Canoas, Brazil3
DNA repair impairment or lack of apoptosis induction may play a role in the resistance of cancer cells to therapeutic
drugs. Among several DNA repair pathways, the nucleotide excision repair (NER) is the most versatile one in repairing
of bulky adducts and other types of damage. This mechanism involves more than 30 proteins, among them the XPD
protein, which is a subunit of the TFIIH complex. Patients deficient in Xeroderma Pigmentosum (XP) genes present high
sensitivity to sunlight and develop skin cancer in early age. In this study, we have utilized SV40-transformed skin fibroblasts from biopsies of XPD, XP combined with CS and TTD patients, as well as MRC5 (human normal fibrosblast) and
HeLa cells to investigate the responses to doxorubicin (DOX), an anti-tumoral drug used in cancer therapy. Cell survival
was measured by the XTT assay after 72 h treatment with DOX. All NER deficient cell lines showed the same sensitivity
to DOX and HeLa cells were the most resistant. The genotoxic effects and the repair kinetics studies were studied by
using the Comet assay. Our results demonstrate that DOX induced concentration-related increase in DNA damage in
NER deficient human fibroblasts that persists during the 3 hours period of post incubation. Alteration in Sub-G1 population and cell cycle arrest was measured by FACS after 48 h, 72 h, and 96 h of DOX treatment. This analysis revealed an
intensive arrest at G2 and formation of 8 n cells for all cell lines. However, XPD cells did not present arrest at G2 and
8n cells formation at dose of 0.6 ?g/mL and for 96h of 0.2 ?g/mL, which could be caused by cell death, as evidenced
by the increased sub-G1 population. XP/CS showed highest number of 8n cells after treatment with 0.2 ?g/mL of DOX.
Our data also showed the involvement of p38 MAPK in cell cycle arrest at G2 after 16 hours treatment indicating that
initial arrest could be induced by activation of p38 pathway. Supported by CNPq, Capes and FAPESP.
P-073
PERSISTENT GENOMIC INSTABILITY BY ARSENIC EXPOSURE IN V79 CHINESE HAMSTER
CELLS
Giulia Sciandrello
Maurizio Mauro; Irene Catanzaro; Fabio Caradonna; Giusi Barbata
University of Palermo, Palermo, Italy
Previously, we demonstrated that acute treatment with arsenic leads mammalian cells to exhibit persistent chromosomal instability and DNA hypomethylation, by performing investigations after about 2 months of subculturing. In order to
evaluate quantitatively the continuing instability during the expanded growth, we carried out cytogenetic, morphologic
and molecular analyses immediately after exposure and every week up to 112 cell generations.
Briefly, V79 Chinese hamster cells were treated with 10 µM sodium arsenite (SA) for 24h; at the end of exposure,
mitotic rounded-up cells were harvested by shake-off and allowed to grow in drug-free medium. The instability markers,
micronucleated and multinucleated cells as well as aneuploid cells, seen just after treatment, reappeared since 60th cell
generation. Metaphases with dicentric chromosomes or telomeric associations characterized cell population since 90th
cell generation. To gain insight into the mechanism involved in perpetuating the unstable phenotype, groups of clones,
stable and unstable, were analysed also for telomerase activity by TRAP assay and for levels of reactive oxygen species
(ROS) measured by ability to oxidize fluorogenic dye. Some of the isolated unstable clones, also bearing chromosomal
end-to-end fusions, maintained telomerase activity and were capable to proliferate accumulating genomic instability
as well as transformed phenotype and spontaneous increased gene mutation oxidative stress associated. Furthermore
these clones showed altered DNA methylation pattern.
On the whole, these results raise the possibility that cell variants induced by the short-term exposure to arsenic probably gain a selective advantage when they are able to epigenetically reprogram their genome and proliferate in an
error-prone mode.
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P-074
THE DEL ASSAY DETECTS CARCINOGENS
Robert H. Schiestl
Genetic instability and DNA deletions are involved in carcinogenesis. We have constructed and/or used assays that
select for DNA deletion events in yeast, human cells and in vivo in the mouse (DEL assays). DEL events in all three
formats are inducible by a wide variety of carcinogens including carcinogens that are negative in the Salmonella assay
and many other short-term tests. Furthermore, the yeast DEL assay correctly differentiates between carcinogen - non
carcinogen structural analogs. With published data the DEL assay shows a sensitivity of 90 % (44/49 carcinogens
detected), a specificity of 75 % (15/20 non-carcinogens negative) and an overall accuracy of 86 % (59/69 compounds
correctly classified) compared to the Salmonella assay that detects only 33% of the same chemicals correctly. DNA
double strand breaks initiate DEL recombination. Additional experiments with 20 alkylating chemicals show that the
DEL assay detects both, Salmonella mutagenic and structural alert as well as clastogenic chemicals and with a different set of 10 chemicals in parallel with the micronucleus assay it shows a high correlation with the micronucleus assay.
A bioluminescent version of the yeast DEL assay has been developed in collaboration with Dr. Jiri Aubrecht of Pfizer
which has been developed into a high throughput screening format as a prescreen to identify the potential carcinogenic
and clastogenic activity of chemicals. More widespread use of the DEL assays may lead to a higher accuracy in predicting the carcinogenic potential of chemicals and may give more mechanistic information about the biologic activity of
carcinogens than current assays.
P-075
THE EFFECT OF ANTIOXIDANTS ON GENETIC INSTABILITY AND CANCER IN ATAXIA
TELANGIECTASIA
Robert H. Schiestl
Ramune Reliene
Ataxia Telangiectasia (AT) is an autosomal recessive disorder characterized by motor dysfunction, chromosomal instability, radiosensitivity, oxidatively stressed phenotype and high incidence of cancer. Our previous studies showed that ATM
deficiency caused significantly elevated levels of DNA deletions in mice. Such deletion events occurring in somatic cells
in pun/pun mouse embryos cause reversion of the pun mutation to wildtype p gene by deletion of a 70 kb DNA fragment
and result in the assembly of a black pigment melanin complex in melanocytes. These deletion events result in black
spots on the gray fur and patches of black cells (eye spots) on the transparent retinal pigment epithelium (RPE).
Since AT cells are oxidatively stressed, we hypothesized that dietary intake of antioxidants may eliminate oxidative
stress in AT and thereby reduce the frequency of DNA deletions and oxidative DNA damage. To test our hypothesis we
crossed ATM+/- pun/pun mice with each other and the dams were treated with the antioxidant N-acetyl-cystein (NAC) in
drinking water during pregnancy. The frequency of DNA deletions and oxidative DNA damage was determined in the
offspring. NAC treatment reduced the levels of DNA deletions as well as oxidative DNA damage in ATM-/- mice to the
wildtype level. Furthermore, continuous NAC treatment significantly prolonged the life of and reduced the frequency of
lymphomas in ATM-/- mice.
This finding suggests that NAC is a prospective?nutritional antioxidant to counteract DNA damage, DNA instability and
cancer in AT.
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P-076
INFLUENCE OF CMF CHEMOTHERAPY ON THE LEVEL OF DNA DAMAGE IN LEUKOCYTES
OF WHOLE BLOOD IRRADIATED IN VITRO BY X-RAYS
Nikolai P. Sirota 1
Elena A. Kuznetsova 1; Irina G. Zakharova 2
Institute of Theoretical and Experimental Biophysics, RAS, Pushchino, Russian Federation1; Hospital of Pushchino Research Center,
Pushchino, Russian Federation2
In the course of chemotherapy, the drugs affect both cancer cells and normal tissue cells. Drugs used in treatment of
patients having breast cancer, such as cyclophosphamide, metotrexate, 5-fluorouracil (CMF), are capable of not only
damaging the DNA structure but also block DNA repair processes. Controlling the level of damage to healthy tissue cells
is necessary for successful treatment. The purpose of the present study was to assess the effect of CMF therapy on the
defense systems of normal cells. The state of leukocyte defense systems was assessed with the use of an additional
genotoxic influence, exposure to X-rays, under in vitro conditions. METHOD: the level of DNA damage in whole blood
leukocytes of cancer patients and healthy donors was determined by the alkaline version of comet assay. The amount
of DNA in the comet tail was expressed in percent of total DNA in the comet (%TDNA). Aliquots (200-500 ?l) of whole
blood were taken from cancer patients 24 hours before and 24 hours after drug infusion. In patient No1, blood samples
were taken during cycles IV and V of chemotherapy, in patient No2 during cycles III and VI. Slides with whole blood
immobilized in agarose were prepared. The slides were irradiated with X-rays at a dose rate 1.12 Gy/min, 200 KV, 20
mA, filters 1mm Al and 1 mm Cu, focus distance 37 cm, at room temperature. RESULTS: for healthy donors, the value
%TDNA varied between 1.0 and 3.6%, the mean value was 2.9±0.5% TDNA (n=25). Irradiation of blood at a dose of 4
Gy induced a response with a mean value of 7.5±3.1% TDNA (n=11). In cancer patient blood samples, the vulnerable
effect of irradiation increased during chemotherapy. For cycle III it was 5.8±1.0%; for cycle IV, 12.4±2.7%; for cycle
V, 14.2±2.2%; for cycle VI, 18.9±2.7%. CONCLUSION: The CMF therapy increases the sensibility of leukocytes to the
action of ionizing radiation, which may be the result of suppression/depletion of the defense systems in healthy cells.
P-077
MEASUREMENT OF P53-SPECIFIC DAMAGE FORMATION/REPAIR IN PERIPHERAL BLOOD
MONONUCLEAR CELLS FOLLOWING IN VITRO EXPOSURE TO MELPHALAN MAY PREDICT
CLINICAL OUTCOME IN PATIENTS WITH MULTIPLE MYELOMA
Soterios A Kyrtopoulos 1; Meletios A Dimopoulos 2; Athanasios Anagnostopoulos 2; Petros P Sfikakis 3
Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece1; Department of Clinical
Therapeutics, University of Athens School of Medicine, Athens, Greece2; First Department of Propedeutic Medicine, University of
Athens School of Medicine, Athens, Greece3
Introduction: Increased DNA damage and slower repair capacity in the p53 gene from blood leukocytes of multiple
myeloma (MM) patients after therapeutic treatment with the nitrogen mustard melphalan correlate with improved outcome.
Purpose: To quantitate the individual levels of p53-specific damage formation and repair in peripheral blood mononuclear cells (PBMC) from MM patients before the therapeutic treatment following in vitro exposure to melphalan and to
search for possible correlations with the clinical outcome.
Methods: p53-specific damage was measured in PBMC from 32 MM patients (23 responders and 9 nonresponders) candidates for high-dose melphalan (HDM) supported by autologous stem cell transplantation (ASCT) following (a) in vitro
treatment with melphalan or (b) therapeutic treatment with HDM (in vivo data).
Results: In both in vitro and in vivo exposures, large interpatient variation (7-fold difference in the in vitro and 13-fold
in the in vivo) was found, indicating that the melphalan-induced biological effect is largely individualized. An excellent
linear correlation was observed between the in vitro and the in vivo data in the same patients (R2=?.83, p<0.0001). In
the in vitro data, a significantly greater DNA damage and a slower rate of repair in the p53 gene were found in patients
who achieved tumor reduction compared to nonresponding patients (Responders: 892.1+254.9 adducts/106 nucleotides
x h, nonresponders: 492.8+173.8 adducts/106 nucleotides x h, p=0.003). Furthermore, longer progression-free survival
correlated with increased DNA damage following in vitro treatment (p=0.0045).
Conclusions: Quantification of p53-specific formation/repair in PBMC from MM patients before the therapeutic treatment
following in vitro exposure to melphalan may be a useful predictive marker for the selection of those patients who are
more likely to benefit from the HDM.
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P-078
DEVELOPMENT AND VALIDATION OF A MULTIPLEX LONG QUANTITATIVE POLYMERASE CHAIN
REACTION TO MEASURE P53-SPECIFIC DAMAGE FORMATION AND REPAIR
Vassilis L Souliotis
Dimitris Typas; Margarita Bekyrou; Soterios A Kyrtopoulos
Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece
Introduction: Increased DNA damage and slower repair capacity in the p53 gene from peripheral blood mononuclear
cells following chemotherapeutic or in vitro treatment with melphalan correlate well with improved clinical outcome in
patients with multiple myeloma.
Purpose: To develop and validate a gene-specific, rapid, quantitative, non-radioactive, sensitive method measuring
damage formation/repair following exposure to chemotherapeutic agents inducing bulky adducts.
Methods: HepG2 cells were treated with various doses of cisplatin (0-200 μg/ml) for 3 h at 37oC. Genomic DNA was
isolated and the multiplex long quantitative PCR (QPCR) was carried out in order to amplify a 7 kb fragment, part of
the p53 gene (from exon 4 to exon 11) and a 500 bp fragment, part of the IFN-β1 sequence (internal standard). The
PCR products were electrophoresed on an agarose gel, stained with SYBR Green and measured with a phosphoimager
documentation and analysis system.
Results: To establish that amplification is proportional to the amount of intact template in the reaction during the exponential phase of PCR amplification, initial experiments were designed to determine (a) the initial DNA concentrations and
(b) the number of PCR cycles that could provide quantitative amplification during PCR using untreated DNA. Moreover,
using various doses of cisplatin, we found that QPCR is a sensitive method for validly estimating DNA damage formation
and repair following exposure of human carcinoma cells (HepG2) to chemotherapeutic agents inducing bulky adducts.
Conclusion: The results presented here suggest that the current protocol of the QPCR method can be used to assess
the damage formation and repair efficiency of chemotherapeutic agents inducing bulky adducts at biologically relevant
doses and to allow a more precise determination of gene-specific repair in disease susceptibility and drug resistance in
the clinical practice.
P-079
THE GLOBAL GENOME REPAIR SUBPATHWAY OF NUCLEOTIDE EXCISION REPAIR PLAYS
A CRUCIAL ROLE IN THE REPAIR OF MELPHALAN-INDUCED LESIONS
Hara G Episkopou1
Vassilis L Souliotis 1; Soterios A Kyrtopoulos1; Meletios A Dimopoulos2; Maria J Fousteri3;
L HF Mullenders3; Petros P Sfikakis4
Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece1; Department of Clinical
Therapeutics, University of Athens School of Medicine, Athens, Greece2; Department of Toxicogenetics, Leiden University Medical
Center, Leiden University, Leiden, Netherlands3; First Department of Propedeutic Medicine, University of Athens School of Medicine,
Athens, Greece4
Introduction: Gene- but not strand-specific repair in the in vivo repair of genes with dissimilar transcription levels (bactin>p53>N-ras>d-globin) was observed in the peripheral blood mononuclear cells from multiple myeloma patients
following therapeutic treatment with melphalan (200 mg/m2). Moreover, the “looseness” of the chromatin structure in
each gene correlated with the level of repair.
Purpose: To examine the relative contribution of transcription-coupled repair (TCR) and global genome repair (GGR) to
the repair of melphalan-induced lesions.
Methods: The melphalan-induced damage formation/repair was studied in the active p53 and the inactive d-globin
genes in normal human fibroblasts, XP-C cells (only TCR, deficient in GGR), CS cells (only GGR, deficient in TCR), and
XP-A cells (completely NER deficient).
Results: In all repair proficient cells, gene-specific repair of melphalan-induced lesions was found: the active p53 gene
was repaired more efficiently than the inactive d-globin gene. The damage formation/repair in the p53 gene is approximately the same in the normal and the CS cells, suggesting that GGR plays a crucial role in the repair of melphalaninduced lesions. Furthermore, we found that the XP-C cells are able to repair DNA damage in the p53, indicating that the
melphalan-induced lesions are targets for TCR as well. It is noted that in the XP-C cells, repair in the p53 is confined to
the transcribed strand and occurs at about half the rate of repair seen in normal cells. No p53-specific repair was found
in the XP-A cells. As for the inactive d-globin gene, as expected, the DNA damage formation and repair is the same in
the normal and the CS cells, while no repair was observed in the XP-C and the XP-A cells.
Conclusion: The GGR subpathway of NER, with little if any contribution of TCR plays a crucial role in the repair of melphalan-induced lesions.
180
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P-080
THE INDUCTION OF MICRONUCLEI, SINGLE-STRAND BREAKS IN DNA AND DNA REPAIR
IN MICE EXPOSED TO 1,3-BUTADIENE BY INHALATION
Rudolf Stetina 1
Pavel Vodička 2; Ludmila Vodičková 2; Petr Šmerák 3; Ivo Barta 3
Faculty of Military Health Sciences, Hradec Kralove, Czech Republic, Hradec Kralove, Czech Republic1; Institute of Experimental
Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic, Prague, Czech Republic2; Charles University,
3rd Medical Faculty,Prague, Czech Republic, Czech Republic3
We analysed the DNA damage in hepatocytes and frequencies of micronuclei in bone-marrow polychromatic erythrocytes in male NMRI mice (2 months old, weight 30-35g) during a subacute inhalation exposure to 1.3-butadiene (28
days, 500 mg/m3) and 28 days after the exposure. The exposure resulted in a moderate increase of concentrations
of 1.3-butadiene in blood, an indicator of internal exposure. The initial increase in micronuclei in the exposed mice
constantly decreased from 20.4±5.1 (day 3) to 15.1±3.2 (day 28) within the exposure period, and from 12.4±5.1 to
4.6±1.6 in the period following termination of the 1,3-butadiene exposure, while micronuclei frequencies in control
animals were significantly lower (from 1.7±1.5 to 4.2±0.8). Single-strand breaks and endonuclease III sensitive sites
in DNA were measured along with ?-irradiaton - specific DNA repair activities in hepatocytes isolated from the exposed
mice. Mild but significant increase in SSB was found in hepatocytes 7 days after the beginning of the exposure and on
the 7th day after the termination of the exposure. ?- irradiation-specific DNA repair rates, reflecting mainly base excision repair, gradually increased during the exposure, being significantly higher compared to control levels at days 7 and
28 of exposure (P=0.005 and P=0.035, respectively), reaching a maximum at day 1 after the termination of exposure
(P=0.003). A significant correlation between DNA repair rates and the 1,3 butadiene concentration in blood (R=0.866,
P=0.050) was found. This finding suggests, that DNA repair is induced in the liver of exposed mice by the exposure to
1,3-butadiene and formation of its metabolites.
Acknowledgements: This study was supported by the grant of Internal Grant Agency of the Czech Ministry of Health
no. NR 8563-5-2005
P-081
SEASONAL VARIABILITY IN GENOTOXIC POTENTIAL OF URBAN AIR PARTICULATE MATTER
Oksana Sevastyanova
Jan Topinka; Zuzana Novakova; Katerina Hanzalova; Blanka Binkova; Radim J. Sram
The main aim of this study was to compare genotoxic potential of organic extracts from urban air particles collected in
various seasons in the center of Prague (Czech Republic). For this purpose we analyzed DNA adduct forming activity of
organic extracts from urban air particles (PM10) in human hepatoma cell line HepG2. DNA adducts were analyzed by
32
P-postlabelling with nuclease P1 enrichment. Concentrations of PM10 were 36.9 mg/m3, 39.0 mg/m3, 62.6 mg/m3 in
summer, autumn and winter, respectively. Corresponding EOM contents were 5.0 mg/m3 (13.9% of PM10), 6.7 mg/m3
(17.2%) and 14.9 mg/m3 (23.8%). The total DNA adduct levels induced by 10 mg EOM/ml were 17.7; 37.2 and 19.5
adducts/108 nucleotides in summer, autumn and winter, respectively. However, when the EOM quantities per m3 were
taken into the consideration summer sample exhibited 3-fold lower genotoxicity than those of autumn and winter, while
difference between autumn and winter samples was not significant: 88.6, 249 and 291 adducts (in relative units),
respectively. Higher concentrations of EOMs induce toxic effects. Although concentration PM10 and EOM content in
autumn were significantly lower than in winter, genotoxic potential of ambient air in autumn and winter was almost
equal. There are significant positive correlations between B[a]P and c-PAH contents in EOM from various sampling periods and total DNA adduct levels detected in the EOM treated samples. These findings support hypothesis that B[a]P and
c-PAH contents in EOMs are the most important factors for their genotoxic potential. Thus, estimation of the genotoxic
potential of ambient air and prediction of health risk should be ?ased mainly on concentration of c-PAHs and biological activity of extracts, while amount of particles and EOM content are not crucial determinants. Supported by Czech
Ministry of Environment grant VaV-SL/5/160/05 and Academy of Science of the Czech Republic grant 1QS500390506.
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P-082
GENOTOXICITY OF VANTEX-A PYRETROID INSECTICIDE IN MAMMALIAN CELLS
Fatma Unal 1
Serkan Yilmaz 2; Deniz Yuzbasıoglu 3; Hüseyin Aksoy 4; Mustafa Çelik 5
Gazi University, Ankara, Turkey1; Gazi University, Ankara, Turkey 2; Gazi University, Ankara, Turkey3; Gazi University, Ankara,
Turkey4; Sutcu Imam University, Kahramanmara, Turkey5
The genotoxic effects of the synthetic pyretroid insecticide vantex (containing 60g/L Gamma-Cyhalothrin) was studied in
mouse bone marrow cells and human lymphocytes culture. 0.63, 1.25, 2.50, 3.75, 5.00 mg/kg concentrations of vantex
were used for in vivo assay and 1.25, 2.50, 3.75, 5.00?g/ml used for in vitro assay. In both test systems, a negative
and a positive control (MMC) were also conducted. Chromosomal aberrations (CAs) and sister chromatid exchanges
(SCEs) were scored in human lymphocytes and CAs was scored in mouse bone marrow cells as genetic endpoints. This
insecticide caused structural and numerical abnormalities in both mammalian cells. These are chromatid and chromosome breaks, chromatid exchanges, dicentric chromosomes, fragments, sister chromatid union and polyploidy. Vantex
increased the frequency of CAs at all concentrations in both test systems. A significant increase was observed for induction of SCE at all treatments compared with the negative control in human lymphocyte culture. The present results
indicate that vantex is genotoxic to cultured human lymphocytes and mouse bone marrow cells.
Keywords:Vantex; insecticide; chromosomal aberrations (CAs); sister chromatid exchange (SCE), human lymphocyte
culture, mouse bone marrow cells.
P-083
IN VIVO AND IN VITRO GENOTOXICITY TESTING OF THE ANTIFUNGAL DRUG TRIFLUCAN
Deniz Yuzbasıoglu
Hüseyin Aksoy; Mustafa Çelik; Fatma Unal; Serkan Yilmaz
In this study the genotoxic effects of antifungal drug triflucan (its active ingredient is flucanazole) was assessed by
using chromosome aberrations in mouse and chromosome aberrations, sister chromatid exchanges and micronuclei in
human lymphocytes. 12.5, 25 and 50 mg/kg concentrations of triflucan were used for in vivo assay and 12.5, 25 and
50 ?g/ml used for in vitro assay. In both test systems a negative and a positive control (MMC) were also conducted. Six
types of structural aberrations were observed; chromatid and chromosome breaks, sister chromatid union, chromatid
exchange, fragment, dicentric chromosomes. Polyploidy was observed as numerical aberration in both test systems. In
in vivo test, triflucan increased the number of CAs dose dependently but this increase was not statistically significant
compared with the negative control. In in vitro assay CA, SCE and MN frequencies significantly increased in a dose
dependent manner compared with negative control. The mitotic and nuclear division indices were not affected by treatments of triflucan. According to these results triflucan may posses clastogenic and mutagenic activity, however, further
information should be conducted.
Keywords: Triflucan, flucanazole, drug, chromosomal aberrations (CAs), sister chromatid exchanges (SCEs), micronucleus (MN).
182
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P-084
SCREENING OF FOOD ADDITIVE BENZOIC ACID FOR ITS GENOTOXIC PROPERTIES IN HUMAN
LYMPHOCYTES IN CULTURE
Serkan Yilmaz
Fatma Unal; Deniz Yuzbasıoglu
In this research chromosomal aberration, sister chromatid exchange and micronucleus test were employed to investigate the in vitro effects of antimicrobial food additive benzoic acid on human chromosomes. Lymphocytes were treated
with various concentrations (50, 100, 200 and 500 ?g/ml) of benzoic acid. And also a negative and a positive control
(MMC) were carried out to determine the validity of the assay. The results of used assays showed that benzoic acid significantly increased the chromosomal aberration, sister chromatid exchange and micronucleus frequency (200 and 500
?g/ml) in a dose dependent manner. Six types of structural aberrations (chromatid and chromosome breaks, chromatid
exchanges, fragments, sister chromatid union and dicentric) and only one type of numerical aberration (polyploidy)
were observed. Also this additive significantly decreased the mitotic index at the highest concentration for 24 h and
100, 200 and 500 ?g/ml for 48 h. This decrease dose dependent as well. However, it did not effect the replication and
nuclear division indices. Therefore, we concluded that benzoic acid is mutagenic, clastogenic, aneugenic and cytotoxic
to human lymphocytes.
Keywords: Benzoic acid, food additive, chromosomal aberrations, sister chromatid exchanges, micronucleus.
P-085
ANTIGENOTOXIC EFFECT OF XANTHOHUMOL DETECTED BY THE MODIFIED COMET ASSAY
IN PRECISION-CUT RAT LIVER SLICES
Janja Plazar
Metka Filipič, Geny M. M. Groothuis
Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia;
Pharmacokinetics and Drug Delivery, Groningen University Institute for Drug Exploration, A. Deusinglaan 1, 9713 AV Groningen,
The Netherlands
Xanthohumol (XN) is the principal prenylated flavonoid present in the hop plant, Humulus lupulus L. Hop is primarily
used to add flavour and bitterness to beer, the main dietary source of XN and related prenyl flavonoids. XN has been
suggested to have cancer chemopreventive activities. It is already known that XN is an antioxidant and inhibits metabolic activation of procarcinogens by inhibiting some of the cytochrome P450 enzymes in vitro in microsomes. There
are no studies so far that show that XN is preventing genotoxic effects in mammalian cells. Therefore, we investigated
antigenotoxic effects of XN in precision-cut rat liver slices. The environmental procarcinogen, polyaromatic hydrocarbon
benzo(a)pyrene (BaP) and the food procarcinogen, heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)
were chosen as genotoxicants. Both are metabolised by cytochrome P450 to final mutagens, forming adducts with DNA.
During the BaP metabolism, also reactive oxygen species are generated that are involved in the activation of stress
responses, while IQ metabolites do not cause an oxidative damage to DNA.
Rat liver slices were exposed to BaP (10-100 μM) and IQ (0.5-2 mM) for 24 hours alone and together with XN at noncytotoxic concentrations (0.001-10 μM). Cytotoxicity of XN was measured by the ATP assay. Genotoxic and antigenotoxic
effects were measured using a modified comet assay, designed to evaluate DNA damage in precision-cut liver slices.
Dose-dependent DNA damage in liver slices induced by the procarcinogens BaP and IQ could be detected by the modified comet assay. Exposure of liver slices to concentrations lower than 10 μM XN for 24 hours did not affect their viability.
All non-cytotoxic concentrations of XN prevented DNA damage, induced by IQ and BaP.
These results indicate that XN exhibits antigenotoxic effects, providing additional evidence for cancer preventive potential of XN, possibly by inhibition of bioactivation of procarcinogens and/or by antioxidant activity.
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ORAL PRESENTATIONS
P-086
PHOTOCHEMICALLY INDUCED DNA EFFECTS IN THE COMET ASSAY WITH EPIDERMAL CELLS
OF THE SKH-1 MICE AFTER SINGLE ADMINISTRATION OF DIFFERENT FLUOROQUINOLONES
AND 8-MOP PLUS UVA EXPOSURE
Uta Wirnitzer
Bayer HealthCare, Wuppertal, Germany
Due to the need for in vivo photogenotoxicity tests, the in vivo photo comet assay was established in epidermal cells
of the SKH-1 mouse. Groups of ten male SKH-1 mice each were treated once orally with vehicle only, 25 mg/kg
Clinafloxacin, 20 mg/kg Lomefloxacin, 200 mg/kg Ciprofloxacin or 200 mg/kg 8-Methoxypsoralene (8-MOP). Thirty
minutes post application half of the mice in each group were exposed to 23.8 J/cm2 UVA. Thereafter the mice were
sacrificed and the alkaline (pH > 13) comet assay applied to their epidermal cells; at the same time after administration
treated, non-irradiated mice were sacrificed and analysed. A negative control group of ten male SKH-1 mice received
the vehicle analogously; half of these were exposed to UVA, half not.
The comet tail length of epidermal cells of treated +UVA exposed mice were statistically significantly increased by 30%
for Clinafloxacin, by 25% for Lomefloxacin and by 24% for Ciprofloxacin as compared to controls (both irradiated and
unirradiated). Treatme?t with 8-Methoxypsoralene +UVA induced a significant reduction of comet tail length by 33%.
Tail intensity and tail moment gave essentially the same results after combined treatment and irradiation, except for
ciprofloxacin: tail intensity was comparable to irradiated controls after ciprofloxacin +UV exposure. Without UVA the tail
lengths of controls and treated mice were comparable. In contrast, tail intensity and tail moment were clearly increased
for all test compounds (including 8-MOP) without irradiation. In conclusion: under the presented experimental conditions the in vivo photo comet assay is able to detect photochemically-induced DNA strand breaks as well as photochemically-induced DNA cross-links.
P-087
PROSTATECTOMY AFFECTS MRNA EXPRESSION OF DNA REPAIR GENES
Andreas Woelfelschneider 1,2
Peter Schmezer 1,2; Claudia Mayer 1,2; Helmut Bartsch 1,2; Odilia Popanda 1,2; Carmen Lilla 1,5;
Jenny Chang-Claude 1,5; Juergen Debus 3,4
German Cancer Research Center (DKFZ), Heidelberg, Germany1; Toxicology and Cancer Risk Factors, Germany2; University of
Heidelberg, Heidelberg, Germany3; Department of Clinical Radiology, Germany4; Clinical Epidemiology, Germany5
There is evidence for considerable interindividual variability in DNA repair. This may affect both individual cancer risk
and response to cancer therapy. To gain insight into the underlying mechanisms, we investigated the constitutive mRNA
expression of several DNA repair genes in prostate cancer patients who received radiotherapy (RT). A cohort of 375
patients were included: 134 without adjuvant therapy, 102 who underwent prostatectomy (PT), 92 who received hormone therapy (HT), and 47 with both PT and HT. Peripheral blood lymphocytes (PBL) were collected from all patients
prior to RT. Total RNA was extracted from PBL and RNA integrity and concentration was determined using an Agilent
2100 Bioanalyzer. mRNA expression of 8 repair and repair-related genes was investigated: APEX1, ATM, BRCA1, BRCA2,
ERCC1, MDM2, TOP3A and TP53. After reverse transcription, mRNA abundance of each gene was measured by quantitative real-time PCR using SYBR-Green fluorescence and their expression relative to the reference housekeeping gene
TBP determined. The results were corrected for variations between single PCR experiments and for different detection
sensitivities of target and reference amplicons. The Kruskal-Wallis test was used to test for differences in gene expression between groups. Gene expression was not associated with age, BMI or smoking habits of the patients. While there
was no significant association of gene expression with HT, mRNA levels of ATM, MDM2 and TOP3A differed significantly
(p= 0.04, 0.003 and 0.003, respectively) between patients with and without PT. These results suggest that the presence
of prostate cancer cells in patients without PT affect cellular DNA repair response of systemic PBL. We are currently
investigating in functional assays whether the observed increase in mRNA expression indicates higher levels of DNA
damage within these cells. Supported in part by Bundesamt für Strahlenschutz, Salzgitter, Germany.
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P-088
THE INFLUENCE OF MICROCYSTIN-LR ON CELL PROLIFERATION, DNA DAMAGE
AND INTRACELLULAR REACTIVE OXYGEN SPECIES FORMATION IN DIFFERENT CELL LINES
Bojana Žegura
Meta Volčič; Tamara Turnšek Lah; Metka Filipič
Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia
Microcystins (MCs) are the most commonly found hepatotoxic cyclic heptapeptides produced by different cyanobacterial
species such as Microcystis aeruginosa. They are inhibitors of protein phosphatases 1 and 2A and are tumor promoters.
Oxidative stress plays an important role in MCLR induced hepatotoxicity. The main target organ of MCs is the liver due
to their uptake via bile acid carriers. However, organic anion transporter polypeptides are expressed not only in the liver
but also in the brain and therefore, also brain can be the target for MCs.
The aim of the present study was to investigate the toxic and genotoxic activity of MCLR on four different cell lines. Using
the MTT assay we observed that MCLR during 96 hour exposure reduced proliferation of human hepatoma (HepG2) and
human colon adenocarcinoma (CaCo-2), while there was no influence on human B lymphoblastoid (NC-NC) and human
astrocytoma (IPDDC) cell line. The genotoxicity of the toxin was evaluated by using the comet assay. MCLR induced
time and dose dependent increase of DNA damage in HepG2 and Caco-2 cells. Strand breaks were transiently present
in DNA helix and reached a maximum level after 4 hours of exposure and declined afterwards. No DNA damage was
observed in NC-NC and IPDDC cells. We further explored whether the toxin increased the level of intracellular reactive
oxygen species (ROS) in exposed cells. Dose and time dependent increase of ROS after the treatment with MCLR was
confirmed spectrophotometrically using a fluorescent probe 2’,7’-dichlorofluorescin diacetate (DCFH-DA) in HepG2 and
CaCo-2 cells, but not in NC-NC and IPDDC cells. These results show that MCLR reduced cell proliferation and induced
DNA damage, probably by increasing reactive oxygen species formation, which was cell type specific. It is not clear why
NC-NC and IPDDC cells are resistant to MCLR effects, but one possible explanation is that it does not enter the cells
because of the absence of active bile acid carriers.
P-089
STANDARD REFERENCE MATERIAL SRM1649A TOXICANTS MAY CONTRIBUTE
TO CARCINOGENESIS THROUGH BOTH GENOTOXIC AND NON-GENOTOXIC MECHANISMS
OF ACTION
Zdenek Andrysik 1,2
Sona Marvanova 1; Pavel Krcmar 1; Katerina Pencikova 1; Jiri Neca 1; Miroslav Ciganek 1; Miroslav
Machala 1; Jan Vondracek 1,2; Alois Kozubik 2; Brinda Mahadevan 3; William M. Baird 3
Veterinary Research Institute, Brno, Czech Republic1; Institute of Biophysics, Brno, Czech Republic2; Oregon State University,
Corvallis, United States3
Exposition of humans to environmental air pollutants is associated with an increased risk of cancer, but due to a complexity of these mixtures it is difficult to assess the contribution of various components to the overall carcinogenic
potential of the mixture. The aim of this study was to characterize both genotoxic and non-genotoxic effects of the
standard reference material SRM 1649a in the model of rat liver epithelial cells WB-F344 and to evaluate contribution
of polycyclic aromatic hydrocarbon (PAH) fraction to the total carcinogenic potential of this complex mixture of air pollutants. SRM 1649a was found to induce AhR-mediated activity from concentration 0.01 mg/ml. At low concentrations
SRM 1649a showed also a potential to contribute to tumor promotion through initiation of a loss of contact inhibition
and proliferation induction in treated cells. Similar effects were observed in cells exposed to the PAH fraction of SRM
1649a. HPLC analysis of 33P-postlabeled DNA revealed adducts formation in cells treated with high dose (2.5 mg/ml) of
crude extract (CE) of SRM 1649a. Although the abundance of DNA adduct following exposure to CE was lower compared
to effects of known carcinogens benzo[a]pyrene and dibenzo[a,l]pyrene, it was associated with activation of apoptosis
and decrease in numbers of treated cells. Neither DNA adduct generation nor apoptosis were detected in cells treated
with PAH fraction of SRM 1649a. In conclusion, the AhR-mediated effects of SRM1649a seem to be a prevalent mode of
action that migt contribute to the overall carcinogenic potency of this mixture, with PAHs being the major contributors.
(Supported by Ministry of Agriculture, No.00002716201, and GA ASCR, No. B6004407).
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P-090
ANALYSIS OF HYPERMETHYLATION IN TUMOUR SUPPRESSOR GENES FOR MOLECULAR
CHARACTERISATION OF BLADDER CANCER
Sonata Jarmalaite 1,2
Kristina Kurgonaite 1; Kestutis Suziedelis 1,3; Pertti Mutanen 2; Kirsti Husgafvel-Pursiainen 2;
Dainius Characiejus 3; Genovefa Chvatovic 3; Feliksas Jankevicius 3
Nature Faculty, Vilnius University, Vilnius, Lithuania1; Finnish Institute of Occupational Health, Helsinki, Finland 2; Institute of
Oncology, Vilnius University, Vilnius, Lithuania3
Hypermethylation in promoter region of tumour suppressor genes (TSGs) is an early and frequent event leading to
inactivation of the genes during carcinogenesis. Bladder cancer (BC) is a highly recurrent cancer, with progression to
muscle invasiveness occurring in 15-30% cases. Molecular characterisation of tumours by means of genetic and epigenetic analysis may facilitate early diagnosis and identification of aggressive BC for radical treatment.
In order to evaluate the suitability of epigenetic biomarkers for molecular characterisation of BC we analysed promoter
hypermethylation in a panel of TSGs involved in cell cycle control, apoptosis and DNA repair. In 64 BC cases, using methylation-specific PCR (MSP), the frequencies of hypermethylation of the RARb, RASSF1A, DAPK, p16 and MGMT genes
were found to be 36%, 28%, 27%, 14%, and 8%, respectively. In general, the frequency of hypermethylation, as well
as the methylation index (the number of genes altered) increased with increasing tumour stage and grade. Significantly
higher risk of hypermethylation of RARb and RASSF1A was observed in tumour stages pT2-4 as compared to pTa, while
the hypermethylation in DAPK was predominant in moderately differentiated (G2) tumours. Also, 3.5 times higher risk
of hypermethylation of RASSF1A was observed in muscle invasive vs non-invasive tumours. Hypermethylation in the
analysed TSGs was not related to the age or sex of the patients. Associations with disease recurrence and progression
as well as sensitivity to endovesical immunotherapy are under evaluation.
Significant associations with stage and grade of bladder tumours as detected in our study suggest that analysis of
hypermethylation in selected TSGs may provide a valuable biomarker for improved diagnosis of BC. Further study will
reveal its relevance for prediction of recurrence and response to treatment.
We thank the Lithuanian State Science and Studies Foundation for financial support.
P-091
IN VITRO EVALUATION OF GENOTOXIC AND NONGENOTOXIC EFFECTS AND CHEMICAL
ANALYSIS OF RIVER SEDIMENT EXTRACTS AND THEIR FRACTIONS
Sona Marvanova 1
Katerina Pencikova 1; Jiri Neca 1; Miroslav Ciganek 1; Miroslav Machala 1; Anton Kocan 2; Jan Vondracek 1,3
Veterinary Research Institute, Brno, Czech Republic1; Institute of Preventive and Clinical Medicine, Bratislava, Slovakia2; Institute
of Biophysics, Brno, Czech Republic3
Combined chemical and in vitro bioassay analyses were performed with crude extracts of the Elbe River sediments
sampled at an industrial region of the Czech Republic, as well as with their neutral, polar and H2SO4/silica-treated fractions. Toxicological assessment focused on the contribution of different classes of contaminants to the aryl hydrocarbon
receptor-mediated toxicity and genotoxicity. The chemical analysis of genotoxic organic compounds like PAHs and their
methylated derivatives and persistent organic pollutants (POPs) with non-genotoxic mode of action like PCBs, PCDDs,
PCDFs and OCPs was performed as well. The HPLC/DAD, GC/MS, HRGC/MS were used as analytical tools. Calculations
of chemically derived induction equivalents (IEQs) related to 2,3,7,8-TCDD revealed that PAHs with molecular weight
252 (benzofluoranthenes, benzo[a]pyrene), 276 (indenopyrene), 278 (dibenzo[a,h]anthracene) and methylated
benz[a]anthracenes (7-/9-methylbenz[a]anthracene) were the major contributors to the overall dioxin-like activity,
while TEQs of POPs were two orders of magnitude lower. This was confirmed by determination of dioxin-like activity
of complex sediment extracts and their fractions in the DR-CALUX bioassay. While neutral fractions, containing both
non-persistent PAHs and POPs, had high dioxin-like activity, the fractions of POPs (H2SO4/silica fractions) elicited only
low dioxin-like activity. Genotoxicity and apoptosis/necrosis induction were evaluated in rat liver epithelial WB-F344
cells by detection of Ser15-phosphorylated p53 and Ser139-phosphorylated histone H2AX (a marker of DNA double
strand breaks) and flow-cytometric measurement of apoptotic/necrotic cells by Annexin V/propidium iodide staining,
respectively. However, dioxin-like activity was significant at considerably lower doses than the events associated with
genotoxicity. [Supported by the EU project ModelKey, grant No. SSPI-CT-2003-511237-2.]
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P-092
TIME COURSE STUDY OF THE CHANGES IN LEUKOCYTIC AND COLONOCYTIC DNA DAMAGE
AND PRENEOPLASTIC ABERRANT CRYPT FOCI OF DMH-INDUCED COLON CARCINOGENESIS
IN F344 RATS
Kyung-Im Jeon 1
Eunju Park ; Bo-Young Seo 1; Hyun-Jin Lee 1; Su -Yeon Kim 1; Sun-Woo Jung 2; Kyung-Hea Lee 3
Dept. of Food and Nutrition, Kyungnam Univeristy, Masan, Korea1; Dept. of Biology, Changwon University, Changwon, Korea2;
Dept. of Food and Nutrition, Changwon Univeristy, Changwon, Korea3
Colon cancer is one of the leading causes of death in both men and women not only in Western countries also in some
Asian countries including Korea. Experimental colon carcinogenesis is a multistep process involving three distinct stages,
initiation, which alters the DNA coding information of a normal cell, followed by promotion and progression, which ultimately result in a phenotypically altered transformed cell. Rat colon carcinogenesis model using 1,2-dimethylhydrazine
(1,2-DMH) and using preneoplastic aberrant crypt foci (ACF) as endpoint marker lesions have been used to assess
the influence of modulatory factors on colon cancer. To understand the changes in DNA damage in leukocytes and
colonocytes and preneoplastic ACF during colon carcinogenesis, F344 male rats were given subcutaneous injection of
1,2-DMH (30mg/kg body weight) once a week for a period of 6 weeks and sacrificed every 4 week for 40 weeks. The
body weight and the relative weights of organs to body weight of DMH injected group were significantly lower than the
saline injected normal control group. DMH induced DNA damage in colonocytes increased significantly at 8, 24, 28, 32th
week compared to the normal control, while DNA damage in leukocytes increased at 8, 24th week. The number of AC or
ACF per colon substantially increased from 76 at 4th to 215 at 40th for ACF and from 170 to 839 for AC throughout the
experimental period. Further study is being carried out to investigate the changes of antioxidant status and glutathionerelated enzyme expression during colon carcinogenesis.
P-093
DIETARY FLAVONOIDS, THEIR TOXICITY AND INFLUENCE ON DETOXYFING SYSTEM
Jasna Franekić Čolic1
Ksenija Durgo 1; Lidija Vukovic 2; Gordana Rusak 3; Maja Osmak 3
Faculty of Natural Science, Zagreb, Croatia1; Faculty of Food Technology and Biotechnology, Zagreb, Croatia2; Ruđer Bošković
Institute, Zagreb, Croatia3
Flavonoids are phytochemicals exhibiting a wide range of biological activities, among which are antioxidant activity, the
ability to modulate activity of several enzymes or cell receptors and possibility to interfere with essential biochemical
pathways. Some of them can induce point mutations as well.
We have examined five flavonoids, three structurally related flavons (quercetin, fisetin, and myricetin) one flavonol
(luteolin) and one glycosilated flavanone (naringin) for their (a) ability to inhibit mitochondrial dehydrogenases as an
indicator of cytotoxic effect, (b) influence on cell membrane permeability (lactate dehydrogenase permeability); (c)
influence on intracellular level of glutathione, and (e) effect on expression of cytochrome CYP1A1. In this study human
cervical carcinoma parental HeLa cells were used and their drug-resistant subline (HeLa CK).
Toxicity of investigated flavonoids was similar in both cell lines. Only quercetin and luteolin have caused induction of glutathione in HeLa CK cell line, while in HeLa cell line glutathione was not inducible. Myricetin, quercetin and luteolin have
caused increased GSTs activity in HeLa cell line, and activity of GSTs in HeLa CK cell line was increased after treatment
with all examined flavonoids. Basal level of cytochrome 1A1 was higher in in resistant, HeLa CK cell line. Quercetin,
fisetin and luteolin have caused induction of CYP 1A1 expression. In resistant HeLa CK cell line only luteolin has caused
slight repression of CYP 1A1 expression. Other flavonoids did not influence on CYP 1A1 expression. In conclusion, small
differences in chemical structure of flavonoids lead to drastic change of their biological effects. These effects are strongly
dependent of cell type as well as test systems used. Extensive studies on structure-function relationship of flavonoids
in different test systems could provide rational approach to drug and chemopreventive agent design.
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P-094
ANTIGENOTOXIC EFFECT OF THE POLYSACCHARIDE BETA-GLUCAN EXTRACTED FROM
AGARICUS BLAZEI AGAINST DAMAGE INDUCED BY BENZO(A)PYRENE
José Pedro Friedmann Angeli 1
Vitor Basso Schul 1; Mario Sergio Mantovani 1; Lucia Regina Ribeiro 2; Marilanda Ferreira Bellini 3; Sandra
de Aguiar Soares 4
Universidade Estadual de Londrina, Londrina, Brazil1; UNESP, Rio Claro, Brazil2; UNESP, São José do Rio Preto, Brazil3; Universidade
Federal do Ceara, Fortaleza, Brazil4
In the last years, there has been a growing interest in the consumption of specific foods or food components that can be
beneficial to health. The aim of the present study was to determine the protective effect of the polysaccharide β-glucan
(BG) against damage induced by the carcinogen benzo(a)pyrene (B(a)P) in human hepatic HepG2 cells (expressing
phase I and II drug metabolizing enzymes). BG was extracted from the cell wall of the mushroom Agaricus blazei and
tested for genotoxicity and antigentoxicity using the comet assay and the micronucleus test with cytokinesis block. The
cells were grown in MEM medium supplemented with 10% fetal bovine serum and maintained in 5% CO2 incubator at
37şC at relative humidity of 95%. The cells were treated with 3 concentrations of BG (7, 21 and 63 µg/ml) in the tests
for genotoxicity and antigenotoxicity. In treatments testing for antigenotoxicity, we used two protocols: a) cells were
treated simultaneously with BG and B(a)P (2.5µg/ml) and b) cells were pre-treated for 24 h with BG and then with
B(a)P for 24 h. The results showed that β-glucan at the concentrations used was not genotoxic. On the other hand, a
protective effect was observed at all concentrations tested with simultaneous treatment, demonstrating a dose-response
relationship in both the comet assay and micronucleus test. Under pre-treatment conditions, only the highest concentration demonstrated a protective effect in both tests. These findings indicate that BG protects against damage caused
by B(a)P and that it is more effective with simultaneous treatment, indicating that BG probably acts by binding and
inactivating the genotoxic agent or via its capacity to scavenge free radicals formed by B(a)P. However, the protective
effect of BG observed in the pre-treatment protocol raises the possibility that it may also modulate phase I or II drug
metabolizing enzymes.
