Genoto ic effect of cadmium in okra seedlings: Comperative

Comments

Transcription

Genoto ic effect of cadmium in okra seedlings: Comperative
985
Journal Home page : www.jeb.co.in  E-mail : [email protected]
JEB
Journal of Environmental Biology
ISSN: 0254- 8704
CODEN: JEBIDP
Genotoxic effect of cadmium in okra seedlings: Comperative
investigation with population parameters and molecular
markers
Semra Soydam Aydin1, Esin Basaran2, Demet Cansaran-Duman3 and Sümer Aras2*
1
Department of Plant and Animal Production, Ulukisla Vocational School University of Nigde, Ulukisla, Nigde, 51900, Turkey
2
Biotechnology Section, Department of Biology, Faculty of Science, University of Ankara, Tandogan, Ankara, 06100, Turkey
3
Biotechnology Institute, University of Ankara, Tandogan, Ankara, 06100, Turkey
*Corresponding Author email : [email protected]
Abstract
Plants are considered as good bioindicators because of their significant role in food chain transfer.
They are also easy to grow, adaptable to environmental stresses and can be used for assaying a
range of environmental conditions in different habitats. Thus, many plant species have been used
as bioindicators. In order to evaluate the genotoxic effect of cadmium, okra (Abelmoschus esculantus
L.) seedlings were treated with different concentrations (30, 60, 120 mg l ­1) of cadmium and
investigated for their population parameters such as inhibition of root growth; total soluble protein
content, dry weight and also the impact of metal on the genetic material by RAPD analysis. Root
growth and total soluble protein content in okra seedlings were reduced with increased Cd
concentrations. RAPD analysis indicated formation of new bands mostly at 60 and 120 mg l­1 Cd
treatments. Altered DNA band patterns and population parameters after Cd treatments suggest
that okra could be used as an indicator to reveal the effects of genotoxic agents.
Publication Info
Paper received:
11 April 2012
Revised received:
26 December 2012
Accepted:
25 January 2013
Key words
Cadmium, DNA damage, Genotoxicity, Okra, RAPD,
Introduction
Okra (Abelmoschus esculentus (L.) Moench),
previously named as Hibiscus esculentus (L.) belonging to
Malvaceae family is cultivated in many tropical, sub-tropical
and Mediterranean countries. As the actual origin of okra
remains unclear, West Africa, India and Southeast Asia
could be considered as the origin of genetic diversity
(Hamon and Sloten, 1989). Okra is an important vegetable
crop in India, West Africa, Southeast Asia, USA, Brazil,
Australia and Turkey (Mahajan et al., 1996).
Knowledge about responses to abiotic stresses may
play significant role in greenhouse cultivation and
production of okra. Heavy metal toxicity is one of the major
abiotic stress which leads to hazardous effects in plants.
Because of their high reactivity, they can directly influence
© Triveni Enterprises, Lucknow (India )
growth, senescence and energy synthesis processes. Heavy
metals could interact with many metabolic functions of
plants. For instance; inhibition of growth processes or
decrease in the activity of the photosynthetic apparatus
often correlated with progression of senescence processes
etc. (Ouzounidou et al., 1995; Sharma and Agrawal, 2010;
Duquesnay et al., 2010) as well as shortened and thickened
or poorly developed roots (Khudsar et al., 2004). Growth
inhibition and senescence stimulation, caused by heavy
metals in excess are intriguing effects, more so, as the
knowledge of their mechanisms can have a great
significance in ecophysiology and medicine (Waldemar,
2007; Amat et al., 2010).
Heavy metals are classified among the most
dangerous groups of anthropogenic environmental
pollutants due to their toxicity and persistence in the
Journal of Environmental Biology, Vol. 34, 985-990, November 2013
Journal of Environmental Biology, November 2013
986
S.S. Aydin et al.
environment. Consequently, the evaluation of the level of
metal deposition is of vital importance for the assessment
of plant exposure (Carreas et al., 2009; Ahmed et al., 2010).
Cadmium (Cd) is a multitarget toxicant for most organisms,
and is a well established human carcinogen. Unlike transition
metals such as iron (Fe+2) and copper (Cu+2) that may
undergo redox cycling and act as potent catalysts in some
of the reactions generating reactive oxygen species (ROS)
(Yuan et al., 2011), Cd does not undergo redox cycling.
However, Cd may induce increased production of ROS by
interfering with metalloproteins involved in cellular redox
or electron transfer processes (Schützendübel et al., 2001),
and in this manner Cd may induce oxidative damage to
cellular macromolecules including DNA (Collin-Hansen et
al., 2005).
Genetic variation due to heavy metal contamination
and use of genetic biomarkers of environmental pollution
has been interesting topic in the literature recently.
Especially, the impacts of pollutants on the genetic
structures of plants have now been demonstrated by several
studies (Ma et al., 2000; Moraga et al., 2002; Ross et al.,
2002; Sokolowski et al., 2002; Yap et al., 2004). The
mutagenic effect of chemicals has been analysed with
different plant systems such as Allium cepa (Fiskesjo, 1997),
Vicia faba (Koppen and Verschaeve, 1996), Trifolium repens
(Citterio et al., 2002) and Tradescantia virginiana (Fomin
et al., 1999) as plants