P-095
ATYPICAL AND HIDDEN WAYS OF CONTAMINATION OF ENVIRONMENT BY GENOTOXIC
COMPOUNDS
Andrej Gajdoš
Dagmar Gajdošová; Lívia Lucová; Zuzana Dietzová; Alexander Hudák
Regional Public Health Authority, Košice, Slovakia
We are made objectivisation of risk of mutagenicity in environment with nature abundance of arsenic compounds in
soil and water of the select region in Eastern Slovakia . The same objectivisation we made in control region without
abundance of nature arsenic compounds.
It was examined two groups of persons from these regions. First group was „exposed“ to nature arsenic from soil and
water. Second - control group were not exposed natural arsenic neither in soil nor in water.
Both of groups were examined to presence of arsenic in urine. It was very surprised result in control group. We are
detected arsenic in urine at control group too.
Therefore we did new detailed questionary search with persons of control group with aim to find the secret way of
arsenic into their urine.
We discovered, that lignit was used as fuel material by people from control region, during 15 years. 10 years ago they
finished with it and they started to use gas. The people added cinder from burned lignit into soil of their vegetable´s
and fruit´s gardens, because they wanted increase quality of soil.
Braun lignit contains more arsenic as black lignit, this fact is true for cinder too. This way arsenic came in on a soil and
a water by cinder, where its occurrence are not natural.
The second step is, that arsenic have been coming in on food-stuffs /vegetables and fruits from gardens/ from soil and
water.
Because braun lignit was being used long time as a fuel in many regions of Slovak Republik /and in other countries of
central Europe/, many people were exposed by arsenic through this way during 15-20 years their´s life.
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P-096
THE ANTIMUTAGENIC EFFECT OF PHENETHYL ISOTHIOCYANATE
Martina Langova 1
Zdenka Polivkova 1; Petr Smerak 1; Jirina Bartova 1; Helena Sestakova 2
3rd Faculty of Medicine, Charles University, Prague, Czech Republic1; National Institute of Public Health in Prague, Prague, Czech
Republic2
Isothiocyanates - biologically active compounds contained in cruciferous vegetables are effective chemoprotective
agents against the carcinogenic effect of different compounds.
The aim: We tested the antimutagenic effect of phenethyl isothiocyanate (PEITC) on mutagenic activity of indirect-acting carcinogens 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) and aflatoxin B1 (AFB1), and direct-acting carcinogen
N-nitroso-N-methylurea ( MNU) by the following methods: Ames test, micronucleus test, chemiluminiscence and blastic
transformation methods. Mutagenic substances were used in vitro at three different concentrations in both strains of S.
typhimurium TA98 and TA100. Each mutagen was combined with four different concentrations of antimutagen.
In vivo experiments: micronucleus, chemiluminiscence and blastic transformation tests were carried out on BALB/c
mice. PEITC was applied for three days in doses of 50mg/kg b.w. Carcinogens were administered on the third day.
Results: In the Ames test an inhibition of mutagenic activity by almost all concentrations of PEITC was proved against
both indirect mutagens, direct effect of MNU was reduced only by the concentration 30µg/plate.
PEITC, administered orally, decreased significantly the number of micronuclei enhanced by AFB1, IQ and MNU.
The protective effect of PEITC on immunity was assessed by the chemiluminiscence and blastic transformation methods.
In all of the experiments, PEITC reparied to a significant degree the immunosuppressive effects caused by all three
carcinogens tested.
Conclusions: Results of this study indicate that PEITC may play an important role in the prevention of carcinogenesis
and restrains immunosupression caused by the carcinogens.
Supported by IGA at the Ministry of Health of the Czech Republic, Project No. 8985-3.
P-097
CYTOTOXICITY AND GENOTOXICITY OF THE FOOD FLAVOURINGS EUGENOL AND ESTRAGOLE
IN V79 AND CHO CELL LINES
Celia Martins 1
Ana Filipa Monteiro 1; Alexandra Maralhas 1; José Rueff 1; Antonio Sebastião Rodrigues 1; Patricia
Saraiva 1; Antonio Laires 1,2
Genetics, Faculty of Medical Sciences-UNL, Lisbon, Portugal1; SABT, Faculty Sciences and Technology-UNL, Lisbon, Portugal2
Eugenol and Estragole are two alkenylbenzene compounds used as food flavourings and present in spices, such as
basil, cinnamon and nutmeg, and tarragon and anise respectively. Eugenol is also used as an antiseptic and analgesic
in destistry. Structural similarities with the class IIB IARC carcinogen safrole raises questions on it’s carcinogenicity.
While eugenol is a flavouring substance authorized by the European Commission for use in foodstuffs, estragole is not.
We evaluated the cytotoxicity of eugenol and estragole using the MTT test in V79 cells and in CHO AA8 cells, and also
in the CHO XRCC1 deficient cell line EM9. Cytotoxicity was observed above 2 mM, in all cell lines, either with a 3h or
24 h incubation period. We also evaluated the genotoxicity of eugenol and estragole in V79 cells using sister chromatid
exchanges (SCEs) and chromossomal aberrations (CAs), with and without rat liver biotransformation (S9). Estragole
induced a dose dependent induction of SCEs, whereas eugenol was negative. Eugenol induced CAs up to 3 mM, with
cytotoxicity at higher doses, and S9 increased CAs, from 0.5 % (control) to 18 % (3 mM) with particular emphasis on
chromatid exchanges. Estragole did not induce CAs. We observed a dose dependent increase of endoreduplication in
V79 cells and in EM9 cells with eugenol and estragole. Our results confirm the genotoxicity of eugenol and estragole in
vitro, and the different results suggest different mechanisms of genotoxicity for these two alkenylbenzene derivatives.
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P-098
THE ROLE OF NATURAL POLYSACCHARIDE AS AGENT WITH ANTIMUTAGENIC/
ANTICARCINOGENIC ACTIVITY
Eva Miadoková 1
Slavomíra Naďová 1; Eva Pražmáriová 1; Viola Dúhová 1; Viera Vlčková 1; Grigorij Kogan 2; Peter Rauko 3
Comenius University, Faculty of Natural Sciences, Department of Genetics, Bratislava, Slovakia1; Istitute of Chemistry, SAV,
Bratislava, Slovakia2; Institute of Experimental Oncology, SAV, Bratislava, Slovakia3
Discovery and exploration of naturally occuring compounds possessing antimutagenic and anticarcinogenic properties have increased in importance in recent years. Yeast cell wall polysaccharides, including b-D-glucans, have been
described as substances capable of providing direct or triggering host-mediated protective effects against the action of
several mutagens, toxicogens and free radicals. b-D-glucans isolated from yeasts, fungi and bacteria belong to the class
of drugs known as biological response-modifiers (BRMs), which modify the host’s biological response by stimulation of
the immune system. The new water-soluble derivative of microbial polysaccharide b-D-glucan, carboxymethyl glucan
(CMG), belongs in such a category.
It was demonstrated on five experimental model systems that biological and consequential medicinal importance of
CMG is based on the combined application with another active compound. In the Saccharomyces cerevisiae antimutagenicity assay CMG significantly reduced ofloxacin-induced mutagenicity. After simultaneous application with methyl
methanesulfonate CMG exhibited antitoxic and antimutagenic effect on recombination-repair-deficient strain of unicellular green alga Chlamydomonas reinhardtii. In the Vicia sativa simultaneous phytotoxicity and anticlastogenicity assay
CMG showed statistically significant anticlastogenic effect against maleic hydrazide induced clastogenicity. In the DNA
topology assay CMG protected pBR322 plasmid DNA from breaks induced by Fe2+ ions, but enhanced damages induced
by H2O2. In bacterial assay on bacteria Salmonella typhimurium strain TA97 it reduced mutagenicity induced by 9-aminoacridine at the highest concentration.
The obtained data unambiguously document that CMG is a polysaccharide with marked biomodulatory effects which
might contribute to cancer prevention and/or therapy.This work was supported by grant APVT-20-002604 and VEGA
grants 1/2337/05, 2/4053/24, 2/4143/04, 1/1311/04
P-099
WHEY PROTEIN INHIBITS OXIDATIVE STRESS INDUCED BY IRON OVERLOAD IN RATS
Hyun-Jin Lee 1
Eunju Park; Kyung-Im Jeon 1; Bo-Young Seo 1; Su-Yeon Kim 1; Hyun-Dong Paik 2; Yoe-Chang Yoon 2
Dept. of Food and Nutrition, Kyungnam Univeristy, Masan, Korea1; Dept. of Food Science and Biotechnology of Animal Resources,
Konkuk University, Seoul, Korea2
Whey, a protein complex obtained from milk, is being touted as a functional product with a number of health advantages. The effects of whey protein on oxidative stress are studied in rats submitted to oxidative stress by iron overload.
Thirty male rats were divided into three groups; control group (regular (500 mg/kg diet) dose of iron + 20% casein),
iron overload group (high (2000 mg/kg) dose of iron + 20% casein), whey protein group (high dose of iron + 10%
casein + 10% whey protein). After 5 weeks, high doses of iron caused a reduced plasma total antioxidant potential
(TRAP) and an increased lipid peroxidation (conjugated dienes), while the supplementation of whey protein could ameliorate the changes induced iron overload. Whey protein increased the level of plasma a-tocopherol corrected by plasma
triglyceride. In addition, iron overload induced DNA damage in leukocytes and colonocytes was fully prevented by whey
protein supplementation. These results demonstrate the antioxidative and antigenotoxic capacity of whey protein in an
in vivo model of oxidative stress induced by iron overload.
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26.6.2006 16:06:01
P-100
HAEMOGLOBIN ADDUCTS AS BIOMARKER OF BACKGROUND EXPOSURE TO ACRYLAMIDE
IN FIVE EUROPEAN COUNTRIES - A PILOT STUDY
Birgit Paulsoon
Anna Vikström 1; Ronnie Davies 1; Per Rydberg 1; Margareta Törnqvist 1; Paul Aston 2; Paul Scheepers 3
Dept. of Environmental Chemistry, Stockholm University, Stockholm, Sweden1; Dept. of Environ. Chemistry, Stockholm university,
Stockholm, Sweden1; AB Biomonitoring Ltd, Cardiff, United Kingdom2; University Nijmegen Medical Centre, Dept. of Epidemiol. &
Biostatistics, Nijmegen, Netherlands3
This study was performed within the EU project BIOMONECS (Biological monitoring of carcinogenic substances) aiming
at transfer of biomonitoring methods from universities to routine labs. One part concerned measurement of background
values to be used as reference values in studies of occupational exposures. Blood, urine and exhaled air were collected
from non-smoking individuals (6 males/6 females) in each of five European countries. Questionnaires on chemical
exposures and life-style factors were completed. Methods for 22 biomarkers were applied in pilot studies in the laboratories involved. One compound studied was acrylamide (AA), a neurotoxic chemical classified as a probable human
carcinogen. AA is metabolized to glycidamide, the assumed genotoxic agent in AA exposure. The finding that heated
food gives a considerable exposure to the public raised concern about associated health risks. In this context these
BIOMONECS samples offered a possibility to investigate, through analysis of haemoglobin adducts, whether differences
in AA-exposure in the European population (non-smokers without occupational exposure) are indicated due to different
dietary habits. Analysis of AA-adducts to the N-terminal valine (N-carbamoylethylvaline) in haemoglobin was performed
using the N-alkyl Edman procedure which leads to detachment of the adducts as fluorinated derivatives. The derivatives
are isolated by extraction and analysed with GC-MS/MS. Standards of the AA-adduct derivatives were synthesized. The
results showed small, non-significant, variations in AA-adduct levels between the 5 countries. The median values per
country varied between 28 and 37 pmol/g globin. The median for the whole population (n=60) was 33 pmol/g globin
(range 17-70) which is in agreement with other studies. This pilot study indicates that differences in the exposure to
dietary AA in the European population are not large.
This project was supported by the European Union (contract no.QLK4-CT-2002-71801).
P-101
ANTIMUTAGENIC AND IMMUNOPROTECTIVE EFFECTS OF LYCOPENE
Zdenka Polivkova 1
Martina Langova 1; Petr Smerak 1; Jirina Bartova 1; Helena Sestakova 2
3rd Faculty of Medicine, Charles University, Prague, Czech Republic1; National Institute of Public Health, Prague, Czech Republic2
A great variety of health benefits, including protection against tumors on different anatomic sites, such as cancers of
lungs, stomach and prostate gland, has been attributed to tomatoes and tomato-based products consumption.
The aim of our work was to determine protective effects of tomato carotenoid lycopene on mutagenicity and immunosuppression caused by two indirect mutagens and carcinogens: 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and aflatoxin B1 (AFB1) and by direct mutagen/ carcinogen N-nitroso-N-methylurea (MNU), using the Ames and micronucleus
tests, chemiluminiscence and blastic transformation methods.
Results: In the Ames test, the significant dose dependent antimutagenic effect of two highest concentrations of lycopene (30µg and 300µg/plate) against three different concentrations of both indirect mutagens AFB1 and IQ was proved
in TA98 and TA100 strains of S. typhimurium. The effect on the direct mutagenicity of MNU was lower and significant
only in the combination of the highest lycopen concentration (300µg/plate) with the lowest concentration of MNU
(10µg/plate). A treatment of mice with three daily doses of lycopene (25mg/kg b.w.) before administration of individual
mutagens leads to a significant reduction of micronuclei numbers in the micronucleus test. The same treatment of mice
leads also to a significant reduction of immunosuppression caused by all three mutagens, as detected by the chemiluminiscence method and the blastic transformation method.
Conclusions: Results of our study indicate that lycopene for its remarkable antimutagenic effect and protective effect
on immunity may have an important role in the prevention of carcinogenesis.
Supported by Grant IGA No. 8985-3, Ministry of Health of the Czech Republic
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P-102
CONTRIBUTION OF STEROLS TO BREAST CANCER CELLS PROLIFERATION
Maria Rybczynska
Blazej Rubis
University of Medical Sciences, Poznan, Poland
Anti-proliferative effects of sitosterol on cancer cells were associated with activation of the sphingomyelin cycle, cell
cycle arrest and stimulation of apoptotic cell death. However, the underlying mechanisms of its action are not fully
understood. To study the mechanism of antiproliferative action of sitosterols, two cell lines estrogen dependent (MCF-7)
and estrogen non-dependent and (MDA-MB-231) were cultured and tested which was supposed to elucidate if the ER
are involved in phytosterols action.
Methods:
The contribution of sterols and their derivatives to the cell proliferation were analyzed by MTT test in a time and dose
dependent manner. The analysis was performed in vitro and MCF-7 and MDA-MB-231 cells were studied.
Results:
It was shown that incubation of cells in the presence of beta-sitosterol resulted in a significant decrease in both cell lines
proliferation by about 60% at the concentration of 2µM. Similar effect in both cell lines was also evoked by cholesterol
but at about 4 times higher concentrations. Beta-epoxy-siosterol did not influence neither MCF-7 nor MDA-MB-231 cells
division while cell incubation with epoxycholesterol resulted in a significant proliferation decrease but only after 72h
incubation time and at concentration of 64µM and higher. A significant cytotoxic effect was also observed when fried
oil, mixture of oxy-sitosterols, was added.Beta-sitosterol showed strong antiproliferative action in breast cancer cells in
vitro while its oxy-derivative, Beta-epoxy-sitosterol showed no contribution to the cell division. Moreover, it was shown
that the specific action of beta-sitosterol did not depend on the presence of estrogen receptor on the cell surface. It is
concluded that the action of beta-sitosterol probably involved some mechanism related with cell membrane rearrangement.
Our results showed that consumption of phytosterols might be an important protecting factor against breast cancer.
P-103
GENOTOXIC POTENTIALS OF SOME VEGETABLES GROWN OVER CHROMIUM RICH SOIL
Manjit Inder Singh Saggoo
Arneet Grewal
Department of Botany, Punjabi University, Patiala, India
An important pathway for the movement of various elements from soil to man and animals is food. Plants can absorb
and accumulate heavy metals and other toxins from soil and can easily pass on to food chain. Chromium, one such
element, in trivalent form it is an essential trace element for glucose metabolism in animals but its hexavalent form is
highly toxic even mutagenic to biota. A toxic substance stored in a plant may make that plant unsafe for consumption.
To evaluate whether the uptake and accumulation of chromium by common vegetables like amaranth, lady’s finger,
pepper, onion, turnip, coriander, radish and spinach has induced genotoxicity in their water extracts, pot experiment was
performed. The plants were raised from seeds in pots containing soil amended with potassium dichromate to make the
final concentration of chromium (Cr) as 1, 10, 50, 100 and 200 µg/g of soil. Plants raised on garden soil alone served
as control. Chromium uptake in the edible parts of these vegetables varied from 8.39±1.73 µg/g DW to as much as
129.46±20.17 µg/g DW which exceeds the safety limits. Chromium is reported to be a week mutagenic agent. The
acquired mutagenicity due to accumulation of chromium by the edible parts of the various vegetables was evaluated
by performing Ames Salmonella assay. The homogenate of leaves of amaranth and spinach and roots of turnip plants
from pots with higher concentration of chromium in soil showed genotoxic abilities in both TA 98 and TA 100 strains of
S. typhimurium. The fruits of lady’s finger, chilli and coriander did not show any genotoxicity. The entry of mutagens in
our system via this route cannot be overlooked.
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P-104
Elsa Dias 1
Water Quality Center, National Institute of Health, Lisbon, Portugal1; Water Quality Center, National Institute of Health, Lisbon,
Portugal2; Center of Human Genetics, National Institute of Health, Lisbon, Portugal3
Human exposure to microcystins from cyanobacterial water contamination has been associated with a high prevalence
of liver cancer. Although microcystin-LR (MCLR) has been recognized as a tumor promoter, knowledge of potential
genotoxic effects is limited and its carcinogenicity mechanisms remain largely unknown. In this work we evaluated the
genotoxic potential of MCLR in Vero cells by the cytokinesis-blocked micronuleus assay. Microcystins extracted from
Microcystis aeruginosa were purified by preparative liquid chromatography and quantified by HPLC-DAD. An extract from
a non-toxic M. aeruginosa strain was used as a negative control. Cells were exposed to toxic (MCLR at 2 - 40 ug.ml-1)
and non-toxic extracts for 24h prior to micronulei analysis. In parallel, clonogenic assay was performed to evaluate cytotoxic effects. Preliminary results of micronuclei analysis in MCLR-treated Vero cells (≥ 20 ug.ml-1) revealed that MCLR
has an aneugenic/clastogenic activity. Results from clonogenic assay showed that cell survival was significantly affected
by MCLR at 40ug.ml-1. Lower toxin concentrations and non-toxic cyanobacteria extract failed to induce cytotoxicity. In
summary, we showed that MCLR presents cytotoxic and genotoxic properties in a permanent cell line. Those findings
support previous suggestions that microcystins might be carcinogenic by a genotoxic mechanism.
(Funded by Foundation for Science and Technology SFRH/BD/10585/2002).
P-105
URINARY MUTAGENICITY IN HEALTHY NON-SMOKING SUBJECTS
Sofia Pavanello 1
Alessandra Pulliero 1; Erminio Clonfero 1; Silvia Lupi 2; Pasquale Gregorio 2
Occupational Health Section, Department of Environmental Medicine and Public Health, University of Padova, Padova, Italy1;
Section of Hygiene and Occupational Medicine, Department of Clinical and Experimental Medicine, University of Ferrara, Ferrara,
Italy2
Urinary mutagenicity is related to the risk of cancer (bladder, colon) in humans. This paper reports the influence of low
environmental exposure on urinary mutagenicity on waking in healthy non-smokers (n=283, 46% males, 20-62 years),
taking into account the critical features of the bacterial assay (histidine content in urinary extracts and inoculum size).
Mutagenic exposure 24 hours before urine collection was checked by questionnaire; urinary mutagenicity with the sensitive YG1024 Salmonella typhimurium strain plus S9 mix. High variability in urinary mutagenicity was found (range
0.02-9.84 rev/ml) and 20 out of 283 (7%) were positive samples (Rs/Rc>=2, samples able to double number of spontaneous revertants). Histidine content (>=0.012micromoles/25ml) and inoculum size (>=3 x 109 bacteria) considerably
affected urinary mutagenicity (p<0.01). The influence of exposure was evident in the subgroup with intake of mutagen-rich meals (p<0.01), also showing higher percentages of positive urine (13% vs 5%, p=0.0252). Indoor exposure
revealed fewer effects -only the percentages of positive urine were significant (14% vs. 5%; p=0.015)- although 28
subjects exposed to fumes from cooking revealed higher urinary mutagenicity (p=0.035) and percentages of positive
urine (25% vs. 5%, p<0.01) than non-exposed subjects. The modest influence of occupational outdoor exposure on
urinary mutagenicity, which also persisted in the linear multiple regression analysis (p=0.067), together with the more
pronounced histidine content (p=0.02) and inoculum size (p<0.01), should be further investigated. Instead, the effects
of diet and indoor exposure disappeared in this statistical analysis. In conclusion, the presence of mutagens in urine
on waking of non-smokers is greatly related to histidine content and inoculum size. However, environmental exposure
(mainly mutagen-rich meals and fumes from cooking) influencing urinary mutagenicity may be considered as moderate
risk factors of bladder and colon cancer in non-smokers.
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P-106
A NOVEL ENDOGENOUS MUTAGEN, AMINOPHENYLNORHARMAN FORMED FROM ABUNDANT
FOOD COMPONENTS, NORHARMAN AND ANILINE
Yukari Totsuka 1
Rena Nishigaki 1; Takashi Sugimura 1; Keiji Wakabayashi 1; Hiroyuki Kataoka 2
National Cancer Center Research Institute, Tokyo, Japan1; Shujitsu University, Okayama, Japan2
Norharman is widely distributed in our environment such as cigarette smoke and cooked foods. This compound is not
mutagenic to Salmonella strains, but becomes mutagenic to S. typhimurium TA98 and YG1024 with S9 mix in the presence of aniline. When a mixture of norharman and aniline is incubated with S9 mix, a mutagenic compound, 9-(4’-aminophenyl)-9H-pyrido[3,4-b]indole [aminophenylnorharman (APNH)] is produced, and converted to the hydroxyamino
derivative, which eventually forms the DNA adduct, dG-C8-APNH through possible ultimate reactive forms with esterification, and this induces mutations. The formation of APNH from norharman and aniline was mediated by CYP3A4
and CYP1A2. APNH induced mutagenicity in TA98 and YG1024, and its values were 187,000 and 1,783,000 revertants
per mg, respectively, in the presence of S9 mix. These values are comparable to those obtained for a mutagenic heterocyclic amine, MeIQx. A long-term carcinogenicity experiment of APNH was conducted. Hepatocellular carcinomas
(HCCs) were found at incidences of 79% and 34% in male and female rats fed 40 ppm of APNH, and colon adenocarcinomas were found at incidences of 9% and 13% in both sexes of rats fed 40 ppm of APNH, respectively. PCR-SSCP
analysis revealed b-catenin gene mutations in 24% of HCCs, and K-ras, b-catenin and Apc gene mutations were found
in 22, 44 and 33% of colon cancers induced by APNH, respectively. Among these, most mutations occurred at G:C base
pairs. APNH was detected in 24 hr urine of rats by simultaneous administration with norharman and aniline. Moreover,
APNH was detected in urine samples from healthy volunteers at levels of 0.021-0.594 ng per 24 hr urine. These results
suggest that APNH is endogenously formed from norharman and aniline in the human body. It is very important to study
the significance of APNH, a novel endogenous mutagenic and carcinogenic compound, in human cancer development.
P-107
DNA DAMAGE AND REPAIR IN GERM CELLS OF PARP1-/- MALE MICE AFTER X-RAY
IRRADIATION
Eugenia Cordelli
Annamaria Fresegna; Alessia D'Alessio; Roberto Ranaldi; Patrizia Eleuter; Paola Villani; Francesca
Pacchierotti
ENEA, Rome, Italy
Poly(ADP-ribose)polymerase-1 (PARP1) is a nuclear protein activated by DNA strand breaks that plays a role in DNA
repair, recombination, proliferation and genomic stability. Germinal cells are characterized by a high expression level
of PARP. PARP1-/- mice, although fertile, show an increased genomic instability but data on the sensitivity of germ
cells to genotoxic stress are not available. Aim of this study was to evaluate differences in spontaneous and X-ray
induced DNA lesions in testicular cells from PARP1-/- and wild-type mice. Furthermore, the kinetics of DNA repair was
also investigated. Testes were irradiated in vivo with 4 Gy X-ray and the level of DNA damage was assessed by comet
assay, immediately or 2, 24, 48 hours and 45 days after irradiation. In addition, the presence of double strand breaks
was assessed by H2AX phosphorylation in round spermatids 2 hours after irradiation. Finally the induction of cytotoxic
effects was evaluated by flow cytometric assessment of apoptotic cells and testicular subpopulations. Results showed
a similar level of DNA damage in unirradiated testicular cells from PARP1-/- and wild-type mice. 4 Gy X-ray induced a
comparable increase of DNA strand breaks in both groups of mice. Results on DNA repair capability suggested a delayed
recovery in PARP1-/- cells as shown by the higher level of residual damage in these cells at short time after irradiation.
This defect was particularly evident in round spermatids. In the same cells a strong induction of double strand breaks
was shown without significant difference between the two strains. Radiation induced cytotoxicity was also evident and
similar in PARP1-/- and wild type mice. In conclusion these data support the hypothesis of a transient role of PARP1 in
DNA repair of single strand breaks in male germ cells.
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P-108
REPRODUCTIVE TOXICITY AFTER EXPOSURE OF YOUNG MALE MICE
TO DI(2-ETHYLHEXYL)PHTHALATE
Małgorzata Dobrzyńska
National Institute of Hygiene, Dept of Radiation Protection and Radiobiology, Warsaw, Poland
Di(2-ethylhexyl)phthalate (DEHP), one of the most commonly used plasticizers, has been shown to leach out from the
plastics into the air, water and ground, and to enter various foods. It has been recognised as an endocrine disruptor
inducting adverse effects in androgen responsive tissue following perinatal exposure of male rodents.
Young, 32-35 days old Pzh:SFIS male mice were exposed by gavage to 2000 mg/kg bw and 8000 mg/kg bw DEHP daily
for 8 weeks, 3 days/week. After the end of exposure,
5 animals per group were killed to examination of testes and epididymides weight, sperm count, motility, morphology
and comet assay in germ cells. Remaining males were caged to two untreated females each. Females was killed 18 days
after finding of a vaginal plug. They were examined for the number of live and dead implantations and the number and
type of gross malformations.
After 8-weeks exposure to 2000 mg/kg bw DEHP the effect on the sperm quantity and quality was not observed.
Treatments to 8000 mg/kg bw of DEHP slightly reduced relative testes and epididymides weights, diminished sperm
count and increased the frequency of sperm head abnormalities. Frequency of DNA damages in male haploid germ cells
was also slightly increased.
In both experimental groups frequency of pregnant females were decreased and there were no effects on the male
fertility and on the number of total implantation. Exposure to higher dose decreased the number of live implantation,
increased the number of dead foetuses and the percent of DML. There were no effects on the body weight of surviving
foetuses. Some of live foetuses showed congenital defects, but results were not statistically significant.
Results confirmed that DEHP is able to induce male-mediated developmental toxicity.
This work was funded by Polish Ministry of Education and Science (2004-2006), Project no 2PO5D 02926 .
P-109
BULKY DNA-ADDUCTS AND POLYMORPHISMS IN METABOLIC GENES IN THE EPIC-SPAIN
COHORT
Antonio Agudo 1
Carlos A González 1; Gabriel Capella 1; Peluso Marco 2; Arnelle Munnia 2; Sala Nuria 3; Nadia Garcia 3; Pilar
Amiano 4; M Dolores Chirlaque 5; Eva Ardanaz 6; M José Sánchez 7; J Ramon Quirós 8
IDIBELL - Catalan Institute of Oncology, Hospitalet de Llobregat, Spain1; CSPO - Scientific Institute of Tuscany Region, Florence,
Italy2; IDIBELL - Oncology Research Institute, Hospitalet de Llobregat, Spain3; Department of Public Health of Gipuzkoa, San
Sebastian, Spain4; Consejería de Sanidad y Consumo, Murcia, Spain5; Public Health Institute, Navarra, Spain6; Escuela Andaluza de
Salud Pública, Granada, Spain7; Consejería de salud y Servicios Sanitarios, Oviedo, Spain8
Background: Although formation of bulky adducts may be associated with exposure to PAH and other aromatic compounds, individuals with similar exposures may have different levels of adducts due to polymorphisms in genes involved
in metabolic activation.
Aims: To assess the association between bulky DNA-adducts and SNPs in genes encoding the metabolic enzymes
CYP1A1, CYP1A2, EPHX1, GSTM1, NAT2, and SULT1A1.
Methods: DNA was obtained using phenol extraction from WBC in 0.5 ml straws of a random sample of 296 subjects
aged 35-64 from the EPIC-Spain cohort. Bulky adducts were measured by the nuclease P1 32P-postlabelling technique.
SNP genotyping was performed in a LightCycler instrument by melting curve analysis of a fluorescently labelled sensor
probe specific for each PCR amplified variant.
Results: Overall the geometric mean of bulky adducts was 5.538×10-9 (adducted nucleotides/normal nucleotides).
Imputed phenotypes were built from known functional meaning of SNPs and haplotype analysis. Increased oxidative
activity was defined by the presence of G variant in the I462V polymorphism of CYP1A1, while decreased oxidative
activity was defined by homozygous variant CC in IVS1-154A>C in CYP1A2. Hydrolysis by EPHX1 was considered slow
for homozygous variant Histidine in Y113H, and fast for the homozygous variant Arginine in H139R. Combination of
both enzymatic activities showed the following adduct levels (×10-9): 3.98 for low, 5.57 for normal, and 7.42 for high
activity (p- trend 0.012). Lower levels of adducts were also observed for slow acetylators defined by variant A in *7A/B
of NAT2: 2.88 as compared with 5.74 in homozygous wild-type subjects, p-value=0.019).
Conclusions: Oxidation by CYP enzymes combined with hidrolysis by mEH seems to be an important pathway of PAH
activation to form reactive diol-epoxides. However, the association with NAT2 suggests the involvement of other aromatic compounds in the formation of bulky adducts.
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P-110
INFLUENCE OF GENETIC FACTORS ON TOLUENE DIISOCYANATE-RELATED SYMPTOMS
Karin Broberg 1
Margareta Littorin 1; Hakan Tinnerberg 1; Anna Axmon 1; Margareta Warholm 2; Agneta Rannug 2
Department of Occupational and Environmental medicine, Lund University, Lund, Sweden1; Department of Work Environment
Toxicology, Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden2
Toluene diisocyanate (TDI) is a highly reactive compound widely used in the production of e.g. polyurethane foams and
paints. Exposure to TDI is well known to cause respiratory symptoms as well as symptoms from the upper airways like
rhinitis and conjunctivitis. Other adverse effects, like toxic reactions have been described and TDI is a possible carcinogenic hazard. Because TDI causes symptoms in only a fraction of the exposed, genetic factors have been proposed to
modify susceptibility. The objective of this study was to characterize whether the associations between different exposure measures for TDI and symptoms of the upper and lower airways were modified by polymorphisms in metabolizing
enzymes or proteins involved in airway inflammation.
A group of workers (152 individuals) exposed mainly to TDI and a reference group (118 individuals) were analyzed for
isocyanate exposure (TDI in air, plasma and urine), genotype (metabolising genes: CYP1A1, GSTM1, GSTM3, GSTP1,
GSTT1, MPO, NAT1, NAT2, and SULT1A1; immune-related genes: CCL5, HLA-DQ05, and TNF) and medical effects
(symptoms of the eyes and upper and lower airways, based on structured interviews).
The strong effect of TDI-exposure on symptoms of the eyes, lower airways and nose bleeding was modified by genetic
factors: gene-exposure interaction was observed for symptoms of the eyes (NAT1, SULT1A1, CCL5), lower airways
(GSTP1) and to minor extent to symptoms of the nose (GSTP1, SULT1A1, CCL5).
The association of certain genotypes with symptoms arising in different tissues suggests common mechanisms of
pathogenesis. Genetic variants influencing both the metabolism of TDI and the immune response seem to be involved
in susceptibility to TDI-associated health effects.
P-111
GENETIC PREDISPOSITION TO LUNG CANCER IN WOMEN FROM UPPER SILESIA, POLAND A PRELIMINARY REPORT FROM AN ONGOING STUDY
Dorota Butkiewicz 1
Marek Rusin 1; Malgorzata Krzesniak 1, Barbara Wlodarczyk 2; Jadwiga Rachtan 3 Department of Tumor
Biology Center of Oncology,
M. Sklodowska-Curie Memorial Institute, Gliwice Branch, Glwice, Poland1; Department of Cancer Epidemiology, Center of Oncology,
M. Sklodowska-Curie Memorial Institute, Gliwice Branch, Gliwice, Poland2; Epidemiology Unit, Center of Oncology, M. SklodowskaCurie Memorial Institute, Cracow, Poland3
Lung cancer is a major cause of mortality and a serious health issue around the world. In Poland, an increasing incidence of lung cancer among women has been observed during the last decade. The epidemiology and etiology of lung
carcinoma appear to differ between men and women but most of the molecular epidemiology studies are based on
male patients. In the present, ongoing work, we investigate genetic factors of lung cancer risk in women from a highly
industrialized and polluted region of Poland. The subject groups collected so far include 155 women with histologically
confirmed lung carcinoma, and 150 healthy, unrelated controls. We sought to assess the influence of two common DNA
repair polymorphisms on lung cancer risk: the (-4)G A substitution in XPA participating in nucleotide excision repair
mechanism and the Ser326Cys amino-acid exchange in hOGG1 coding for base excision repair protein, 8-oxoguanine
DNA glycosylase. The polymorphisms are detected using PCR-RFLP assay. The frequency of the alleles in our population was similar to that reported in studies from other European countries. A preliminary crude OR (95% CI) analysis,
performed according to histology, age and smoking, showed no association between the polymorphisms studied and
lung cancer risk in women from Silesia region.
Additionally, we search for germline mutations in TP53 gene in selected patients who show family history of cancer or/
and suffered from other type of cancer. The screening for mutations in exons 2-11 of the TP53 is performed by direct
DNA sequencing of PCR products. Among 26 cases chosen until now for the analysis, one patient was a carrier of 190:
CCT>CGT mutation causing Pro Arg substitution in exon 6. Six common TP53 polymorphisms also have been found.
Supported by the Polish State Committee for Scientific Research grant no PZB-KBN 090/P05/13.
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P-112
MUTAGEN SENSITIVITY OF WORKERS EXPOSED TO LOW LEVELS OF IONIZING RADIATION
ASSESSED BY MICRONUCLEUS ANALYSIS AND FISH
Fabio Carbone 1,2
Patrizia Hrelia 1; Francesca Maffei 1; Sabrina Angelini 1; Giorgio Cantelli Forti 1; Hannu Norppa 2
Department of Pharmacology, University of Bologna, Bologna, Italy1; New Technologies and Risks, Finnish Institute of Occupational
Health, Helsinki, Finland2
Since the genotoxic mechanisms of bleomycin (BLM) and ionizing radiation (I.R.) are somewhat similar, it could be
expected that subjects sensitive to one of these agents are also sensitive to the other. In vitro sensitivity to BLM ("mutagen sensitivity") has been associated with an increased risk of various human cancers. We have studied BLM-sensitivity
by the cytokinesis-block micronucleus (MN) assay using fluorescence in situ hybridisation (FISH) to detect centromeres
(C+) and telomeres (T+) in the MN.
Baseline MN frequency in binucleated lymphocytes (BNCs) was significantly higher in 21 radiological workers (mean±D.
S., 8.67±2.92) exposed to low levels of I.R. than in 21 controls matched for sex, age, and smoking status (5.81±2.23;
P=0.003). When only MN harboring chromosomal fragments were taken into account (C-T+ and C-T- MN), the mean
frequency of C-MN/1000 BNCs was two times higher in the exposed group (3.25±1.47) than in the controls (1.54±0.77;
P=0.001), whereas no significant differences were seen in C+MN/1000 BNCs.
The level of genotoxic damage induced by BLM treatment, calculated as subtraction of the number of MN/1000 BNCs
in the untreated culture from that in the treated one, was slightly but not significantly higher in the exposed group
(9.55±3.72) than in the controls (7.14±4.96; P=0.081). The C-MN/1000 BNCs frequencies were significantly higher in
the treated than in the untreated cultures, both in the exposed (11.96±4.53 vs 3.25±1.47; P=0.0001) and controls
(8.86±4.57 vs 1.54±0.77; P=0.0001). No difference in C+MN/1000 BNCs was found.
Even though our findings are limited by the small number of subjects, they suggest that FISH analysis improves the
specificity of the MN assay in detecting clastogenic effects after exposure to low levels of I.R. and treatment with BLM.
We are presently examining differences in BLM-sensitivity profile between the radiation-exposed and the controls.
P-113
MTHFR GENE POLYMORPHISM AS RISK FACTOR OF VASCULAR DISEASE
Zuzana Cermakova 1
T.Lhotanova1; A.Maluskova1,2
Dept. Of Blood Banking, Faculty Hospital Ostrava1; Medical - Social Faculty Ostrava, Czech Republic2
Introduction: Hyperhomocysteinaemia is mentioned in lots of studies as a risk factor for cerebrovascular, peripheral
vascular and coronary heart disease. Elevated level of plasma homocystein can result from methylenetetrahydrofolate
reductase (MTHFR) 677C/T and MTHFR 1298A/C gene polymorphisms.
Methods: 190 patients (58 men, 132 women; age 44,8±16) with vascular disease (thrombosis, coronary artery disease) were investigated in our department. Blood samples were collected during 12 months, plasma homocystein was
determined by gas chromatography method (Schimadzu, Japan). DNA was extracted from fresh blood using NucleoSpin
Blood Kit (Machery-Nagel, Germany). MTHFR 677C/T and 1298 A/C gene polymorphism were detected by real time
PCR protocol with commercial kits Gene MTHFR (Genetica, Czech Republic), using Rotor Gene 3000 (Corbett Research,
Australia).
Results: In 190 patients (58 men, 132 women, age 44.8±16) from region North Moravia (Caucasians) were 14.2%
homozygotes for MTHFR 1298CC, 14.7% homozygotes for MTHFR 677TT. 18.4% patients were compound heterozygotes
for C677T/A1298C. In patient´s group with wild genotype for the MTHFR 677 and MTHFR 1298 were homocystein level
10.45± 4.39 µmol/l, patients that have some of aforementioned MTHFR polymorphism have had slightly increased level
of plasma homocystein – 11.57±5.75 µmol/l (p=0.15;RR=1.97; chi2 = 2.02).
Conclusion: In our study patients with MTHFR polymorphism are at increased risk of hyperhomocysteinaemia (RR=1.97)
that may represent an important genetic risk factor of vascular disease.
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P-114
CHROMOSOME INSTABILITY AND FOLATE GENE POLYMORPHISMS IN YOUNG MOTHERS
OF DOWN SYNDROME INDIVIDUALS
Fabio Coppede 1
Gabriele Siciliano 1; Ilaria Fontana 2,3; Lucia Migliore 2,3; Alessia Bonelli 2,3; Renato Colognato 2,3; Stefania
Bargagna 2,4; Guia Astrea 2,4
Department of Neurosciences, University of Pisa, Pisa, Italy 1; Dept. of Human and Environmental Sciences, University of Pisa,
Pisa, Italy3, Scientific Institute "Stella Maris", Calambrone, Pisa, Italy 4
We recently observed associations between combinations of polymorphisms in the methylenetetrahydrofolate reductase
(MTHFR 677C>T and 1298A>C) and reduced folate carrier (RFC-1 80G>A) genes and the risk of a Down syndrome (DS)
pregnancy in young Italian women. Others observed associations between a methionine synthase (MTR 2756A>G) gene
polymorphism and the risk of a DS offspring in Italy. Moreover, in a separate study, we observed an increased frequency
of both binucleated micronucleated cells (BNMN) and chromosome malsegregation events in peripheral lymphocytes of
young mothers of DS individuals (DS mothers) in respect to controls. Impairments in folate metabolism can result in
DNA damage and hypomethilation, thus we speculated on a possible correlation between folate gene polymorphisms
and the observed predisposition to chromosome instability.
Aim of the present study was to evaluate cytogenetic characteristics, measured by means of the micronucleus assay, in
peripheral lymphocytes of a group of women (n = 28) who had a DS child in young age (< 35 years) and in a control
group (n = 35), and to correlate them with MTHFR 677C>T and 1298A>C, RFC-1 80G>A and MTR 2756A>G polymorphisms.
We observed an increased frequency of MNBN in the DS mothers group compared to the control group (17,85 + 8,99 vs
10,28 + 4,53; P<0,0001), and, in the general population, a clear correlation between years of age and BNMN frequency
(P<0,02) and a borderline significant correlation between the frequency of BNMN and the MTHFR 677C>T polymorphism
(P = 0.054).
Present results indicate that DS mothers are more prone to chromosome instability than control mothers; moreover the
contribution of folate gene polymorphisms to the frequency of MNBN cannot be excluded. Further studies are ongoing
in a larger sample-size population.
P-115
EFFECT OF POLYMORPHISM IN THE DNA REPAIR GENE HOGG1 (SER326CYS) ON PHENOTYPE
Soňa Gurská
Timea Farkašová; Alena Gábelová
Cancer Research Institute, Bratislava, Slovakia
The product of hOGG1 gene, 8-oxoguanine DNA glycosylase 1 (hOGG1), is DNA repair enzyme that excises highly mutagenic lesion 8-oxoguanine (8-oxoG) from DNA by base excision repair pathway. Mice deficient in OGG1 accumulate GC
to TA transversions, and in humans the mutations in this gene are associated with increased risk of lung cancer in smokers. Several polymorphisms in the hOGG1 sequence have also been identified. The polymorphism resulted in change
of Ser to Cys (codon 326, exon 7) is present in population with highest frequency (~ 40%). The aim of our work was
to characterize the influence of this sequence variation on phenotype.To evaluate the impact of this genetic variation
on the phenotypic expression, the response of three cell lines, HeLa (Ser/Ser, wt), SiHa (Ser/Cys, heterozygote) and
C-33A (Cys/Cys, homozygote variant) to oxidative damage induced by visible light-excited methylene blue was studied.
Various biomarkers of exposure including cell growth activity, cytotoxicity, cumulative DNA, RNA and protein synthesis
as well as the expression of P53 and hOGG1 proteins were analyzed.
After induction of specific DNA damage the growth activity was markedly diminished in homozygote variant cells (C33A) in comparison to other cell lines. The differences between variants (HeLa, SiHa and C-33A cells) were observed
on the level of DNA and RNA syntheses. Interestingly, the intensity of DNA and RNA syntheses was significantly higher
in treated than in control cells in all cell lines. In the intensity of protein syntheses there were no differences between
cells. Increased expression of P53 and hOGG1 proteins after induction of specific DNA damage was observed only in
HeLa cells (wt).
Obtained results signalize that Ser326Cys polymorphism could have impact on the function on hOGG1 protein.
This work was supported by Science and Technology Assistance Agency under the contract No. APVT-51-015-304.
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P-116
GENETIC POLYMORPHISMS IN FOLATE METABOLISM AND THEIR ASSOCIATION WITH
CHROMOSOMAL ABERRATIONS AND SISTER CHROMATID EXCHANGES
Iiris Heilimo 1
Hannu Norppa 1; Hilkka Järventaus 1; Ari Hirvonen 1; Heidi Maunu 1; Päivi Rosenström 1; Katja Metsola 1;
Jarno Tuimala 2; Anne Kiuru3; Carita Lindholm 3
Finnish Institute of Occupational Health, Helsinki, Finland1; CSC, the Finnish IT Center for Science, Espoo, Finland2; STUK Radiation
and Nuclear Safety Authority, Helsinki, Finland3
Genetic polymorphisms in genes affecting xenobiotic metabolism, DNA repair, or folate metabolism may influence the
level of chromosome damage. Folate metabolism plays an essential role in preventing genome instability by affecting
DNA methylation and nucleotide pools. We examined the effects of polymorphisms of two main enzymes affecting folate
metabolism, methylenetetrahydrofolate reductase (MTHFR) Ala222Val and methionine synthase (MTR) Asp919Gly, on
the level of chromosomal aberrations and sister chromatid exchanges in Finnish data combined from eight different
studies (n=602). The genotype-phenotype relationships were modeled using Poisson regression. The effects were corrected for study, age, sex, smoking, and occupational exposure to chemicals. Our results indicate that homozygosity for
the variant allele of MTHFR is associated with an increased number of chromatid gaps (RR=1.22, 95% CI=1.07–1.38).
In contrast, homozygosity for the variant allele of MTR seems to be linked to a decrease in the number of chromatid-type
breaks (RR=0.78, 95% CI=0.64–0.95). MTHFR determines the balance between the different forms of folate by catalyzing 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate (methylTHF). MTR catalyzes the transfer of methyl
base from methylTHF to homocysteine, converting it to methionine. Methionine is further modified into S-adenosyl
methionine which is the universal methyl donor in DNA methylation. Reduced activity of MTHFR, associated with the
homozygous variant MTHFR genotype, decreases the amount of methylTHF and thus methionine synthesis. High MTR
activity, possibly associated with homozygous MTR variant genotype, should increase methionine levels. Thus, subjects
homozygous for the MTHFR variant allele or carrying the MTR wild-type allele are expected to have decreased levels of
methionine and DNA methylation, leading to an increased level of chromosomal aberrations. [Supported by EU QLK4CT-2000-00628 and Finnish Work Environment Fund]
P-117
LIFESTYLE FACTORS MODULATE GENETICALLY-PREDISPOSED CANCER IMMUNOSURVEILLANCE
Kazue Imai
Tomonori Hayashi; Yukari Morishita; Kei Nakachi
The immune system may be the body’s last line of defense against cancer development, besides its relevant role in
defending against infectious diseases. We have succeeded in identifying two NKG2D haplotype blocks in the natural killer
receptor complex (NKC) gene region, based on a genotype-phenotype association analysis within a prospective cohort
study among a Japanese general population. Each of the NKG2D haplotype blocks generated two major haplotype alleles
closely related to low (LNK1, LNK2) or high natural cytotoxic (NK) activity (HNK1, HNK2), and these different activity
levels were in turn associated with increased or decreased risk of cancer development. From the standpoint of cancer
prevention, we studied the influences of various lifestyle factors on NK activity in groups with different NKG2D haplotypes. Specifically, we compared NK activity of current smokers and non-smokers with the same NKG2D haplotypes, in
order to simulate the effects of quitting smoking on NK activity by genetic background. We found as a result that quitting smoking might effectively enhance NK activity among those with a certain haplotype. Other lifestyle factors were
found to differently affect NK activity depending on the NKG2D haplotypes. Our findings should be useful for identifying
immunologically high-risk individuals and will also provide an important hint for future individualized immuno-prevention, namely whether NK activity-enhancing effects of lifestyle coincide or differ among individuals with different NKG2D
haplotypes. In addition, since the NKG2D receptor is expressed on several T cells and also involved in T-cell functions,
we examined the association between the NKG2D haplotypes and other immunological markers measured in this cohort
study, such as CD4/CD8 T cells and blast formation of lymphocytes in the presence of PHA.
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P-118
LEVELS OF 2-THIOTHIAZOLIDINE-4-CARBOXYLIC ACID (TTCA) AND EVIDENCE OF EFFECT
MODIFICATION OF POLYMORPHISMS OF GLUTATHIONE-RELATED GENES IN VULCANIZATION
WORKERS IN THE SOUTHERN SWEDEN RUBBER INDUSTRIE
Lena S Jönsson
Karin Broberg; Ulf Bergendorf; Anna Axmon; Margareta Littorin; Bo AG Jönsson
Dep. of Laboratory Medicine, Division of Occupational and Envionmental Medicine, Lund University, Lund, Sweden
Background/aims:Workers in the rubber industry are exposed to a complex mixture of hazardous substances and have
increased risk of developing several diseases, e.g. different cancers. However, there is no up to date survey examining the exposure in the Swedish rubber industry. One of the toxic compounds in the industry is carbon disulfide (CS2),
which is biotransformed to 2-thiothiazolidine-4-carboxylic acid (TTCA). TTCA is used as a biomarker of CS2 exposure,
but there seems to exist inter- and intraindividual variability; this could partly be due to genetic variation. The aim of
the study was to determine TTCA levels and the modifying effects of glutathione-related genes in a group of Swedish
rubber workers.
Method:Urine was collected from both exposed workers and unexposed controls during the last four hours of a work
shift. The level of TTCA in urine was analyzed by liquid chromatography tandem mass spectrometry. Genotyping of the
single nucleotide polymorphisms GCLC-129, GCLM-588, GSTA1-52, GSTP1-105 and GSTP1-114 and deletions of GSTM1
and GSTT1 was performed with real-time PCR or ordinary PCR and subsequent agarose electrophoresis.
Results:We found the highest levels of TTCA among workers curing industrial and consumer rubber goods with salt
bath, hot air, microwaves or fluid-bed. Furthermore, the level of TTCA differed significantly depending on genotype in
the analysis of GSTM1 and GSTT1 among exposed workers but not among unexposed controls. We observed no differences in TTCA levels when analyzing the impact of GCLC-129, GCLM-588, GSTA1-52, GSTP1-105 and GSTP1-114
polymorphisms.
Conclusions:The present study demonstrates relatively high levels of TTCA in urine from rubber workers in southern
Sweden. Polymorphisms in GSTM1 and GSTT1 modified the levels of TTCA.
P-119
XRCC1 ARG399GLN GENETIC POLYMORPHISM IN A TURKISH POPULATION
Bensu Karahalil
Neslihan Aygun Kocabas
Gazi University Pharmacy Faculty Toxicology Department, Ankara, Turkey
Humans are routinely exposed to mutagenic and carcinogenic chemicals. These chemicals can form DNA adducts in
vivo and thus lead to DNA damage. The integrity of most of the so-damaged DNAs is typically restored as a consequence of the action of certain DNA-repairing enzymes. In several DNA repair genes, polymorphisms may result in
reduced repair capacity, which has been implicated as a risk factor for various types of cancer. XRCC1 is a base excision repair protein that plays a central role in the repair of DNA base damage and strand breaks. Amongst the known
genetic polymorphisms of the DNA-repair genes x-ray repair cross-complementing groups 1 and 3 (XRCC1 and XRCC3)
have been studied most commonly. The frequency of the polymorphic alleles varies among populations, suggesting an
ethnic distribution of genotypes. There has been no information on inter-individual variability of Arg399Gln genotype
in the Turkish population. We evaluated the allelic frequencies of Arg399Gln genotype in healthy Turkish population by
Polymerase Chain Reaction-Restriction Fragment Polymorphism (PCR-RFLP) analysis to enable to show inter-individual
differences and compare to other populations.
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P-120
GENETIC VARIATION IN XRCC1 AND XRCC3 GENOTYPES AND RISK OF GLIOMA
AND MENINGIOMA
Anne Kiuru 1
Carita Lindholm 1; Sirpa Heinävaara 1; Taina Ilus 1; Päivi Jokinen 1,2; Anssi Auvinen 1,2; Tiina Salminen 2;
Beatrice Malmer 3; Maria Feychting 4; Christoffer Johansen 5; Minouk Schoemaker 6
STUK-Radiation and Nuclear Safety Authority, Helsinki, Finland1; Tampere School of Public Health/University of Tampere, Tampere,
Finland2; Umeĺ University Hospital, Umeĺ, Sweden3; Karolinska Institute, Stockholm, Sweden4; Danish Cancer Society, Copenhagen,
Denmark5; Institute of Cancer Research, Surrey, United Kingdom6
Background: DNA repair genes XRCC1 (X-ray repair cross-complementing group 1) and XRCC3 (X-ray repair crosscomplementing group 3) are involved in the repair of DNA damage after ionising radiation, which is the only established
exposure associated with the risk of brain tumours. Therefore, we tested a hypothesis that polymorphisms of the XRCC1
and XRCC3 genes have an effect on the risk of glioma and meningioma.
Material and Methods: DNA samples from 722 glioma (325 glioblastoma [GBM]) cases, 545 meningioma cases, and
1600 controls were collected from Denmark, Finland, Sweden, and the United Kingdom. Genotypes of DNA repair genes
XRCC1 (Arg194Trp, Arg280His, and Arg399Gln) and XRCC3 (Thr241Met) were determined from leukocyte DNA using
polymerase chain reaction-based methods. Logistic conditional analysis (SAS) was applied to evaluate the effect of
these polymorphisms on the risk of glioma and meningioma.
Results: Statistical testing of our results indicated that homozygous carriers of the XRCC1 codon 399Gln variant allele
had a 1.5-fold increased risk of GBM compared to homozygous wild type 399Arg allele carriers. The same tendency was
also detected in other types of glioma and in meningioma. However, heterozygous carriers of the XRCC3 codon 241Met
variant allele were associated with a significantly decreased risk of GBM compared with homozygous carriers of 241Thr
wild type allele. Other studied genotypes did not have an effect on the risk of glioma or meningioma.
Conclusions: Our data suggests that individuals with XRCC1 homozygous variant 399Gln allele may have an increased
risk of GBM, and on the other hand that the XRCC3 241Met variant allele in combination with a wild type allele could
have a protective impact on GBM.
P-121
INHERITED BREAST CANCERS
Petra Kopecká 1
Josef Kopecký 1; Eva Šilhánová 2; P. Plevová 2
Mamocentrum Femina s.r.o., Ostrava, Czech Republic1; FNsP Ostrava, Ostrava, Czech Republic2
Introduction: 5-10% of breast cancers are inherited. BRCA 1,2 mutations are associated with 50-80% lifetime risk of
breast cancer and 20-40% risk of ovarian cancer and predispose also to higher risk of additional primary cancer.Present
mamological screening is not able to cover this patient group. Comprehensive programme for high risk women is not
available. Therefore we tried to create an algorithm of clinical and genetic examination, genetic testing and advanced
care of positively tested patient based on interdisciplinary cooperation. Methods: 5160 patients have been examined at
our surgery. 51 of them were referred to initial genetic counseling session.41 individuals have already underwent examination, 10 women hesitate,4 refused.Gene testing has been done at 17 cases. In 4 women testing can not be provided.
We consider the counseling by other specialists (gynecologist, plastic surgeon, psychologist etc.) also very important.
Results: 9 patients are BRCA1, 2 negative.Two women and one man are mutation carriers. Other 5 patients are sill waiting for the results. Having data about other 8 individuals, genetics expert consider their cancers as disease clustered in
family.8 women are not appropriate for genetic examination because of not verifiable family cancer history.
Conclusion: Advanced comprehensive follow up should be offered to the patients in inherited breast cancer risk.
Extended, more radical surgical procedure is appropriate. Surgeon should evaluate the benefit prophylactic contralateral
mastectomy. Premenopausal prophylactic oophorectomy is recommended
as it can reduce the breast cancer risk. Mutation carriers should undergo mammography annualy, USG examination
and tumor markers assessment every six months, examination by gynecologist and gastroenterology specialist etc.
Medical,social and economical benefit from early treatment is well known.
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P-122
GST GENE POLYMORPHISMS AND SUSCEPTIBILITY TO ASBESTOS AND SMOKING RELATED
LUNG DISEASES
Mari Kukkonen 1
Ari Hirvonen 1; Simo Kaleva 1; Tapio Vehmas 1; Matti Huuskonen 1; Harri Vainio 1; Päivi Piirilä 2
Finnish Institute of Occupational Health, Helsinki, Finland1; Helsinki University Hospital, Helsinki, Finland2
In addition to lung cancer, tobacco smoke is causally linked to various lung diseases such as emphysema. On the other
hand, occupational exposure to asbestos fibres results in pulmonary fibrosis and pleural diseases. Despite the intensive research during recent years, the impact of genetic variation on individual susceptibility to develop asbestos and
smoking related lung diseases has remained quite unknown. We investigated the roles of GSTM1, GSTT1, and GSTP1
(Ile104Val) polymorphisms in the non-malignant pulmonary diseases in a Finnish study population comprising of 575
asbestos-exposed workers (564 men and 11 women) and 2105 population controls. The asbestos-exposed patients
were divided into five groups based on HCRT-scoring: fibrosis (n = 68), emphysema (n = 147), pleural disease (n =
181), combined fibrosis and emphysema (n = 70) and marked adhesions (n = 109). The GST genotypes were determined by PCR and RFLP-based methods, and logistic regression analyses were used in the statistical evaluations. The
GSTM1 genotype did not significantly affect the risk for pulmonary diseases, whereas the GSTT1 null genotype halved
the risk to develop pleural disease (OR = 0.5; 95% CI = 0.3-1.0). The effect of the GSTT1 null genotype was very similar when workers with other pulmonary diseases were used as referents instead of the population controls (OR = 0.4;
95% CI = 0.2-0.7). Although no statistically significant associations between the GSTP1 genotype and the studied lung
diseases were found, the GSTP1 Val104Val-genotype was almost twice as common among healthy controls (8.3%) than
among patients with marked adhesions (4.6%; OR = 0.4; 95% CI = 0.1-1.0) or fibrosis (4.5%; OR = 0.4; 95% CI =
0.1-1.2 ). Our results support the hypothesis, that the GST genotypes modify the individual susceptibility to asbestos
and smoking related pulmonary diseases.
This study was supported by the Finnish Work Environment Fund.
P-123
GENETIC POLYMORPHISMS IN THE MDR-1 GENE ENCODING FOR P-GLYCOPROTEIN DRUG
TRANSPORTER AND SUSCEPTIBILITY TO COLORECTAL CANCER
Barbara Pardini 1,2
Roberto Barale 1; Pavel Vodicka 2; Elena Tulupova 2; Alessio Naccarati 2; Ludmila Vodickova 2,4; Jan
Novotny 3
Department of Biology, University of Pisa, Pisa, Italy1; Institute of Experimental Medicine, Academy of Sciences of Czech Republic,
Prague, Czech Republic2; First Medical Faculty, Charles University, Prague, Czech Republic3; National Institute of Public Health,
Prague, Czech Republic4
There is a high interest in relationships between a particular genetic background, the environment and susceptibility to
cancer. Colorectal cancer (CRC) is one of the most frequent tumors in the world. A diet rich in meat and poor in fruits
and vegetables, alcohol consumption and other lifestyle factors in association with an “adverse” genetic background,
could be risk factors increasing the development of CRC.
Many metabolising enzymes, involved in the detoxification of xenobiotics, toxins, carcinogens, and drugs, have known
allelic variants. These genetic polymorphisms may modulate the individual response to environmental insults and to
potentially dangerous compounds that reach the cellular compartment, affecting the risk to various cancers.
Aim of the study is to investigate the role of genetic polymorphisms in the MDR1 gene (C3435T and G2677T,A) in a
cohort of 500 CRC patients and healthy controls from Czech Republic, a country with one of the highest CRC incidence
worldwide. Our preliminary results suggest a role of C3435T SNP in modulating CRC risk (for individuals with at least
one variant allele: OR=1.25, 95 % CI=0.96-1.63).
The MDR1 gene (Multidrug Resistance Transporter 1) encodes for P-Glycoprotein (P-gP), a transporter protein playing
a prominent role in detoxification phase. C3435T is the most frequent SNP for MDR1 and may influence P-gP expression and activity in duodenum. The TT genotype was previously correlated with lower expression levels of P-gP in
intestine. This polymorphism may modulate expression and activity of P-gP, influencing intake of dangerous substrates.
Xenobiotics will consequently be able to enter the cell, but with less efficient elimination. For this reason, potentially
harmful compounds or metabolic intermediates will remain longer in direct contact with the cell, thus increasing the risk
of damaging biological molecules, including DNA.
Acknowledgement: GAČR project n° 371 is acknowledged
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P-124
IMPACT OF GENETIC VARIATION IN GLUTATHIONE-RELATED GENES ON BODY BURDEN
OF METHYLMERCURY
Karin Schläwicke 1
Karin Broberg 1; Staffan Skerfving 1; Ulf Strömberg 1; Bengt Vessby 2; Göran Hallmans 3
Department of Laboratory Medicine, Division of Occupational and Environmental Medicine, Lund University, Lund, Sweden1;
Department of Public Health and Caring Sciences, Uppsala University, Uppsala, Sweden2; Department of Public Health and Clinical
Medicine, Umeĺ University, Umeĺ, Sweden3
Exposure to toxic methylmercury (MeHg) through fish consumption is a large problem worldwide, which has led to
governmental recommendations of reduced fish consumption and blacklisting of fish from contaminated seas. The
elimination half-times of MeHg vary greatly between individuals. One possible explanation is hereditary differences in
MeHg metabolism. Knowledge about the reasons for this variation would be of great importance for improving the risk
estimates for MeHg. MeHg is eliminated from the body as glutathione (GSH) conjugates, and polymorphisms in GSHsynthesizing or GSH-conjugating enzymes may influence the elimination capacity. We have in an earlier study shown
the impact of polymorphisms in glutamyl-cysteine ligase (GCL) and glutathione S-transferase Pi-1 (GSTP1) on MeHg
retention (Custodio et al., 2004). Here, we have performed a similar study on 300 individuals from northern Sweden.
Subjects selected had a high consumption of fish (lean/fat fish 2-3 times per week or more) based on questionnaires.
Total mercury in erythrocytes (mainly MeHg), plasma polyunsaturated phospholipids (as a proxy for fish intake), and
genotyping was performed. We found that GCL and GSTP1 genotype modified the elimination of EryHg for individuals in
the highest exposure groups. These results stress the importance of genetic factors for MeHg metabolism.
P-125
POLYMORPHISMS IN NON HOMOLOGOUS END-JOINING GENES ASSOCIATED WITH BREAST
CANCER RISK AND CHROMOSOMAL RADIOSENSITIVITY.
Petra Willems 1
Hubert Thierens 1; Anne Vral 1; Kathleen Claes 1; Ans Baeyens 1; Kim De Ruyck 1; Rudy Van Den
Broecke 2; Amin Makar 3
University of Ghent, Gent, Belgium1; Ghent University Hospital, Ghent, Belgium2; Middelheim Hospital, Antwerpen, Belgium3
Enhanced chromosomal radiosensitivity (CR) has been observed in a significant number of breast cancer patients. Since
ionising radiation induces double-strand breaks (DSB), polymorphisms in DSB repair genes could be involved in genetic
predisposition to breast cancer. Non homologous end-joining (NHEJ) is the major DSB repair pathway in mammalian
cells.
We investigated the association of 6 single nucleotide polymorphisms (SNP) in NHEJ repair genes (Ku70, Ku80, DNAPK and XRCC4) with breast cancer in a population-based case-control setting. The population of breast cancer patients
was composed of a group of 60 familial patients and a group of 90 non-selected patients. The association between a
SNP and breast cancer predisposition was estimated by comparing both groups to a matched population of healthy
controls and calculating the odds ratio (OR) with its 95% confidence interval (95% CI). Of the examined SNP, 2 showed
positive results. The OR of C-61G in Ku70 in the familial patients was significantly higher than 1 (OR=2.53; 95% CI:
1.22; 5.24) indicating a positive relation between the presence of the SNP and familial breast cancer. The OR of the
unselected patients was 1.59 (O.81,3.12) suggesting an analogue association but the statistical significance of the OR
disappeared. For G69506A in Ku80, the unselected patients had a borderline significant OR of 2.14 (1.01,4.54) while
the familial patients demonstrated a higher, significant OR of 3,35 (1.68,6.68).
The CR of both patient populations also was investigated by our research group. The CR observed in breast cancer
patients was more pronounced in the familial group compared to the unselected patient group (61% vs. 26% with low
dose rate micronucleus assay; 45% vs. 35% with G2 assay using the 90th percentile of the normals as cut-off point for
radiosensitivity). These results point to a possible relationship between the presence of the variant risk alleles, breast
cancer incidence and chromosomal radiosensitivity.
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P-127
MICRONUCLEI INDUCTION IN TRADESCANTIA TETRADS BY PYRETHROIDS
Rafael Villalobos-Pietrini 1
Ana Rosa Flores-Márquez 1; Sandra Gómez-Arroyo 1; Omar Amador-Mu?oz 1; Miguel Angel RicoRodríguez 2; Stefan M. Waliszewski 3
Instituto de Medicina Forense / Universidad Veracruzana, Veracruz, Mexico 1; Centro de Ciencias de la Atmosfera / Universidad
Nacional Autónoma de Mexico, Mexico, Mexico 1; Centro de Estudios Academicos sobre Contaminacion Ambiental / Universidad
Autonoma de Queretaro, Queretaro, Mexico 2; Instituto de Medicina Forense / Universidad Veracruzana, Veracruz, Mexico3
In recent years insecticides containing pyrethroids have been widely used in houses because of their low to moderate
toxicity to organisms. In order to evaluate the genotoxic effects of pyrethroids, we exposed cuttings of Tradescantia
with young inflorescences in closed chambers for 0, 2, 4, 6, 8, 10 and 12 h to the vapors of three electrical vaporizers
containing the pyrethroid, esbiothrin or d-allethrin: Baygon small plates (Johnson), Raid small plates (Bayer) and Raid
45 nights. This totaled 36 hours with the pollen mother cells exposed in prophase I and the tetrads at the end of microsporogenesis where the micronuclei were detected. We obtained slopes for the three replicates of the controls (positive
and negative) and of each experimental group. The results with Baygon small plates and Raid small plates did not differ
from the negative control (p>0.05). Only with Raid 45 nights were the results significantly different from the negative
control, but this was the only pyrethroid mixed with a solvent – dibutyl hydroxy toluene. The other two insecticides were
only impregnated to the small plates without any solvent. It seems the micronuclei were induced by the solvent, paradoxically considered an inert compound. Other authors have found chromosome aberrations in alveolar macrophages
of rats treated with the same pyrethroid which also contained the same inert solvent – dibutyl hydroxy toluene. The
positive control was another insecticide, maleic hydrazide, which induced significant frequencies of micronuclei.
P-128
MODULATION OF B[A]P-INDUCED EXPRESSION OF CYTOCHROME P450 AND PHASE II
ENZYMES BY PLANT PHENOLS IN MOUSE EPIDERMIS
Wanda Baer-Dubowska
Violetta Krajka-Kuzniak; Hanna Szaefer
B[a]P is the most extensively studied polycyclic aromatic hydrocarbon and recognized carcinogen ubiquitous in human
environment. This carcinogen is metabolized to large number of metabolites including phenols, arene oxides, quinones,
dihydrodiols, and diol epoxides. Our earlier studies showed that naturally occurring protocatechuic, chlorogenic and
tannic acids and resveratrol, besides scavenging reactive B[a]P metabolites, modulate the activity of cytochrome P450
and phase II enzymes in mouse epidermis. .
The aim of the present study was to examine the effect of these compounds on the expression of CYP1A1/1A2, CYP1B1,
GST pi and theta in female BALB/C mouse epidermis by Western blot assays. The activities of GST and NQO1 were also
evaluated.
Phenolics were applied topically at the dose of 16 µmoles one hour before a single application of 400 nmoles of B[a]P.
The constitutive expression of all tested enzymes was observed, however their expression levels were different.
Topical application of initiation dose of B[a]P increased mainly the level of CYP1A1/1A2 and in to less extent CYP1B1.
Pretreatment of mice with tannic acid one hour before B[a]P decreased the B[a]P-induced protein level of both cytochrome P450 isozymes. The all tested compounds did not affect the total GST activity. B[a]P enhanced the level of GST
theta, but only tannic acid decreased the expression of this GST isoform. The most striking effect was observed in case
of NQO1. Both B[a]P and all tested phenolic compounds significantly induced the activity of NQO1. Resveratrol was the
most potent inducer of this enzyme. The results of this study indicate that plant phenols, particularly tannic acid and
resveratrol may modulate the B[a]P activation and play certain role in their anticarcinogenic activity.
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P-129
THE EFFECT OF ORAL ADMINISTRATION OF BEETROOT JUICE ON THE ACTIVITY
AND EXPRESSION OF ENZYMES METABOLIZING XENOBIOTICS IN DMBA-INDUCED RAT LIVER
AND MAMMARY GLAND
Hanna Szaefer
Wanda Baer-Dubowska; Violetta Krajka-Kuzniak
It was shown, that beetroot extract in vivo inhibited lung and skin cancer in mouse. 7,12-Dimethylbenzanthracene
(DMBA) is a powerful initiating and /or promoting agent, particularly in experimental breast cancer models.
In this study the effect of beetroot juice on the activities and expression of isozymes cytochrome P450 and II phase
enzymes in rat liver and mammary gland was investigated.
Female Sprague-Dawley rats were given by gavage with 2,5 ml of beetroot juice for 28 consecutive days. On the 27th
and 28th days animals were treated i.p. with (DMBA) in the dose of 10 mg/kg body weight.
In rat liver the significant decrease of EROD (30%) (the marker of CYP1A1) and MROD (29%) (the marker of CYP1A2)
activities were observed after beetroot juice treatment in comparison with the control group. DMBA induced EROD
(~23 fold) and MROD (~6 fold) activities. The same effect was observed after treatment with beetroot juice and DMBA.
Western blot analysis with CYP1A1 and CYP1B1 specific antibodies showed significantly increased in the levels of hepatic
and mammary gland CYP1A1 and CYP1B1 in animals treated with DMBA alone or in combination with beetroot juice.
Administration of beetroot juice enhanced the activities of GST (103%) and NQO1 (41%) in rat liver. In this organ DMBA
induced both tested II phase enzymes. Similarly, enhanced activity of GST and NQO1 was observed in animals receiving
DMBA and beetroot juice. The most marked effect was found for GST alpha protein in liver. In mammary gland expression of GST alpha and mu was not statistically changed after treatment with beetroot juice. These results indicated that
beetroot juice may affect selectively the expression of enzymes involved in the activation and detoxification of DMBA.
However this effect might be tissue specific.
P-130
EVALUATION OF THE DNA DAMAGE BY CHEMICAL CARCINOGENS AND ITS MODULATION
BY CLOUDY APPLE JUICE.
Ewa Ignatowicz
Zaneta Kazmierczak
Department of Pharmaceutical Biochemistry, University of Medical Sciences, Poznan, Poland
The aim of the study was to examine the effect of the DNA damage in the rat blood leukocytes, caused by chemical
carcinogens and its modulation by oral administration of cloudy apple juice (CAJ) rich in phytochemicals of diversified
biological activity, mainly antioxidant. CAJ was given by gavage to male Wistar or female Sprague-Dawley rats in the
dose of 2,5 ml/animal for 28 consecutive days. On the day 27, diethylnitrosamine (DEN), carbon tetrachloride (CCl4) and
potassium bromate (KBrO3) were administered i.p. to the male rats. Females obtained i.p. 7,12-dimethylbenzanthracene
(DMBA). DNA strand breaks were assessed by the alkaline comet assay. All tested carcinogens induced substantial damage to leukocyte nuclear DNA to approx 170% of control. Apple juice to some extent (by approx. 20%) protected DNA
in case of DEN and KBrO3. After DMBA and CCl4 administration no effect was found in animals receiving the apple juice
in comparison to animals receiving carcinogens alone.
The obtained results suggest the comet assay performed in whole blood might be used as surrogate biomarker of DNAdamaging caused by diverse carcinogens and evaluation of potential protective agents.
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P-131
ANTIGENOTOXIC APPLE POLYPHENOLS MODULATE GENE EXPRESSION IN HUMAN COLON
ADENOMA CELLS AS DETERMINED WITH A CUSTOM-MADE CDNA MICROARRAY FOR
TOXICOLOGICAL DEFENSE AND STRESS RESPONSE
Selvaraju Veeriah 1
Nina Habermann 1; Thomas Hofmann 1; Stefanie Klenow 1; Julia Sauer 1; Frank Böhmer 1; Stefan Wölfl 2;
Beatrice Louise Pool-Zobel 3
Institute of Molecular Cell Biology, Medical Faculty, Friedrich-Schiller-University, Drackendorfer Str. 1, D-07747 Jena, Germany1;
Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg,
Germany2; Department of Nutritional Toxicology, Institute for Nutrition, Friedrich-Schiller-University, Dornburger Str. 25, D-07743
Jena, Germany3
Background: An important mechanism of antigenotoxicity is the induction of phase II, detoxifying enzymes. Apples
contain significant amounts of polyphenols which are antigenotoxic and could act chemoprotective by this mechanism.
Aims: To assess whether apple polyphenols induce expression of genes related to toxicological defense and stress
responses in human colon adenoma (LT97) cells and to elucidate whether this could contribute to chemoprotection in
colon cells. Materials and Methods: An extract containing apple polyphenols (AE) was produced and analytically characterized by HPLC. LT97 cells were treated with non-cytotoxic concentration of AE which delivered 7.5 µM phloridzin (major
AE-polyphenol) to the cell culture medium. After 24 h treatment, total RNA was extracted. First-strand cDNA were
synthesized from 2 µg of total RNA with a primer incorporating a T7 RNA polymerase promoter. After second-strand
cDNA synthesis cRNA amplification was performed with RNA polymerase. The cRNA was used as a template to prepare
labeled cDNAs with Cy3-dUTP and Cy5-dUTP. Labeled cDNAs were hybridized to custom cDNA microarrays spotted with
300 genes related to toxicological defense. Differential gene expression was analyzed by GenePix pro v6.0 software.
The average intensity of each probe set was used for normalization based on the “ratio of median”. Results: Expression
of several genes (ABCC2, ARNT, BRCA2, CYP24A1, GSTA4, GSTM2, GSTT2, NFKB2, TRAF1, UGT1A6, UGT2B10, UGT2B7
and CYP11A, PCNA) was changed as a result of AE treatment. The pattern of changes explains previously observed
antigenotoxic effects by AE toward genotoxic compounds, which could better be detoxified as a result of AE treatment. Conclusions: Our novel approach of studying this specific profile of gene expression in preneoplastic human cells
provides a rapid method to identify gene targets that may enhance cellular resistance toward genotoxins and thus to
identify agents that can lead to chemoprotection.
P-132
ASSESSMENT OF THE GENOTOXICITY ASSOCIATED WITH KILNS USED FOR THE MANUFACTURE
OF RED BRICK AND OF PESTICIDES
Guillermo Cabrera
R.M.G. Durán; A.G. Perales; L.M. Pérez; G.L. Martínez
Universidad Autonoma de Querétaro, Querétaro, Mexico
Among the more dangerous pollutants to which man is exposed are different kind of organic compounds, including
polychlorinated biphenyls, polycyclic aromatic hydrocarbons and pesticides, which represent a potential hazard to the
environment and humans. In the state of Querétaro, México, one of the main environmental problems are the emissions
from kilns used in the manufacture of red brick. As an example, there is one town with an area of 2 km2, a population
of 4,500 people and there are inside the town 250 kilns. Some of the important pollutants found in the emissions are
PCBs (over 100 ppm) and PAHs. On the other hand, in some places in Mexico, where they export fruits and vegetables
the use of pesticides is very high compared with the average in this country. For example, in the town of Jacona, state
of Michoacán, México, the most common pesticides they are using are chlorpyrifos, methamidophos, methyl parathion,
mancozeb, endosulfan and malathion. The purposes of this study were a) to investigate the genotoxic effect of emissions from kilns and of the pesticides themselves and those found in soil and spring water, b) to compare the results
in different bioassays, and c) to validate the plant assays for the determination of the genotoxicity of these pollutants.
The Trad-MCN assay was used to find out the genotoxicity of pesticides and a battery of assays consisting of human,
mice, plant and bacterial cells was used to evaluate the genotoxicity of pollutants present in the emissions from kilns.
The genotoxicity of all the tested pesticides and soil extracts was significantly different from the negative control (Tap
water), and the spring water was not mutagenic. The genotoxicity of the pollutants present in the emissions was different in different assays. The plant assays (Trad-MCN and Trad-SHM), which are cheap, quick and simple, resulted as
good as other assays.
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P-133
Elsa Dias 1
Water Quality Center, National Institute of Health, Lisbon, Portugal1; Water Quality Center, National institute of Health, Lisbon,
Portugal2; Center of Human Genetics, National Institute of Health, Lisbon, Portugal 3
Human exposure to microcystins from cyanobacterial water contamination has been associated with a high prevalence
of liver cancer. Although microcystin-LR (MCLR) has been recognized as a tumor promoter, knowledge of potential
genotoxic effects is limited and its carcinogenicity mechanisms remain largely unknown. In this work we evaluated the
genotoxic potential of MCLR in Vero cells by the cytokinesis-blocked micronuleus assay. Microcystins extracted from
Microcystis aeruginosa were purified by preparative liquid chromatography and quantified by HPLC-DAD. An extract from
a non-toxic M. aeruginosa strain was used as a negative control. Cells were exposed to toxic (MCLR at 2 - 40 ug.ml1) and non-toxic extracts for 24h prior to micronulei analysis. In parallel, clonogenic assay was performed to evaluate
cytotoxic effects. Preliminary results of micronuclei analysis in MCLR-treated Vero cells (above 20 ug.ml-1) revealed that
MCLR has an aneugenic/clastogenic activity. Results from clonogenic assay showed that cell survival was significantly
affected by MCLR at 40ug.ml-1. Lower toxin concentrations and non-toxic cyanobacteria extracts failed to induce cytotoxicity. In summary, we showed that MCLR presents cytotoxic and genotoxic properties in a permanent cell line. Those
findings support previous suggestions that microcystins might be carcinogenic by a genotoxic mechanism.
(Funded by Foundation for Science and Technology SFRH/BD/10585/2002)
P-134
GENOTOXICITY OF NANOSIZED TITANIUM DIOXIDE
Ghita C-M Falck
Hanna Sandvik; Satu Suhonen; Hannu Norppa
Finnish Institute of Occupational Health, Helsinki, Finland
Nanotechnology is a rapidly increasing area of industry providing a wide variety of nano-structured materials for pharmaceutics, cosmetics, biomedical products, and other uses. Characteristic features of nanoparticles are their small size
(ř<100nm) and ability to readily traverse cell membranes. As nanoscale materials possess novel and unique physicochemical properties, they may also have unpredictable health effects. Since the applications of nanoparticles are rapidly
expanding, there is an urgent need to understand more about the possible genotoxicity of nanoparticles. With a few
exception, information on this topic is lacking. Nanoparticles might be genotoxic directly or indirectly via oxidative stress
and inflammatory responses. Studies in lungs cells, the expected primary target of nanoparticle effects, are especially
expected to be informative. There are some indications that nanoparticles such as nanosized titanium dioxide (TiO2) can
elicit inflammation and evoke the generation of reactive oxygen species and lipid peroxidation products. The objective
of this study was to examine the potential genotoxicity of nanosized titanium dioxide in pulmonary cells in vitro by the
analysis of DNA strand breaks and micronuclei (MN). Human bronchial epithelial cells (BEAS) were cultured in the presence of eight different concentrations (1-100 µg/cm˛) of titanium(IV)oxide nanopowder (rutile, 99.5%). DNA strand
breaks were assessed with the single cell gel electrophoresis (comet) assay. The induction of MN was examined by the
cytokinesis-block method 24, 48, and 72 h after TiO2 treatment. According to our preliminary results, titanium dioxide
induced a dose-dependent increase in DNA damage, although the effect was not high by absolute means. No clear
positive effects were observed in the micronucleus assay. Our results suggest that rutile, a phase of TiO2 nanoparticles
known to be a relatively inactive photocatalyst and poor in generating reactive oxygen species, is slightly genotoxic in
vitro.
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P-135
INVESTIGATION OF GENOTOXIC EFFECTS OF HOSPITAL WASTE WATERS IN BACTERIA
AND PRIMARY RAT HEPATOCYTES
Franziska Ferk 1
Armen Nersesyan 1; Robert Mader 1; Wolfram Parzefall 1; Christoph Buchmann 1; Siegfried Knasmüller 1;
Maria Fuerhacker 2
Medical University of Vienna, Institute of Cancer Research, Vienna, Austria1; Boku University of Natural Resources and Applied Life
Sciences, Vienna, Austria2
Aim of the study was the investigation of genotoxic effects caused by waste waters of a hospital before and after a
waste water treatment pilot plant (activated sludge with membrane separation). The water samples represented the
raw waste water of the total hospital (TH) and of the oncological ward (OW). The samples were tested in Salmonella/
microsome mutagenicity assays with TA98 and TA100 with and without metabolic activation as well as in single cell gel
electrophoresis assays (SCGE) with primary rat hepatocytes. In bacterial assays negative results were observed in all
experiments, but acute bacteriocidal/ bacteriostatic effects which were seen with the highest water volumes (2ml /plate)
were reduced in the effluent of the waste water treatment.
Induction of DNA-migration was detected in the SCGE assays but no significant differences were seen between DNAmigration caused by TH- and OW-samples. The genotoxic activities of the waters were significantly (25%-50%) reduced
after treatment, but further purification with activated carbon did not result in a additional decrease of the genotoxic
potential. Furthermore, experiments were conducted in which the hepatocytes were treated simultaneously with H2O2
(100µM) and the water samples. We found that raw waste waters caused a reduction of H2O2 induced DNA-migration
(24%-38%), while after treatment the inhibition of DNA-migration was significantly decreased. This phenomenon is
probably due to the presence of crosslinking agents, which were used as cytostatic drugs (platinum compounds) and
were present in the OW-samples and were reduced after treatment. Additional experiments showed that quaternary
ammonium compounds (QAC), namely benzalkonium chloride (BAC) and dimethyl dibenzylammonium chloride (DDAC),
which are commonly used in hospitals as disinfectants cause DNA-migration. These findings indicate that part of the
effects seen in the hospital waste waters may be due to the presence of these compounds.
P-136
URINARY NAPHTHALENE AND PHENANTHRENE AS BIOMARKERS OF EXPOSURE
TO POLYCYCLIC AROMATIC HYDROCARBONS IN THE STEEL INDUSTRY.
Steve Rappaport 1
J. Sobus 1; S. Waidyanatha 1; Andrej Gajdoš 2; Daniela Tarabčáková 2
School of Public Health, University of North Carolina, North Carolina, United States1; Regional Public Health Authority, Košice,
Slovakia2
Polycyclic aromatic hydrocarbons (PAH) represent a class of compounds that have long been associated with mutagenic
and carcinogenic effects among workers in the steel industry, particularly those engaged in coke production. Most studies of cytogenetic effects of PAH among steel workers have relied upon air levels of particulate (4- to 6-ring) PAHs for
quantitative assessments of exposure. Since air concentrations of particulate PAHs tend to be very low, such assessments of exposure are difficult and require expensive equipment. Although the smaller 2- and 3-ring PAHs, which exist
primarily in the vapor phase, tend to be the most abundant PAHs arising from a given source, they have rarely been
used to assess PAH exposures. Here, we consider the utility of unmetabolized naphthalene (NAP, 2-rings) and phenanthrene (PHE, 3-rings) in urine as biomarkers of exposure to PAH among steel workers. NAP has been shown to be carcinogenic in the lungs of rats and mice and PHE is the smallest PAH to possess a bay region, a feature often associated
with carcinogenicity in 4- to 6-ring PAHs, such as benzo(a)pyrene. Using solid-phase microextraction (SPME) followed
by gas chromatography-mass spectrometry, we measured NAP and PHE in the urine of 58 steel workers (28 coke-oven
workers and 30 workers from other jobs) and 29 regional control subjects in the Slovak Republic. Median levels of both
compounds were significantly greater in steel workers than in control workers (NAP: 0.075 vs. 0.016 mg/l; PHE: 0.012
vs. 0.004 mg/l). We also found relatively high correlation between levels of NAP and PHE among all subjects (Spearman
r = 0.56). We are currently comparing urinary levels of NAP and PHE with the numbers of chromosome aberrations
measured in these workers and expect to present these results at the meet
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P-137
CHROMOSOMAL ABERRATIONS ASSOCIATED WITH EXPOSURE TO PAHs
Dagmar Gajdošová 1
Daniela Tarabčáková 1; Lívia Lucová 1; Vladimír Rimár 1; Keneth Mund 2; Thomas Birk 2
Regional Public Health Authority, Košice, Slovakia1; Applied Epidemiology, Inc., Amherst MA, United States2
The human health effects of Polycyclic Aromatic Hydrocarbons (PAH’s) have not been adequately studied to allow sound
quantitative risk estimation. Appropriate data are needed for air quality regulatory actions (currently debated in US and
EU). Government scientists in Košice, Slovak Republic maintain a unique and previously untapped reservoir of historical,
quantitative air exposure data on PAHs from a large coke plant which began production in 1965. The plant produces
coke that is used in the manufacture of steel; coke-oven gases are burned in the plant as fuel; and other by-products
are sold or processed as waste.
Occupationally exposed workers at the coke plant in Košice, Slovakia are at potentially greater cancer risk for their exposure to PAHs. No association was found with age, duration of employment, or smoking. That we did not find evidence
of a strong trend in percent chromosomal aberration with duration of exposure is consistent with current understanding
of lymphocyte physiology and P53 function. This is a cell population that is exquisitely sensitive to genotypic damage
and that responds typically by undergoing apoptosis, thus precluding accumulation of multiple anomalies with increasing
exposure duration. Accordingly, we caution on the use of the CALPL methodology in analyses of exposure dose – cancer
risk relationships.
P-138
VALIDATION OF URINARY EXCRETION OF CYCLOPHOSPHAMIDE AS A BIOMARKER
OF EXPOSURE BY STUDYING ITS RENAL CLEARANCE AT HIGH AND LOW PLASMA
CONCENTRATIONS IN CANCER PATIENS
Maria Hedmer 1
Maria Albin 1; Bo AG Jönsson 1; Peter Höglund 2
Occupational and Environmental Medicine, Lund University, Lund, Sweden1, Clinical and Experimental Pharmacology, Lund
University, Lund, Sweden2
Background/aims: Cyclophosphamide (CP) is an alkylating agent classified as a human carcinogen. Health care workers
handling this drug may be exposed during e.g. preparation or administration. Cyclophosphamide is readily absorbed by
inhalation and by dermal uptake. A biomarker, CP in urine, has frequently been used to assess the occupational exposure to CP, but has not been fully validated. The aim of this study was to investigate if the proportion of the CP dose
that is excreted in urine (renal clearance) is stable over different plasma drug concentrations and other pharmacokinetic
parameters, e.g. urine flow.
Method: Pharmacokinetics of CP were studied in 16 breast cancer patients that were treated with postoperative adjuvant
chemotherapy including CP. Plasma and urine from the patients were collected at different occasions up to 12 days after
the dose. Urine was collected during four-hour periods and blood was sampled at the end of each period. Analysis of CP
was performed by liquid chromatography tandem mass spectrometry. The limit of detection for CP in urine and plasma
was 0.01 and 0.02 ng/ml, respectively. The precisions of the developed methods were determined to less than 8%.
Results: The administered doses of CP in absolute amounts ranged between 800 and 2240 mg. Mean renal clearance of
CP was 8.6 (confidence interval 6.5-10.7) ml/min and was not significantly dependent of the plasma drug concentration.
However, a significant correlation between renal clearance and urine flow was observed. There was a large inter-individual variation in the plasma and urine concentrations even when the same doses were given.
Conclusions: Cyclophosphamide in urine can be continued to be used as a biomarker to monitor occupational exposure
to CP, however the inter-individual variability of excretion of CP in urine, and its dependency on urine flow must be taken
into consideration in future applications.
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P-139
INVESTIGATION ON THE RELATIONSHIP BETWEEN IL-4 OR IL-4 RECEPTOR GENETIC
POLYMORPHISMS AND ALLERGY-RELATED IMMUNOLOGIC STATUS IN ADULT EMPLOYEES
AT SOCIAL WELFARE FACILITIES
Yong Heo 1
Kyu Dong Ahn 2; Hyoung Ah Kim 3; Sang Hoon Kim 4
Catholic University of Daegu, College of Natural Sciences, Dept. Occupational Health, Kyongsan si, Kyongbuk, Korea1;
Soonchunhyang University, College of Medicine, Dept. Preventive Medicine, Asan si, Chungnam, Korea2; The Catholic Univeristy of
Korea, College of Medicine, Dept. Preventive Medicine, Seoul, Korea3; Eulji University, School of Medicine, Dept. Internal Medicine,
Seoul, Korea4
Genetic variations on IL-4 receptor alpha chain or IL-4 promotor have been associated with atopic diseases, and demonstrated mostly with children. The association was investigated with adult employees at welfare facilities for children
or aged peoples. Skin prick tests and methacholine airway response were performed against 25 different aero-allergens
to 22 employees from 4 social welfare facilities. Polymorphism was examined for IL-4 receptor alpha chain (Gln576Arg)
and IL-4 promotor (C589T) by RFLP. We examined plasma levels of IgG2, IgG4, IgE, histamine, IL-4 and IFNγ production from peripheral blood mononuclear cells. Positive percent on the skin prick test was 50% (11 persons); 4 against
house dust mite, 3 against both mite and house dust, 2 against cockroach, 2 against other allergens. Frequency of
Gln576Arg polymorphism was not different between the skin positive (4 among 11 subjects) and skin negative subjects (5 among 11 subjects). Frequency of C589T polymorphism was 1 among the positives and 4 among the negative
subjects. Level of IgG4 was upregulated in the positives (0.82±0.20 mg/ml) compared with the negatives (0.65±0.12
mg/ml). Level of IgE was higher in the positives (1025±534 ng/ml) than the negatives (358±200 ng/ml). Percent of
eosinophils was higher in the positives (2.8±0.5) than that of the negatives (1.5±0.2). No significant difference in
IL-4 and IFNγ production was observed between the groups. Even though no association of the genetic mutation with
the allergen positivity in adults, this study demonstrated that workers at the social welfare facilities could suffer from
allergic diseases. [Supported grant No. R01-2004-000-10427-1 from the Basic Research Program of the Korea Science
& Engineering Foundation]
P-140
OCCUPATIONAL EXPOSURE TO POLYCYCLIC AROMATIC HYDROCARBONS (PAHS)
IN THE RUBBER INDUSTRY
Bo AG Jonsson 1
Christian H Lindh 1; Hans Kromhout 2; Roel Vermeulen 2
Division of Occupational and Environmental Medicine, Lund, Sweden1; Division of Environmental and Occupational Health, Institute
of Risk Assessment Sciences, Utrecht, Netherlands2
Background: Workers in the rubber industry are exposed to a large number of toxic substances and have increased risk
of developing several diseases such as cancer. The aim of this study was to assess the exposure to PAH in the rubber
industry in the Netherlands. This was performed through analysis of urinary 1-hydroxypyrene (1-HP), a commonly used
biomarker of PAH exposure.
Methods: One hundred and two workers employed in the Dutch rubber industry were investigated. Urine samples were
collected on a work-free Sunday and the consecutive working days Tuesday, Wednesday and Thursday. 1-HP was analysed by liquid chromatography with fluorescence detection. The limit of detection (LOD) was set at 0.1 ng/ml urine.
Results: The median urinary 1-HP on Sunday, Tuesday, Wednesday and Thursday were 0.12 (range <LOD-0.88), 0.24
(range <LOD-0.91), 0.20 (range <LOD-1.38) and 0.22 (range <LOD-0.88) µmol/mol creatinine, respectively. There
were significant differences between the levels at all working days compared to the levels at the Sunday (p<0.001;
Wilcoxon signed rank test).
Conclusions: Urinary levels of 1-HP from the rubber workers were generally rather low. However, the results do indicate
an overall increase in urinary 1-HP levels due to exposures in the contemporary rubber industry. Mean levels found in
this study are approximately 4 to 5 times lower than currently found among well-protected coke oven workers in The
Netherlands.
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P-141
DEVELOPMENT OF FOUNDRY TECHNOLOGIES: GENOTOXIC RISK OF FURANE MOULDING
MIXTURES
Jaromira Kusová1
Lubomír Dobiáš1; Irena Mikulenková1; Hana Tomášková1; Milan Adamčík2
Institute of Public Health, Ostrava, Czech Republic1; Institute of Hygiene, Ostrava, Czech Republic2
In foundry technologies, an application of moulding mixtures based on furan resins is used worldwide. These technologies are considered to be ecological. However, iron and steel founding represents an industrial source of wide spectrum
of harmful substances having an important fraction of genotoxic contaminants.
Methods.This study was focused on the relationship among an external dose of hazardous compounds (chemical identification of selected compounds in personal air), their internal dose (chemical identification of compounds’ metabolites in
urine) and genotoxic effect on human organism (chromosomal aberrations’ detection in blood lymphocytes) in exposed
and non-exposed groups. Comparison between groups was analyzed using Wilcoxon rank-sum test.
Results. Significant differences were found between the control group and the foundry workers on area of exposures
in air. For biomarkers in urine, the significant difference was noted between the control group and founders in levels of
metabolite of benzo/a/pyrene. No significant differences in the level of metabolite of furfuryl alcohol were detected.
All values of above-mentioned parameters, usually used to evaluating the endangerment of human health, ranged deep
below the hygienic exposure limits for occupational environment. However, cytogenetic analysis of blood lymphocytes
showed two significantly different groups. The control group corresponds to the non-exposed adult population. Founders
value was higher and gives an evidence for the increased exposure to genotoxic substances. No statistically significant
difference in this indicator was found between smokers and non-smokers.
Conclusion. Even though the foundry plant using furan technology has met the limits for external and internal dose of
observed compounds, founders belong to the group with higher exposure to genotoxic factors. This study didn’t confirm
furan derivates as a dominant genotoxic factor.
P-142
QUARTZ AND CARCINOGENICITY RISK ON THE WORKPLACES: BIOMONITORING RESULTS
Hana Lehocká 1,2
Tomáš Adamus 1; Ivona Závacká 1; Lubomír Dobiáš 1,2; Jaromíra Kůsová 2
University of Ostrava, Ostrava, Czech Republic1; Institute of Public Health, Ostrava, Czech Republic2
In the presented study, we summarize the data of the biomonitoring of the genotoxic potential of the quartz in the
ambient air on the workplaces of the coal mines, quarrying and stone processing. From the evaluation of results, it is
possible to deduce the following conclusions:
1. Quartz play an major role as determinant of the genotoxic potencial in complex mixture of fibrogenic respirabile
airborne particles in ambient air at the workplaces of:
● Quarrying and stone processing (of the respirabile dust concentration 0.18 –2.16 mg/m3, quartz-containing in the
dust 30 %).
● Coal mines (average value of the respirabile dust concentration 2.68 mg/m3, quartz-containing in the dust 3 %
and lower).
2. The comparision of the chromosomal aberrations levels in peripheral lymphocytes of workers professionally exposed
to industrial dust in mines and quarrying and stone processing demonstrates, the existence of differences versus
control level of %AB.C. in the Czech Republic. The frequencies of chromosomal aberrations were significantly higher
in exposed groups, than in the control. The differences were not significant between each groups
3. The results indicate a significant increase in the formation of chromosomal aberration in the group of workers professionally exposed to dust with content of quartz, in the early period of exposure (about 30 % HAE). The results
show evidence of high dose non – linearity of chromosomal aberration dose-response. (The study was supported by
a grant No. NJ/6578-3.)
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P-143
MUTAGENICITY TESTS AS A TOOL FOR EFFECTIVE EVALUATION OF BIODEGRADATION
OF POLLUTANTS
Kateřina Malachová 1
Zuzana Pavličková 1; Čeněk Novotný 2; Tomáš Cajthaml 2
Faculty of Science,University of Ostrava, Ostrava, Czech Republic1; Institute of Microbiology, Academy of Sciences, Praha,
Czech Republic2
Bacterial mutagenicity tests, Ames test and Chromotest, were used for evaluation of the effectivity of degradation of
PAHs and synthetic dyes. During microbial degradation, mutagenic intermediates can be formed. Efficiency of the degradation technologies was determined on the basis of mutagenicity measured. Other factors affecting the formation of
mutagens were determined. Degradation of PAHs was investigated in two independent studies. Mutagenicity evaluation
showed that in the biodegradation processes studied using a bacterial consortium or composting process, mutagenic
products were detectable with Ames test and SOS Chromotest in soil elutriates. The amount of mutagens was changing in time and in dependence on the degradation conditions, namely aeration. Formation of mutagenic intermediates
was studied also in a two-step biodegradation of monoazo dye Reactive Orange 16 and anthraquinone dye Disperse
Blue 3. It was demonstrated that a two-step degradation was necessary to achieve complete decolorization. After the
degradation with activated sludge, the mutation potential of both dyes decreased by one half. The complete elimination
of mutagenicity was accomplished in the second step where the fungus Irpex lacteus was used. Bacterial mutagenicity
tests can be used for monitoring the production of mutagens during biodegradation and for detection of the reduction
of dye mutagenic potential by the degradation process. The tests enable us to compare the efficiency of biodegradation
processes in terms of their effect on the mutation potential of organic pollutants. Genotoxicity tests can be used for
optimization of biodegradation technologies.
The study was supported by and Grant agency of the Czech Republic No. 526/00/1303 a No. 104/05/2296.
P-144
MEASUREMENT OF DNA ADDUCTS AS BIOMARKERS OF GENOTOXIC EXPOSURE
TO BENZO[A]PYRENE USING A LC-MS/MS TECHNIQUE
Caroline Marie 1
Jean-Luc Ravanat 1; Alain Favier 1; Emilie Vieu 2; Marie Marques 2; Anne Maître 2
CEA Grenoble, Grenoble, France1; Faculté de Médecine de Grenoble, Grenoble, France2
Introduction
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants representing the first family of
carcinogenic pollutants in professional environment. It is now well established that some of these molecules including
benzo[a]pyrene (BaP) are responsible for the apparition of cancers. It is therefore of major importance to evaluate
individual exposure to carcinogenic PAHs in order to improve health risk assessment. In addition to the classical determination of atmospheric concentrations of PAHs and urinary 1-hydroxyprene, we are interested in measuring specific
urinary metabolites and DNA adducts of BaP.
Methods
A high performance liquid chromatography coupled to fluorescence detection (HPLC-Fluo) method has been set-up for
the determination of urinary 3-hydroxybenzo[a]pyrene (3-OHBaP) and BaP tetrol. A HPLC coupled to tandem mass
spectrometry (HPLC-MS/MS) method was optimised to detect the different DNA adducts of B[a]P.
Results
The limit of detection of the HPLC-Fluo method for 3-OHBaP is 0.4 ng/L of urine subsequently to a double-step procedure
of purification and concentration of urinary samples. Such sensitivity is not improved using tandem mass spectrometric
detection. Optimisation of the detection of BaP tetrol is in progress. HPLC-MS/MS allows a highly sensitive and specific
detection and quantification of eight stable and depurinating DNA adducts of BaP. These include five DNA adducts of
the carbocation pathway and three adducts arising from the BaP-diol-epoxyde pathway. The limits of detection range
from 1 to 40 injected fmoles.
Conclusion
In the near future, our developed analytical tools will be applied to measure BAP metabolites, DNA adducts and DNA
oxidative damages in urinary samples obtained from volunteers professionally exposed to PAHs. The results should be
of main importance to identify pertinent biomarkers in order to measure exposure to BaP and assess health risks of
workers which may orientate preventing actions.
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P-145
GENOTOXIC EVALUATION OF THE CO-EXPOSITION TO INDUSTRIAL AIR POLLUTANTS
BY THE IN VITRO COMET ASSAY ON MACROPHAGES ALVEOLAR CELLS AND LUNG CELLS
OF SPRAGUE-DAWLEY RAT
Daniel Marzin 1,4
Fabrice Nesslany 1; Ludovic le Hegarat 1,2; Frank le Curieux 3,4
Institut Pasteur de Lille, Lille, France1; Faculté des Sciences Pharmaceutiques et Biologiques, Lille, France2; Institut Pasteur de Lille,
Lille, France3; Faculté des Sciences Pharmaceutiques et Biologiques, Lille, France4
Background: Numerous epidemiological studies have demonstrated association between air pollution and adverse health
effects including cardiovasculary diseases, asthma and cancer. The southern coast of the North sea in France is characterised by the proximity of industrial activity that has produced high quantity of particulate matter. Particulate matter
are a complex mixture of components including metals and organic aromatic compounds, that may interact to produce
synergistic, antagonistic, or additive effects. In order to determine the genotoxic potential induced by the co-exposition
to industrial air pollutants, DNA strand breaks were measured in vitro by the alkaline single-cell gel electrophoresis
(comet assay) in two different cells populations: the alveolar macrophages and the lung cells of Sprague-Dawley rat.
Method: After recovery of the healthy lungs of an anaesthetized Sprague-dawley rat, alveolar macrophages were collected by broncho-alveolar lavage and lung cells were isolated by collagenase 1A (0,1%) digestion. The compounds selected
for the study were found in significant proportion in particulate matter in the area around the city of Dunkerque: seven
organic compounds (styrene, 2-, 3- and 4-chloronitrobenzene, 2-, 3- and 4-nitrotoluene) and three metals (hematite
(Fe2O3), aluminium oxide (Al2O3) and manganese oxide (MnO2)). In the first part of the work, cells were treated durng
3 hours with each pollutant, and in the second part one metal and one organic compound were added together in the
culture media and cells were incubated for 3 hours. Results: For the studied non cytotoxic concentrations, no clear DNA
damage induced by organic compounds was detected by the comet assay in both cell types. Regarding metals, only
hematite was genotoxic in alveolar macrophages. Results from co-exposition studies will also be presented.
P-146
CHROMOSOME LESIONS IN FISHERMEN WHO PARTICIPATED IN THE CLEAN-UP
OF THE PRESTIGE OIL SPILL
Gemma Monyarch
MA Rigola; JP Zock; Y Torralba; L Bouso; F Gómez; H Verea; F Pozo Rodríguez; MD Coll; JA Barbera;
C Fuster; G Rodríguez Trigo
Grupo SEPAR-Prestige, Sociedad Espańola de Neumología y Cirugía Toracíca (SEPAR), Spain
In November 2002, the oil tanker Prestige wrecked and produced high contamination of the coast of Galicia (Spain).
To evaluate potential genotoxic effects in lymphocytes of the fishermen exposed, we analyzed presented chromosome
instability.
We report preliminary results including 43 clean-up work fishermen and 37 non-exposed from 800 fishermen that
were previously interviewed about the details of their cleaning-up activities. The fishermen never-smokers, fertile and
in good health were included in the exposed (E) group (>15 days of cleaning activities at least four hours per day)
and non-exposed (NE) group who did not participate in clean-up work. The collection of the samples and the analysis
were performed between July 2004 and February 2005. Peripheral lymphocytes were cultured and chromosome spread
stained with Leishman. About 100 randomly selected metaphases stained were analyzed in each case.
Any chromosome lesions were found in 32 (74%) and 19 (51%) of exposed and non-exposed, respectively (P=0.03).
Comparison of cytogenetic data between E and NE groups showed significant differences for (i) the proportion of metaphases with chromosome lesions (E:62/4521; NE:33/3884; P<0,04), and (ii) the proportion of chromosome lesions
(E:68/4521; NE:37/3884; P<0,03). Moreover, in our study, we also detected an increase of structural chromosome in
E vs. NE groups. We observed three rings, twenty-six markers and eight metaphases with multiple structural abnormalities in fishermen exposed; while in non-exposed, no rings, four markers and one multiple structural abnormalities
were found.
Our findings point towards an association between clean-up work of the Prestige oil spill and an increased chromosomal
instability in lymphocytes one to two years after the exposure.
Financial support: FIS (03/1685), SEPAR and Red Respira (RTIC 03/11)
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P-147
Beatriz Pérez-Cadahia 1,2
Josefina Méndez 1; Blanca Laffon 1,2; Eduardo Pásaro 2; Joao Paulo Teixeira 3; Susana Silva 3;
Joana Roma-Torres 3; Olga Mayan 3
Department of Cell and Molecular Biology, University of A Coruńa, A Coruńa, Spain1; Toxicology Unit, University of A Coruńa,
A Coruńa, Spain2; National Institute of Health, Environmental Health and Toxicology Department, O Porto, Portugal3
In the environment of tyre producing plants presence of several organic solvent vapours and airborne particulate matter
has been characterized. In addition, elevated mutagenicity and genotoxicity have been observed in air and particulate
samples. The objective of this study was to determine the genotoxic effects in a group of individuals engaged in the
production of rubber tyres from a Portuguese factory. Urinary thioethers were measured as biomarker of exposure.
Peripheral blood samples were collected from 32 exposed workers and 32 controls, and micronucleus (MN) test and
sister chromatid exchanges (SCE) were performed to evaluate the genotoxicity. Microsomal epoxide-hydrolase codons
113 and 139 polymorphisms were determined to estimate the expected activity of the enzyme. Thioethers excretion was
found significantly higher in rubber workers. No significant difference was observed in MN or SCE frequencies between
exposed and controls. Higher levels of cytogenetic rates were obtained in epoxide-hydrolase expected low activity
donors with regard to medium and high activity individuals.
P-148
INDUCTION OF MICRONUCLEI IN LYMPHOCYTES OF FOUNDRY WORKERS DUE TO CHRONIC
EXPOSURE TO EXTREMELY-LOW FREQUENCY ELECTROMAGNETIC FIELDS
Claudia Schwarz 1
Hugo W Rüdiger 1; Elisabeth Kratochvil 1; Marietta Weninger 1; Petra Hartbauer 1; Otto Zierer 2,3
Medical University of Vienna, Division of Occupational Medicine, Vienna, Austria1; Vertical Galva, Augsburg, Germany3
Exposure to extremely-low-frequency (3 – 300 Hz) electromagnetic fields (ELF-EMF) is ubiquitous in private households
(domestic appliances, power transmission lines) and industrial workplaces (melting furnaces operated by magnetic
induction, inductive welding). With respect to the genotoxic potential of ELF-EMF we wanted to investigate putative
DNA-damaging effects of chronic exposure to ELF-EMF of high field intensities on the basis of a collective of foundry
workers operating inductive melting furnaces.
DNA damage related to ELF-EMFs was assessed in terms of DNA strand breaks (alkaline comet assay), micronuclei
(cytokinesis-blocked micronucleus assay), dicenctric chromosomes (chromosome analysis), and sister chromatid
exchanges. Blood was collected from foundry workers after being exposed for several years to ELF-EMF generated by the
inductive melting furnace. The same collective was used for control purposes because the melting furnace was heated
using gas as of a certain time (blood and urine samples were drawn two months afterwards). At this second point levels
of heavy metals in blood, serum, and urine were determined, because our first visit in the factory revealed additional
exposures to heavy metals, dust, and heat.
The frequency of micronuclei was fourfold elevated in ELF-EMF exposed workers as compared to controls, DNA strand
breaks were only marginally elevated, and there were no detectable alterations of dicentric chromosomes and sister
chromatid exchanges. A control study 10 weeks after the ELF-EMF exposure had ceased revealed a marked reduction
of micronuclei frequency, although twofold elevated levels still persisted.
We conclude that chronic exposure to ELF-EMF induces genotoxic effects in vivo which may persist for at least 10
weeks.
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P-149
AN EVALUATION OF CYTOGENETIC DAMAGE IN A POPULATION EXPOSED TO URANIUM MINES
RESIDUES
Maria João Silva 1
Maria Guida Boavida 1; Ana Carla Sousa 1; Paula Costa 1; Anabela Dias 1; Octávia Monteiro Gil 2; Patricia
Cardoso Painço 2; Luisa Pedro 2; Joao Cardoso 3; Luis Santos 3
Paulo Nogueira 4; José Marinho-Falcao 4
Center of Human Genetics, National Institute of Health Dr. Ricardo Jorge, Lisbon, Portugal1; Department of Radiological Protection
and Nuclear Safety, Nuclear and Technological Institute, Sacavém, Portugal2; Laboratory of Ionizing Radiation Metrology, Nuclear
and Technological Institute, Sacavém, Portugal3; National Health Observatory, National Institute of Health Dr. Ricardo Jorge,
Lisbon, Portugal4
The mine and milling complex of “Urgeiriça”, localized near a small town in the Centre of Portugal was recently closed
down leaving behind an open-air tailing pile of 2 500 000 tons. Preliminary environmental characterization of the area
showed high levels of uranium and heavy metals contamination. The present study was conducted to evaluate the
existence of potential cytogenetic damage in peripheral blood lymphocytes of individuals with long-term residence in
the vicinity of Urgeiriça mines and its residues. Additionally, DNA repair competence of radiation-induced DNA damage
was evaluated by the challenge assay. The exposed group consisted of non-smoking males, aged 45-64 years, living
near Urgeiriça mines (n=32). Inhabitants of the same region but without the influence of uranium mines or ores were
selected as controls (n=33). Translocations and unstable chromosome aberrations (dicentrics, acentric fragments and
rings) analyses using FISH with whole-chromosome paint probes for chromosomes 1, 2 and 4 were used to assess
cytogenetic damage in lymphocytes. For each individual, 2000 metaphases were scored for spontaneous aberrations
while 700 were analysed to determine the aberration frequencies after a 2 Gy challenging dose of gamma-radiation. No
significant difference was detected in the frequency of spontaneous chromosome aberrations between the exposed and
the control groups (p>0,05). However, in response to in vitro irradiation the exposed group presented a significantly
decreased frequency of total aberrations (p=0.0004), either translocations (p<0.0001) or unstable aberrations (p=0.02
for dicentrics), when compared to the control group. These results are suggestive of an adaptive response in the lymphocytes of individuals with long-term exposure to uranium mines residues. However, a direct correlation between the
finding of an adaptive response and a health effect cannot be presently established for the exposed population.
(Work funded by the Portuguese Ministry of Health)
P-150
CYTOGENETIC MONITORING OF WORKERS OCCUPATIONALLY EXPOSED TO IONISING
RADIATION
Grazina Slapsyte 1
Jurate Mierauskiene 1; Inga Januskeviciute 1; Birute Griciene 1,2
Vilnius University, Vilnius, Lithuania1; Radiation Protection Centre, Vilnius, Lithuania2
The Ignalina Nuclear Power Plant (INPP) and outside workers constitute the largest group of workers occupationally
exposed to low doses of ionising radiation in Lithuania. In 2004, the average annual individual dose of these workers
was 1.53 mSv. Maximum doses were obtained during outages and constitute 19.16 mSv for INPP and 29.41 for outside
workers. The aim of the present study was to analyse chromosome aberration (CA) frequencies in lymphocytes of INPP
workers having relatively high individual doses recorded in 2004 year.
35 male INPP employees (mean age 45 years) were included into the study. Their occupational radiation exposure
resulted mainly from external γ-rays. 8 workers had an incorporation of 60Co or 137Cs radionuclides contributing to the
annual committed effective dose with less than 0.1 mSv. Average cumulative dose of the workers was 219 mSv. The
mean annual dose averaged over the three-year-period preceding blood sampling was 11.1 mSv. 64 unexposed healthy
male donors approximately matched by age were used as controls.
Heparinized venous blood samples were taken and cultures initiated according to the standard procedures. Generally 500
first division metaphases per individual were analysed. CA analyses revealed no significant differences between radiation workers and controls. The mean CA frequency in radiation workers was 1.72±0.21 CA/100 cells, and 1.65±0.15
in controls. The average yield of dicentrics per 100 cells was 0.09±0.04 and 0.08±0.02 respectively. However, significant increase in total CA frequency was recorded for radiation workers with incorporated radionuclides as compared to
those without internal radiation exposure (2.79±0.37 vs. 1.40±0.22 CA/100 cells, P<0.0001) and controls (P=0.0125).
Significant differences were also found for total chromosome-type aberrations, chromosome and chromatid breaks. The
differences were not significant for dicentrics. The estimated increase of chromosome aberrations may be attributed to
internal radiation exposure.
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P-151
CYTOGENETIC ANALYSIS OF BUCCAL, NASAL AND UROTELIAL HUMAN CELLS
Ljudmila P. Sycheva
A.N.Sysin Research Institute of Human Ecology and Environmental Health, Moscow, Russian Federation
Micronucleus test on human exfoliated cells is a perspective method of an estimation of complex effect of environmental and industrial mutagens. This method is non invasive; it is more correct for the prognosis of carcinogenic effect
so about 90% malignant tumors occurs from epithelial cells; more simple and less expensive. Improvement of the
approach consists in analysis of cells of two or more organs; a quantitative estimation of all conditions of a nucleus
cells: cytogenetic parameters (micronuclei, bridges and protrusions); indirect parameters of proliferation (binucleated
cells and cells with central nuclear grooves) and apoptosis. We have improved techniques of reception of preparations
exfoliated buccal, nasal and urothelial cells, have carried out analysis the cytogenetic status buccal and nasal epithelium
of 205 conditionally healthy Moscow inhabitants (employees of office), and investigated urothelial cells of 20 person on
10 cariological parameters. Rough background levels of these parameters on 1000 cells have made: micronuclei- 0,24;
0,35; 0,31 accordingly; protrusions-0,29; 1,51; 0,53; binucleated cells - 1,06; 2,29; 1,21; cells with nuclear grooves
- 1,7; 2,84; 1,95 etc. At comparative analysis of parameters in groups of people working in different working conditions,
their non-uniform distribution is marked. Among 17 groups it is revealed 3 groups with 60% people having one or two
parameters with elevated boundary values (x±3 St.dev.) and 2 groups with 40 % people having exceeded boundary
values of these parameters. In other study differences of frequency of cytogenetic parameters in two groups of children
of living in Vietnam territories with a different level of dioxine pollution are received.
P-152
MODIFICATION OF SENSITIVITY OF CELLS FROM HEALTHY DONORS TO MUTAGEN EXPOSURE
IN VITRO AFTER VITAMINS INTAKE
Ekaterina S. Voronina 1
Viktoria A. Nikitina 1; Ali K. Zhanataev 2; Andrey D. Durnev 2
Research Center for Medical Genetics, Russian Academy of Medical Sciences, Moscow, Russian Federation1; State Zakusov Institute
of Pharmacology, Russian Academy of Medical Sciences, Moscow, Russian Federation2
Spontaneous and induced in vitro mutagenesis was studied in blood cells of healthy donors who had taken different
vitamin complexes (VC): VC1 – for 30 days, VC2 and VC3 – for 14 days. The whole blood samples were taken before
and after VC intake and were cultivated during 54 hours. Mutagens (dioxidine 0.01, 0.03 and 0.1 mg/ml, bleomycine
0.1 and 1.0 U/ml, CdCl2 0.2 mg/ml) were added to cultures after 50 hours of cultivation. At each variant of the experiment 200-600 metaphases were analyzed (total >120 000 cells). VC intake did not influence on the level of spontaneous cytogenetic damage. Total number of aberrant cells and ratio of different types of aberrations agreed with current
data on spontaneous mutagenesis in Russian population. VC1 and VC2 contained vitamins in doses greatly exceeding
the recommended daily allowance (RDA). Intake of these VCs either increased the resistance of peripheral blood cells
to mutagens or had no significant effect. The increasing of cell resistance was more evident when VCs were taken for a
longer period. Modification of cells sensitivity to mutagen exposure depended not only on duration of VC intake but also
on mutagen concentration. VC3 contained vitamins in doses 20-30% exceeding RDA. VC3 intake did not influence on
effect of dioxidine and increased cells sensitivity to cytogenetic effect of bleomycine (0.1 U/ml). Intake of VC2 did not
modify cells sensitivity to bleomycine. At all variants of the experiment significant changes in ratios between different
types of chromosome aberrations were not observed. Thus, intake of vitamins in exceeding RDA doses did not influence
on the level of spontaneous mutagenesis and in most cases decreased human cells sensitivity to cytogenetic effects of
chemical mutagens. The effect depended on duration of VC intake, their quantitative and qualitative composition, kind
of mutagen and its concentration.
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P-153
A NEW MECHANISM FOR THE DIFFERENTIAL ANTICARCINOGENIC EFFECTS OF N-3 AND N-6
POLYUNSATURATED FATTY ACIDS
Gerrit Alink1
Ivonne M.C.M. Rietjens 1; Vincent A. van Beelen 1; Jac M.M.J.G. Aarts 1; Astrid Reus 1; Hans Mooibroek 2;
Lolke Sijtsma 2; Dirk Bosch 3
Chair Toxicology, Wageningen University, Wageningen, Netherlands1; Agrotechnology and Food Innovations B.V, Wageningen,
Netherlands2; Plant Research International, Wageningen, Netherlands 3
Background/Aims:The mechanisms by which n-3 polyunsaturated fatty acids (n-3 PUFAs) reduce cancer risk are still a
matter of debate. N-6 PUFAs, in contrast to n-3 PUFAs, seem to have no chemopreventive characteristics. As in earlier
studies an oxidative stress-based mechanism was suggested (Dommels et al., 2003), the aim of this research was to
test whether differential induction of electrophile-responsive element (EpRE)-regulated genes (i.e. phase II, antioxidant)
could be a mechanism to explain the different effects of n-3 and n-6 PUFAs.
Methods:Eicosapentaenoic acid (EPA; n-3), docosahexaenoic acid (DHA; n-3), and arachidonic acid (AA; n-6) were
tested for their induction of EpRE-regulated genes in a mouse hepatoma cell line, using: a luciferase reporter cell line,
real-time RT-PCR, and enzyme activity assays.
Results:The results from the reporter assay showed that the n-3 PUFAs EPA and DHA can induce EpRE-regulated gene
expression, whereas the n-6 PUFA arachidonic acid (AA) is hardly able to do so. It appears that n-3 PUFAs need to be
oxidized to induce EpRE-regulated gene expression, as the antioxidant vitamin E was able to partially inhibit the n-3
PUFA induced dose-dependent effect. Real-time RT-PCR and enzyme activity assays of EpRE-regulated genes confirmed
these findings.
Conclusion:We suggest that the induction of EpRE-mediated phase II genes by n-3 PUFAs may be
which n-3 PUFAs, in contrast to n-6 PUFAs, are chemopreventive and anticarcinogenic. This is, as
first time that an oxidative-stress-based mechanism is proposed that can explain the differential
and n-6 PUFAs.Dommels, Y. E., Haring, M. M., Keestra, N. G., Alink, G.M., VanBladeren, J. & Van
Carcinogenesis 24, 385-392.
a major pathway by
far as we know, the
effects between n-3
Ommen, B. (2003).
P-154
ANTIGENOTOXIC EFFECT OF BASIL (OCIMUM BASILICUM L.) IN MICROBIAL SHORT-TERM
TESTS
Branka Vukovic-Gacic
Tanja Beric; Biljana Nikolic; Jasna Stanojevic; Draga Simic; Jelena Knezevic-Vukcevic
Laboratory for Microbiology, Faculty of Biology, Serbia and Montenegro
Sweet basil (Ocimum basilicum L.) is employed as a folklore remedy for a wide spectrum of ailments in many traditional
medicines. The mutagenic and antimutagenic properties of essential oil of basil (EO) and its major constituent, terpenoid
alcohol Linalool, were examined in bacterial tests. In preliminary experiments both basil derivatives showed no mutagenic effect, with or without metabolic activation, in the Salmonella/microsome assay in TA100. Antimutagenic effect
against spontaneous and t-BOOH-induced mutagenesis was examined in mismatch repair (MMR) deficient and repair
proficient Escherichia coli K12 strains. In wild type strain SY252 the reduction of t-BOOH-induced mutagenesis was
49% for EO and 36% for Linalool. The spontaneous mutagenesis was reduced in its MMR deficient counterpart (mutH)
and inhibition ranged from 27% for EO to 44 % for Linalool. Reduction of spontaneous and t-BOOH-induced microsatellite instability was obtained in mutH and repair proficient strains (30-35% of inhibition). Inhibitory potential of EO and
Linalool was also tested in E. coli WP2 strain deficient in induction of antioxidative enzymes (oxyR). The reduction of
t-BOOH-induced mutagenesis was 30% for EO and 60% for Linalool. Antigenotoxic potential of Linalool was tested in
Comet assay on Saccharomyces cerevisiae. Linalool exhibited protective capacity against H2O2-induced comets, more
in pre-treatment than in co-treatment experiments. Results obtained with EO, Linalool and Vitamin E, used as a model
antioxidant, indicate that antimutagenic and antigenotoxic potential of basil derivatives could be attributed to their
antioxidative properties.
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P-155
OXIDATIVE STRESS-RELATED DNA DAMAGE AND P53 MODULATION IN BENZO[A]PYRENE
EXPOSED HUMAN HEPATOMA HEPG2 CELLS
Jinhee Choi
Sun-Yong Park; Ji-Yeon Rho
University of Seoul, Seoul, Korea
Oxidative stress-related DNA damage and p53 modification were investigated Benzo[a]pyrene toxic mechanisms to
identify potential biomarkers for Benzo[a]pyrene monitoring and risk assessment in human hepatoma HepG2 cells.
Benzo[a]pyrene exposure induced decreased cell viability and increased antioxidant enzyme activity and DNA damage.
The p53 protein activation seemed to have been a downstream response to the Benzo[a]pyrene-induced DNA damage,
which suggests that p53 plays important roles in defense against Benzo[a]pyrene-induced genotoxicity. The response
of phosphorylated p53 towards Benzo[a]pyrene exposure could be more sensitive than that of normal p53. Activation
of p53 following DNA damage in turn acts as a transcriptional regulator of several target genes. One of the main targets p21 protein, a gene that encoded the Cdk inhibitor was induced by Benzo[a]pyrene exposure. The p53 mRNA
level increased after the cells were treated with Benzo[a]pyrene, as well as following p53 protein level induction, which
suggests that Benzo[a]pyrene-stimulated p53 accumulation may also be induced transcriptionally. Benzo[a]pyreneinduced NF-kB binding activity was also observed. Oxidative DNA damage and p53 accumulation seem to be related
to Benzo[a]pyrene toxicity, however, such that their potential as biomarkers for Benzo[a]pyrene monitoring and risk
assessment need to be validated in the context of their specificity and sensitivity. Acknowledgement: This work was
supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD)( Grant No. KRF-2005204-D00009) and by the grant from Korean Ministry of Environment through Ecotechnopia21 project (Grant No.091041-025 ). Corresponding author : Jinhee Choi
P-156
OXIDATIVE STRESS CAUSED BY SUBWAY PARTICLES
Hanna Karlsson
Lennart Möller
Department of Biosciences and Nutrition, Huddinge, Sweden
Background: Epidemiological studies have shown an association between airborne particles and a wide range of adverse
health effects, including cancer and cardiovascular diseases. Oxidative stress is considered to be an important mechanism behind these health effects. The knowledge regarding how particles from various sources differ in their ability to
induce oxidative stress is still limited.
Aim: The aim of this study was to compare particles generated from tire-road wear and collected from a street and a
subway station, regarding their ability to form 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and intracellular reactive oxygen species (ROS).
Method: 2’-deoxyguanosine (dG) in solution and cultured human lung cells (A549) were exposed to particles, or water
extracts from particles. The formation of 8-oxodG in dG-solution and in cells was analyzed using HPLC with EC detection.
Intracellular ROS in human lung cells were analyzed using a fluorescent dye in combination with a flow cytometer.
Results: All particles oxidized dG in solution. The subway particles were most potent, mainly due to non-soluble components, followed by particles from street, mainly due to soluble substances. Only the subway particles caused formation
of 8-oxodG in cultured human lung cells. Similarily, only the subway particles formed measurable intracellular ROS.
Conclusion: Subway particles can induce oxidative stress in cultured human lung cells, likely due to redox-active surfaces of the iron-rich subway particles. This give cause of concern since concentrations of particles have shown to be
5-10 times higher in the subway in Stockholm compared to a very busy street in the city.
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P-157
EFFECT OF ACTIVE AND PASSIVE SMOKING ON OXIDATIVE DNA DAMAGE MEASURING
BY 8-OXO-7,8-DIHYDRO-2’-DEOXYGUANOSINE LEVELS IN HUMAN LEUKOCYTE
Maura Lodovici
University of Florence, Pharmacology, Florence, Italy
Tobacco smoke contains numerous compounds that generate reactive oxygen species, which can damage DNA directly
or indirectly via increased inflammation, thus promoting carcinogenesis in smokers. However, data regarding the effects
of exposure to environmental tobacco smoke (ETS) on non-smokers are still limited and controversial. Therefore, we
decided to evaluate the effect of ETS on human leukocytes and to estimate the correlation of oxidative DNA damage with
exposure measuring by plasma cotinine levels. Since lifestyle factors can also influence oxidative DNA damage, we also
investigated the effect of physical activity, body mass index, alcohol, and tea and coffee consumption. Oxidative DNA
damage, measuring by 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodGuo) levels was detected in DNA of leukocytes
of healthy donors (30 smokers, 29 non-smokers, and 28 ETS-exposed subjects). Non-smokers had significantly (P =
0.00007) lower 8-oxodGuo levels (5.94 ± 0.87 x 10-6 dG; mean ± SE) compared with smokers (19.85 ± 4.75 x 10-6
dG; mean ± SE). Subjects exposed to ETS had higher, but not significantly (P = 0.074 by univariate analysis) mean
value of 8-oxodGuo compared with non-smokers (9.18 ± 1.53 x 10-6 dG; mean ± SE). On the contrary, multiregression
analysis indicated that the increase of 8-oxodGuo levels induced by ETS was significant (P = 0.045) and that coffee
and tea consumption reduced DNA oxidation (P = 0.0053). Moreover, oxidative DNA damage was positively correlated
with plasma cotinine levels in ETS-exposed subjects (r = 0.47, P < 0.01) and was increased by age in non-smokers and
ETS-exposed subjects (P = 0.049). The results seem to confirm that ETS exposure is capable to induce oxidative DNA
damage in leukocytes and that coffee and tea consumption might partially protect against oxidation damage induced
by smoking.
P-158
PROTECTIVE EFFECT OF PLANT ANTIOXIDANTS AGAINST T-BOOH INDUCED DNA DAMAGE
AND MUTAGENESIS IN PROKARYOTIC AND EUKARYOTIC TESTS IN VITRO
Dragna Mitic-Culafic1
Biljana Nikolic 1; Branka Vukovic-Gacic 1; Jelena Knezevic-Vukcevic 1; Bojana Zegura 2; Metka Filipic 2
Faculty of Biology University of Belgrade, Belgrade, Serbia and Montenegro1; National Institute of Biology, Department of Genetic
Toxicology and Cancer Biology, Ljubljana, Slovenia2
The aim of this study was to investigate the protective effects of antioxidative substances from plants: eucalyptol,
linalool and myrcene against oxidative DNA damage and mutagenesis in bacterial and mammalian cells in vitro. The
bacterial assay was performed with repair proficient strains of E. coli K12 (SY252) and E. coli WP2 (IC185) and on the
oxyR mutant of E. coli WP2 (IC202), deficient in induction of antioxidative enzymes. Experiments were performed as cotreatment with oxidative mutagen (t-butyl-hydroperoxide (t-BOOH)) and antioxidant substances . The comet assay with
human hepatoma cell line (HepG2 cells) was used to measure the potential of antioxidants to reduce t-BOOH induced
DNA damage. The cells were A) pre-treated with different concentrations of antioxidants (0, 0.01, 0.1 and 1 μg/ml) for
21 hours and than co-treated with antioxidants and t-BOOH (1μM) for 20 minutes and B) the cells were co-treated with
antioxidants (0, 0.01, 0.1 and 1 μg/ml) and t-BOOH (1μM) for 20 minutes.
In the bacterial assay reduction of t-BOOH-induced mutagenesis was obtained in all tester strains with all tested substances. The highest inhibition of mutagenesis was obtained in IC202 strain (40% for eucalyptol, 0.05 mg/p.p.; 60%
for linalool, 1mg/p.p., and 69% for myrcene, 1 mg/p.p.), indicating that their antimutagenic potential is based primarily
on their antioxidative properties. In the comet assay linalool and eucalyptol significantly reduced t-BOOH-induced DNA
damage in HepG2 cells in both approaches used, while myrcene only slightly decreased t-BOOH induced DNA strand
breaks. However, the higher effect against t-BOOH induced DNA damage was observed when the cells were pretreated
with the oxidants for 21 hours prior to the exposure to t-BOOH (protocol A). This shows that eucalyptol, linalool and
myrcene can act as reactive oxidant scavengers and probably increase antioxidant cell pool especially when the cells
are pretreated with antioxidants.
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P-159
-866 G/A POLYMORPHISM OF THE UNCOUPLING PROTEIN 2 IN RELATION TO CANCER RISK
Susan Nowell1
Kelly Mercer 1; Carolyn Wise 2; Fred F. Kadlubar 2; Luke Ratnasinghe 2; Christine B. Ambrosone 3; Nicholas
P Lang 4
University of Arkansas for Medical Sciences, Little Rock, United States1; Natiional Center for Toxicological Research, Jefferson,
United States 2; Roswell Park Cancer Institute, Buffalo, United States3; Central Arkansas Veteran's Healthcare System, Little Rock,
United States4
Production of reactive oxygen species (ROS) is a generalized cellular response to a variety of stimuli, including exposure to environmental mutagens. Elevated cellular ROS has been implicated in the etiology of several human diseases,
and recent studies have supported a role for ROS in breast cancer risk. Uncoupling proteins (UCP) are encoded by
nuclear DNA and expressed in the inner mitochondrial membrane, where they function to translocate protons into the
mitochondrial matrix. UCP1 is the most extensively characterized isoform and is found in brown adipose tissue where it
plays a key role in regulating thermogenesis. The UCP isoform 2 (UCP2) is the most ubiquitously expressed form, but its
physiological function is less clear. In animal studies, however, UCP2 has been demonstrated to modulate mitochondrial
ROS and to play a role in decreasing the activation of mitochondrial-mediated apoptosis. UCP2 plays a role in several
disease states, including the metabolic syndrome (obesity, type II diabetes) and atherosclerosis. Recently, expression
of UCP2 was correlated with the degree of neoplastic changes in colorectal cancer.
A functional single nucleotide polymorphism (SNP) in the promoter region of the UCP2 gene (-866 G/A) results in
increased protein expression for individuals with the A variant. This SNP has been associated with leptin resistance,
diabetes mellitus and body weight regulation. The effect of this SNP on breast and colorectal cancer risk in case-control
studies will be presented.
P-160
SIX WEEKS OF CHLORELLA SUPPLEMENTATION HAS PARTIAL EFFECT ON BIOMARKERS
OF ANTIOXIDANT STATUS IN KOREAN MALE SMOKERS
Sun-Hee Lee 1
Hae Jin Kang 1; Yoo Kyoung Park 1; Jeong Woo Jeon 1; Hye-Jin Lee 2; Eun-Jae Jeon 2; Chang-sook Kim 2;
Myung-Hee Kang 2
Kyung-Hee University, Seoul, Korea1; Hannam University, Deajeon, Korea
2
Chlorella is a genus of unicellular green algae belonging to the Phylum Chlorophyta. Chlorophytes comprise a major
component of the phytoplankton. It is a popular food supplement in Asia and is marketed as a nutritional supplement.
Smoking increases oxidative damage to lipids, proteins and DNA which are potential underlying processes in the pathogenesis of many diseases. We evaluated whether 6 weeks of chlorella supplementation to smokers can be protective
against oxidative damage in a randomized double-blinded placebo-controlled trial, with a 1-wk run-in period before
treatment. Fifty-two smokers were given 18 capsules of chlorella containing 6.3 g every day for 6 weeks. Chlorella
supplementation increased plasma vitamin C (44.4%), α-tocopherol (15.7%) and significant reduction was observed
on DNA damage, measured using single cell gel electrophoresis (COMET assay). Although Six weeks of chlorella supplementation resulted in a significantly decrease in lymphocytes DNA damage, placebo supplementation also reduced
the measures of lymphocyte DNA damage. Putting these results together, chlorella supplementation showed conserving
plasma antioxidant nutrient status in the subjects, while no independent effect was observed in the status of lymphocyte
DNA damage. Therefore, our result suggests partial supportive role of chlorella as an anti-oxidant supplement.
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P-161
IRON OVERLOAD INDUCES OXIDATIVE DNA DAMAGE IN RAT LEUKOCYTES AND COLONOCYTES
Kyung Im Jeon 1
Eunju Park 1; Su-Yeon Kim 1; Bo-Young Seo 1; Hyun-Jin Lee 1; Beatrice L Pool-Zobel 2
Dept. of Food and Nutrition, Kyungnam Univeristy, Masan, Korea1; Dept. of Nutritional Toxicology, Friedrich-Schiller-University,
Jena, Germany2
Iron exposure is directly associated with pathogenesis of many disorders including atherosclerosis, cancer, inflammation, possibly via production of reactive oxygen species (ROS). These induce oxidative damage to lipids, proteins and
DNA. The purpose of this study was to evaluate the effect of iron overload on oxidative DNA damage in rat leukocytes
and colonocytes. Male Sprague Dawley rats were fed semi-synthetic diets containing regular (50mg/kg diet) or high
(2,000mg/kg) doses of iron for 6 weeks. The high dose of iron induced DNA breaks, oxidized bases (formamido-pyrimidine DNA glycosylase-sensitive sites) and oxidized pyrimidine base (endonuclease III sensitive sites) in rat leukocytes.
Iron overload increased significantly colonocytic DNA damage by 37%. In addition, we observed that iron overload
correlated reduced total antioxidant status (TRAP) and increased lipid peroxidation (conjugated dienes) in rat plasma.
These results indicated that iron overload impaired antioxidant and oxidants balance and markedly induces oxidative
DNA damage in rat leukocytes and colonocytes.
P-162
ANTIOXIDANT AND ANTICANCER ACTIVITIES OF ACETON EXTRACT FROM STYELA CLAVA
Bo-Young Seo 1
Eunju Park 1; Eun-Sil Jung 2; Hae-Ryong Park 2; Seung-Chul Lee 2
Dept. of Food and Nutrition, Kyungnam Univeristy, Masan, Korea 1; Dept. of Food Science and Biotechnology, Kyungnam University,
Masan, Korea2
Styela clava (also called as rough sea squirt or leathery tunicate) is thought to be native to the northwest Pacific including Korea and widely distributed in parts of northwestern Europe, North America and Australia. To evaluate Styela clava
as a potential bioactive agent, antioxidant activity of aceton extracts from Styela clava (whole, tunic and substance)
was tested by measuring scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and inhibitory effect of H2O2
induced DNA damage using comet assay. Also, anticancer activity on human colon cancer cell (HT-29) was investigated
by MTT reduction assay. The DPPH radical scavenging activity (RSA) increased as concentration increased from 1 to 10
mg/ml in all tested samples. The extract from tunic part scavenged the free radical most effectively. The 200 uM H2O2
induced DNA damage was inhibited with Styela clava aceton extract in dose dependent manner in human leukocytes.
The maximum inhibition was by 62.8, 78.3 and 62.1% at the concentration of 50 ug/ml of whole, tunic and substance
extracts, respectively. The aceton extracts from S. clava were also found to inhibit the growth of human colon cancer
cell. The cell proliferation rates decreased to 26.9, 12.0 and 30.6% at the concentration of 500 ug/ml of whole, tunic and
substance extracts, respectively. These results support that aceton extracts from S. clava may be a potential candidate
as a possible antixodiant and chemotherapeutic agent.
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P-163
ANTIOXIDATIVE AND CYTOTOXIC ACTIVITY OF RED BEET (BETA VULGARIS) IN COMBINATION
WITH INDUCERS OF OXIDATIVE STRESS
Piotr Szweda
1,2
T. Friedberg 2; M.J. Paine 2; L. McLaughlin 2; E. Polson 2; A. Stenhouse 2; J. Lewandowska, A. Bartoszek 3
Dept. Food Chemistry, Technology and Biotechnology, Gdansk University of Technology, Gdansk, Poland 1; Biomedical Research
Centre, University of Dundee, Level 5, Ninewells Hospital and Medical School, Dundee, United Kingdom2; Dept. of Pharmaceutical
Technology and Biochemistry, Gdansk University of Technology, Gdansk, Poland3
Environmental pollutants, inflammation, oncological ailments as well as cancer therapy stimulate the exposition of
human organism to ROS. The importance of controlling the oxidative stress for human health prompted massive
research on the natural antioxidants. We centered our studies on red beet (Beta vulgaris) juice (RBJ) since it is a rich
source of antioxidants “ready for consumption” and is regarded in Eastern European folk tradition as a health promoting
factor. The protective effect was evaluated in 2 human colon cancer, 2 human breast cancer and mouse leukaemia cell
lines using RBJ concentrations 0.1 – 7.5% [v/v], hence doses that can be expected in alimentary tract after consumption
of this vegetable. Despite documented lack of toxicity in vivo, RBJ turned out to be quite toxic to cultured tumor cells
(EC50
4% [v/v] for 72 hr exposition). However, in cells exposed to hydrogen peroxide, RBJ at concentrations 1 –
7.5% significantly decreased cytotoxic effects. Commercial products based on red beet (e.g. powdered soup) enhanced
hydrogen peroxide toxicity when used at equivalent doses. Preincubation of cells with RBJ did not enhance protective
effect, hence it stemmed from chemical ROS scavenging rather than induction of endogenous cellular mechanisms. In
contrast to hydrogen peroxide, in cells treated with doxorubicin, a cytostatic stimulating ROS formation, the protection was seen only for RBJ concentrations below 1%, higher doses potentiated drug cytotoxicity. In ongoing studies,
we compare cytotoxic vs. genotoxic effects of doxorubicin in combination with RBJ. We plan also to investigate stress
response in cells treated with doxorubicin +/- RBJ at a gene level by means of GEArray containing 96 genes indicative
for stress and toxicity pathways.
This work was partially funded by ECNIS Network of Excellence; ECNIS Training Fellowship enabled Piotr Szweda’s
research at Biomedical Research Centre, University of Dundee.
P-164
A COMPARATIVE STUDY ON THE EFFECT OF ALGAL AND FISH OIL ON VIABILITY AND CELL
PROLIFERATION OF HUMAN CACO-2 CELLS, AND ABERRANT CRYPT FOCI FORMATION IN RAT
Vincent A. van Beelen 1
Ivonne M.C.M. Rietjens 1; Gerrit M. Alink 1; Johannes Roeleveld 1; Hans Mooibroek 2; Lolke Sijtsma 2;
Raoul J. Bino 3; Dirk Bosch 3
Chair of Toxicology, Wageningen University, Wageningen, Netherlands1; Agrotechnology and Food Innovations B.V., Wageningen,
Netherlands2; Plant Research International, Wageningen, Netherlands3
Background/Aims:The main sources of n-3 polyunsaturated fatty acids (PUFAs) are fish oils. However, because of
contamination with environmental pollutants, over-fishing, and rapidly increasing demands for n-3 PUFAs, alternative
processes for PUFA-production are currently being exploited and further studied; one of them is the exploitation of PUFA
sources like microalgae.
The aim of this research was to test PUFA-rich micro-algal oils in vitro for antiproliferative properties in human colon
adenocarcinoma Caco-2 cells, comparing these results with the properties of fish oil, and to study the anti-carcinogenic
properties of algal and fish oil in vivo.
Methods:Oils derived from Crypthecodinium cohnii, Schizochytrium sp. and Nitzschia laevis, three commercial
oil capsules, and menhaden fish oil were used in cell viability and proliferation assays with Caco-2 cells. The n-3
acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were used as a comparison. A fish oil and an
oil were hydrolyzed to mimic the in vivo situation. An animal test was conducted to compare algal and fish oil for
anticarcinogenic properties on the formation of azoxymethane-induced aberrant crypt foci (ACF).
algal
fatty
algal
their
Results:The hydrolyzed fish and algal oil showed a comparable dose-dependent decrease in cell viability and cell proliferation while almost no effect was observed of the non-hydrolyzed samples in Caco-2 cells. The inhibitory effects of the
hydrolyzed samples were equal to that of the total amount of free fatty acids. Oxidative stress was shown to play a role
in the anti-proliferative properties of EPA and DHA, as vitamin E could partially reverse the EPA/DHA-induced effects.
Compared to corn oil, algal oil decreased significantly the number of ACF in the rat, in the same order of magnitude
as fish oil.
Conclusion:The results of the present study suggest that algal oil has the same cancer preventive properties as fish
oil.
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P-165
NOVEL SALMONELLA TESTER STRAINS HIGHLY SENSITIVE TO PHOTOCHEMICAL SOURCES
AND OXIDATIVE DAMAGE
Masami Yamada
Keiko Matsui; Takehiko Nohmi
Induction of DNA damage as a consequence of exposure to UV light has been established as the major cause of skin
cancer. DNA molecules absorb photon energy directly for wavelength < 320 nm, leading to well characterized mutagenic DNA damage. Alternatively, endogenous or exogenous chemicals (sensitizers) may absorb light, with the potential
of subsequent energy or electron transfer, leading indirectly to DNA damage. The types of DNA damage indicate the
involvement of reactive oxygen species (ROS). 8-Hydroxyguanine (8-OH-G) is one of the major DNA lesions induced by
the ROS and has potential to induce mutations and can be generated spontaneously or induced by a variety of chemicals
including photochemicals. The mutM-gene product, i.e., 8-OH-G DNA glycosylase, plays an important role in removing
8-OH-G in DNA in Escherichia coli. Salmonella typhimurium is a relative species of E. coli and widely used for mutagenicity assay called as Ames test. Strain TA1535 carries hisG46 missense mutation, efficiently detects mutations occurring
at G:C base pairs. It is also deficient in the excision repair system (DuvrB) and has an rfa mutation, which increases the
permeability of bacteria. In order to determine the roles of photochemical reactivation, we disrupted the mutM gene in
the TA1535 strain. The resultant strain YG3001, was eight-, four- and five-times more sensitive to the mutagenicities of
methylene blue plus visible light, or neutral red plus visible light and 2-nitrofluorene than TA1535, respectively, while the
mutagenicity of 4-nitroquinoline N-oxide was similar regardless of the mutM gene existence (Mutat. Res., 393, 233-246,
1997). In addition, the strain revealed that benzo[a]pyrene generates ROS with visible light and exhibited mutagenicity
without usual metabolic activation (Env. Mol. Mutagen., 46, 141-149, 2005). The constructed strain could be potentially
useful for monitoring of oxidative mutagens in environment with its high sensitivity.
P-166
A BACTERIAL MUTATION (AMES) ASSAY DESIGN FOR COMPARATIVE ASSESSMENT
OF MUTAGENS
Mark Ballantyne
Ann Gradwell
Covance Laboratories Ltd., Harrogate, United Kingdom
Comparative assessments of mutagenic chemicals using regulatory compliant genotoxicity assay designs has always
been problematic, as these are designed to provide qualitative rather than quantitative assessments of mutagenicity.
This issue is particularly relevant when assessing the relative mutagenicity of pyrollised tobacco products (i.e. smoke
condensates), which are known to contain complex mixtures of mutagens. We have therefore investigated an Ames
study design using increased numbers of replicate treatments, aimed at achieving comparison between smoke condensates that would reliably detect a 30% difference in mutagenic responses. Here we describe the methodology, the
process of selecting appropriate replicate numbers and the evaluation criteria, and provide some preliminary data to
demonstrate the ability of this methodology to detect differences between smoke condensate mutagenic responses.
Using Ames data from responding strains with a typical/characteristic cigarette smoke condensate, calculations were
performed to estimate the number of replicates per dose that would be required to give 80% power to detect a 30%
difference in response at the 5% (two-sided) level of significance. Replicate plate numbers per dose of 12, 4 and 19
were required for Salmonella typhimurium strains TA98, TA100 and TA1537 respectively. Ames assay experimentation
was then performed using these replicate numbers at appropriate dose levels, selected as lying on or near to the linear part of the smoke condensate dose-response curve, with a reference smoke condensate both neat and diluted to
70% strength. The data from these increased replicate treatments was assessed at each dose level using a two-sided
t-test. In each strain the 70% strength smoke condensate showed significantly lower responses (p < 0.05) at most or
all dose levels, indicating that this methodology has sufficient power to detect the known 30% difference between the
condensates, for each of the strains tested.
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P-167
AUTOMATED MICRONUCLEUS DETECTION IN VIVO AND IN VITRO, DIFFERING PERSPECTIVES
Ann T Doherty
Julie E Hayes; Mike R O Donovan
AstraZeneca, Cheshire, United Kingdom
Automated micronucleus detection promises to be a useful tool for routine genotoxicology testing. We are evaluating
the use of the MetaSystems MetaCyte™ and MicroNuc™ programs for detecting micronuclei in both in vivo and in vitro
assays.
Work was conducted using bone marrow samples from both rats and mice dosed with either cyclophosphamide (72
μmol/kg rat and 233 μmol/kg mouse) or water control. Initial results with the MetaCyte™ spot counting program showed
that micronuclei signals were being masked by the bright fluorescent signals of the nucleated cells in the bone marrow. Therefore, the bone marrow was filtered to remove the nucleated cells (Romagna and Staniforth 1989). A significant induction of MIE was then detected using MetaCyte™ with both May-Gruenwald Giemsa stain and acridine orange
stain.
An in vitro micronucleus assay using rapidly dividing L5178Y cells has been developed as an early genotoxicity screen.
The cytokinesis block method was not used for L5178Y cells due to their continuous division with a cell cycle of 10 ˝
hours, therefore the preparations are mononuclear and not binucleate. To increase further the throughput of the assay
automation of micronuclei detection would be required.
Micronuclei were induced in L5178Y cells with 4NQO (0.001 mMol) and the classifier for the MicroNuc™ program modified
to detect mononuclear cells instead of binucleate cells. With this modified classifier we were able to detect significant
numbers of micronuclei in the 4 NQO treated cells.
Further work is in progress in conjunction with MetaSystems to develop and refine new classifiers specific to the cell
type characteristics that we are using in our micronucleus assays.
Reference:Romagna F and Staniforth CD. Mut Res 213 (1989) 91-104
P-168
CONTINUED EVALUATION OF THE LITRON XCELL MN KITTM FOR THE FLOW CYTOMETRIC
ENUMERATION OF MICRONUCLEI WITH L5178Y MOUSE LYMPHOMA CELLS. TREATMENT WITH
DEXAMETHASONE AND STAUROSPORINE.
Patricia Ellis 1
Robert Rees 1; Anthony Lynch 1; Joanne Collins 1; Antonia Booth 1; Claire Moore 1; James Harvey 1;
Svetlana Avlasevich 2; Steve Dertinger 2
Glaxosmithkline, Ware, United Kingdom1; Litron Laboratories, New York, United States2
The prototype XCell MN KitTM (Litron Laboratories) uses sequential staining to differentiate DNA fragments derived from
apoptotic/necrotic cells and true micronuclei, using flow cytometry. We have previously compared the performance of
this assay with conventional microscopy for direct (MMS) and indirect (DMBA) acting genotoxins, with promising results.
Here, we have extended our studies to include two apoptosis-inducers, dexamethasone and staurosporine.
L5178Y cells were treated for 3 hrs in the absence of S9-mix in replicate studies (n=3) with dexamethasone (250 to
400 µg/mL) and staurosporine (0.025 to 0.4 µg/mL). All treatment and MMS positive control cultures were set up in
triplicate, whilst four DMSO vehicle control cultures were included. All cultures were harvested after 20 hrs incubation and toxicity was assessed using relative survival (RS). Only cultures with 40% RS or above were selected for flow
cytometry or microscopic analysis.
Results with dexamethasone showed clear negative responses for both manual and flow cytometry analysis, with comparable % micronuclei (%MN) values and fold increases for both methodologies (all <1.7 fold cf DMSO control). However
data for staurosporine were inconsistent, and showed higher %MN and fold increases using flow cytometry compared
with manual analysis (up to 32.7 for flow cytometry and 2.3-fold with manual analysis). Cytotoxicity measurements,
using ethidium monoazide (EMA) and 7-amino actinomycin D (7-AAD) staining, showed comparable but not marked
decreases in cell viability following staurosporine treatment. The higher %MN values observed with flow cytometry may
reflect the maintenance of cell membrane integrity at cell harvest inhibiting apoptotic body staining with EMA. Therefore
following cell lysis, DNA fragments become incorrectly stained as MN. This hypothesis, together with any compound
specific effects, will require further investigation before the final assay acceptance criteria are reviewed.
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P-169
POSITIVES IN IN-VITRO-CLASTOGENICITY-TESTS AND THEIR BIOLOGICAL RELEVANCE.
ANALYSIS OF TESTS SUBMITTED WITH DRUG SUBSTANCES TO THE BFARM IN THE PERIOD
1998-2005.
Roland Froetschl
Peter Kasper
Federal Institute for Drugs and Medical Devices, Bonn, Germany
Mouse-Lymphoma-Test (MLA) and chromosome aberration test (CAb) in V79, CHO and CHL cells and human lymphocytes
(hL) are the current standards in in-vitro-clastogenicity testing of drug substances. These tests are knowingly prone to
frequent positive results with questionable biological relevance as already put into focus earlier (Müller & Kasper, Mut.
Res. 464 19-34, (2000), Snyder & Green Mut. Res. 488 151-169 (2001)). This analysis of in vitro clastogenicity tests
submitted to the BfArM during the years 1998 - 2005 updates these earlier data evaluations.
578 tests were submitted with 433 drug substances. 34% of all substances were positive in at least one in vitro clastogenicity test. Similar distribution of positive tests was found among all test models (MLA 32%, CAb-hL 23%, CAb-V79
27%, CAb-CHO 30%, CAb-CHL 33%). In contrast only 10% were positive in the AMES and 6% in the in vivo tests.
In most cases positives in in vitro clastogenicity tests were judged biologically irrelevant. 10% of AMES-test positives
were also positive in-vivo, which was similar to 11% in vivo positives of the in vitro clastogenic substances. For 42 in
vitro clastogenics additional in vivo studies were performed. In 40 cases these additionals were negative and only 2
positive.
This quite frequent positive results in standard in vitro clastogenicity tests and similar distribution of frequencies in all
in vitro clastogenicity models compared to the rest of the standard battery suggests that currently used test systems
for in vitro clastogenicity might be oversensitive. Most in vitro positives were judged biologically irrelevant and it seems
prudent to reevaluated the standard guideline protocols focusing on possibilities to eliminate at least some of the irrelevant positives.
P-170
STATISTICAL ANALYSIS OF MOUSE LYMPHOMA ASSAY DATA AND ITS CORRELATION
TO CLASTOGENICITY PREDICTION BY THE CHROMOSOMAL ABERRATION TEST
Kristin Gritzko
Michael Kraft; Gregory Rodrigo; Thomas Becker
BSL Bioservice Scientific Laboratories GmbH, Planegg (Munich), Germany
The Mouse Lymphoma Thymidine Kinase Gene Mutation Assay (MLA) using L5178Y cells is an established in vitro mammalian cell test for detecting the genotoxic activity of chemicals, pharmaceuticals and medical devices. Among the
determination of gene mutations in the tk-locus the MLA provides an indication of clastogenic effects of the test items.
In the Genotoxicity testing strategy the chromosomal aberration test (CA) is performed as an additional in vitro mammalian cell test to examine the potential of clastogenicity due to its higher sensitivity compared to the MLA. Therefore,
the CA verifies the obtained hints of clastogenic effects in the MLA. Here the results of test materials (n=46), which
were evaluated in the MLA and in most cases in the CA, too, during the last two years at BSL BIOSERVICE, Scientific
Laboratories GmbH, Planegg/Munich, Germany are presented. The results are discussed with respect to the nature as to
the mutagenicity and the clastogenicity of the test items. The hints for clastogenicity observed in the MLA were verified
with the results of the CA. Statistical analysis of the data is presented.
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P-171
THE AMES MPF 98/100 ASSAY: NOVEL MUTAGENICITY TESTING IN LIQUID MICROPLATE
FORMAT USING S. TYPHIMURIUM TA98 AND TA100
Sini Flückiger-Isler
Markus Kamber
Xenometrix, Allschwil, Switzerland
Genetic toxicity testing has moved towards the earlier stages of drug discovery in order to identify genotoxic liabilities of new compounds in the pipeline. Scaled-down versions of the original Ames plate incorporation test using the
S. typhimurium strains TA98 (frameshifts) and TA100 (base-pairs) are often used for this purpose. Because in early
development many compounds are available in very small quantities, a liquid microplate version with these strains was
developed to decrease compound consumption and to increase the through-put of the assay.
TA98 is already successfully used in the Ames II assay, in combination with TAMix, a mixture of strains to detect basepair mutations. TA100 with its high spontaneous reversion rate was as yet not suitable for the microplate format with
its 48-well limit.
We were able to decrease the spontaneous reversion rate of TA100 to a level low enough to be used in the microplate
format without loss of sensitivity. The mutagenic response to 13 reference compounds, examined in TA100 and in the
Ames II TAMix cultures, resulted in comparable results: Seven compounds were stronger mutagenic in TA100 than in
TAMix, and both strains showed similar responses with one compound. One compound had a weaker effect in TA100.
As expected, one compound was detected by TAMix only and three chemicals showed no mutagenic activity in both
strains.
The new Ames MPF 98/100 test by Xenometrix using a liquid format and 384-well microplates offers a time and costeffective pre-regulatory alternative to the plate incorporation method. As both assays use the same Salmonella strains,
TA98 and TA100, results can be compared with existing data sets. The new test kit including ready-to-use media and
bacteria enables rapid screening of a large number of compounds and consumes six times less test substances and
consumables than the plate incorporation method, and reduces hands-on time.
P-172
CLASTOGENICITY, PHOTO-CLASTOGENICITY OR PSEUDO-PHOTO-CLASTOGENICITY:
GENOTOXIC EFFECTS OF ZINC OXIDE IN THE DARK, IN PRE-IRRADIATED OR
SIMULTANEOUSLY-IRRADIATED CHINESE HAMSTER OVARY CELLS
David Kirkland1
Eric K Dufour2; Gerhard J Nohynek2; Hervé Toutain2; Tirukalikundram Kumaravel1
Covance Laboratories Ltd, Otley Road, Harrogate HG3 1PY, United Kingdom, Harrogate HG3 1PY, United Kingdom1; L'Oreal
Research & Development, Worldwide Safety Department, 92600 Asnieres, France, 92600 Asnieres, France2
Zinc oxide (ZnO), a widely used ingredient in dermatological preparations and sunscreens, is clastogenic in vitro. It is
slightly more clastogenic in the presence of UV than in the dark. In order to clarify whether this is a genuine photogenotoxic effect, we investigated the clastogenicity of ZnO (mean particle size: 100 nm) in CHO cells in the dark (D),
under pre-irradiation (PI, i.e. UV irradiation of cells followed 2-3 hr later by treatment with ZnO) and under simultaneous irradiation conditions (SI, i.e. ZnO treatment concurrent with UV irradiation). In terms of cytotoxicity, SI > PI > D.
As expected, ZnO produced a concentration-related increase in chromosome aberrations (CA) in the dark. Under both
PI and SI conditions, ZnO was clastogenic at significantly lower concentrations (approximately 2- to 4-fold) and produced higher CA frequencies than in the dark. SI exposures produced a higher incidence of CA than PI. However, the
incidences of CA at equitoxic exposures were nearly identical under PI and SI conditions. The modest increase in the
clastogenic potency of ZnO + UV contrasts with the results observed with genuine photo-clastogenic agents, such as
8-MOP, where clastogenic potency under SI exposure was >15,000-fold higher than in the dark or under PI conditions.
Our data suggest that minor increases in clastogenic response under standard conditions of photo-genotoxicity testing
do not necessarily represent an effect due to a photo-reactive pathway, but may be due to an increased sensitivity of
the test system subsequent to UV irradiation. Accordingly, the definition of in vitro photo-genotoxicity for substances
that are clastogenic in the dark requires urgent attention, particularly when taking into account the high rate of false
positive results in in vitro clastogenicity tests, as well as the absence of validated in vivo tests that could distinguish
genuine from pseudo-photo-clastogens.
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P-173
MUTAGENICITY OF THE AMBIENT AIR IN OSTRAVA
Jaromira Kusová
Hana Miturová; Hana Tomášková; Lubomír Dobiáš
Institute of Public Health, Ostrava
Introduction. Genetic toxicology testing is able to find substances that can damage DNA and chromosomes of cells.
These processes could result to the initiation and progression of cancer. Real complex mixtures from environment
include except from measured contaminants a lot of unknown chemical components. The interactions among all components are complicated not only on chemical, but even more on the biological level. So the testing of biological effect
is an important moment for environmental safety. The most widely used test to identify mutagenic/ genotoxic effect of
chemical or their complex mixtures is Salmonella (Ames) test.
Methods. Urban air samples taken in Ostrava were tested in Ames test and concurrently chemical analysis of hygienically relevant carcinogenic and mutagenic compounds was carried out. The results of chemical analysis (benzene,
trichlorethene, arsenic, nickel, cadmium, selected polycyclic aromatic hydrocarbons – PAHs – including benzo/a/pyrene,
particulate matter PM10) and results of Ames test were compared. Association between pollutants and between their
presence in air samples and results of Ames test were analyzed by correlation analysis.
Results
Significant correlations were confirmed for the presence of some compounds being measured in urban air: benzo/a/
pyrene and PAHs (r=0.95); arsenic, nickel and PM10 (r=0.77). Correlations for observed carcinogenic or mutagenic
substance and biological (genotoxic) effect of air samples in Ames test were weaker.
Discussion and Conclusions
Results of the study confirmed that interactions among components of complex mixture with a number of potentially
genotoxic substances are complicated. The closest correlation were found for biological effect and PAHs.
P-174
GENOTOXICOLOGICAL STUDY AIMED AT ANTIMUTAGENIC/ANTICARCINOGENIC
AND BIOMODULATORY EFFECTS OF EXTRACT FROM ARTICHOKE CYNARA CARDUNCULUS L.
EVALUATION
Slavomíra Naďová 1
Eva Miadoková 1; Eva Miadoková; Viera Vlčková 1; Viola Dúhová 1; Mária Trebatická 1; Ján Grolmus 1;
Peter Rauko 2; Ľuboš Čipák 2; Pavel Mučaji 3; Daniel Grančai 3
Comenius University, Faculty of Natural Sciences, Department of Genetics, Bratislava, Slovakia1; Institute of Experimental
Oncology, SAV, Bratislava, Slovakia2; Comenius University, Faculty of Pharmacy, Department of Pharmacognosy and Botany,
Bratislava, Slovakia3
The potential antimutagenic/anticarcinogenic and biomodulatory effects of the plant extract from artichoke Cynara
cardunculus L. involucral bracts, rich mainly on flavones apigenin (4´,5,7-trihydroxyflavone) and luteolin (3´,4´,5,7tetrahydroxyflavone) and their derivatives glycosides apigenin-7-glucoside and luteolin-7-glucoside were evaluated on
five genetic model systems.
The extract was applied together with diagnostic mutagens in the Ames assay in the presence and/or absence of metabolic activation on four bacterial Salmonella typhimurium strains TA97, TA98, TA100 and TA102; in the toxicity and
antimutagenicity assay on the yeast strain Saccharomyces cerevisiae D7; in the simultaneous phytotoxicity and anticlastogenicity assay on a plant species Vicia sativa; in the sex-linked recessive lethal test in Drosophila melanogaster.
Because of its potential biomodulatory effect we performed also the cell revitalization assay, in which we evaluated the
incidence of the artichoke extract on the murine leukemia L1210 cells affected by commercial cytotoxic/cytostatic drug
cisplatin (platidium).
Depending on the experimental model system and used genotoxic agent, the extract exerted versatile activities. It
exhibited antimutagenic, but also comutagenic or anticlastogenic effect. Moreover, in the cell revitalization assay was
revealed its cytotoxic/cytostatic enhancing potential.
The issue of changing the traditional cytostatic therapy by combining conventional chemotherapeutics with the defined
natural compounds may result in enhancing cytostatic therapeutic effects of cytostatics. Results obtained in the cell
revitalization assay are promising for more efficient anticancer therapy.
This work was supported by grant APVT-20-002604 and VEGA grants 1/2337/05, 2/4053/24, 2/4143/04, 1/1311/04.
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P-175
THE DETECTION OF IN VIVO MICRONUCLEI BY FLOW CYTOMETRY AND MICROSCOPIC
ANALYSIS IN PERIPHERAL BLOOD
Ingrid Pontén 1
Eva-Lena Härnwall 1; George Bolcsfoldi 1; Mike O'Donovan 2; Ann Doherty 2; Julie Hayes 2; Jo Cunliffe 2;
Jan Grawé 3
AstraZeneca R&D Södertälje, Safety Assessment Sweden, Södertälje, Sweden1; AstraZeneca R&D Alderley Park, Safety Assessment
UK, Mereside, United Kingdom2; Cellanalyslab, Rudbecklaboratoriet/C5, Uppsala Universitet, Uppsala, Sweden3
A flow cytometric method for detection of micronuclei induced with cyclophosphamide in rat peripheral blood was
investigated. Flow cytometry data were compared with levels obtained in the conventional bone marrow micronucleus
test and with peripheral blood smears scored microscopically. The study was performed on two sites, each using the
flow cytometric method based on Abramsson- Zetterberg et al 1999, but with different in equipment (triple-laser
FACSVantage SE and a triple laser LSR, at SAS and SAUK respectively).
Cyclophosphamide (72, 23, 7.2 μmol/kg) and vehicle (water) were dosed to groups of 3 male Wistar Han rats and bone
marrow sampled from the 23 and 7.2 μmol/kg groups after 24 hours. Peripheral blood was sampled from all groups
pre-dose, 24, 48, 72 and 96 hours after dosing and blood smears prepared for microscopic analysis using supravital
staining, Proudlock et al 1997. Replicate blood samples were analysed by flow cytometry.
Results from the 23 and 7.2 μmol/kg groups indicated a significant induction of micronuclei in bone marrow, with a
three fold increase above control at 7.2 μmol/kg. No bone marrow data were generated concurrently at 72 μmol/kg as
extensive historical data existed in each laboratory, therefore historical control was used.
Flow cytometric and microscopic analysis detected significant levels of micronuclei in immature erythrocytes in the
peripheral blood after 48 hours at 72 and 23 μmol/kg but not at 7.2 μmol/kg.
Micronuclei detection showed lower sensitivity in peripheral blood than in bone marrow. Results from testing new staining parameters and sampling time points (24, 30 and 48 hours) to increase the sensitivity for the low dose, will be
presented.
References:Abramsson-Zetterberg et al. Mutation Research 1999; 423:113-124.
Proudlock et al. Mutation Res. 392 (1997) 243-249.
P-176
THE NEWLY DEFINED ACCEPTANCE AND EVALUATION CRITERIA FOR THE MOUSE LYMPHOMA
ASSAY AND THEIR IMPACT AND RELEVANCE
Albrecht Poth
Hans-Eric Wollny; Susanne Kunz; Wolfgang Völkner
RCC Cytotest Cell Research GmbH, Rossdorf, Germany
In the present study historical data from 313 mouse lymphoma studies (microwell method) performed in the years
2003-2005 at RCC were investigated in respect to the newly acceptance and evaluation criteria as defined in the workshop reports of the IWGT working group. First the defined acceptable background mutant frequencies for negative/vehicle (50-170 x 10-6) were investigated for these studies. In 23 studies either the vehicle control or the solvent control
were out of the defined range where 19 studies were beyond and 4 studies below the range. Secondly our data were
investigated in respect to the newly defined acceptance criteria for the positive controls (GEF, IMF300) and compared
to our previously used criteria (mutation factor of 2.0). MMS, used at RCC as a positive control without S9 mix in the
pulse and in the prolonged treatment, indicate that in 94 studies at least one of the newly defined criteria could not be
fulfilled, even if the mutation factor of 2 was reached or exceeded.
A further investigation includes test items which were classified as genotoxic by the mutation factor approach compared
to the newly defined evaluation criteria for the definition of a positive effect. In 37 out of 126 studies (29%) performed
in 2005 at RCC, a positive result was obtained. 20 studies showed a positive response at pulse treatment, 17 at the
prolonged treatment. In 4 studies the global evaluation factor 126 could not be reached even if the mutation factor of
2.0 was reached or exceeded (from 2.0 to 3.0).
The current presentation will discuss the impact and the relevance of the newly defined acceptance and evaluation
criteria for the mouse lymphoma test based on RCC´s historical data and eventually a re-consideration of certain definitions.
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P-177
AUTOMATION OF SCORING PROCEDURE BY FLOW CYTOMETRY FOR THE IN VITRO
MICRONUCLEUS TEST.
Gregory Rodrigo
Margit Oppong; Thomas Becker
BSL Bioservice Scientific Laboratories GmbH, Planegg (Munich), Germany
Cytogenetic assays in vitro allow evaluating potential clastogenic or aneugenic effects caused by physical or chemical
factors, directly by observing and classifying chromosomal aberrations (OECD 473) or with an alternative approach by
quantifying micronuclei (Draft OECD 487). Micronuclei induction in cells is a sensitive and specific parameter for assessing the genotoxic potential of a test compound. Compared to the chromosome aberrations test, the micronucleus test is
easier and can be evaluated more objectively. However, the manual scoring of each slide is a monotonous, exasperating
and time consuming procedure. Automated systems have been developed but many of them are based on automatic
slides analysis coupled to an image processing software system. At BSL Bioservice Scientific Laboratories GmbH, an
automated method using a flow cytometric technique has been adapted and improved to perform genotoxicity screening. Here, a comparison between the FACS® automatic analysis and the manual scoring of acridine orange (AO) stained
slides, based on the cytokinesis block micronucleus assay (CBMN), is presented. In spite of variations of sensitivity,
a high degree of concordance is observed between the results obtained by the two methods. Moreover, flow analysis
permits the scoring of samples faster, within about 1 to 5 minutes per sample. Therefore, this automated processing is
a powerful tool for the assessment of genotoxic potential of drugs by the in vitro micronucleus assay.
P-178
EVALUATION OF AN AUTOMATED SCORING SYSTEM (CELLOMICS) FOR THE IN VITRO
MICRONUCLEUS ASSAY IN CHO-K1 CELLS
Andrew Scott 1
Paul Carmichael 1; Sophie Malcomber 1; Sharon Maskell 1; Claire Moore 1; Sam Windebank 1; Dolores
Diaz 2
Safety & Environmental Assurance Centre, Unilever, Sharnbrook, Bedfordshire, MK44 1LQ, United Kingdom1; Cerep Inc., 15318 NE
95th Street, Redmond, Washington 98052, United States2
The in vitro micronucleus (IVM) assay is a standard genotoxicity assay that is used to assess the potential for compounds to induce chromosome damage in cultured mammalian cells. As with other tests for chromosome damage, the
IVM assay uses gram quantities of compound and significant amounts of personnel and time resources. Therefore, the
application of automated technologies to this assay is desirable. Traditionally, micronucleus detection in the IVM assay
is undertaken by manual scoring methods. Automation of this phase of the assay has the potential to reduce the time
required to generate data. The Cellomics Micronucleus bioapplication software allows for the automated and rapid
quantification of binucleated cells and micronuclei required for the assay. Additionally, the Cellomics-based IVM assay is
performed using 96-well plate format and as such has dramatically reduced compound requirements (milligram quantities of material). The aim of this study was to evaluate the performance of the Cellomics-based IVM assay compared
to results generated using manual scoring methods for a diverse set of 30 compounds.
We have conducted an evaluation study at Cerep Inc. with a range of 21 mutagenic and 9 non-mutagenic compounds
with varying modes of action (results determined from studies in which standard micronucleus scoring methods were
used). All compounds were coded by Unilever before being sent to Cerep for genotoxicity assessment. Overall, the concordance of the Cellomics-based IVM assay with the IVM assay using manual scoring methods was 83.3% (specificity
was 88.9% and sensitivity 80.1%).
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P-179
THE IMPORTANCE OF PREMATURE CENTROMERE DIVISION IN ASSESSING GENTOTOXIC RISK
OF CYTOSTATICS
Vladan Peter Bajic 1
Biljana Spremo Potparevic 2; Lada Gordana Zivkovic 2; Ninoslav Djelic Djelic 2; Zorka Milicevic Milicevic 3
Institute for Biomedical Research, Galenika pharmaceuticals, Belgrade, Serbia and Montenegro1; Department of Biology, Faculty of
Pharmacy, Belgrade, Serbia and Montenegro2; Institute for Nuclear Research Vinca, Lab.mol.biol.endocrinology, Belgrade, Serbia
and Montenegro3
The assessment of the genotoxic risk of antitumor agents shows specificity to other chemical agents that have mutagenic and/or genotoxic potential, i.e., antitumor agents can induce de novo tumors (second tumors) in patients that
have been cured of a primary tumor after chemotherapy. The incidence of premature centromere division in chromosome instability syndromes and various tumors have suggested that premature centromere division (PCD) is a primary,
yet an unspecific, manifestation of genomic instability that can be induced by xenobiotics.
The main goal of our research was to establish if PCD induced by cycloheximide (genomic instability) could predispose
human peripheral lymphocytes to an increased rate of chromosomal aberrations and micronucleus induction in peripheral blood lymphocytes exposed to increasing doses of the investigated substances 8-Cl-cAMP, Mitomycin C and Taxol.
Comparing the differences between experimental groups with and without the induction of PCD we have seen that there
is a increase of chromosomal aberrations in the experimental group with the induction of PCD, exposed to 8-Cl-cAMP in
a dose of 15µM, Taxol in a dose 0,2µM and Mytomicin C in a dose of 0,6 µM. Results from the micronucleus test hasn’t
shown any statistical changes in micronuclei frequencies between experimental groups with and without the induction
of PCD.
Our results show that PCD per se is a manifestation of latent genomic instability and can influence a change (increase)
in the frequency of chromosomal aberrations by substances that destabilize microtubule organization of the spindle
(Taxol, 8-Cl-cAMP) and clastogenic agents (Mitomycin C) in peripheral blood lymphocytes.
On the basis of the performed investigation we can conclude that PCD should be included as a parameter of genomic
instability for an adequate genotoxic risk assessment of cytostatics.
P-180
INDUCTION OF LACZ MUTATIONS IN MUTAMOUSE CAN DISTINGUISH CARCINOGENIC FROM
NON-CARCINOGENIC ANALOGUES OF DIAMINOTOLUENES AND NITRONAPHTHALENES
Carol Beevers
Mark Ballantyne; David Kirkland
Covance Laboratories Ltd, Harrogate, United Kingdom
2,4-diaminotoluene (2,4-DAT) is a liver carcinogen in rats and mice whereas 2,6-DAT is not. Both are genotoxic in
vitro. Tests for mutations in transgenic mice, UDS, DNA damage and enhancement of initiated foci in vivo have shown
some discrimination between these 2 analogues, but only after oral administration. 1- and 2-nitronaphthalene (1- and
2-NNT) are also both genotoxic in vitro, although, unlike 2,4- and 2,6-DAT, do not require metabolic activation. There
is some evidence the 2-NNT may be able to induce liver and bladder tumours, and there is some evidence that 1-NNT
is not carcinogenic to rats or mice, but none of the data are convincing. When tested for induction of LacZ mutations
in Muta™Mouse after topical exposure (human occupational exposure route) at their maximum tolerated doses, 2,4DAT induced a positive response in liver and a marginal response in kidney, whereas 2,6-DAT was negative. 2-NNT
also induced a positive mutagenic response in liver, and a marginal response in bladder, whereas 1-NNT was negative.
Neither 2,4- nor 2,6-DAT induced mutations at the site of application (skin) as might be expected for chemicals requiring activation by liver enzymes. 2-NNT, which is a direct acting mutagen in vitro, gave a marginal response for induced
mutation at the site of application but 1-NNT was negative. This study shows that investigation of induction of LacZ
mutations after topical application in vivo can provide useful data to help discriminate potentially carcinogenic from
non-carcinogenic chemicals that are mutagenic in vitro. Robust carcinogenicity data are needed to determine whether
2-NNT can induce tumours in the liver and bladder.
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P-181
SMOKING AND CANCER RELATIONSHIP
Liana Monica Deac 1
Dana Coza 1; Fl. Nicula 2
“Professor Dr. Iuliu Moldovan“ Institute of Public Health Cluj-Napoca, Romania1; “Professor Dr. I. Chiricuta“ Oncological Institute
Cluj-Napoca, Romania2
INTRODUCTION:According to data from WHO, the incidence for Lifetime Risk for prematured death for smokers is 1 in
15 males and 1 in 41 females . 30% of cancer mortality belongs to the smoking habit.The incidence for cancer in 2004
in Romania was 287 new cases which indicates a 30 % increase of incidence compared to 5 years ago. Therefore, a
follow-up survey was conducted.
MATERIAL AND METHOD: A questionnaire survey of smokers with cancer was conducted in several surgical units and in
the Oncological Institute in Cluj, Romania. The survey was performed from 2004-2006.
RESULTS AND ANALYSIS: From the 850 surveys, 635 (75%) were smokers and 215 were passive smokers. Among the
smokers, 530 (84%) smoked cigarettes and 105 (16%) smoked pipes or cigars. From the cigarettes smokers : 200
smoked high tar cigarettes and 10-20 cigarettes per day for about 5 years; 250 smoked a pack of low tar cigaretts per
day over 5 years and 80 smoked more then 1 pack of high tar cigarettes per day over 10 years. The age and gender of
cancer cases were: 20-40 years old with 110 males and 40 females; 40-60 years old with 250 males and 120 females;
> 60 years old with 230 males and 70 females. 28 % of cancers were lung and broncho-pulmonary and from these
around 50% are from high tar cigarettes smokers. 25 % are from passive smokers with a variety of cancers.
CONCLUSIONS: Smoking represented a significant risk factor for cancer, especially in the group of middle aged persons
(29,4%). The data also indicate that the passiv smokers have much higher risk for cancer than expected. Their concurrent exposure to enviromental mutagenes may be a contributing factor.
Key words: cancer, smoker, passive smoker, risk factors
P-182
GENOTOXICITY AND HEAVY METALS ASSESSMENT IN MARINE SEDIMENTS OF FOLLONICA
GULF (THYRRENIAN SEA, CENTRAL ITALY)
Stefania Frassinetti 1
Marco Cini 1; Clara Della Croce 1; Leonardo Caltavuturo 1; Marco Carlo Mascherpa 1;
Leonardo Lampugnani 2
Institute of Biology and Agricultural Biotechnology (IBBA) NRC, Pisa, Italy1; Institute of Chemical and Physical Processes (IPCF)
Research Area of Pisa, Italy2
Marine sediments represent a sink for anthropogenic contaminants. Heavy metals in marine sediments can affect the
ecosystem through bioaccumulation processes and are potentially toxic for environment and human health.The aim of
this study was to evaluate the quality of waters from the Gulf of Follonica, an important area of the Central Thyrrenian
Sea, by two different experimental procedures. Sediments were collected at five sites. Among these, four sites belong
to polluted areas of agricultural or industrial origin and the last site to uncontaminated coastal area. Samples sediments
were tested for genotoxicity employing short-term assay in yeast cells of Saccharomyces cerevisiae. Cellular survival,
mitotic gene conversion and point reverse mutation were measured.
As, Cd, Cr, Cu, Ni, Pb concentrations were determined by atomic absorption spectroscopy (ETAAS). Results demonstrated cytotoxic effects in three out of five sites examined. Mutagenic effects were also found in two sites. Chemical
analyses showed that heavy metal contamination reaches high levels in four sites. Particularly, Cr appears to be potentially the most dangerous pollutant, with concentrations three times higher than limit values. These results suggest an
anthropogenic contamination probably of industrial origin. Cd concentrations, even if below the approved limit values,
were generally higher than the background ones, suggesting an agricultural pollution. Considering that several heavy
metals are known to be mutagenic or carcinogenic compounds, the metal contamination may also explain some of
evidenced genotoxic results.
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P-183
ASSESSMENT OF DNA DAMAGE IN WHITE BLOOD CELLS OF HEALTH HUMAN DONORS USING
THE ALKALINE COMET ASSAY AND CHROMOSOME ABERRATION ANALYSIS
Verica Garaj-Vrhovac
Nevenka Kopjar; Snježana Ramić; Davor Želježić
Assessment of normal levels of DNA damage in the general population is essential for the proper interpretation of data
obtained by monitoring of populations occupationally or accidentally exposed to known or potentially genotoxic agents.
The present study was undertaken to investigate how the variability in baseline damage in white blood cells from healthy
human donors is associated with external and internal factors. Altogether 114 healthy donors, randomly selected from
the general population of the Republic of Croatia, participated in the study. Two sensitive biomarkers: the alkaline comet
assay and the chromosome aberration (CA) test were applied. The results point to inter-individual differences, indicating
different genome sensitivity. As revealed by both assays, smoking habit mostly influenced the background levels of DNA
damage. Gender and age did not significantly contribute to the pattern of DNA migration in white blood cells. Although
the highest levels of primary DNA damage were recorded in blood samples collected in autumn, smoking status mostly
influenced them. The results of chromosome aberration analysis also confirmed that smoking significantly influences the
total number as well as the percentage of CA recorded in lymphocytes. Age was found to influence the total number of
acentric fragments in subjects aged between 40 and 49 years. Significantly increased incidence of chromatid breaks,
total number of CA and the percentage of aberrant cells were recorded in blood samples taken in autumn, but they
were mostly influenced by smoking habit. Statistical evaluation of the data confirmed that a positive correlation exists
between DNA migration and the number of long-tailed nuclei found with the comet assay and the total number of chromosome aberrations. The data obtained can serve as control values in forthcoming biomonitoring studies.
P-184
INDIVIDUAL GENETIC VARIABILITY IN BIOTRANSFORMATION ENZYMES CAN MODULATE
RISK FOR EXPOSED PEOPLE
Alexandra Horská
Jana Tulinská; Ladislava Wsólová; Mária Dušinská
Introduction: Inhaled asbestos dust can persist in human lung tissue and start a cascade of processes potentially
resulting in inflammation, fibrosis or carcinogenesis. These alterations are due to the mechanic damage of lung cells
or/and due to the bioactivation of asbestos fibers in cells, however, the detailed mechanism is not fully discovered yet.
Individual response to environmental agents such as asbestos can be influenced by biotransformation/detoxification
capability of organism determined by DNA sequence variations in genes for metabolizing enzymes.
Methods: We performed a molecular epidemiological study in asbestos cement factory. Altogether 130 subjects were
investigated: 61 long term exposed workers, 49 town-controls and 21 control subjects working in administration of the
same factory. Participants were interviewed by detailed questionnaires and all workers underwent clinical examination.
Venous blood was used to measure biomarkers of exposure, effect and individual susceptibility. Genetic polymorphisms
of biotransformation enzymes (GSTM1/T1/P1, EPHX1 and NQO1) were analysed.
Results: Asbestosis has developed in 44.3% of workers. Fibrotic plaques have appeared in 55.6% of subjects with
asbestosis. Interstitial fibrosis was found in 74.1% of subjects with asbestosis. 33.3% of subjects were carriers of both
attributes of the disease. Frequency of GSTP1*b allele was significantly decreased in the group of workers with asbestosis (p=0.044). Frequency of low activity mEH genotype was decreased in workers with fibrotic plaques (p=0.025) and
in workers with at least one attribute of asbestosis (p=0.014). Distribution of genotypes in group of workers with and
without developed asbestosis was not influenced by smoking habits.
Conclusions: We suppose that polymorphisms responsible for biotransformation may modify individual risk for asbestos
exposed people.
Supported by EC contracts QLK4-CT-1999-01629
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P-185
TRANSPLACENTAL GENOTOXICITY AND HEALTH RISK ASSESSMENT OF PROCHLORAZ
AND TRICHLORFON
Su-Yin Chiang 1
Kuen-Yuh Wu 2; GP Chang-Chien 3; Ching-Tang Kuo 4; Hsiu-Ching Wu 5
Graduate Institute of Chinese Medical Science/China Medical University, Taichung, Taiwan 1; Institute of Environmental Health and
Occupational Medicine/National Health research Institute, Zhunan, Taiwan 2; Super Micro Mass research and technology center/
Cheng-Shiu University, Kaohsiung, Taiwan3; Graduate Institute of Environmental Medicine/China Medical University, Taichung,
Taiwan4; School of Post Baccalaureate Chinese Medicine/China Medical University, Taichung, Taiwan5
Content: Pesticides will increase crop productivity and improve crop quality, however, the use of pesticides possess
potential health risk to human. Prochloraz is a commonly used imidazole fungicide. Trichlorfon, an organophosphate
pesticide, is one of the most frequently used insecticides. In this study, we conduct health risk assessment for Taiwan
consumers to residues of pesticides, prochloraz and t richlorfon. We first examined the potential genotoxic effects of
prochloraz and trichlorfon in pregnant ICR mice and their fetuses using Micronuclei assay. The pregnant mice were
given by intraperitoneal injection (i.p.) or gavage with prochloraz (at highest dose of 200mg/kg, ip) or trichlorfon (at
highest dose of 150mg/kg, ip; 250mg/kg, gavage) from gestation day 7 through 17 every other day. Treatment of
prochloraz and trichlorfon did not induce a statistically significantly increase in frequencies of micronuclei, a marker for
chromosome damage, in maternal and fetal reticulocytes. The toxicological profiles for prochloraz and trichlorfon were
compiled and updated from articles in TOXNET, IPCS, EXTOXNET, WHO, EPA website, etc. Based on the Taiwan food
consumption data, the highest Pesticide residue! concentrations and the current regulatory limits in crops, fruits and
vegetables provided by Council of Agriculture in Taiwan, the hazard indexes (HI) for prochloraz and trichlorfon for customers were calculated to be both lower than one. Using Monte Carlo Simulation, the upp! er 95% confidence limits for
HI for prochloraz and trichlorfon were also less than one. Whereas, cancer risks of prochloraz for the current standards
and residue concentrations were greater than 1 x 10-6 except for root vegetables. The results suggest that the current
regulatory standards for prochloraz should be lowered, and those for trichlorfon are adequate to protect the public
health. These results can serve as scientific tool for Taiwan government to make scientific sound public policy.
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P-187
EFFECTS OF ANIMAL AND PLANT METABOLISM IN THE MUTAGENICITY INDUCED
BY ORGANOPHOSPHORUS INSECTICIDES IN SALMONELLA TYPHIMURIUM
Sandra Gómez-Arroyo 1
Liliana Sánchez-Estrada 1; Josefina Cortés-Eslava 1; Rafael Villalobos-Pietrini 1; Concepción
Moreno-Zenteno 1; Saúl Flores-Maya 2
Centro de Ciencias de la Atmosfera / Universidad Nacional Autonoma de Mexico, Mexico, Mexico1; Facultad de Estudios
Superiores-Iztacala / Universidad Nacional Autonoma de Mexico, Mexico, Mexico2
The organophosphorus insecticides are a very serious source of toxic substances for humans due to the widespread
application mainly in Mexican agriculture. Besides, these compounds can be activated by animal and plant metabolism
and produce mutagens. To have a general idea of the genetic risk involved, one can use several biological test systems.
Thus, the aim of this study was to evaluate the metabolic ability of both the S9 animal fraction and the S10 Vicia faba
fraction by the promutagen activation of the organophosphorus insecticides – methamidophos, methyl azinphos, omethoate and methyl parathion – using the Ames reversion mutagenicity assay with Salmonella typhimurium TA98. The
assay was carried out with and without metabolic activation by adding to bacteria, mixtures of S9 mammalian microsomal enzymes or Vicia faba S10 mixtures and several concentrations of each insecticide. Methamidophos, omethoate
and methyl parathion did not induce direct acting mutagenesis or indirect-acting mutagenesis when S9 or S10 mixtures
were added. Of the three, methyl parathion had great toxicity. Methyl azinphos did not have direct or indirect mutagenic
activity with S9; however when S10 was added, the revertants increased in relation to the concentration. This means
it is a promutagen activated by the plant metabolism to induce frameshift mutations characteristic of the TA98 strain.
The positive controls 2-aminofluorene and 4-nitro-o-phenylenediamine increased mutagenicity significantly when the
enzymatic mixtures S9 and S10 were added, respectively.
Acknowledgment to PAPIIT (Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica, UNAM) project
No. IN227305.
P-188
A FULLY AUTOMATED HIGH-CONTENT-SCREENING IMAGING SYSTEM FOR COMET ASSAY
Jérôme Sallette 1
Françoise Soussaline 1; Alexandre Papine 1; Jean-Philippe Belaidi 2; Laurent Marrot 2; Jean-Roch Meunier 2
IMSTAR S.A., PARIS, France1; Safety Department, Life Science Research, L'Oréal, Aulnay sous Bois, France2
COMET assay is an increasingly important test in genotoxicity testing as well as biomonitoring and basic research (Tice
et al., 2000; Collins, 2004; Moller and Loft, 2004) Regulation in Europe is about to make it compulsory for large-scale
screening of chemical compounds. Yet, conventional methods for COMET assay screening are essentially manual or
semi-automated, thus limiting screening to small numbers of comets (generally, 50 to 100 nuclei). IMSTAR S.A., a Parisbased high technology company, has developed a unique technology leading to a robust, fully automated High Content
Screening solution (instrument and software). This novel concept enables studies on large numbers of comets, typically
hundreds to thousands, without any human intervention.
This technique has been validated using different biological samples (immortalized cell line, human keratinocytes from
primary cultures or reconstructed skin), submitted to various kinds of genotoxic stress, such as H2O2 or UVA, UVB.
Our results confirm that this new automated system optimizes accuracy, reproducibility, versatility and ease of use for
systematic, fully automated analysis of COMET assays. Moreover, such an approach will be of particular interest for the
development of methods in genotoxicity as an alternative to animal testing.
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P-189
THE CYTOTOXIC EFFECT OF BEE VENOM ON HUMAN LYMPHOCYTES
Martina Djurinec
Bee venom is a very complex mixture of active peptides, enzymes, and amines which can cause proliferation and death
of human lymphocytes. Literature data have shown that certain concentrations stimulate proliferation or necrosis. The
aim of this study was to investigate the cytotoxic effect of bee venom on peripheral blood lymphocytes, depending on
different concentrations and time of exposure.
Trypan-blue dying was used to distinguish between cells which enter necrosis and viabile cells.
Cells were treated with 0.71, 1.42, 2.85, 5 and 10 μg/ml concentrations of bee venom for 30, 60 and 120 min.
In the first? 30 min of treatment the cell survival for all concentrations ranged from 90% to 100%; in 60 min of treatment the cell survival ranged from 77% to 88% and in 120 min it ranged from 69% to 75%.
Control samples yielded 100 % cell survival for the 30 min treatment, 80% for the 60 min treatment and 75% for
the 120 min treatment. No significant differences were observed between the control and treated samples.
As bee venom might inhibit tumor growth by different mechanisms, these preliminary results of the cytotoxic effects
could be used for further investigation.
P-191
THE MOUSE SPLENOCYTE - A POTENTIAL TOOL FOR DETERMINING THRESHOLDS FOR
ANEUGENIC RESPONSES
Guy Steiblen 1
Catherine Pallen 1; Thierry Orsičre 2; Alain Botta 3,2; Daniel Marzin 3
Bayer CropScience (Sophia Antipolis), Sophia Antipolis, France1; Medical school of Marseille, Marseille, France2; Institut Pasteur de
Lille, Lille, France3
As aneugenic compounds act according to an indirect mechanism of genotoxicity, it’s been suggested that these compounds exhibit a threshold response. We have developed an in vitro cytokinesis-blocked micronucleus (CBMN) test
using cells that would allow the characterization of aneugenic compounds and a direct comparison of in vitro and in
vivo threshold calculations. We evaluated the use of mouse splenocytes (MS) as a suitable alternative to the human
lymphocyte (HL) for the in vitro identification of aneugens, as both cell types belong to the immune system. In addition, MS are highly exposed to compounds across the blood stream and are easy to obtain in large quantities. First, we
standardized the mouse and human in vitro CBMN protocols. Second, we compared the sensitivity of MS and HL towards
two aneugens, paclitaxel and nocodazole, as concerns their threshold responses. The MS appeared to be slightly more
sensitive to paclitaxel than the HL, while with nocodazole, the sensitivity was similar for both cell types. Both compounds
generated mono and poly-micronucleated cells in mono or bi-nucleated MS and HL. These results show that the MS
may be a suitable alternative to the HL for in vitro aneugen identification. Further comparisons between these cell types
are needed using others aneugenic compounds. Comparison of thresholds obtained in vivo in the MS versus the in vivo
micronucleus assay in mouse bone marrow should then be performed to assess the suitability of the MS for aneugenic
risk assessment and threshold calculation.
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P-192
INDUCTION OF CYP1A1 MRNA AND PROTEIN EXPRESSION BY THE ANTICANCER DRUG
ELLIPTICINE
Dagmar Aimova 1
Marie Stiborova 1; Pavel Soucek 2; Eva Frei 3
Charles University in Prague, Prague, Czech Republic1; National Institute of Public Health, Prague, Czech Republic2; German Cancer
Introduction: Ellipticine is a potent antineoplastic agent used in the therapy of several cancers and has multiple cellular targets. Among these, the inhibition of topoisomerase II after intercalation into DNA was considered the most
important property for its toxicity. We found that ellipticine also forms covalent DNA adducts in vitro and in vivo and
that the formation of adducts is dependent on ellipticine activation by cytochromes P450 (CYP) and peroxidases. 13Hydroxyellipticine formed by CYPs of 3A and 1A subfamilies is responsible for formation of the major DNA adduct.
Expression levels of these enzymes are, therefore, crucial for ellipticine pharmacological efficiencies. Here, we evaluate
the potential of ellipticine to influence the expression of CYP1A1/2 in rats, the animals found to be suitable to mimic
the fate of ellipticine in humans.
Results and discussion: The expression of hepatic CYP1A1/2 proteins in rats of both sexes treated with ellipticine is
strongly induced by ellipticine. The expression levels of these enzymes in treated rats are more than one order of magnitude higher than those in control animals. An increase in CYP1A1/2 protein expression correlates with an increased
EROD activity, a marker for CYP1A1/2, and with the oxidation of Sudan I, a marker for the CYP1A1 activity. The increase
in expression of CYP1A1 protein and its enzymatic activity caused by ellipticine corresponded to elevated CYP1A1 mRNA
levels. However, levels of CYP1A2 mRNA were unaffected. The CYP1A1/2 protein induction is strongly dependent on the
dose of ellipticine administrated to the rats and it is transient. The amount and the activity of the induced CYP1A1/2
decreased to the basal level in two weeks after exposure. The results indicate that a long treatment of humans with
ellipticine might stimulate its pharmacological efficiency against cancer diseases, if the CYP induction also occurs in the
target organs of therapy.
GACR 203/06/0329; ME CR 1M4635608802, MSM0021620808
P-193
THE ENVIRONMENTAL POLLUTANT AND CARCINOGEN 3 NITROBENZANTHRONE AND ITS
HUMAN METABOLITE 3 AMINOBENZANTHRONE ARE POTENT INDUCERS OF RAT HEPATIC
CYTOCHROMES P450 1A1/2 AND NQO1
Helena Dracinska 1
Marie Stiborová 1; Volker M. Arlt 2; David H. Phillips 2; Eva Frei 3; Heinz H. Schmeiser 3
Charles University in Prague, Prague, Czech Republic1; Institute of Cancer Research, Sutton, United Kingdom2; German Cancer
Introduction: 3-Nitrobenzanthrone (3-NBA) is a suspected human carcinogen occurring in diesel exhaust and air pollution. Its reductive counterpart, 3-aminobenzanthrone (3-ABA), has been found in urine samples of salt mine workers
occupationally exposed to diesel emissions. Both these compounds were investigated for their ability to induce biotransformation enzymes in rat liver and the influence of such induction on DNA adduct formation.
Results and discussion: Rats were treated (i.p.) with 0.4, 4 or 40 mg/kg body weight of 3-NBA or 3-ABA. When hepatic
cytosolic fractions from rats treated with 40 mg/kg body weight of 3-NBA or 3-ABA were incubated with 3-NBA, DNA
adduct formation, measured by 32P-postlabelling analysis, was 10-fold higher in incubations with cytosols from pretreated rats than with controls. The increase in 3-NBA-derived DNA adduct formation corresponded to a dose-dependent
increase in protein levels and enzymatic activity of NAD(P)H:quinone oxidoreductase (NQO1). NQO1 is the major
enzyme reducing 3-NBA in human and rat livers. Incubations of 3-ABA with hepatic microsomes of rats treated with
3-NBA or 3-ABA (40 mg/kg body weight) led to an up to 12-fold increase in 3-ABA-derived DNA adduct formation compared to controls. The observed stimulation of DNA adduct formation by both compounds was attributed to their potential to induce protein expression and enzymatic activity of cytochromes P450 1A1 and/or -1A2 (CYP1A1/2), the major
enzymes responsible for 3-ABA activation in human and rat livers. Collectively, these results demonstrate for the first
time that by inducing hepatic NQO1 and CYP1A1/2, both 3-NBA and 3-ABA increase the enzymatic activation of these
two compounds to reactive DNA adduct forming species, thereby enhancing their own genotoxic potential. Supported
by GACR (303/05/2195) and the Ministry of Education of the Czech Republic (MSM0021620808).
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P-194
COMPARATIVE ANALYSIS OF GENE EXPRESSION IN THE TISSUES OF MICE EXPOSED
TO ARSENATE OR DIMETHYLARSINIC ACID IN DRINKING WATER.
Marafante Erminio1
Cimino Reale G 1; Casati B 1; Collotta A 1; Brustio R 1; Vhater M 2
EC Joint Research Centre, ispra, Italy1; Karolinska Instituet, Stockholm, Sweden2
The health consequences of chronic arsenic exposure include various forms of cancer, e.g. skin, lungs, urinary bladder
and kidney. Reported mechanisms or mode of action of arsenic include enzyme inhibition, including DNA repair enzymes
and enzymes involved in cell cycling, oxidative stress, modification of DNA methylation, interactions with nuclear receptors, and un-coupling of cellular respiration. Apparently the mode of action is highly dose-dependent. There seems to
be a vide variation in susceptibility to arsenic toxicity. Known risk modifying factors include gender, age, genetic predisposition, nutrition and metabolism of arsenic. Arsenic is metabolized by a series of reduction and oxidative methylation reactions, obviously modulating the toxicity and dimethyl arsinic acid (DMA) is the major metabolites found in the
urine of populations exposed to arsenic in drinking water. In the present study we investigated the modulation of gene
expression in tissues of mice chronically exposed to equivalent doses of 1 mg/L of arsenic as AsV or DMA in drinking
water for four months. We used the total genome Applied Biosystems Microarrays of 28Kgenes to identify gene families
and pathways. Major differences occurred among gender and tissues following both the exposure to arsenate and DMA,
suggesting that toxicological mechanisms of arsenic is related to metabolism and strongly differentiate between male
and female mice.
P-195
CHARACTERISATION OF THE P53 GENE EXPRESSION RESPONSE INDUCED
BY BENZO(A)PYRENE-DIOL-EPOXIDE IN MAMMALIAN CELLS
Sarah L Hockley
Ian Giddings; Volker M Arlt; David H Phillips
Institute of Cancer Research, Sutton, United Kingdom
Exposure to genotoxic carcinogens such as benzo(a)pyrene B(a)P results in activation of the tumour suppressor protein
p53. This protein transcriptionally regulates genes that trigger downstream cellular processes such as cell cycle arrest,
apoptosis and DNA repair. To gain further insight into the cellular response to carcinogens and to identify carcinogeninduced gene expression alterations that are specific to the p53 response, 22K cDNA microarrays have been used to
investigate gene expression alterations in a pair of isogenic cell lines that differ in p53 status, after exposure to the ultimate carcinogenic metabolite of BaP, benzo(a)pyrene-diol-epoxide (BPDE). DNA adduct analysis and cell cycle analysis
were also performed on the cells. Exposure to BPDE (0-1 µM) for up to 24 h resulted in 79 and 86 altered transcripts
in the p53-WT and p53-null cells, respectively. In both cell lines cell cycle regulation genes were significantly over-represented in the BPDE-expression profiles, however these genes differed between the two cell lines. Apoptotic signalling
was also significantly over-represented in the p53-WT BPDE-expression profiles but this was not evident in the p53-null
cells. A t-test comparison of the two cell lines identified 25 genes with significantly different expression including BAX,
CDKN1A and members of the TNF receptor superfamily. Thirty-four genes were commonly altered in both cell lines, a
number of which are linked to chromatin remodelling (e.g. histone genes). In both cell lines BPDE exposure resulted
in an accumulation of the cells in the S phase of the cell cycle indicating that this effect is independent of p53 signalling. Similar levels of BPDE-DNA adducts were detected in the p53-WT and p53-null cells. These results provide further
insight into the transcriptional signature of genotoxic insult and identify p53-dependent and p53-independent cellular
effects in response to BPDE, further characterising the role of this tumour repressor in the DNA damage response.
237
26.6.2006 16:06:09
P-196
GLOBAL ANALYSIS OF GENE EXPRESSION IN ACETAMINOPHEN-INDUCED MICE
HEPATOTOXICITY
Jin Seok Kang
Ki Kyung Jung; Soo Kyung Suh; Joohwan Kim; Tae Gyun Kim; Bang Hyun Kim; Woo Sun Lee; Youn
Kyoung Jeong; Ye Mo Koo; Hyun-Joo Kim; Hai Kwan Jung; Sue Nie Park
National Institute of Toxicological Research, Korea Food and Drug Administration, Seoul, Korea
To clarify and understand the hepatotoxic mechanism of acetaminophen (APAP), we investigated the global gene expression in mouse liver treated with APAP. ICR mice were orally treated with acetaminophen (0, 50, 500 mg/kg) and were
sacrificed at 6, 24 and 72 h after treatment. Hepatic gene expression was assessed using high-density olignucleotide
microarrays (Applied Biosystem). In high dose treatment group, there was centrilobular necrosis at 6 and 24 h after
treatment with it, along with elevation of serum ALT and AST, but not evident at 72 h. However, there were no alterations in histopatholgical changes and serum biochemistry at low dose. Hierachical and K-means clustering after two-way
ANOVA analysis showed most alterations of gene expression occurred at 6 h, and these were diminished gradually.
Using pathway analysis, we found APAP modulated the expression of many genes encoding for apoptosis, steroids
biosynthesis, Ca signaling pathway, cell cycle, cysteine and glutathione metabolism, glycolysis, MAPK pathway and
so on. In conclusions, APAP induced global gene alterations, along with the changes of histopatholgical findings and
serum biochemistry, and it suggests pathway analyses will lead to a better understanding of the molecular mechanism
of APAP-induced liver injury, providing their potential importance in propagating toxicogenomics profiles. It would also
facilitate to discover new toxicological biomarkers and provide a new evaluation method for safety of toxic substances
such as APAP.
P-197
CYTOTOXICITY OF THE ANTICANCER DRUG ELLIPTICINE IN HUMAN LEUKEMIA
AND NEUROBLASTOMA CELL LINES
Jitka Poljakova 1
Marie Stiborova 1; Jan Hrabeta 1,2; Tomas Eckschlager 1,2; Eva Frei 3
Charles University in Prague, Prague, Czech Republic1; University Hospital Motol, Prague, Czech Republic2; German Cancer
Introduction: Ellipticine and several of its more soluble derivatives exhibit promising results in the treatment of osteolytic breast cancer metastases, myeloblastic leukemia and tumors of brain. The main reason for the interest in ellipticines for clinical purposes is their high efficiencies against several cancers, their rather limited toxic side effects and
their complete lack of hematological toxicity. Nevertheless, ellipticine is a potent mutagen. The target of this study
was to investigate the cytotoxicity of ellipticine towards human leukemia and neuroblastoma cell lines and its ability to
generate ellipticine-DNA adducts in these cells.
Results and discussion: We found that ellipticine is toxic to two model human leukemia cell lines, HL-60 and CCRF-CEM
cells in culture and to human neuroblastoma cells (human parental IMR-32, UKF-NB-3 and UKF-NB-4 cell lines) and
their daughter derived cells that were resistant to Vincristine, Doxorubicine and Cis-platin. After exposition of the cells
to this drug, ellipticine-derived DNA adducts are generated. The IC50 values for ellipticine are in a range of 0.26 – 4.67
μM. Toxicity of ellipticine towards the leukemia and neuroblastoma cells correlates with levels of ellipticine-derived DNA
adducts generated in these cells. Expression of enzymes activating ellipticine in leukemia and neuroblastoma cell lines
is investigated. The results indicate the formation of covalent DNA adducts by ellipticine as one of the multiple modes
of antitumor action of ellipticine for these cancer cells. The pre-treatment of UKF-NB-3 and UKF-NB-4 neuroblastoma
cells with inhibitors of histone deacethylase, valproate and trichostatin A, resulted in an dose-dependent increase in
the toxicity of ellipticine to these cells which correlated with an increase in ellipticine-DNA adduct formation, predominantly in UKF-NB-3 cells. UKF-NB-3 neuroblastoma cells were more sensitive to valproate and trichostatin A than the
UKF-NB-4 line.
GACR 203/06/0329; ME CR MSM0021620813
238
26.6.2006 16:06:09
P-198
BIOACTIVATION OF BENZO(A)PYRENE IN AHR KNOCK OUT MICE
Steen Mollerup
Carlos Sagredo; Steinar Ovrebo; Ingrid V Botnen; Rita Baera; Einar Eilertsen; Aage Haugen
Section for Toxicology, National Institute of Occupational Health, Oslo, Norway
Ahr knock out mice have been shown to be resistant to tumorigenesis after exposure to benzo(a)pyrene [B(a)P]. Here,
the effect of Ahr genotype on B(a)P bioactivation after oral exposure was studied. Ahr wild type (Ahr+/+), heterozygous
(Ahr+/-) and homozygous knock out (Ahr-/-) mice were orally exposed to a single dose of B(a)P (100 mg/kg) for 24 hrs,
with corn oil as vehicle. The mice were sacrificed and lung, liver, spleen, kidney, heart, and blood samples were collected. Gene expression was measured by real time RT-PCR. Protein and DNA adducts, and free metabolites of B(a)P,
were quantitated by fluorescence HPLC. Cyp1a1 gene expression was significantly increased in lung and liver of Ahr+/+
and Ahr+/- B(a)P exposed mice. Steady state Cyp1a1 gene expression (mice exposed to corn oil only) was low, but differed Ahr gene-dose dependently in both lung and liver (Ahr+/+ > Ahr+/- > Ahr-/-). Cyp1b1 expression was also increased
in the lungs of B(a)P exposed Ahr+/+ and Ahr+/- mice, but did not appear to be induced in the liver of B(a)P exposed
mice regardless of the genotype. Benzo(a)pyrene-diol-epoxide-I-1 (BPDE-I-1) and BPDE-II-2 were the protein adducts
formed at the highest levels after B(a)P exposure. An inverse relation between Ahr gene-dose and protein adduct levels
was found (Ahr-/- > Ahr+/- > Ahr+/+) in all tissues. DNA adducts were measured in liver, confirming the protein adduct
findings. Also, higher levels of free B(a)P-metabolites were found in Ahr-/- mice compared to Ahr+/+ and Ahr+/- mice in
all tissues, in addition to plasma. Finally, significantly higher levels of unmetabolized B(a)P were found in all tissues
of Ahr-/- mice. In conclusion, an apparent paradox of higher adduct levels in Ahr-deficient than in wild type mice was
found after oral B(a)P exposure. This may, however, at least partly be explained by a delayed bioctivation of B(a)P in
mice lacking the Ahr.
P-199
POLY(ADP-RIBOSE) POLYMERASE DEFICIENCY DOES NOT INFLUENCE THE MUTAGENESIS
OF ETHYLNITROSUREA IN LIVER AND TESTIS FROM TRANSGENIC MICE
Maria J. Silva
Henriqueta Louro; Ines Faustino; Ana Carla Sousa; Anabela Dias; Maria Guida Boavida
Center of Human Genetics, National Institute of Health Dr. Ricardo Jorge, Lisbon, Portugal
Poly (ADP-ribose) polymerases(PARP) are a family of proteins linked to several DNA damage cellular responses.
Accumulating evidence indicates that PARP-1 participates in base excision repair, single- and double-strand break repair
and cell death. In response to a genotoxic insult, in vitro PARP-1 deficiency results in genetic instability, increased levels
of sister chromatid exchanges, micronuclei, gene amplification and other mutational or recombination events. In the
present work we evaluated the in vivo mutagenic effects of PARP-1 deficiency on the frequency and spectrum of mutations induced by N-Ethyl-N-Nitrosourea(ENU). Groups of homozygous PARP-1-/-LacZ and PARP-1+/+LacZ plasmid-based
transgenic mice were injected with 120 mg/kg of ENU or saline. After 30 days, the LacZ mutant frequencies(MF) and
mutation spectra(MS), obtained by restriction analysis, were determined in mouse liver and testis. The spontaneous MF
was similar in both organs of PARP-1+/+ and PARP-1-/- mice(P>0.15). ENU-treatment induced an approximately 3-fold
significant increase in MF in liver and testis of PARP-1-/- and PARP-1+/+ mice(liver P= 0.002; testis P=0.00004) and there
was no significant difference between both mouse lines. Analysis of the MS revealed that the majority of ENU-induced
mutations were point mutations with a small fraction of gross deletions and insertions in both organs of both mouse
lines. In summary, no detectable effect of PARP-1 deficiency was found either in the MF or in the pattern of mutations
(gross deletions/insertions versus point mutations) suggesting a nonessential role for PARP-1 on the repair of ENUinduced DNA lesions. These findings are in contrast with previous results showing that methyl-nitrosurea induces more
deletions/insertions in PARP-1-/- than in PARP-1+/+ mouse testis, suggestive of a protective role of PARP-1 against the formation of deletion mutations in vivo. (Funded by Foundation for Science and Technology, POCTI/2000/MGI/ 34270).
239
26.6.2006 16:06:11
P-200
AN EXPERIMENTAL MODEL OF POTENTIAL TRANSPLACENTAL GENOTOXICITY
OF FLUCONAZOLE
Darko Markovic 1
Boris Mildner 1; Zeljko Ferencic 1; Glojnaric Ines 1; Ranko Stojkovic 2; Suzana Borovic-Sunjic 2; Ana Marija
Jazbec 3; Tomislav Dobranic 4; Aleksandra Fucic 5
PLIVA Research Institute Ltd, Zagreb, Croatia1; Rudjer Boskovic Institute, Zagreb, Croatia2; Faculty of Forestry, Zagreb, Croatia3;
Faculty of Veterinary Medicine, Zagreb, Croatia4; Institute for Medical Research and Occupational Health, Zagreb, Croatia5
Chemical genotoxicity is evaluated using batteries of sensitive test. However, not a single test does evaluate transplacental genome damage in young organisms. The aim of this study was to develop a model for testing transplacental
cytogenetic damage in newborn and three-week old mice. The model comprises biochemical (glutathione peroxidase
(GPX), malonaldehyde (MDA)), genotoxic (in vivo micronucleus) and histological (apoptosis) levels. Dams (Balbc/c)
were exposed to physiological saline (PS), cyclophosphamide (CP) (10mg/kg) and fluconazole (FN) (12 mg/kg). The
compounds were administered i.p. in three consecutive daily doses on days 12-14 of pregnancy. Blood samples were
taken before and after the treatment (48h after initial dose). Neonatal genotoxicity in pups was analysed from 6-18h
after delivery and tissues were taken for analysis. Late genotoxic effects were investigated using the challenge assay
in pups administered CP on day 19 of birth. Blood was sampled before and 48h after treatment. MN frequency in dams
was significantly above the control values in the CP treated group. In the groups treated with PS and FN there were no
significant differences. In the neonatal pups a significant increase in MN frequency was observed in the neonates from
dams treated with CP and FN. These significant increases in frequency of MN sustained without change in pups until
the day 19 after delivery. The CP challenge assay showed potency to distinguish difference in repair capacity possibly
caused by intrauterine exposure. Significant decreases in the concentrations of MDA and GPX in the livers of neonatal
pups in groups treated with CP and FN were detected. Animals treated with CP and FN showed no differences in the
number of renal and hepatic apoptotic cells. The pilot study results indicate the importance of the model for testing the
transplacental genotoxicity of chemical agents.
P-201
THE GENOTOXIC POTENTIAL AND CHEMOPREVENTIVE EFFECTS OF TWO COMMONLY USED
ANTI-ABORTION HERBAL MEDICINES ON ENU-INUDCED TRANSPLACENTAL GENOTOXICITY
IN MICE
Hsiu-Ching Wu 1
Chiang Su-yin; Mei-Yi Lin 2; Jun-Ming Chen 3; Chien-Liang Fang 4; Hui-Feng Huang 5; Su-Yin Chiang 5
School of Post Baccalaureate Chinese Medicine/China Medical University, Taichung, Taiwan1; Department of Traditional Chinese
Medicine/Chia-Yi Christian Hospital, Chiayi, Taiwan2; Chen Jun-Ming TCM clinics, Taipei, Taiwan3; Department of Traditional Chinese
Medicine/China Medical University Hospital at Pei-Kang, Pei-Kang, Taiwan4; Graduate Institute of Chinese Medical Science/China
Medical University, Taichung, Taiwan5
“Thirteen Herbs An-Tai In” (THATI) and “Bow-Tai-Tang” (BTT) are two commonly used anti-abortion herbal medicines.
Due to their extensive use, it is of great interest regarding their genotoxic and anti-genotoxic potential to pregnant
women and their offspring. In this study, the potential genotoxic and chemopreventive effects of THATI and BTT in
pregnant ICR mice and their fetuses were examined by Micronuclei assay. For genotoxicity group, aqueous extracts of
THATI and BTT were given by gavage from gestation day (GD) 12 to 17. For chemoprevention group, N-ethyl-N-nitrosourea (ENU) was given by intraperitoneal injection (ip) at a dose of 90mg/kg on GD 17 two hours after the treatment
of THATI and BTT from GD 12 to 17. Blood samples were collected on GD 19 to examine the frequencies of micronuclei,
a marker for chromosome damage, in maternal and fetal reticulocytes (RET). There is no statistically significant difference among the THATI and BTT experimental groups and the negative control group in maternal body weight gain and
RET/normochromatic erythrocytes (NCE) ratio. However, RET/NCE ratios in fetuses were markedly higher than those
in pregnant dams. Treatment of THATI at the dose of 17.6 g/kg and 5.9 g/kg and BTT at the dose of 9.8 g/kg did not
induce a statistically significantly increase in frequencies of micronuclei in maternal and fetal reticulocytes. Moreover,
pretreatment with BTT but not THATI resulted in statistically significant decrease in the frequencies of ENU-induced
micronucleated reticulocytes, with an inhibition ratio of about 30% and 20%, in the maternal blood and fetal blood,
respectively. These data suggests that BTT but not THATI possess the chemopreventive effect with no observable cytotoxicity in ICR mice and their fetuses.
240
26.6.2006 16:06:11
P-202
PREGNANT WOMEN WHO SMOKE HAVE HIGHER LEVELS OF PLACENTAL POLYCYCLIC
AROMATIC HYDROCARBON (PAH)-DNA ADDUCTS COMPARED TO NON-SMOKERS:
AN IMMUNOHISTOCHEMICAL EVALUATION
Miriam C. Poirier 1
Paul Sirajuddin 1; Margaret Pratt 1; Radim J Sram 2; David K Manchester 3
National Cancer Institute, NIH, Bethesda, MD, United States1; Laboratory of Genetic Ecotoxicology, Institute of Experimental
Medicine, Prague, Czech Republic 2; University of Colorado School of Medicine, Denver, Colorado, United States3
Epidemiologic studies have linked parental exposure to chemicals, including PAHs in tobacco smoke, with childhood
leukemias. The placental cytotrophoblast (CT) and syncytiotrophoblast (ST) cells that line the chorionic villi actively
metabolize xenobiotics. DNA extracted from the chorionic villi has been shown to contain PAH adducts, but the cellular
localization was not previously confirmed. Here, PAH-DNA adducts have been localized in CT and ST cells by immunohistochemical (IHC) staining withantiserum elicited against anti-(+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-benzo[a]pyrene
(BPDE)-modified DNA, and quantified by Automated Cellular Imaging System (ACIS). Semi-quantitation of DNA adduct
levels was accomplished by comparison with a standard curve consisting of human keratinocytes exposed for 1 hr to 0,
0.053, 0.153 or 0.331 µM BPDE. BPDE-DNA adducts determined in keratinocyte DNA extracted and assayed by competitive chemiluminescence immunoassay (CIA) showed good correlation (r2 =0.99) with adducts in paraffin embedded keratinocytes measured by IHC/ACIS. Paraffin-embedded human placentas (n=14), stained with anti-BPDE-DNA,
showed nuclear CT and ST cell staining that was confirmed specific through use of antiserum absorbed with the BPDEDNA immunogen. For the human placentas, comparison with the standard curve revealed PAH-DNA adduct values of
173 ± 35/108 nucleotides (mean ± SD) for 7 smokers and 58 ± 15/108 nucleotides for 7 non-smokers (p<0.02, MannWhitney and Student’s t test). The study showed that PAH-DNA adducts are highly-concentrated in the CT and ST cells
that metabolize PAHs, less-concentrated in macrophages, and not detectable in the remaining areas of the placental
tissue. The study also showed strong evidence of a smoking effect, with PAH-DNA adduct formation significantly higher
in smokers compared to non-smokers.
241
26.6.2006 16:06:11
AUTHORS INDEX
AUTHORS INDEX
Aarts Jac M.M.J.G.
P-153 O-045
Abdel-Rahman Sherif Z.
P-052
Abrahamsson-Zetterberg Lilianne O-004 O-004
Adamčík Milan
P-141
Adamus Tomáš
P-142
Agnoletto Mateus H
P-072
Agrawal Meenakshi
O-114
Agudo Antonio
P-109
Ahn Kyu Dong
P-139
Ahr Hans J.
O-016
Aimova Dagmar
P-192
Aiub Claudia
O-024
Akesson Bjorn
O-042
Akiyama Yoshimitsu
O-057
Aksoy Hüseyin
P-083 P-082
Akyuz Nuray
P-026
Albertini Richard J.
O-120
Albin Maria
P-138
Alink Gerrit M
O-040 P-038 P-164 P-153
Aliyazicioglu Yuksel
P-042
Alvur Muhlise
P-042
Amador-Muñoz Omar
P-127
Amarante-Mendes G P
P-072
Ambrosone Christine B.
P-159
Amiano Pilar
P-109
Anagnostopoulos Athanasios
P-077
Andersen Sonja
O-020
Anderson Diana
P-032
Andreoli Cristina
P-039
Andrianopoulos Constantinos
P-040
Andrysik Zdenek
O-080 P-089 O-060
Angelini Sabrina
P-112 P-112 P-062
Anna Livia
P-002 P-010
Aoki Yasunobu
O-059 O-114
Arbillaga Leire
O-018
Ardanaz Eva
P-109
Arlt Volker M
O-054 P-195 P-193
Aston Paul
P-100
Astrea Guia
P-114
Au William W.
O-108 O-109
Aubrecht Jiri
O-013
Aurelie Laurent
P-188
Auvinen Anssi
P-120
Aventis Sanofi
O-113
Avlasevich Svetlana
O-003 P-168
Axmon Anna
P-110 P-118
Aygun Kocabas Neslihan
P-119
Azim-Araghi Ali
P-024
Azqueta Amaia
O-018 P-061
Bačun-Družina Višnja
P-059
P-020
Badouard Carine
Bæra Rita
P-198
Baer-Dubowska Wanda
P-129 P-128
Baeyens Ans
P-125
Baginska Agata
P-069
Bagnasco Maria
O-093
Baird William M.
P-089 O-061 O-060
Bajic Vladan Peter
P-179
Balansky Roumen
O-093
Balia Cristina
P-004
Ballantyne Mark
P-166 P-180
Banaszkiewicz Zbigniew
P-046
Bańkowski Robert
O-082
Barale Roberto
O-001
Barale Roberto
P-123
Barbata Giusi
P-073
242
Barberà Ja
Bargagna Stefania
Barta Ivo
Bartoszek A.
Bartova Jirina
Bartsch Helmut
Batista Luis Fz
Baumann Cindy
Bavorova Hana
Bebenek Katarzyna
Becker Thomas
Bedir Abdulkerim
Beevers Carol
Bekyrou Margarita
Belaidi Jean-Philippe
Beland Frederick A.
Belinsky Steven
Bell Douglas A.
Bellini Marilanda Ferreira
Bennicelli Carlo
Bensimon Aaron
Bergendorf Ulf
Beric Tanja
Beskid Olena
Bétous Rémy
Bialkowski Karol
Bielen Aleksandra
Bilgici Birşen
Billinton Nick
Bingham Sheila
Binkova Blanka
Bino Raoul J.
Birk Thomas
Blanco Luis
Blasiak Janusz
Boavida Maria Guida
Boehden Gisa S
Boersma Marelle G
Boffeta Paolo
Bogaards Jan Jp
Böhmer Frank
Bolcsfoldi George
Bolognesi Claudia
Bonassi Stefano
Bonatto Diego
Bonelli Alessia
Booth Antonia
Borovic-Sunjic Suzana
Borská Lenka
Bosch Dirk
Botnen Ingrid V
Botsivali Maria
Botta Alain
Bouso L
Brabec Marek
Bradman A.
Brendel Martin
Britton Julie A
Broberg Karin
Browning Brian
Brustio R
Bryce Steven
Budzinski Hélène
Buffler P.
Buchmann Christoph
Burgaz Sema
P-146
P-114
P-080
P-163 P-003
P-101 O-096
P-087 O-094 P-087
P-072
P-026 P-041
P-007
O-032
P-177 P-170
P-042
P-180
P-078
P-188
P-047
O-078
O-110 O-106
P-094
O-093
O-030
P-118
P-154
O-047 P-021
O-030
P-046
P-060
P-042
O-088
O-041
O-051 P-081
P-164
P-137
O-032
P-063
P-199 P-149
P-041
O-040
O-121
O-040
P-131
P-175
P-004
O-046
P-043 P-051
P-114
P-168
P-200
P-017
P-164 P-153
P-198
P-033
P-191
P-146
P-017
O-052
P-043 P-051
O-114
P-124 P-110 P-118
O-043
P-194
O-003
O-105
O-052
P-135
P-006
Burgaz Sema
P-044
Bürkle Alexander
P-041
Butkiewicz Dorota
P-069 P-111
Cabrera Guillermo
P-132
Cachot Jérôme
O-105
Cajal Santiago Ramón
O-032
Cajthaml Tomáš
P-143
Cakmak Gonca Demircigil
P-006
Cakmak Gonca Demircigil
P-044
Caldecott Keith
O-029
Cammerer Zoryana
O-002 O-006
Camoirano Anna
O-093
Canitrot Yvan
O-032
Cantelli Forti Giorgio
P-112 P-001 P-062
Cantero Gloria
P-045
Capella Gabriel
P-109
Capp Jean-Pascal
O-032
Caradonna Fabio
P-039 P-073
Carbone Fabio
P-001 P-112 P-062
P-051
Cardone Jaqueline M
Cardoso João
P-149
Carmichael Paul
P-178
Cartiglia Cristina
O-065
Carvalho Heleotonio
P-072
Casati B.
P-194
Castellà Maria
O-049
Catalán Julia
P-023
Catanzaro Irene
P-073
Cazaux Christophe
O-030
Cazaux Christophe
O-032
Cebulska-Wasilewska Antonina
O-051 P-066
Cegla Roksana
P-049
Çelik Mustafa
P-082
Çelik Mustafa
P-083
Ceppi Marcello
O-089
Cermakova Zuzana
P-113
Cerna Milena
P-007 P-17
Cetindag Faik
P-006
Cetindag Faik
P-044
Ciardelli Roberta
O-055
Cieślak Anna
P-003
Ciganek Miroslav
P-089 O-060 P-091
Cimino Reale G
P-194
Claes Kathleen
P-125
Clonfero Erminio
P-105 O-075
Cnubben Nicole Hp
O-040
Coll Md
P-146
Collins Andrew R
P-061
Collins Joanne
P-168
Collotta A
P-194
Colognato Renato
P-114
Conti Chiara
O-030
Coppedè Fabio
P-114
Cordelli Eugenia
P-107
Cornelius Michael G.
P-005
Cortés Felipe
P-045
Cortés-Eslava Josefina
P-187
Corvi Raffaella
O-015
Coskun Erdem
P-006 P-044
Costa Paula
P-149
Courter L.A.
O-061
Coza Dana
P-181
Crebelli Riccardo
P-031
Csekeo Attila
P-002 P-010
Cunliffe Jo
P-175
Czaplicki Jerzy
O-030
26.6.2006 16:06:12
AUTHORS INDEX
Čipák Ľuboš
P-174
D‘Agostini Francesco
O-093
D‘Alessio Alessia
P-107
Davies Ronnie
P-100
De Boeck Marlies
O-002 O-012
De Bont Kelly
O-055
De Flora Silvio
O-093 O-065 O-096
De Kok Theo M
O-045
De Palma Giuseppe
O-126
De Ruyck Kim
P-125
De Waard Pim
O-045
Deac Liana Monica
P-181
Debus Juergen
P-087
Decordier Ilse
O-055
Demarini David M
O-038
Demopoulos Nikos A.
P-040
Dertinger Stephen
O-003
Dertinger Steve
P-168
Dessen P.
O-027
Dettbarn Gerhard
O-098
Di Marco Barbara
O-065
Diallo M S
P-022
Dias Anabela
P-199 P-149
Dias Elsa
P-104 P-133
Diaz Dolores
P-178
Dietrich Cornelia
O-080
Dietzová Zuzana
P-095
Dimopoulos Meletios A
P-079 P-077
Divi Rao L
O-037
Djelic Ninoslav Djelic
P-179
Djurinec Martina
P-189
Dobiáš Lubomír
P-141
Dobiáš Lubomír
P-142 P-141
Dobranic Tomislav
P-200
Dobrzyńska Małgorzata
P-108
Doerge Daniel R.
P-047
Dogliotti Eugenia
O-019
Doherty Ann T
P-167 P-175
Domínguez Inmaculada
P-045
Dostal Miroslav
O-056
Dracinska Helena
P-193
Duann Lan-Tyi
P-027
Duburs Gunars
P-070
Dufour Eric K
P-172
Dúhová Viola
P-174 P-098
Durán R.M.G.
P-132
Durgo Ksenija
P-093
Durnev Andrey D.
P-152
Dušinská Mária
P-008 P-184
Dziaman Tomasz
P-046
P-008
Džupinková Zuzana
Eckschlager Tomas
P-197
Edinsel Hayriye
P-006 P-044
Eguchi Hidetaka
P-011
Eilertsen Einar
P-198
Elespuru Rosalie K.
O-101
Eleuteri Patrizia
P-107
Elhajouji Azeddine
O-002
Ellinger-Ziegelbauer Heidrun
O-016
Ellis Patricia
P-168
El-Zein Randa A
O-050
Eng Sybil
O-114
Episkopou Hara G
P-079
Erminio Marafante
P-194
Eskenazi B.
O-052
Eszter Nagy
P-016
Etzel Carol J
O-050
Evenson Don
O-098
Evenson Donald P.
O-099
Falck Ghita C-M
P-134 P-023
Faldikova Ludmila
O-004
Falque-Gonzalez Cesar
P-032
Fang Chien-Liang
P-201
Farkašová Timea P-009
P-009
Farmer P.B. O-074 O-074 P-066 O-051 O-072
Farrar Diane
P-032
Faust Dagmar
O-080
Faustino Inês
P-199
Favier Alain P-144
P-144
Felzenszwalb Israel
O-024
Fenech Michael O-012
O-012
Ferencic Zeljko
P-200
Ferguson Lynnette R.
O-043
Ferk Franziska
P-135
Feychting Maria
P-120
Fiala Zdeněk
P017
Filipic Metka
P-158 P-088 P-055
Flamma Floriana
P-039
Flores-Márquez Ana Rosa
P-127
Flores-Maya Saúl
P-187
Flückiger-Isler Sini
O-171
Foksinski Marek
P-046
Fontana Ilaria
P-114
Fousteri Maria J
P-079
Francois Sichel
P-188
Franekić Čolić Jasna
P-093
Franklin Jamey
O-050
Frassinetti Stefania
P-182
Fred C.
O-097 O-122
Freeman Kathryn M.
P-053
Frei Eva
P-192 O-077 P-193 P-197
Fresegna Annamaria
P-107
Frieauff Wilfried
O-109 O-009
Friedberg T.
P-163
Friedmann Angeli José Lourenço
P-094
Froetschl Roland
P-169
Fucic Aleksandra
P-029 P-030
Fuerhacker Maria
P-135
Fujikawa Kazuo
O-025
Furlong C.
O-052
Fuster C
P-146
Gábelová Alena
P-009 O-062 P-115
Gabriels Joseph
O-109
Gackowski Daniel
P-046
Gajdoš Andrej
P-136 P-095
Gajdošová Dagmar
P-136 P-137 P-095
Gamboa Da Costa Gonçalo
P-047
Gammon Marilie D
O-114
Gangarosa Lisa
O-038
Garaj-Vrhovac Verica
P-183 P-190
Garcia Nadia
P-109
García-Díaz Miguel
O-032
Garte Seymour
O-106
Gaskell Margaret
P-024
Gaspar Jorge F.
P-047
Gaudet Mia
O-114
Georgiadis Panagiotis
P-048
Giddings Ian
P-195
Giefing M
O-087 P-049
Giordano Elena
P-004
Glatt Hansruedi
O-090 O-089 O-125
Gmuender Hans
O-017
Godschalk Roger Wl
O-023
Gómez F
P-146
Gómez-Arroyo Sandra
P-187 P-127
Goncharova Rose I.
P-070
González Carlos A
P-109
Gradwell Ann
P-166
Grajek Włodzimierz
P-003
Granath F.
O-097 O-122
Grančai Daniel
P-174
Grawé Jan
P-175
Greenfield Teri
O-056
Gregorio Pasquale
P-105
Grewal Arneet
P-103
Griciene Birute
P-150
Gritzko Kristin
P-170
Grolmus Ján
P-174
Gruz Petr
O-021 P-050
Grzesiuk Elzbieta
P-065 P-057
Guecheva Temenouga N
P-072
O-092 O-090
Guengerich F. Peter
Guerin Adele
P-052
Guialis Apostolia
P-067
Gundy Sarolta
O-076
Gungor Nejla
O-023
Gunter Marc J
O-037
Gurská Soňa
P-009 P-115
Gut Ivan
O-068
Guz Jolanta
P-046
Gyorffy Erika
P002 P-010
Gyori Zoltan
P-002 P-010
Habermann Nina
P-131
Haglund Johanna
O-074 O-070
Haldsrund Renate
O-104
Hallmans Göran
P-124
Hamatani Kiyohiro
P-011
Hanova Monika
O-112 P-019 O-126
Hanzalova Katerina
P-081
Harley K.
O-052
Härnwall Eva-Lena
P-175
Harris Curtis C.
O-064
Hartbauer Petra
P-148
Hartung Thomas
O-015
Harvey James
P-168
Hastwell Paul
O-088
Haugen Aage
P-198
Haumont Dominique
O-055
Hayashi Makoto
P-054
Hayashi Tomonori
P-015 O-124 O-111 P-117
O-122
Hayata Isamu
O-025
Hayes Julie E
P-167 P-175
Healey Katherine
O-026
Hedmer Maria
P-138
Heilimo Iiris
P-116
Hein David
O-107
Heinävaara Sirpa
P-120
Hemminki Kari
P-001 O-112 P-019 P-062
Henriques João Antonio Pegas
P-051 P-072
P-043
Heo Yong
P-139
Herceg Zdenko
O-077 O-118
Hernández-Valero María A.
P-052
Hertz-Picciotto Irva
O-056
Hiraku Yusuke
O-031
Hirvonen Ari
P-122 O-067 O-125 P-116
Hockley Sarah L
P-195
Hoffmann George R.
P-053
Hoffmann Jean-Sébastien
O-030 O-032
Hofmann Thomas
P-131 O-033
Höglund Peter
P-138
Hökelek Murat
P-042
Holland Nina
O-052
Holmila Reetta
P-002
Honma Masamitsu
P-054
Hoogenboezem Wim
P-038
Hoogenboom Ron
O-045
Horská Alexandra
P-184
Hou Sai-Mei
P-071
Hrabeta Jan
P-197
Hrelia Patrizia
P-001 P-112 P-062
Hreljac Irena
P-055
243
26.6.2006 16:06:12
AUTHORS INDEX
Huang Hui-Feng
P-201
Hubackova Miluse
O-068
Hübner Claudia
O-043
Hudák Alexander
P-095
Huen K.
O-052
Husgafvel-Pursiainen Kirsti
P-002 P-090
Huuskonen Matti
P-122 O-067
Chadt Jiří
P-056
Chan Cecilia
O-022
Chang-Claude Jenny
P-087
Chang-Chien Gp
P-185
Characiejus Dainius
P-090
Chen Jun-Ming
P-201
Chen Ming-Fong
P-027
Cheng Tsung Jen
P-027
Chiang Su-Yin
P-185 P-201
Chirlaque M Dolores
P-109
Choi Jinhee
P-155
Choi Jung-Hwa
P-037
Churchwell Mona I.
P-047
Chvatalova Irena
O-056
Chvatovic Genovefa
P-090
Ide Hiroshi
O-097 O-095
Ignatowicz Ewa
P-130
Ichinohe Kazuaki
O-025
Illes Natasha
O-029
Ilus Taina
P-120
Imai Kazue
O-124 O-111 P-117 O-057
Ines Glojnaric
P-200
Ivicevic-Bakulic Tomislav
P-029
Izzotti Alberto
O-093 O-065
Jacob Juergen
O-098
Jankevicius Feliksas
P-090
Januskeviciute Inga
P-150
Janz Christine
P-068
Jarmalaite Sonata
P-090
Jarmuz M
O-087
Järventaus Hilkka
O-125 P-116
Jascone Lia
O-024
Jawien Arkadiusz
P-046
Jaworski Albert
P-057
Jazbec Ana Marija
P-200
Jeon Eun-Jae
P-160
Jeon Jeong Woo
P-160
Jeon Kyung Im
P-161 P-037 P-009 O-092
Jeong Youn Kyoung
P-196
Jeppsson Marina C
P-012
Jeurissen Suzanne
O-040
Jianxin Duan
P-016
Jo Cheon-Ho
O-117
O-056
Joad Jesse P.
Johansen Christoffer
P-120
Johansson Clara
P-013
Jokinen Päivi
P-120
Jones Lovell A.
P-052
Jonsson Bo AG
P-140 P-012 P-138 P-118
Jönsson Lena S
P-118
Jung Eun-Sil
P-162
Jung Hai Kwan
P-196
Jung Ki Kyung
P-196
Jung Sun-Woo
O-092
Kadlubar Fred F.
P-126 P-159
Kagawa Nao
O-025
Kahraman Hakki
P-042
Kaila Stella
P-048
Kaleva Simo
P-122 O-067
Kalina Ivan
O-051 P-066
Kamber Markus
O-171
Kang Hae Jin
P-160
Kang Jin Seok
P-196
Kang Myung-Hee
P-160
244
Kapka Lucyna
P-034
Karahalil Bensu
P-119
Karlsson Hanna
P-156
Kasai Hiroshi
O-039 O-124 O-063
Kasper Peter
P-169
Kasuba Vilena
P-030
Kataoka Hiroyuki
P-106
Katyal Satchin
O-029
Kauffmann A.
O-027
Kaur Balvinder
P-024
Kavli Bodil
O-020
Kawai Kazuaki
O-039 O-063
Kawanishi Shosuke
O-031
Kazmierczak Zaneta
P-130
Kiechle Markus
O-022
Kim Bang Hyun
P-196
Kim Hyoung Ah
P-139
Kim Hyun-Joo
P-196
Kim Chang-Sook
P-160
Kim Joohwan
P-196
Kim Sang Hoon
P-139
Kim Su -Yeon
O-092 P-037 P-161 P-009
Kim Tae Gyun
P-196
Kirchhoff Tomas
O-032
Kirkland David
P-172 P-180 O-083 O-087
Kirsch-Volders Micheline P-014 O-053 O-055
O-002 O-011
Kiuru Anne
P-120 P-116
Kleinjans Jos C
O-053 O-014
Klenow Stefanie
P-131 O-033
O-023
Knaapen Ad M
Knasmüller Siegfried
P-135
Knezevic-Vukcevic Jelena
P-158 P-154
Knöbel Yvonne
O-033
Knudsen Lisbeth E
O-053 P-035 P-036
Kocabas Neslihan Aygun
P-044 P-006
Kocan Anton
P-091
Kodet Roman
O-068
Kogan Grigorij
P-098
Koizumi Tomoko
P-054
Kokubo Kiyoko
O-021
König Jochen
O-017
Koo Ye Mo
P-196
Kopecká Petra
P-121
Kopecký Josef
P-121
Kopjar Nevenka
P-030 P-058 P-183 P-059
Kosalec Ivan
P-059
Kostic Szilard
P-002 P-010
Kostka Grażyna
O-082
Kotova N
P-022
Koukouves George
P-024
Kouloumenta Asimina
P-040
Kowalczyk Pawel
P-060
Kozubik Alois
O-080 P-089
Kraft Michael
P-170
Krajka-Kuzniak Violetta
P-129 P-128
Kratochvil Elisabeth
P-148
Krcmar Pavel
O-080 P-089 O-060
Kroes Robert
O-083 O-084
Krokan Hans E.
O-020
Krokje Aase
O-102
Kromhout Hans
P-140 O-115
Kruszewski Marcin
P-034
Krzesniak Malgorzata
P-069 P-111
Kubo Yoshiko
P-015
Kujawski Maciej
P-049
Kukkonen Mari
P-122 O-067
Kuldorff Martin
O-037
Kumar Rajiv
P-001 P-071 P-062
Kumaravel Tirukalikundram
P-172
Kunkel Thomas
O-032
Kunz Susanne
P-176
Kuo Ching-Tang
P-185
Kurgonaite Kristina
P-090
Kuricova Miroslava
O-126
Kusmierek Jaroslaw T
P-065
Kůsová Jaromira
P-141 P-173 P-142
Kusunoki Yoichiro
P-015 O-124 O-111
Kuznetsova Elena A.
P-076
Kwan M.
O-052
Kyrtopoulos Soterios AP-078 P-024 P-079 P-048
P-077 P-067
Lacigová Silvie
O-069
Laffon Blanca
P-018
Laffon Blanca
P-147
Lah Tamara Turnšek
P-088 P-055
Laires Antonio
P-097
Laitinen Tarja
O-066
Lal Ash
O-005
Lambert Bo
P-071
Lang Nicholas P
P-159
Langova Martina
P-101 O-096
Laubitz Daniel
P-060
Law Mac
O-105
Le Curieux Frank
P-145
Le Hegarat Ludovic
P-145
Le Menach Karine
O-105
Le Pla Rachel C
P-024
Lee Byung-Mu
O-116
Lee Han-Woo
P-037
Lee Hye-Jin
P-160
Lee Hyo-Min
O-117
Lee Hyun-Jin
P-037 P-161 P-009 O-092
Lee Kyung-Hea
O-092
Lee Seung-Chul
P-162
Lee Sun-Hee
P-160
Lee Woo Sun
P-196
Lehocká Hana
P-142
Lennart Möller
P-016
Lewandowska J.
P-163
Lewis Leeann
O-043
Lilla Carmen
P-087
Lim Duck S
O-116
Lin Mei-Yi
P-201
Lindh Christian H
P-012 P-140
Lindholm Carita
P-120 P-116
Linseisen Jakob
O-035
Littorin Margareta
O-063 P-110 P-118
Liu Yu-Ying
O-125
Lněničková Zdena
O-123
Lodi Vittorio
P-004
Lodovici Maura
P-157
Loft Steffen
O-095 O-096
Longobardi Mariagrazia
O-065
Lopez De Cerain Adela
O-018 P-061
Lopez Mirtha S
O-050
Louro Henriqueta
P-199
Lucas Joe Nathan
P-030
Lucová Lívia
P-137 P-095
Ludwicki Jan Krzysztof
O-082
Lukačko Pavol
P-009
Łukowicz Jolanta
P-003
Lupi Silvia
P-105
Lupski Jim
O-029
Lynch Anthony
P-168
Maas Hannie L.
P-038
Maciejewska Agnieszka M
P-065
Mader Robert
P-135
Maekawa M.
O-063
Maffei Francesca
P-001 P-112 P-062
Magnus Zeisig
P-016
Mahadevan Brinda P-089 O-061 O-060 O-057
26.6.2006 16:06:12
AUTHORS INDEX
Macháčková Zuzana
P-009
Machala Miroslav
O-080 P-089 O-061 O-060
P-091
Maître Anne
P-144
Makar Amin
P-125
Maki Mayumi
P-015
Malachová Kateřina Malachová
P-143
Malcomber Sophie
P-178
Malinowski Mariusz
P-063
Malmer Beatrice
P-120
Manchester David K
P-202
Manini Paola
O-126
Mantovani Mario Sergio
P-094
Marafante E.
P-194
Maralhas Alexandra
P-097
Marco Peluso
P-109
Marcos Ricardo
O-049
Marczynski Bolek
O-126
Marie Caroline
P-144
Marinho-Falcão José
P-149
Markovic Darko
P-200
Marques M. Matilde
P-047
Marques Marie
P-144
Marrot Laurent
P-188
Martínez G.L.
P-132
Martino Angela
P-039
Martins Celia
P-097 P-047
Martins Vanda
P-047
Marvanova Sona
O-080 P-089 O-060 P-091
Marzin Daniel
P-191 P-191
Maskell Sharon
P-178
Mateos Santiago
P-045
Mateuca Raluca
P-014
Mathilde Lechevrel
P-188
Matsuda T.
O-063
Matsui Keiko
P-165
Matsui S.
O-063
Matsumoto Tsuneya
O-025
Maunu Heidi
P-116
Mauro Maurizio
P-039 P-073
Mayan Olga
P-018 P-147
Mayer Claudia
P-087
Mazzei José Luis
O-024
Mcginnis Claudia
O-109
Mckinnon Peter
O-029
Mclaughlin L.
P-163
Meinl Walter
O-090
Menck Carlos F
P-072
Méndez Josefina
P-018 P-147
Mercati Francesco
P-039
Mercer Kelly
P-159
Mercke Per
O-042
Merlo Domenico Franco
O-089
Merlo Franco
O-054
Metsola Katja
P-116
Meunier Jean-Roch
P-188
Miadoková Eva
P-174 P-098
Mielzynska Danuta
P-034
Mierauskiene Jurate
P-150
Migliore Lucia
P-114
Michael Fenech
P-014
Michiels S.
O-027
Mikulenková Irena
P-141
Milcová Alena
O-123
Mildner Boris
P-200
Milić Mirta
P-058
Milicevic Zorka Milicevic
P-179
Min Kyung-Jin
O-117
Minarovits Janos
P-002 P-010
Miskov Snjezana
P-029
Mitic-Culafic Dragana
P-158
Miturová Hana
P-173
Mollard Lucie
P-020
Möller Lennart
P-165 P-013 P-028
Møller Peter
O-096
Mollerup Steen
P-198
Monteiro Ana Filipa
P-097
Monteiro Gil Octávia
P-149
Monyarch G
P-146
Mooibroek Hans
P-164 P-153 N
Moore Claire
P-178 P-168
Moreno Jenifer
O-049
Moreno-Zenteno Concepción
P-187
Morishita Yukari
P-015 O-124 P-117
Mrhalova Marcela
O-068
Muckel Eva
O-090
Mučaji Pavel
P-174
Mueller Lutz
O-084
Mullenders, L Hf
P-079
Mund Keneth
P-137
Munday Karen
O-043
Munnia Arnelle
P-109
Mutanen Pertti
P-090
Myazin Andrey E.
P-064
Myung Seung-Woon
O-117
Naccarati Alessio
O-112 P-019 P-123 O-126
Naďová Slavomíra
P-174 P-098
Nagamura Hiroko
P-015
Nagasaki Hiromi
O-057
Nagy Eszter
P-028 P-016
Nair Jagadeesan
O-093 O-094 P-087
Nakachi Kei P-015 O-124 O-111 O-110 P-117
P-011 O-057
Nakamura H.
O-063
Nakamura Shingo
O-025
Natarajan A.T.
O-058 O-060
Neca Jiri
P-089 O-060 P-091
Nersesyan Armen
P-135
Nesslany Fabrice
P-145
Neugut Alfred I
O-114
Nicula Fl.
P-181
Nieminuszczy Jadwiga
P-065 P-057
Niimi Naoko
P-050
Nikitina Viktoria A.
P-152
Nikolic Biljana
P-158 P-154
Nilsson Robert
P-056
Nishigaki Rena
P-106
Nogueira Paulo
P-149
Nohmi Takehiko
O-021 P-165 P-050
Nohynek Gerhard J
P-172
Nomura Taisei
O-099 O-100
Norppa Hannu P-112 P-134 O-125 P-116 P-023
Norris Michelle
O-105
Novakova Zuzana
P-081
Novotná Božena
O-127
Novotný Čeněk
P-143
Novotny Jan
O-112 O-068 P-019 P-123
Nowell Susan
P-159
Nucci Maria Concetta
P-004
Nuria Sala
P-109
Nygren Jonas
P-013
O Donovan Mike R
P-167
Ocadlikova Dana
P-007
O‘Donovan Mike
P-175
Oesch Franz
O-091
Oghiso Yoichi
O-025
Okobia Michael
O-106
Okuyucu Ali
P-042
Olinski Ryszard
P-046
Oliveira Nuno G.
P-047
Önning Gunilla
O-042
Ono Tetsuya
O-025
Oplustil Frantisek
P-025
Oppong Margit
P-177
Orsière Thierry
P-191
Orta Manuel Luis
P-045
Osmak Maja
P-093
Øvrebø Steinar
P-198
Özmen Zeliha Cansel
P-042
Pabel Ulrike
O-090
Pacchierotti Francesca
P-107
Paik Hyun-Dong
P-009
P-149
Painço Patricia Cardoso
Paine M.J.
P-163
Pallen Catherine
P-191
Panek Agnieszka
P-066
Papine Alexandre
P-188
Pardini Barbara
O-112 P-019 P-123 O-126
Park Eunju
P-037 P-161 P-009 P-162 O-092
Park Hae-Ryong
P-162
Park Sue Nie
P-196
Park Sun-Yong
P-155
Park Yoo Kyoung
P-160
Parzefall Wolfram
P-135
Pásaro Eduardo
P-018 P-147
Pasero Philippe
O-030
Pastor Nuria
P-045
Pastorková Anna
P-017
Pastwa Elzbieta
P-063
Patrinou-Georgoula Meropi
P-067
Paulsson B.
O-122 P-100 P-126
Pavanello Sofia
P-105 O-075
Pavlica Vesna
P-058
Pavličková Zuzana
P-143
Pedersen Marie
O-053 P-035 P-036
Pedro Luisa
P-149
Peijnenburg Ad
O-045
Pencikova Katerina
P-089 O-060 P-091
Penders Eric J.M.
P-038
Pennisi Tanya Melissa
O-065
Perales A.G.
P-132
Pereira Paulo
P-104 P-133
Pérez L.M.
P-132
Pérez-Cadahía Beatriz
P-018 P-147
Peszyńska-Sularz Grażyna
P-003
Petermann Ivonne
O-043
Phillips D.
O-071 P-195 P-193 O-070
Philpott Martin
O-043
Piasek Anita
P-003
Piirilä Päivi
P-122 O-067
Pillaire Marie-Jeanne
O-030
Pingarilho Marta
P-047
Plazar
P-085
Pletsa Vassiliki
P-048 P-067
Plevová P
P-121
Poirier Miriam C
O-037 P-202
Polakova Veronika
O-112 P-019 O-062
Polivkova Zdenka
P-101 O-096
Poljakova Jitka
P-197
Poloncová Katarína
O-062
Polson E.
P-163
Pomahačová Renata
O-069
Pomerantseva Marina D.
P-064
Pontén Ingrid
P-175
Pool-Zobel Beatrice L
P-161 O-033 P-131
Popadiuk Stefan
P-003
Popanda Odilia
P-087
Poplawski Tomasz
P-063
Popov Teodor
O-051
Portier Christopher
O-119
Poth Albrecht
P-176
Potparevic Biljana Spremo
P-179
Poznan M
O-087
245
26.6.2006 16:06:13
AUTHORS INDEX
Pozo Rodríguez F
P-146
Pratt Margaret
P-202
Pražmáriová Eva
P-098
Prochazka Gabriela
O-062
Pulay Attila
O-076
Pulliero Alessandra
P-105 O-075
Quik Joris T.K.
P-038
Quirós J Ramon
P-109
Racek Jaroslav
O-069
Rafter Joseph
O-036 O-044
Rachtan Jadwiga
P-111
Ramayia Lylyvati K.
P-064
Ramic Snjezana P-029 P-030 P-058 P-183 P-059
Ranaldi Roberto
P-107
Rank Jette
O-103
Rannug Agneta
P-110
Rappaport Steve
P-136
Raschke Marian
O-033
Ratnasinghe Luke
P-159
Rauko Peter
P-174 P-098
Ravanat Jean-Luc
P-144 P-020
Rees Robert
P-168
Reliene Ramune
P-075
Remenar Eva
O-076
Restle Anja
P-068
Reus Astrid
P-153
Rho Ji-Yeon
P-155
Ribeiro Lucia Regina
P-094
Ribeiro Pinto Luis Felipe
O-024
Rico-Rodríguez Miguel Angel
P-127
Rietjens Ivonne M.C.M.
P-164 P-153 O-040
Rigola Ma
P-146
Rimár Vladimír
P-137
Roach Jonathan
P-024
Rodrigo Gregory
P-177 P-170
Rodrigues Antonio Sebastião
P-097
Rodríguez Trigo G
P-146
Roelants Mathieu
P-014
Roeleveld Johannes
P-164
Roggieri Paola
P-004
Roma-Torres Joana
P-018 P-147
Roos Wynand
P-067
Rosenström Päivi
P-116
Rosselli F.
O-027
Rosselli Filippo
P-067
Rossner Pavel O-089 O-047 P-007 O-114 P-021
Rössner, Jr. Pavel
O-123
Rossnerova Andrea
O-047 P-021
Rothman Nathaniel
O-037
Rotteveel Serge G.P.
P-038
P-071 O-026
Routledge Michael N
Rozalski Rafal
P-046
Rozgaj Ruzica
P-030
Rubes Jiri
O-004 O-100
Rubis Blazej
P-102
Rüdiger Hugo W
P-148
Rueff José
P-097 P-047
Rusak Gordana
P-093
Rusin Marek
P-069 P-111
Rušavý Zdeněk
O-069
Ryabokon Nadezhda I.
P-070
Rybar Roman
O-004
Rybczynska Maria
P-102
Rydberg Per
P-100
Rydzanicz Malgorzata
P-049 P-049
Ryeom Tai-Kyung
O-117
Ryk Charlotta M
P-071
Rzeszowska-Wolny Joanna
P-070
Sabbioni Gabriele
O-125
Saffi Jenifer
P-072
Saggoo Manjit Inder Singh
P-103
246
Sagiv Sharon
O-114
Sagredo Carlos
P-198
Saia Bruno Onofrio
O-075
Saifi Mustafa
O-029
Saini Francesca
P-031
Sakamoto Hiroko
P-054
Sakuraba Mayumi
P-054
Sallette Jérôme
P-188
Salminen Tiina
P-120
Sandberg T I
P-022
Sandler Robert S
O-038
Sandvik Hanna
P-134 P-023
Sanchez Arancha
O-032
Sánchez M José
P-109
Sánchez-Estrada Liliana
P-187
Santella Regina M
O-114 O-094
Santonen Tiina
P-023
Santos Luis
P-149
Saraiva Patricia
P-097
Sarasin A.
O-027
Sassa Akira
P-050
Sauer Julia
P-131 O-033
Savela K.
O-063
Sawa R.
O-063
Sciandrello Giulia
P-039 P-073
Scott Andrew
P-178
Scuric Zorica
O-022
Segerbäck D
P-022
Segesdi Judit
P-002 P-010
Seidel Albrecht
O-058 O-098
Seo Bo-Young P-037 P-161 P-009 P-162 O-092
Sepai Ovnair
O-125
Sestakova Helena
P-101 O-096
Sevastyanova Oksana
O-080 P-081
Sfikakis Petros P
P-079 P-077
Shaikhaev Gadjiramazan O.
P-064
Shaughnessy Daniel T
O-038
Shelling Andrew
O-043
Shevchenko Vladimir A.
P-064
Shuichi Adachi
P-016
Schabath Matthew B
O-050
Scheepers Paul
P-100
Schiestl Robert H
O-022 P-075 P-074
Schläwicke Karin
P-124
Schliebe Barbara
O-038
Schmeiser Heinz H.
P-193 P-005
Schmezer Peter
P-087
Schmid Oliver
O-073
Schoemaker Minouk
P-120
Schoket Bernadette
P-002 P-010
Schuetz Petra
O-073
Schul Vitor Basso
P-094
Schunck Christian
O-007
Schwarz Claudia
P-148
Siala Konrad
O-069
Siciliano Gabriele
P-114
Sijtsma Lolke
P-164 P-153
Sikora Anna
P-057
Silva Maria Joao
P-104 P-133 P-199 P-149
Silva Susana
P-018 P-147
Simic Draga
P-154
Singh Rajinder
P-024
Sinha Rashmi
O-037 O-034
Siniscalchi Ester
P-031
Sintorn Ida-Maria
O-010
Siomek Agnieszka
P-046
Sirajuddin Paul
P-202
Sirota Nikolai P.
P-076
Skerfving Staffan
P-124
Slapsyte Grazina
P-150
Slupphaug Geir
O-020
Slyskova Jana
P-019
Smerak Petr
P-101 O-096
Smid Jiri
P-007
Smith Elizabeth C
O-026
Soares Sandra De Aguiar
P-094
Sobus J.
P-136
Sofia Pavanello
P-105
Solansky Ivo
O-047 P-021
Soltesz Ibolya
P-002 P-010
Soucek Pavel O-112 P-192 O-068 O-126 O-111
Souliotis Vassilis L
P-078 P-024 P-079 P-077
Sousa Ana Carla
P-199 P-149
Soussaline Françoise
P-188 O-008
Spatz A.
O-027
Speit Guenter
O-071 O-073
Spenkelink Bert
P-038
Spitz Margaret R
O-050
Sram Radim J P-202 O-053 O-051 O-056 O-047
P-081 O-089
Stagi Elena
O-089
Stambrook Peter J.
O-079
Stankevicins Luiza
O-024
Stanojevic Jasna
P-154
Steiblen Guy
P-191
Steiner Claudia
O-033
Stenhouse A.
P-163
Stephanou Georgia
P-040
Stetina Rudolf
O-069 P-025 P-080 O-126
Stiborova Marie P-192 O-077 O-092 P-197 P-193
Stojkovic Ranko
P-200
Stožický František
O-069
Strömberg Ulf
P-124
Sudhölter Ernst Jr
O-040
Sueoka Eisaburo
O-028
Sugimura Takashi
P-106
Suh Soo Kyung
P-196
Suhonen Satu
P-134
Sunter Osman
P-006 P-044
Surrallés Jordi
O-049
Suter Willi
O-002
Suvaci Duygu Erol
P-042
Suziedelis Kestutis
P-090
Svecova Drahomira
O-004
Svihalkova-Sindlerova Lenka
O-080
Svoboda Peter
O-039
Sycheva Ljudmila P.
P-151
Sylla A
P-022
Szaefer Hanna
P-129 P-128
Szekely Gabor
O-076
Szpila Anna
P-046
Szukala K
O-087
Szweda P.
P-163
Szyfter Krzysztof
O-068 P-049 O-087
Šafranić Amalija
P-059
Šilhánová Eva
P-121
Šmerák Petr
P-080
Šrám Radim J.
P-021 O-123 O-127
Švecová Vlasta
O-123
Taga Masataka
P-011
Tager I.
O-052
O-106 O-108
Taioli Emanuela
Takahashi Y.
O-063 P-054
Takeji Takamura-Enya
P-016
Tanaka Kimio
O-025
Tanaka Satoshi
O-025
Tarabčáková Daniela
P-136 P-137
Taylor Jack
O-038
Teitelbaum Susan L
O-114
Teixeira Joao Paulo
P-018 P-147
Terrados Gloria
O-032
Terry Mary Beth
O-114
26.6.2006 16:06:14
AUTHORS INDEX
Thierens Hubert
P-125
Thybaud Véronique
O-085 O-113
Tinnerberg Håkan
P-110
Tomášková Hana
P-141 P-173
Topinka Jan
O-080 O-127 O-056 P-081
Törnqvist M.
O-122 P-100 O-097
Torralba Y
P-146
Totsuka Yukari
P-106
Toutain Hervé
P-172
Trebatická Mária
P-174
Trefil Ladislav
O-069
Trosko James
O-118
Tudek Barbara
P-060
Tuffnell Derek
P-032
Tuimala Jarno
P-116
Tulinská Jana
P-184
Tulupova Elena
O-112 P-019 P-123
Turner P C
P-022
Tweats David
O-086 O-085
Typas Dimitris
P-078
Tyrakis Michael
P-040
Umbach David M
O-038
Ünal Fatma
P-083 P-084 P-082
Upham Brad
O-081
Urbanek Katarzyna
O-082
Uysal Mehmet
P-042
Vaclavikova Radka
O-068
Vhater M
P-194
Vainio Harri
P-122 O-067
Vaitiekunaite Rasa
P-069
Valovičová Zuzana
O-062
Van Beek Teris A
O-040
Van Beelen Vincent A.
P-164 P-153
Van Delft Joost H
O-053 O-018
Van Den Broecke Rudy
P-125
Van Den Oord J.J.
O-027
Van Herwijnen Marcel H
O-053
Van Leeuwen Danitsja M
O-053
Van Ravenzwaay Bennard
O-048
Van Schooten Frederik J
O-023 O-073 O-045
Varvařovská Jana
O-069
Veeriah Selvaraju
P-131 O-033
Vehmas Tapio
P-122 O-067
Verea H
P-146
Vermeulen Roel
O-037 P-140
Vessby Bengt
P-124 P-013
Veznik Zdenek
O-004
Vieu Emilie
P-144
Vikström Anna
P-100
Villalobos-Pietrini Rafael
P-187 P-127
P-107
Villani Paola
Vincent Paget
P-188
Vineis Paolo
O-072 O-108 O-113 O-106
Violante Francesco Saverio
P-004
Vlčková Viera
P-174 P-098
Vodicka Pavel O-112 P-019 P-123 O-126 P-056
P-080
Vodickova Ludmila O-112 P-019 P-123 O-126
P-080
Volčič Meta
P-088
Völkner Wolfgang
P-176
Vondracek Jan O-080 O-081 P-089 O-060 P-091
Voronina Ekaterina S.
P-152
Vral Anne
P-125
Vuković Lidija
P-093
Vukovic-Gacic Branka
P-158 P-154
Waidyanatha S.
P-136
Wakabayashi Keiji
P-106
Waliszewski Stefan M.
P-127
Walmsley Richard M
O-088
Warholm Margareta
P-110
Wasielwska Antonina Cebulska
O-051
Weiland Christina
O-016
Wendt Maria
O-017
Weninger Marietta
P-148
Whitelaw Donald
P-032
Wiadrowska Bożena
O-082
Wierzbicka Malgorzata
P-049
Wiesmueller Lisa
P-026 P-068 P-041
Wiessler Manfred
P-005
Wild C P
P-022 P-071 O-026
Willems Petra
P-125
Windebank Sam
P-178
Winn Richard N
O-105
Winnepenninckx V.
O-027
Wirnitzer Uta
P-086
Wise Carolyn
P-159
Wlodarczyk Barbara
P-111
Woelfelschneider Andreas
P-087
Wolfe Kevin J.
P-052
Wölfl Stefan
P-131
Wollny Hans-Eric
P-176
Wsólová Ladislava
P-008 P-184
Wu Hsiu-Ching
P-185 P-201 P-027
Wyatt Natalie P
P-032
Xu Zong-Li
O-038
Yamada Masami
O-021 P-165
Yan Huifang
O-125
Yap Poh-Sin
O-056
Yılmaz Serkan
P-082
Yılmaz Serkan
P-083
Yılmaz Serkan
P-084
Yoon Yoe-Chang
P-009
Yuasa Yasuhito
O-080 O-057
Yurdakul Zafer
P-042
Yüzbaşıoğlu Deniz
P-083 P-084 P-082
Zabielski Romuald
P-060
Zajc Irena
P-055
Zajicova Atanaska
O-004
Zakharova Irina G.
P-076
Závacká Ivona
P-142
Zegura Bojana
P-158
Zeisig Magnus
P-028
Zeljezic Davor
P-029 P-030
Zhanataev Ali K.
P-152
Zhang Fangfang
O-114
Zidzik Jozef
P-066
Zierer Otto
P-148
Zijno Andrea
P-031
Zivkovic Lada Gordana
P-179
Zock JP
P-146
Žegura Bojana
P-088 P-055
Želježić Davor
P-183
247
26.6.2006 16:06:14
26.6.2006 16:06:15
LIST
OF PARTICIPANTS
26.6.2006 16:06:16
Abrahamsson-Zetterberg Lilianne
Alink Gerrit
Swedish Food Administration
Toxicology
Box 622
75126 Uppsala
Sweden
[email protected]
Wageningen University
Toxicology
Tuinlaan 5
6703 HE Wageningen
Netherlands
[email protected]
Agudo Antonio
Aliyazicioglu Yuksel
Catalan Institute of Oncology
Epid. and Cancer Registry
Avda. Gran Via Km 2,7 s In
8907 L´hospitalet De Llob.
Spain
[email protected]
Ondokuz Mayis University
Biochemistry
Kurupelit
55139 Samsun
Turkey
[email protected]
Ahr Hans
Alvur Muhlise
Bayer Healthcare AG
Molecular and Special Toxicology
Aprather Weg
42096 Wuppertal
Germany
[email protected]
Biochemistry
Kurupelit
55139 Samsun
Turkey
[email protected]
Aimova Dagmar
Anderson Diana
Faculty of Science, Charles University
Department of Biochemistry
Albertov 2030
12840 Prague 2
Czech Republic
[email protected]
The University of Bradford
Dept. of Biomedical Sciences
Richmond Road
BD7 1DP Bradford
United Kingdom
[email protected]
Aiub Claudia
Andreoli Cristina
Salzburg University
Cell Biology
Imbergstrasse 13, 1 stock
A-5020 Salzburg
Austria
[email protected]
Bat-Italia Spa
Research
Amsterdam 147
144 Rome
Italy
[email protected]
Akesson Bjorn
Andrianopoulos Konstadinos
Lund University
Biomedical Nutrition
POBox 124
SE-22100 Lund
Sweden
[email protected]
University of Patras
Biology
RION
26500 Patras
Greece
[email protected]
Aksoy Hüseyin
Andrysik Zdenek
Gari University
Biology
Tehnikokullar
6500 Ankara
Turkey
[email protected]
Veterinary Research Institute
Department of Toxicology
Hudcova 70
621 00 Brno
Czech Republic
[email protected]
Albertini Richard
Angelini Sabrina
University of Vermont, Genetic Toxicology Lab
Pathology
656 Spear Street
5401 Burlington, Vermont
United States
[email protected]
University of Bologna
Pharmacology
via Irnerio, n 48
40126 Bologna
Italy
[email protected]
250
26.6.2006 16:06:16
Anna Livia
Baan Robert
Fodor Jozsef National Center For Public Health
Department of Molecular Environmental Epidemiology
Gyali ut 2-6.
1097 Budapest
Hungary
[email protected]
WHO - International Agency For Research On Cancer
Carcinogen Identification and Evaluation Group
150, cours Albert Thomas
69008 Lyon
France
[email protected]
Aoki Yasunobu
Baer-Dubowska Wanda
National Institute For Environmental Reserch
Reasearch Center For Environmental Risk
16-2 Onogawa
305-8506 Tsukuba
Japan
[email protected]
Poznan University of Medical Sciences
Pharmaceutical Biochemistry
Grunwaldzka 6
60-780 Poznan
Poland
[email protected]
Arbillaga Leire
Bajic Vladan
University of Navarra
Food Sciences and Toxicology
Irunlarrea 1
31008 Pamplona
Spain
[email protected]
Galenika Pharmaceuticals
Department For Biomedical Research
Batajnicki drum b.b.
11080 Belgrade
Serbia and Montenegro
[email protected]
Arlt Volker Manfred
Ballantyne Mark
Section of Molecular Carcinogenesis
Institute of Cancer Research
Brookes Lawley Building
SM2 5NG Sutton, Surrey
United Kingdom
[email protected]
Covance Laboratories Ltd.
Genetic & Molecular Toxicology
Otley Road
HG3 1PY Harrogate
United Kingdom
[email protected]
Assennato Giorgio
Barale Roberto
University of Bari
Dimimp
Piazza G.Cesare-Policlinico
70124 Bari
Italy
[email protected]
University of Pisa
Biology
Via S. Giuseppe n.22
56100 Pisa
Italy
[email protected]
Au William
Barbata Giuseppa
Uversity of Texas Medical Branch
Preventive Medicine
700 Harborside drive
77555-111 Galveston
United States
[email protected]
Universitŕ Di Palermo
Biologia Cellulare E Dello Sviluppo
Viale delle Scienze - Parco d'Orleans
90128 Palermo
Italy
[email protected]
Aubrecht Jiri
Bartoszek Agnieszka
Pfizer
Safety Sciences
Eastern Point Rd
6340 Groton
United States
jiri.aubrecht@pfizer.com
Gdansk University of Technology
Dept. of Pharmaceutical Technology and Biochemistr
Narutowicza 11/12
80-952 Gdansk
Poland
[email protected]
Autrup Herman
Basaran Ahmet
Public Health
Enviromental and Occupational Medicine
Vennelyst Blvd
8000 Aarhus
Denmark
[email protected]
Hacettepe University
Siraselviler Cod. 48/8
34433 Istanbul
Turkey
[email protected]
251
26.6.2006 16:06:18
Basaran Nursen
Beric Tanja
Hacettepe University
Siraselviler Cad. 48/8
34433 Istanbul
Turkey
[email protected]
Laboratory for Microbiology
Studentski trg 16
11000 Belgrade
[email protected]
Baumann Cindy
Bialkowski Karol
Universitäts Frauenklinik
Enodokrinologie/Onkologie
Prittwitzstr. 43
89075 Ulm
Germany
[email protected]
Collegium Medicum, Nicolaus Copernicus University
Department of Clinical Biochemistry
Karlowicza 24
85-092 Bydgoszcz
Poland
[email protected]
Baumeister Manfred
Binderup Mona-Lise
Boehringer Ingelheim Pharma GmbH and Co. KG
Non-Clinical Drug Safety
Birkendorfer Str. 65
88397 Biberach
Germany
[email protected]
Danish Institute of Food and Veterinary Research
Department of Toxicology and Risk Assessment
Moerkhoej Bygade 19
DK-2860 Soeborg
Denmark
[email protected]
Bavoux Clarisse
Bingham Sheila
BERPC
62 rue d'Hauteville
75010 Paris
France
[email protected]
MRC
Dunn Human Nutrition Unit
Hills Road
CB22XY Cambridge
United Kingdom
[email protected]
Bedir Abdulkerim
Blanco Luis
Biochemistry
Kurupelit
55139 Samsun
Turkey
[email protected]
Spanish Research Council
Centro de Biologia Molecular Severo Ochoa
Campus Universidad Autonoma
28049 Canto Blanco, Madrid
Spain
[email protected]
Belinsky Steven
Boffetta Paolo
Lovelace Respiratory Research Institute
Lung Cancer
2425 Ridgecrest Drive SE
87108 Albuquerque
United States
[email protected]
IARC
150 cours Albert Thomas
69008 Lyon
France
[email protected]
Bell Douglas
Bolognesi Claudia
National Institute of Environmental Health Science
Laboratory of Molecular Genetics
PO Box 12233
27709 Research Triangle Park
United States
[email protected]
National Institute for Reasearch on Cancer
Environmental Carcinogenesis Unit
L.go Rosanna Benzi, 10
16132 Genova
Italy
[email protected]
Béres Erzsébet
Bonassi Stefano
Lab International Hungary Research Institute
Mammalian Mutagenesis
Veszprém-Szabadságpuszta
8200 (8201 PBOX.: 384) Veszprém
Hungary
[email protected]
National Cancer Research Institute
Molecular Epidemoilogy
Largo Rosanna Benzi, 10
I-16132 Genova
Italy
[email protected]
252
26.6.2006 16:06:18
Bonatto Diego
Cammerer Zoryana
Instituto de Biotecnologia
Universidade De Caxias Do Sul
Rua Francisco Getulio Vargas 1130 – Bloco 57
95070-560 Caxias Do Sul
Brazil
[email protected]
Novartis Pharma AG
Genetic Toxicology and Safety Pharmacology
Auhafenstrasse
4002 Basel
Switzerland
[email protected]
Botsivali Maria
Carbone Fabio
National Hellenic Research Foundation
Institute of Biological Research and Biotechnology
48 Vassileos Constantinou Avenue
11635 Athens
Greece
[email protected]
Department of Pharmacology
Via Irnerio 48
40126 Bologna
Italy
[email protected]
Broberg Karin
Carmichael Paul
Lund University
Occupational and Environmental Medicine
Lund University
22185 Lund
Sweden
[email protected]
Unilever
SEAC
Sharnbrook
MK44 1LQ Bedfordshire
United Kingdom
[email protected]
Brunborg Gunnar
Cebulska-Wasilewska Antonina
Norwegian Institute of Public Health
Chemical Toxiology
Geitmyrsveien 75
403 Oslo
Norway
[email protected]
Collegium Medicum Jagiellonian University
Chair of Epidemiology and Preventive Medicine
Kopernika 7
31-034 Krakow
Poland
[email protected]
Butkiewicz Dorota
Cemeli Eduardo
Center of Oncology, M.Sklodowska-Curie Institute
Dept. of Tumor Biology
Wybrzeze Armii Krajowej 15
44-101 Gliwice
Poland
[email protected]
University of Bradford
Biomedical Sciences
Richmond Road
BO18 3AN Bradford
United Kingdom
[email protected]
Cabrera Guillermo
Cermakova Zuzana
Universidad Autonoma de Querétaro
Cerro de las Campanas s/n,Col Las Campanas
76010 Querétaro
Mexico
[email protected]
University Ostrava
Medical-Social Faculty
Syllabova 19
703 00 Ostrava
Czech Republic
[email protected]
Cachot Jérôme
Clemenes Julie
University of Le Havre
Laboratory of Ecotoxicology
25 rue Philippe Lebon, B.P. 540
76058 Le Havre
France
[email protected]
Covance
Otley Road
Harrogate, N. Yorkshire
United Kingdom
[email protected]
Caldecott Keith
Colacci Annamaria
University of Sussex
Genome Damage and Stability Centre
Science Park Road
BN19RQ Falmer Brighton
United Kingdom
[email protected]
Environmental Protection Agency
Excellence Environmental Carcinogenesis
Viale Filopanti 20
40126 Bologna
Italy
[email protected]
253
26.6.2006 16:06:19
Collins Joanne
Černá Milena
GlaxoSmithKline
Genetic Toxicology
Park Road
SG12 ODP Ware
United Kingdom
[email protected]
Charles University
Centrum Prev. Medicine
Ruská 87
10000 Praha
Czech Republic
[email protected]
Coppedè Fabio
Dafou Dimitra
University of Pisa
Department of Neurosciences
Via Roma 67
56126 Pisa
Italy
[email protected]
University College London
Gynaecological Oncology
46 Cleveland street
W1T 4JF London
United Kingdom
[email protected]
Cordelli Eugenia
De Boeck Marlies
ENEA
Section of Toxicology and Biomedical Sciences
CR Casaccia, via Anguillarese 301
60 Rome
Italy
[email protected]
J&J Pharmaceutical Research & Development
Genetic & In Vitro Toxicology
Turnhoutseweg 30
2340 Beerse
Belgium
[email protected]
Cornelius Michael
De Flora Silvio
Karolinska Institute
Biosciences and Nutrition
Novum
S-14 157 Huddinge
Sweden
[email protected]
University of Genoa
Department of Health Sciences
Via A. Pastore
I-16132 Genoa
Italy
[email protected]
Corsi Patrizia
De Kok Theo
University of Bari
Farmacologia E Fisiologia Umana
Piazza Giulio Cesare N. 11
70124 Bari
Italy
labo@fisiol.uniba.it
University Maastricht
Health Risk Analysis and Toxicology
P.O. Box 616
6200 MD Maastricht
Netherlands
[email protected]
Corvi Raffaella
Deac Liana Monica
IHCP, JRC of the European Commission
ECVAM
Via E. Fermi 1
21020 Ispra (Va)
Italy
[email protected]
Public Health Institute
Epidemiologie
Louis Pasteur 6
Clui-Napola
Romania
[email protected]
Coskun Erdem
Decordier Ilse
Gazi University, Faculty of Pharmacy
Toxicology
Hipodrom
6330 Ankara
Turkey
[email protected]
Vrije Universiteit Brussel
Laboratorium Voor Cellulaire Genetica
Pleinlaan 2
1050 Brussels
Belgium
[email protected]
Czene Stefan
Dechariaux Huguette
Astrazeneca R-D Södertälje
Safety Assessment/Sag
15185 Södertälje
Sweden
[email protected]
Ministry of Health
DGS
14 avenue Duquesne
75350 Paris
France
[email protected]
254
26.6.2006 16:06:20
Dejmková Eva
Dogliotti Eugenia
Institute of Experimental Medicine, AS CR
LGE
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
Istituto Superiore di Sanità
Environment and Primary Prevention
299 Viale Regina Elena
161 Rome
Italy
[email protected]
DeMarini David
Doherty Ann
US Environmental Protection Agency
Environmental Carcinogenesis Division
B143-06
27711 Research Triangle Park, NC
United States
[email protected]
Astrazeneca Pharmaceuticals
Genetic Toxicology, Safety Assessment
Mereside, Alderley Park, Macclesfield
SK10 4TG Cheshire
United Kingdom
[email protected]
Dertinger Stephen
Dominguez Inmaculada
Litron Laboratories
Research
200 Canal View
14623 Rochester
United States
[email protected]
Faculty of Biology University of Sevilla
Cell Biology
Avda Reina Mercedes 6
41012 Sevilla
Spain
[email protected]
Dias Elsa
Donner Maria
National Institute of Health
Water Quality Center
Av. Padre Cruz
1649-016 Lisbon
Portugal
[email protected]
Dupont Haskell Laboratory
BMT/Genetic Toxicology
1090 Elkton Road
19714-0050 Newark, Delaware
United States
[email protected]
Diaz Pohl Cecilia
Dostal Miroslav
Astra Zeneca R&D Sodertalje
Safety Assessment/Genetic Toxicology
SE-15185 Sodertalje
Sweden
[email protected]
LGE
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
Djurinec Martina
Dracinska Helena
Institute for Medical Research and Occupational Health
Mutagenesis Unit
Ksaverska 2
10000 Zagreb
Croatia
[email protected]
Albertov 2030
12840 Prague 2
Czech Republic
[email protected]
Dobiáš Lubomír
Dziaman Tomasz
Medico-Social Faculty of Ostraviensis University
Genetic Toxicology and Genetic
Syllabova 19
700 30 Ostrava
Czech Republic
[email protected]
Collegium Medicum Nicolaus Copernicus University
Karlowicza 24
85-092 Bydgoszcz
Poland
[email protected]
Dobrzyńska Małgorzata
Džupinková Zuzana
National Institute of Hygiene
Dept of Radiation Protection and Radiobiology
24 Chocimska Str
00-791 Warsaw
Poland
[email protected]
Research Base of Slovak Medical University
Experimental and Applied Genetics
Limbová 12
833 03 Bratislava
Slovakia
[email protected]
255
26.6.2006 16:06:20
Ekström Christina
Evenson Don
Astrazeneca R-D Södetälje
15185 Södertälje
Sweden
christina.ekströ[email protected]
South Dakota State University
Biology and Microbiology
807 32nd Ave
57006 Brookings, SD
United States
[email protected]
Elespuru Rosalie
Falck Ghita
FDA/CDRH
Division of Biology
HFZ-110 9200 Corporate Blvd
MD 20850 Rockville
United States
[email protected]
Finnish Institute of Occupational Health
New Technologies and Risks
Topeliuksenkatu 41a A
FI-00250 Helsinki
Finland
ghita.falck@ttl.fi
Elliott Barry
Farkasova Timea
Syngenta CTL
Human Safety
Alderley Park
SK10 4TJ Macclesfield
United Kingdom
[email protected]
Cancer Research Institute
Dep. of Mutagenesis and Carcinogenesis
Vlarska 7
83391 Bratislava
Slovakia
[email protected]
Ellis Patricia
Farmer Peter
GlaxoSmithKline
Genetic Toxicology
Park Road
SG12 ODP Ware
United Kingdom
[email protected]
Biocentre, University of Leicester
Cancer Biomarkers and Prevention Group
University Road
LE1 7RH Leicester
United Kingdom
[email protected]
El-Zein Randa
Faust Dagmar
The University of Texas Md Anderson Cancer Center
Epidemiology
1155 Pressler Street, #1340
77030 Houston
United States
[email protected]
University of Mainz
Institute of Toxicology
Obere Zahlbacherstr. 67
55131 Mainz
Germany
[email protected]
Emeny Jean
Fenech Michael
University of Leicester
Biocentre
University Road
LE1 7RH Leicester
United Kingdom
[email protected]
CSIRO Human Nutrition
Genome Health Nutrigenomics
Gate 13 Kintore Avenue
5000 Adelaide
Australia
[email protected]
Episkopou Hara
Ferguson Lynnette
48, Vassileos Const. Ave.
11635 Athens
Greece
[email protected]
University of Auckland
Nutrition
85 Park Road, Grafton
1001 Auckland
New Zealand
[email protected]
Erminio Marafante
Ferk Franziska
EC Joint Research Centre
IHCP
Via Fermi 1
21020 Ispra
Italy
[email protected]
Medical University Vienna, Institute of Cancer Research
Toxicology
Borschkegasse 8a
1090 Vienna
Austria
[email protected]
256
26.6.2006 16:06:21
Filipic Metka
Fucic Aleksandra
National Institute of Biology
Dep. of Genetic Toxicology and Cancer Biology
Vecna pot 111
1000 Ljubljana
Slovenia
metka.fi[email protected]
Institute for Medical Research and Occup Health
Ins Med Res Occup Health
Ksaverska c 2
10000 Zagreb
Croatia
[email protected]
Foksinski Marek
Fujikawa Kazuo
Karlowicza 24
85-092 Bydgoszcz
Poland
[email protected]
Kinki University
Faculty of Science and Technology
Kawakae 3-4-1
855-5702 Higashiosaka
Japan
[email protected]
Franekic Čolic Jasna
Gabelova Alena
Faculty of Food Technology and Biotechnology
Department of Biochemical Engineering
Pierottijeva 6
10000 Zagreb
Croatia
[email protected]
Mutagenesis and Carcinogenesis
Vlarska 7
83391 Bratislava
Slovakia
[email protected]
Frassinetti Stefania
Gackowski Daniel
NRC National Research Council
IBBA
Via Moruzzi 1
56124 Pisa
Italy
[email protected]
Karlowicza 24
85-092 Bydgoszcz
Poland
[email protected]
Frei Eva
Gajdoš Andrej
Deutsches Krebsforschungszentrum
Molecular Toxicology
Im Neuneheimer Feld 280
69120 Heidelberg
Germany
[email protected]
Regional Public Health Authority
Genetic Toxicology
Ipelska 1
042 20 Košice
Slovakia
[email protected]
Frieauff Wilfried
Gajdošová Dagmar
Novartis Pharma
SP&A / Genetic Toxicology
MUT-2881.507
CH-4002 Basel
Switzerland
[email protected]
Regional Public Health Authority
Genetic Toxicology
Ipelska 1
042 20 Košice
Select Country
[email protected]
Friedmann Angeli José Pedro
Gallo Valentina
Universidade Estadual de Londrina
Biologia General
Rua Governador Valadares 641
86061150 Londrina
Brazil
[email protected]
Fondazione I.S.I.
Viale Settimio Severo, 65
10133 Torino
Italy
[email protected]
Froetschl Roland
Gamboa Da Costa Goncalo
Federal Institute for Drugs and Medical Devices
Genetic and Reproductive Toxicology
Kurt-Georg-Kiesinger-Allee 3
53175 Bonn
Germany
[email protected]
The Institute of Cancer Research
Cotswold Road
SM2 5NG Suton Surrey
United Kingdom
[email protected]
257
26.6.2006 16:06:22
Garaj-Vrhovac Verica
Gritzko Kristin
Mutagenesis Unit
Ksaverska 2
10000 Zagreb
Croatia
[email protected]
Bsl Bioservice Scientific Laboratories GmbH
In Vitro
Behringstr. 6
82152 Planegg
Germany
[email protected]
Georgiadis Panagiotis
Gromadzinska Jolanta
48 Vassileos Constantinou Ave.
11635 Athens
Greece
[email protected]
Nofer Institute of Occupational Medicine
8 Teresy st.
91348 Lodz
Poland
[email protected]
Gervais Veronique
Grummt Tamara
Biologie Servier
Biologie Servier
Route de Saran 905
45520 Gidy
France
[email protected]
Uba
Toxicology
H-Heine str. 12
8645 Bad Elster
Germany
[email protected]
Giefing Maciej
Gruz Petr
Institute of Human Genetics
Pol Acad Sci
Strzeszynska 32
60-479 Poznan
Poland
[email protected]
National Institute of Health Sciences
Genetics and Mutagenesis
Kamiyoga, Setagaya-Ku 1-18-1
158-8501 Tokyo
Japan
[email protected]
Gichner Tomáš
Guarrera Simonetta
Institute of Experimental Botany AS CR
Dep. of Genetics
Na Karlovce
16000 Praha 6
Czech Republic
[email protected]
Fondazione I.S.I.
Villa Gualino - V.le Settimio Severo 65
10133 Torino
Italy
[email protected]
Glatt Hansruedi
Guengerich F. Peter
German Institute of Human Nutrition (DIfE) Potsdam
Department of Nutritional Toxicology
Arthur-Scheunert-Allee 114-116
14558 Nuthetal
Germany
[email protected]
Vanderbilt University School of Medicine
Biochemistry
23rd & Pierce Avenues
37232-0146 Nashville, TN
United States
[email protected]
Gómez-Arroyo Sandra
Guizon Isabelle
Centro de Ciencias de La Tmosfera, Unam
Ciencias Ambientales
Circuito Exterior Ciudad Universitaria
4510 México
Mexico
[email protected]
Sanofi Aventis
Drug Safety Evaluation
13 Quai Jules Guesde
94403 Vitry Sur Seine
France
isabelle.guizon@sanofi-aventis.com
Grico Nina Sabine
Gundy Sarolta
Johannes Gutenberg University Mainz
Obere Zahlbacher Str. 67
55131 Mainz
Germany
[email protected]
National Institute of Oncology
Cytogenetics
Rath Gy. 7-9.
1122 Budapest
Hungary
[email protected]
258
26.6.2006 16:06:23
Gungor Nejla
Hanke Wojciech
Maastricht University
Department of Health Risk Analysis and Toxicology
P.O. Box 616
6200 MD Maastricht
Netherlands
[email protected]
8 teresy st.
91348 Lodz
Poland
[email protected]
Gurská Soňa
Hánová Monika
Laboratory of Mutagenesis and Carcinogenesis
Vlárska 7
833 91 Bratislava
Slovakia
[email protected]
Department of Genetic and Molecular Toxicology
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
Guz Jolanta
Hanzalová Kateřina
Karlowicza 24
85-092 Bydgoszcz
Poland
[email protected]
LGE
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
Gyorffy Erika
Harris Curtis
Fodor Jozsef National Center for Public Health
Dept. of Molecular Environmental Epidemiology
Gyali ut 2-6.
1097 Budapest
Hungary
[email protected]
National Cancer Institute
Lab. Human Carcinogenesics
37 Convent Dr
20892 Bethesda, MD
United States
[email protected]
Haglund Johanna
Hasnaà Haddouk
Stockholm University
Environmental Chemistry
SvanteArrheniusvag 12
106 91 Stockholm
Sweden
[email protected]
CIT
Mutagenese
BP 563
27005 Evreux
France
[email protected]
Hainaut Pierre
Haugen Aage
International Agency for Research on Cancer
Molecular Carcinogenesis & Biomarkers Group
150 Cours Albert Thomas
69372 Cedex 08 Lyon
France
[email protected]
National Institute of Occupational Health
Section For Toxicology
PO Box 8149 Dep
33 Oslo
Norway
[email protected]
Haldsrud Renate
Hayashi Tomonori
Norwegian University of Science and Technology
Department of Biology
Høgskoleringen 5
7491 Trondheim
Norway
[email protected]
Radiation Effects Research Foundation
Radiobiology/Molecular Epidemiology
5-2, Hijiyama Park, Minami Ward
732-0815 Hiroshima
Japan
[email protected]
Hamatani Kiyohiro
Hayes Julie
5-2 Hijiyama Park, Minami-ku
732-0815 Hiroshima
Japan
[email protected]
AstraZeneca Pharmaceuticals
Genetic Toxicology, Safety Assessment
Mereside, Alderely Park, Macclesfield
SK10 4TG Cheshire
United Kingdom
[email protected]
259
26.6.2006 16:06:24
Hedmer Maria
Hernández-Valero María A.
Lund University
Lund University Hospital
SE-221 85 Lund
Sweden
[email protected]
The University of Texas MD Anderson Cancer Center
Epidemiology
1515 Holcombe Blvd. Unit 639
77030 Houston
United States
[email protected]
Heijink Liesbeth
Hetland Ragna Bogen
Solvay Pharmaceuticals
Non-Clinical Drug Safety
CJ van Houtenlaan 36
1381 CP Weesp
Netherlands
[email protected]
Division of Environmental Medicine
Geitmyrsveien 75
NO-0403 Oslo
Norway
[email protected]
Heilimo Iiris
Hiraku Yusuke
Topeliuksenkatu 41 a A
FI-00250 Helsinki
Finland
iiris.heilimo@ttl.fi
Mie University Graduated School of Medicine
Environmental and Molecular Medicine
2-174 Edobashi
514-8507 Tsu, Mie
Japan
[email protected]
Hein David
Hirvonen Ari
University of Louisville School of Medicine
Pharmacology and Toxicology
500 South Preston Street
40292 Louisville
United States
[email protected]
FI-00250 Helsinki
Finland
ari.hirvonen@ttl.fi
Heo Yong
Hockley Sarah
Catholic University of Daegu
Occupational Health
330 Kumrak 1 ri, Hayang eup
712-702 Kyongsan Si, Kyongbuk Province
Korea
[email protected]
Molecular Carcinogenesis
15 Cotswold Road
KT19 9EB Sutton
United Kingdom
[email protected]
Herbold Bernd
Hoffmann George
Bayer Healthcare AG
Gebäude 514
P.O. Box 101709
42096 Wuppertal
Germany
[email protected]
Holy Cross College
1 College Street
1610 Worcester, MA
United States
[email protected]
Herceg Zdenko
Hoffmann Sebastien
International Agency for Research on Cancer
Molecular Carcinogenesis
Cours Albert Thomas
69372 Lyon
France
[email protected]
IPBS CNRS
Genetic Instability and Cancer
205 route de narbonne
31077 Toulouse
France
[email protected]
Herlová Jana
Holland Nina
LGE
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
University of California, Berkeley
School of Public Health
759 University Hall
94517-7360 Berkeley
United States
[email protected]
260
26.6.2006 16:06:25
Honarvar Naveed
Ide Hiroshi
RCC
In Vivo Genotoxicity
In den Leppsteinswiesen 19
64380 Rossdorf
Germany
[email protected]
Graduate School of Science, Hiroshima University
Department of Mathematical and Life Sciences
Kagamiyama 1-3-1
739-8526 Higashi-Hiroshima
Japan
[email protected]
Honma Masamitsu
Ignatowicz Ewa
Division of Genetics and Mutagenesis
1-18-1 Kamiyoga
158-8501 Setagaya
Japan
[email protected]
University of Medical Sciences
Department of Pharmaceutical Biochemistry
Grunwaldzka 6
60-780 Poznan
Poland
[email protected]
Horská Alexandra
Imai Kazue
Research Base of Slovak Medical University
Department of Experimental and Applied Genetics
Limbová 12
83303 Bratislava
Slovakia
[email protected]
Radiation Effects Research Institute
5-2 Hijiyama-Park Minami-ku
732-0815 Hiroshima
Japan
[email protected]
Hreljac Irena
Izzotti Alberto
Dept of Genetic Toxicology and Cancer Biology
Vecna pot 111
1000 Ljubljana
Slovenia
[email protected]
University of Genoa
Health Sciences
Via Pastore 1
I-16132 Genoa
Italy
[email protected]
Hynes Geoffrey
Jarmalaite Sonata
Huntingdon Life Sciences
Genetic Toxicology
Eye Research Centre, Occold
IP23 7PX Suffolk
United Kingdom
[email protected]
Vilnius University
Nature Faculty
Ciurlionio 21
3101 Vilnius
Lithuania
[email protected]
Chadt Jiří
Jaworski Albert
LGE
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
Intitute of Biochemistry and Biophysics, PAS
Molecular Biology
Pawinskiego 5A
02-106 Warsaw
Poland
[email protected]
Chiang Su-Yin
Jeppsson Marina
China Medical University
Institute of Chinese Medical Science
91 Hsueh-Shih Road,
40402 Taichung
Taiwan
[email protected]
Lund University
Dept. of Occupational and Environmental Medicine
University Hospital
SE 221 85 Lund
Sweden
[email protected]
Chvátalová Irena
Jeurissen Suzanne
LGE
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
Toxicology
Tuinlaan
6703 HE Wageningen
Netherlands
[email protected]
261
26.6.2006 16:06:26
Johansson Clara
Kapka Lucyna
Karolinska Institutet
NOVUM
14157 Huddinge
Sweden
[email protected]
Institute of Occupational Medicine and Enviromental
Genetic Taxicology
Koscielna 13
41-200 Sosnowiec
Poland
[email protected]
Jonsson Bo
Karahalil Bensu
University Hospital
22185 Lund
Sweden
[email protected]
Gazi University Faculty of Pharmacy
Toxicology Department
Hipodrom
6330 Ankara
Turkey
[email protected]
Jönsson Lena S
Karlsson Hanna
Lund University
Depart. of Occupational and Environmental Medicine
Lund University Hospital
SE 221 85 Lund
Sweden
[email protected]
NOVUM
14157 Huddinge
Sweden
[email protected]
Joosten Harrie
Kasai Hiroshi
Organon
Toxicology and Drug Disposition
P.O. Box 20
5340 BH Oss
Netherlands
[email protected]
Univ. of Occupational and Environmental Health
Department of Environmental Oncology
1-1 Iseigaoka, Yahatanishi-ku
807-8555 Kitakyushu
Japan
[email protected]
Jurén Tina
Kawai Kazuaki
Department of Biosciences and Nutrition at NOVUM
Hälsovägen 7
SE-141 57 Huddinge
Sweden
[email protected]
University of Occupational and Environmental Health
Department of Environmental Oncology
1-1 Iseigaoka, Yahatanishi-ku
807-8555 Kitakyushu
Japan
[email protected]
Kadlubar Fred
Kirkland David
Natl. Center for Toxicol Research
Pharmacogenomics
3900 NCTR Road
72079 Jefferson
United States
[email protected]
Covance Laboratories Ltd.
Consulting
Otley Road
HG3 1PY Harrogate
United Kingdom
[email protected]
Kamber Markus
Kirsch-Volders Micheline
Xenometrix
R&D
Gewerbestr. 25
CH-4123 Allschwil
Switzerland
[email protected]
Laboratory of Cellular Genetics
Pleinlaan 2
1050 Brussels
Belgium
[email protected]
Kang Jin Seok
Kiuru Anne
National Institute of Toxicological Research
Genetic Toxicology
5 Nokbun-dong, Eunpyung-ku
122-704 Seoul
Korea
[email protected]
Stuk-Radiation and Nuclear Safety Authority
Dep. of Research and Environmental Surveillance
Laippatie 4
881 Helsinki
Finland
anne.kiuru@stuk.fi
262
26.6.2006 16:06:27
Kleinjans Jos
Kroekje Aase
Universiteitssingel 50
862736 Maastricht
Netherlands
[email protected]
Høgskoleringen 5
7491 Trondheim
Norway
[email protected]
Knezevic-Vukcevic Jelena
Kroes Robert
Faculty of Biology
Laboratory of Microbiology
Studentski trg 16
11000 Belgrade
[email protected]
Utrecht University
Institute for Risk Assessment Sciences
Yalelaan 1
NL - 3508 TD Utrecht
Netherlands
[email protected]
Knudsen Lisbeth E.
Krokan Hans E.
University of Copenhagen
Environmental Health
OesterFarmagsgade 5, room 5.2.12
1014 Copenhagen
Denmark
[email protected]
Dept. Cancer Res. Mol. Med.
Erlings Skjalgson's gt. 1
N-7006 Trondheim
Norway
[email protected]
König Jochen
Kueppers Monika
Genedata
Genedata Expressionist Business Group
Postfach
4016 Basel
Switzerland
[email protected]
Johannes Gutenberg-University
55131 Mainz
Germany
[email protected]
Kopecka Petra
Kukkonen Mari
Mamocentrum Femina
Surgery
Poštovní 11
72200 Ostrava
Czech Republic
[email protected]
Finnish Institute of Occupational Health (FIOH)
Health and Work Ability
250 Helsinki
Finland
mari.kukkonen@ttl.fi
Kopjar Nevenka
Kůsová Jaromíra
Institute for Medical Research and Occup. Health
Mutagenesis Unit
Ksaverska C. 2
HR-10 000 Zagreb
Croatia
[email protected]
Institute of Public Health
Dep. of Genetic Toxicology
Partyzanske nam. 7
702 00 Ostrava
Czech Republic
[email protected]
Kowalczyk Paweł
Kyrtopoulos Soterios
IBB PAS
Molecular Biology
Pawinskiego 5a
02-106 Warsaw
Poland
[email protected]
48 Vassileos Constantinou Avenue
11635 Athens
Greece
[email protected]
Kreimer Aimée
Laitinen Tarja
National Cancer Institute, Nat. Inst. of Health
6130 Executive Blvd, Suite 2131
20892-7333 Bethesda, Maryland
United States
[email protected]
Geneos Ltd
P.O. Box 25, Tukholmankatu 2
250 Helsinki
Finland
tarja.laitinen@geneos.fi
263
26.6.2006 16:06:28
Lambert Richard
Lodovici Maura
Glaxosmithkline
Genetic Toxicology
Park Road
SG12 ODP Ware, Herts
United Kingdom
[email protected]
University of Florence
Pharmacology
Viale Pieraccini 6
50134 Florence
Italy
maura.lodovici@unifi.it
Langova Martina
Loft Steffen
3rd Faculty of Medicine, Charles University
Division of General Biology and Genetics
Ruska 87
100 00 Prague 10
Czech Republic
[email protected]
University of Copehagen
Environmental and Occupational Health
Oesterfarimagsgade 5
1014 Copenhagen
Denmark
[email protected]
Lee Byung-Mu
Lopez De Cerain Adela
College of Pharmacy, Sungkyunkwan University
Division of Toxicology
Chunchundong 300
440-746 Suwon
Korea
[email protected]
University of Navarra
Food Sciences & Toxicology
Irunlarrea 1
31008 Pamplona
Spain
[email protected]
Lee Se-Hoon
Lorge Elisabeth
The Catholic Univ. of Korea College of Medicine
Dept. of Preventive Medicine
505 Banpodong, Seocogu
137-701 Seoul
Korea
[email protected]
Biologie Seruier
Route de Saran 905
45520 Gidy
France
[email protected]
Lehocká Hana
Lynch Anthony
Institute of Public Health Ostrava
Department of Occupational Medicine
Partyzánské nám. 7
702 00 Ostrava
Czech Republic
[email protected]
GlaxoSmithKline
Genetic Toxicology
Park Road
United Kingdom
[email protected]
Linseisen Jakob
Madsen Peter Lynge
German Cancer Research Centre
Clinical Epidemiology
Im Neuenheimer Feld 280
69120 Heidelberg
Germany
[email protected]
H. Lundbeck A/S
Molecular Toxicology
Ottiliavej 9
2500 Valby
Denmark
[email protected]
Littorin Margareta
Maffei Francesca
Lund University
221 85 Lund
Sweden
[email protected]
Pharmacology
Via irnerio, n 48
40126 Bologna
Italy
[email protected]
Lnenickova Zdenka
Mahadevan Brinda
LGE
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
Oregon State University
Environmental and Molecular Toxicology
1007, ALS Building
97331 Corvallis
United States
[email protected]
264
26.6.2006 16:06:29
Machala Miroslav
Martus Hans-Joerg
Hudcova 70
621 00 Brno
Czech Republic
[email protected]
Novartis Pharma AG
Exploratory Development
MUT-2881.5.43
4002 Basel
Switzerland
[email protected]
Maitre Anne
Marvanova Sona
University Joseph Fourier Grenoble 1
Faculte de Medecine de Grenoble
Domaine de La Merci
38700 La Tronche
France
[email protected]
Hudcova 70
621 00 Brno
Czech Republic
[email protected]
Majer Bernhard
Marzin Daniel
Austria Tabak GmbH & Co KG
Gallaher GR&D
Hansnerstr. 127
1160 Vienna
Austria
[email protected]
Institut Pasteur Calamette
Toxicology
Pr. Calmette 1
59019 Lille Cedex
France
[email protected]
Malachová Kateřina
Mateuca Raluca
Faculty of Science, University of Ostrava
Biology and Ecology
30. Dubna 22
70103 Ostrava
Czech Republic
[email protected]
Vrije Universiteit Brussel (VUB)
Cell Genetics
Pleinlaan 2
B-1050 Brussels
Belgium
[email protected]
Malinowski Mariusz
Mathar Ursula
Medical University of Lodz
Department of Molecular and Medical Biophysics
Mazowiecka 6/8
92-215 Lodz
Poland
[email protected]
Bayer AG
Governmental & Product Affairs
Building K9
51368 Leverkusen
Germany
[email protected]
Marcos Ricardo
Mauro Maurizio
Universitat Autonoma de Barcelona
Genetics and Microbiology
Campus de Bellaterra
8193 Cerdanyola Del Valles
Spain
[email protected]
University of Palermo
Cellular and Developmental Biology
Viale delle Scienze
90128 Palermo
Italy
[email protected]
Marie Caroline
Merlo Domenico Franco
CEA-Grenoble
DRFMC/SCIB/LAN
17 rue des Martyrs
38054 Grenoble
France
[email protected]
National Cancer Research Institute
Epidemiology and Biostatistics
Largo R. Benzi, 10
16132 Genova
Italy
[email protected]
Martins Célia
Miadoková Eva
Faculty of Medical Sciences, UNL
Genetics
Rua da Junqueira 96
1349-008 Lisbon
Portugal
[email protected]
Faculty of Natural Sciences, Comenius University
Genetics
Mlynska dolina B1
84215 Bratislava
Slovakia
[email protected]
265
26.6.2006 16:06:30
Mielzyńska Danuta
Mueller Lutz
Institute of O. Medicine and Enviromental Health
Genetic Toxicology
Koscielna 13
41200 Sosnowiec
Poland
[email protected]
F. Hoffmann-La Roche
Toxicology
Bldg. 73/311b
4070 Basel
Switzerland
[email protected]
Milcová Alena
Myazin Andrey
LGE
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
Vavilov Institute of General Genetics
Laboratory of Radiation Genetics
Gubkina str., 3
119991 Moscow
Russian Federation
[email protected]
Milner John
Naccarati Alessio
National Cancer Institute
NSRG
6130 Exec. Blvd, Suite 3160 MSC 7328
20895 Rockvill, MD
United States
[email protected]
Dep. of Genetic and Mol. Toxicol.
Vídenská 1083
14220 Praha 4
Czech Republic
[email protected]
Mitic-Culafic Dragana
Nagy Eszter
Faculty of Biology University of Belgrade
Microbiology
Studentski trg 3/II
11000 Belgrade
[email protected]
NOVUM
14157 Huddinge
Sweden
[email protected]
Möller Lennart
Nair Jagadeesan
CNT, NOVUM
SE-141 57 Huddinge
Sweden
[email protected]
German Cancer Research Center (DKFZ)
Toxicology & Cancer Risk Factor
69120 Heioelberg
Germany
[email protected]
Mollerup Steen
Nakachi Kei
National Institute of Occupational Health
Toxicology
Gydas vei 8, PO Box 8149 Dep.
33 Oslo
Norway
[email protected]
5-2 Hijiyama Park, Minami-ku
732-0815 Hiroshima
Japan
[email protected]
Monyarch Gemma
Natarajan A.T.
Universitat Autonoma de Barcelona
Unitat de Biologia Cel lular. Facultat de Medicina
Campus Universitat Autonoma de Barcelona
8193 Bellaterra, Cerdanyola de Valles. (Barcelona)
Spain
[email protected]
University of Tuscia
Dept. Agrobiology
Via San Camillo de Lellis
1100 Viterbo
Italy
[email protected]
Morishita Yukari
Nieminuszczy Jadwiga
Radiation Effects Research Foudation
5-2 Hijiyama Park, Mimami Ward
732-0815 Hiroshima
Japan
[email protected]
Institute of Biochemistry and Biophysics PAS
Molecular Biology
Pawinskiego 5A
02-106 Warszawa
Poland
[email protected]
266
26.6.2006 16:06:31
Nohmi Takehiko
Oesch Barbara
Division of Gemetics and Mutagenesis
1-18-1 Kamiyoga
158-8501 Setagaya-Ku
Japan
[email protected]
University of Mainz
D-55131 Mainz
Germany
[email protected]
Nomura Taisei
Oesch Franz
Osaka University
Graduate School of Medicine
2-2 Yamada-Oka, Suita
565-0871 Osaka
Japan
[email protected]
University of Mainz
D D-55131 Mainz
Germany
[email protected]
Norppa Hannu
Oinonen Teija
41 aA
FI-00250 Helsinki
Finland
hannu.norppa@ttl.fi
Orion Pharma
Toxicology
P.O. Box 65
2101 Espoo
Finland
[email protected]
Nováková Zuzana
Oláh Béla
LGE
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
Lab International Hungary Research Centre
Microbial Mutagenetic Unit
H-8200 (8201 PBOX.:348) Veszprém
Hungary
[email protected]
Novotná Božena
Olinski Ryszard
LGE
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
Collegium Medicum, Nicolaus Copernicus University,
Clinical Biochemistry
Karlowicza 24,
PO-85-092 Bydgoszcz Bydgoszcz
Poland
[email protected]
Nowell Susan
Önning Gunilla
University of Arkansas for Medical Sciences
Environmental and Occupational Health
4301 W. Markham Street, #820
72205 Little Rock
United States
[email protected]
Lund University
Biomedical Nutrition
Box 124, Chemical Center
221 00 Lund
Sweden
[email protected]
Nygren Jonas
Oscar Kisilu-Mutu
Orion Pharma
Toxicology
P.O. Box 65
2101 Espoo
Finland
[email protected]
Radial Service Presse
Av.maboto N 3 Q/mososo C/limete
243 Kinshasa
Congo (DRC)
[email protected]
O'Donovan Mike
Paget Vincent
AstraZeneca R&D
Safety Assesment
Alderley Park
NG14 6AG Macclesfield
United Kingdom
[email protected]
Centre Francois Baclesse
Grecan
Avenue du General Harris
14076 Caen Cedex 05
France
[email protected]
267
26.6.2006 16:06:32
Panek Agnieszka
Pavlíčková Zuzana
Institute of Nuclear Physics PAN
Radiation and Environmental Biology
Radzikowskiego 152
31-342 Kraków
Poland
[email protected]
Faculty of Science, University of Ostrava
Biology and Ecology
30. dubna 22
70103 Ostrava
Czech Republic
[email protected]
Pardini Barbara
Pedersen Marie
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
Institute of Public Health, University of Copenhag
Occupational and Environmental Health
Øster Farimagsgade 5A, 5.2.14
DK- 1014 København K
Denmark
[email protected]
Park Eunju
Pedersen Peder Bjarne
Kyungnam University
Food and Nutrition
449 Wolyoung-dong
631-701 Masan
Korea
[email protected]
Novozymes A/S
Safety & Toxicology
Krogshoejvej 36
2880 Bagsvaerd
Denmark
[email protected]
Park Yoo Kyoung
Pegas Heriques João Antonio
Kyung Hee University
Dept. Medical Nutrition
Hoegi 1 dong
130-701 Seoul
Korea
[email protected]
Federal University of Rio Grande Do Sul
Biophysics
Av. Bento Gonçalves, 9500 Prédio 43422
90501-970 Porto Alegre
Brazil
[email protected]
Parry Elizabeth
Pénzes Mária
Universtiy of Wales Swansea
SA2888 Swansea
United Kingdom
[email protected]
Lab International Hungary Research Centre
Mammalian Mutagenetic
H-8200 (H-8201 PBOX.:348) Veszprém
Hungary
[email protected]
Parry James
Peplonska Beata
University of Wales
Medical School - Margam
SA2888 Swansea
United Kingdom
[email protected]
8 Teresa st.
91348 Lodz
Poland
[email protected]
Pastorková Anna
Pérez Cadahía Beatriz
National Institute of Public Health
Centr Environ Hlth
Šrobárova 48
10042 Prague
Czech Republic
[email protected]
University of a Coruña
Toxicology Unit
Campus Elviña, s/n
15005 A Coruña
Spain
[email protected]
Paulsson Birgit
Pezzotti Annamaria
Stockholm University
Dept. of Environmental Chemistry
Svante Arrhenius väg 12 C
SE-10691 Stockholm
Sweden
[email protected]
Fondazione I.S.I.
10133 Torino
Italy
[email protected]
268
26.6.2006 16:06:34
Phillips David
Poljakova Jitka
Brookes Lawley Building
Cotswold Road
SM2 5NG Sutton
United Kingdom
[email protected]
Albertov 2030
12840 Prague 2
Czech Republic
[email protected]
Piazza Marie
Pontén Ingrid
Bioreliance, Invirogen Corporation
Innovation Park, Hillfords Road
FK94NF Stirling
United Kingdom
[email protected]
AstraZeneca
15185 Södertälje
Sweden
Ingrid [email protected]
Plas Gina
Pool-Zobel Beatrice
Lab. Cellulaire Genetica
Pleinlaan 2
B-1050 Brussel
Belgium
[email protected]
Friedrich Schiller University Jena
Institute for Nutrition
Dornburger Str. 25
7743 Jena
Germany
[email protected]
Plazar Janja
Portier Christopher
Department of Genetic Toxicology and Cancer Biolog
Vecna pot 111
6000 Ljubljana
Slovenia
[email protected]
NIEHS
111 T.W. Alexander Dr.
21109 Research Triangle Park
United States
[email protected]
Pletsa Vassiliki
Poth Albrecht
48 Vassileos Constantinou Ave.
11635 Athens
Greece
[email protected]
RCC
In Vitro Genotoxicity
64380 Rossdorf
Germany
[email protected]
Poirier Miriam
Pover Paul
National Cancer Institute, Bldg 37 Rm 4032 NIH
Carcinogen-DNA Interactions Section
37 Convent Drive, MSC-4255
20892-4255 Bethesda, Maryland
United States
[email protected]
Perceptive Instruments Ltd
Steeple Bumpstead
CB9 7BN Haverhill
United Kingdom
[email protected]
Poláková Veronika
Pytlinska Ewa
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
8 Teresy st.
91348 Lodz
Poland
[email protected]
Polivkova Zdenka
Rafter Joseph
3rd Faculty of Medicine, Charles University
Division of General Biology and Genetics
Ruska 87
100 00 Prague 10
Czech Republic
[email protected]
NovumHuddinge University Hospital
S-141 86 Huddinge
Sweden
[email protected]
269
26.6.2006 16:06:35
Rank Jette
Rosa Fabio
Roskilde University
Dep. Environ. Techn. Soc.Studies
P.O.Box 260
DK-4000 Roskilde
Denmark
[email protected]
Fondazione I.S.I.
10133 Torino
Italy
[email protected]
Ravanat Jean-Luc
Rössner Pavel
CEA-Grenoble
DRFMC/SCIB/LAN
17 rue des Martyrs
38054 Grenoble
France
[email protected]
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
Restle Anja
Rössner, Jr. Pavel
Universitäts Frauenklinik
Endokrinologie/Onkologie
Prittwitzstr. 43
89075 Ulm
Germany
[email protected]
LGE
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
Riley Sue
Rössnerová Andrea
Covance
Otley Road
Sl63UD Harrogate, N. Yorkshire
United Kingdom
[email protected]
LGE
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
Robinson Sharon
Routledge Michael
GlaxoSmithKline
Genetic Toxicology
Park Road
SG12 ODP Ware
United Kingdom
[email protected]
University of Leeds
Molecular Epidemiology Unit
The LIGHT Laboratories
LS2 9JT Leeds
United Kingdom
[email protected]
Rodrigo Gregory
Rozalski Rafal
BSL Bioservice Scientific Laboratories GmbH
In Vitro
Behringstr. 6
82152 Planegg
Germany
[email protected]
Karlowicza 24
85-092 Bydgoszcz
Poland
[email protected]
Rodriguez Antonio
Rubes Jiri
Laboratorios Dr. Esteve, S.A.
Toxicology
Av. Mare de Deu de Montserrat, 221
8041 Barcelona
Spain
[email protected]
Genetics and Reproduction
Hudcova 70
621 00 Brno
Czech Republic
[email protected]
Roesems Sam
Rusin Marek
Lab. Cellulaire Genetica
Pleinlaan 2
B-1050 Brussel
Belgium
[email protected]
Center of Oncology, M.Sklodowska-Curie Institute
Dept. of Tumor Biology
Wybrzeze Armii Krajowej 15
44-101 Gliwice
Poland
[email protected]
270
26.6.2006 16:06:36
Ryabokon Nadezhda
Sandberg Tove
Centre of Oncology, M. Sklodowska-Curie Mem. Inst.
of Exp. and Clin. Rad.
Webrzeze Armii Krajowej 15
44101 Gliwice
Poland
[email protected]
NOVUM, Karolinska Institutet
Dept of Biosciences and Nutrition
Hälsovägen 7
S-141 57 Huddinge
Sweden
[email protected]
Rybar Roman
Sandvik Hanna
Department of Genetics and Reproduction
Hudcova 70
621 00 Brno
Czech Republic
[email protected]
250 Helsinki
Finland
hanna.sandvik@ttl.fi
Rybczynska Maria
Sanchez Arancha
University of Medical Sciences In Poznań
Department of Clinical Chemistry
Przybyszewskiego 49
60-355 Poznan
Poland
[email protected]
Spanish Research Council
Centro De Biologia Molecular Severo Ochoa
Campus Universidad Autonoma
28049 Canto Blanco, Madrid
Spain
[email protected]
Rydzyński Konrad
Santella Regina
Director General
Sw. Teresy 8
91-348 Łódź
Poland
[email protected]
Columbia Univeristy
Environmental Health Sciences
701 West 168th St
10032 New York
United States
[email protected]
Ryeom Tai-Kyung
Sarasin Alain
Korea Food and Drug Administration
Risk Assessment Research Department
Nokbun-Dong 5
122-704 Seoul
Korea
[email protected]
Gustave Roussy Institute
Genomes and Cancers
39, rue camille Desmoulins
94805 Villejuif
France
[email protected]
Ryk Charlotta
Sarrif Awni M
Department of Biosciences and Nutrition
Halsovagen 7-9
141 57 Huddinge, Stockholm
Sweden
[email protected]
ECETOC
Toxicology
Av E Van Nieuwenhuyse 4
B-1160 Brussels
Belgium
[email protected]
Saffi Jenifer
Sciandrello Giulia
Lutheran University of Brazil
Laboratory of Genetic Toxicology
Av. Farroupilha, 8001
92450900 Canoas
Brazil
jenifer.saffi@ulbranet.com.br
University
Dep. of Cellular and Developmental Biology
Viale delle Scienze, edif.16
90128 Palermo
Italy
[email protected]
Saggoo M.I.S.
Scott Andrew
Punjabi University, Patiala
Botany
147002 Patiala
India
[email protected]
Unilever
SEAC
Sharnbrook
MK44 1LQ Bedford
United Kingdom
[email protected]
271
26.6.2006 16:06:37
Segerbäck Dan
Schwarz Claudia
Department of Biosciences and Nutrition
NOVUM
S-14157 Huddinge
Sweden
[email protected]
Medical University of Vienna
Division of Occupational Medicine
Waehringer Guertel 18-20
1090 Vienna
Austria
[email protected]
Seidel Albrecht
Silva Maria J.
Prof. Dr. Gernot Grimmer-Foundation
Biochemical Institute for Environ. Carcinogens
Lurup 4
22927 Grosshansdorf
Germany
[email protected]
National Institute of Health "Dr. Ricardo Jorge"
Center of Human Genetics
Av Padre Cruz
1649-016 Lisbon
Portugal
[email protected]
Sevastyanova Oksana
Singh Raj
Institute of Experimental Medicine AS CR
LGE
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
Biocentre, University of Leicester
Cancer Biomarkers and Prevention Group
University Road
LE1 7RH Leicester
United Kingdom
[email protected]
Schiestl Robert
Sinha Rashmi
UCLA
Pathology
650 Charles E. Young Dr.
90049 Los Angeles, CA
United States
[email protected]
NCI/USA
Nebidleg
6120 Executive blud
20857 Rockville
United States
Schläwicke Karin
Siomek Agnieszka
Lund University
Depart.of Occupational and Environmental Medicine
University Hospital
SE 221 85 Lund
Sweden
[email protected]
Karlowicza 24
85-092 Bydgoszcz
Poland
[email protected]
Schmuczerová Jana
Sipinen Ville E.
LGE
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
Mikt
Geitemyrsueien
403 Oslo
Norway
[email protected]
Schoket Bernadette
Sirota Nikolai
Natl Inst Environ Hlth, Natl Ctr Publ Hlth
Molecular Environmental Epidemiology
Gyáli út 2-6
H-1097 Budapest
Hungary
[email protected]
Institute Theoretical&Experimental Biophysic
Radiation Molecular Biology
Institutskay 3
142290 Pushchino
Russian Federation
[email protected]
Schreck Ilona
Slapsyte Grazina
Institute for Toxikology
Johannes Gutenberg University Mainz
Obere Zahlbacher Str. 67
55131 Mainz
Germany
[email protected]
Vilnius University
Botany and Genetics
M.K. Ciurlionio 21/27
LT-03101 Vilnius
Lithuania
[email protected]
272
26.6.2006 16:06:38
Slupphaug Geir
Sram Radim
Cancer Research and Molecular Medicine
Erling Skjalgssons gt 1
N-7006 Trondheim
Norway
[email protected]
LGE
Videnska 1083
142 20 Prague
Czech Republic
[email protected]
Slyšková Jana
Stambrook Peter
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
University of Cincinnati Medical Center
Cell Biology
3125 Eden Avenue
45267 Cincinnati, Ohio
United States
[email protected]
Sobala Wojciech
Steenwinkel Marie-José
8 Teresy st.
91348 Lodz
Poland
[email protected]
Tno Quality of Life
Physiological Sciences
Utrechtseweg 48
3700 AJ Zeist
Netherlands
[email protected]
Pavanello Sofia
Steiblen Guy
University of Padova
Environmental Medicine and Public Health
Via Giustiniani 4
35128 Padova
Italy
sofi[email protected]
Bayer Cropscience
Experimental Toxicology
355, rue Dostoďevski
6903 Sophia Antipolis
France
[email protected]
Souček Pavel
Stemp Gill
Šrobárova 48
10042 Prague 10
Czech Republic
[email protected]
GlaxoSmithKline
Genetic Toxicology
Park Road
United Kingdom
[email protected]
Souliotis Vassilis
Stepnik Maciej
48, Vassileos Constantinou
11635 Athens
Greece
[email protected]
8 Teresy st.
91348 Lodz
Poland
[email protected]
Soussaline Françoise
Stetina Rudolf
IMSTAR
60 Rue Notre Dame Des Champs
75006 Paris
France
[email protected]
Faculty of Military Health Sciences
Trebesska 1575
500 01 Hradec Kralove
Czech Republic
[email protected]
Speit Guenter
Stiborova Marie
University of Ulm
Human Genetics
Oberer Eselsberg M25
D - 89070 Ulm
Germany
[email protected]
Albertov 2030
128 40 Prague 2
Czech Republic
[email protected]
273
26.6.2006 16:06:40
Stverakova Olga
Takeiri Akira
LGE
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
Chugai Pharmaceutical Co., Ltd.
Safety Assessment Dept.
1-135 Komakado
412-8513 Gotemba Shizuoka
Japan
[email protected]
Sueoka Eisaburo
Tarantini Adeline
Faculty of Medicine, Saga University
Internal Medicine
5-1-1 Nabeshima
849-8501 Saga
Japan
[email protected]
Universite Joseph Fourier Grenoble 1
Faculte de Medecine de Grenoble
Domaine de la Merci
38700 La Tronche
France
[email protected]
Suter Willi
Thybaud Véronique
Novartis AG
SP&A
MUT-2881.2.35
CH 4002 Basel
Switzerland
[email protected]
Sanofi Aventis
Drug Safety Evaluation
13 Quai Jules Guesde - BP14
94403 Vitry Sur Seine
France
veronique.thybaud@sanofi-aventis.com
Sycheva Ludmila
Topinka Jan
A.N.Sysin Research Institute of Human Genetic
Laboratory of Genetic Monitoring
Vilnusskaya, 7-2, 663
119992 Moscow
Russian Federation
[email protected]
Genetic Ecotoxicology, LGE
Videnska 1083
14220 Prague
Czech Republic
[email protected]
Szweda Piotr
Totsuka Yukari
Gdansk University of Technology
Food Chemistry, Technology and Biotechnology
Gabriela Narutowicza 11/12
80-952 Gdansk
Poland
[email protected]
National Cancer Center Research Institute
Cancer Prevention Basic Research Institute
1-1, Tsukiji 5 Chome, Chuo-ku
104-0045 Tokyo
Japan
[email protected]
Szyfter Krzysztof
Trosko James
Institute of Human Genetics
Department of Environmental Mutagenesis
Strzeszynska 32
60479 Poznan
Poland
[email protected]
Michigan State University
Peds/Human Development
246 Food Safety and Toxicology
48824 East Lansing
United States
[email protected]
Švecová Vlasta
Tulupova Elena
LGE
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
Taioli Emanuela
Tweats David
University of Pittsburgh
Epidemiology
5150 Centre Ave
15232 Pittsburgh
United States
[email protected]
University of Wales-Swansea
Genetics
Singleton park
SA2 8PP Swansea
United Kingdom
djtweats@fish.co.uk
274
26.6.2006 16:06:40
Umar-Tsafe Nasir
Veeriah Selvaraju
University College of Technology & Mngmnt Malaysia
School of Health Sciences
Block C, Jalan Eqestrian 13/52, Section 13
40100 Shah Alam, Selangor
Malaysia
[email protected]
Institute for Nutrition
Department of Nutritional Toxicology
Dornburger Street 25
7743 Jena
Germany
[email protected]
Ünal Fatma
Velemínský Jiří
Gazi University
Biology
Fen-Edebiyat Fak.
6500 Ankara
Turkey
[email protected]
Institute of Experimental Botany AS CR
Dep. of Genetics
Na Karlovce 1a
16000 Praha 6
Czech Republic
[email protected]
Urbanek Katarzyna
Verschaeve Luc
National Institute of Hygiene
Enviromental Toxicology
Chocimska 24
00-791 Warsaw
Poland
[email protected]
VITO
Environmental Toxicology
Boeretang 200
2400 Mol
Belgium
[email protected]
Van Beelen Vincent
Vikse Rose
Toxicology
De Dreijen-tuinlaan
6703 HE Wageningen
Netherlands
[email protected]
Environmental Medicine
Geitmyrsveien 75
403 Oslo
Norway
[email protected]
Van Leeuwen Danitsja
Villalobos-Pietrini Rafael
PO Box 616
6200 MD Maastricht
Netherlands
[email protected]
Universidad Nacional Autonoma de Mexico
Centro de Ciencias de La Atmosfera
Circuito Exterior, Ciudad Universitaria
4510 Mexico
Mexico
[email protected]
Van Ravenzwaay Bennard
Vineis Paolo
BASF AG
Product Safety, Toxicology - Gv/T - Building Z 470
Carl-Bosch-Str. 38
67056 Ludwigshafen
Germany
[email protected]
Fondazione I.S.I.
Epidemology and Life S.
Viale Settimio Severo 65
10133 Torino
Italy
[email protected]
Van Schooten Frederik-Jan
Vodička Pavel
Universiteit Maastricht
Universiteitssingel 50
6229 ER Maastricht
Netherlands
[email protected]
Genetic and Molecular Toxicology
Vídeňská 1083
14220 Prague
Czech Republic
[email protected]
Vaňková Jolana
Vodičkova Ludmila
LGE
Vídeňská 1083
142 20 Prague
Czech Republic
[email protected]
Šrobákova 48
100 48 Praha
Czech Republic
[email protected]
275
26.6.2006 16:06:42
Voelkner Wolfgang
Wiegand Hans-Juergen
RCC
Managment
64380 Rossdorf
Germany
[email protected]
Degussa AG
Corporate Environment, Safety, Health, Quality
Bennigsenplatz 1
40474 Dnesseldorf
Germany
[email protected]
Vondracek Jan
Wiesmueller Lisa
Institute of Biophysics AS CR
Laboratory of Cytokinetics
Kralovopolska 135
61265 Brno
Czech Republic
[email protected]
University of Ulm
Department of Obstetrics and Gynaecology
Prittwitzstrasse 43
89075 Ulm
Germany
[email protected]
Voronina Ekaterina
Willems Petra
Research Center for Medical Genetics Rams
Laboratory of Mutagenesis
Moskvorechie, 1
115478 Moscow
Russian Federation
[email protected]
Ghent University
Anatomy, Embryology, Histology and Medical Physics
Pasteurlaan 2
9000 Gent
Belgium
[email protected]
Vrijhof Hendrik
Williams Lucinda
ECETOC
Secretariat
Avenue E. Van Nieuwenhuyse 4 box 6
1160 Brussels - Auderghem
Belgium
[email protected]
Covance
Otley road
Harrogate, N.Yorkshire
United Kingdom
[email protected]
Vukovic-Gacic Branka
Wirnitzer Uta
Faculty of Biology
Laboratory of Microbiology
Studentski trg 16
11000 Belgrade
Select Country
[email protected]
Genetic Tox.
Bayer Healthcare AG
Aprarher Weg
42096 Wuppertal
Germany
[email protected]
Walmsley Richard
Woelfelschneider Andreas
The University of Manchester
Faculty of Life Sciences
jackson's Mill, Sackville Street
M60 1QD Manchester
United Kingdom
[email protected]
German Cancer Research Center (DKFZ)
Toxicology and Cancer Risk Factors
69115 Heidelberg
Germany
[email protected]
Weber Elisabeth
Wolff Thomas
ARC Seibersdorf Research GmbH
Toxikologie
2444 Seibersdorf
Austria
[email protected]
Danzingerstrasse 50
85758 Garching
Germany
[email protected]
Weiss Carsten
Wu Kuen-Yuh
Research Centre Karlsruhe (FZK)
Institute of Toxicology and Genetics
Hermann von Helmholtzplatz 1
76344 76344 Eggenstein Leopoldshafen
Germany
[email protected]
National Health Research Institutes
Division of Environmental Health and Occupational
Keyan 35
350 Zhunan Town, Mioali County
Taiwan
[email protected]
276
26.6.2006 16:06:43
Yamada Masami
Zijno Andrea
Genetics and Mutagenesis
1-18-1, Kamiyoga, Setagaya
1588501 Tokyo
Japan
[email protected]
Istituto Superiore di Sanità
Department Environment and Primary Prevention
Viale Regina Elena 299
I-00161 Rome
Italy
[email protected]
Yilmaz Serkan
Gazi University
Biology
Teknikokullar
6500 Ankara
Turkey
[email protected]
Yuasa Yasuhito
Tokyo Medical and Dental University
Molecular Oncology
1-5-45, Yushima, Bunkyo-ku
113-8519 Tokyo
Japan
[email protected]
Yüzbasioglu Deniz
Gazi University
Biology
Fen-Edebiyat Fak. Teknikokullar
6500 Ankara
Turkey
[email protected]
Závacká Ivona
Medico-Social Faculty of Ostraviensis University
Genetic Toxicology
Syllabova 19
700 30 Ostrava
Czech Republic
[email protected]
Zegura Bojana
Dept of Genetic Toxicology and Cancer Biology
Vecna pot 111
1000 Ljubljana
Slovenia
[email protected]
Zeisig Magnus
NOVUM
14157 Huddinge
Sweden
[email protected]
Zeljezic Davor
Division for Mutagenesis
Ksaverska 2
10000 Zagreb
Croatia
[email protected]
277
26.6.2006 16:06:43
NOTES:
278
26.6.2006 16:06:44

36th Annual Meeting of the European Environmental Mutagen Society

Transcription

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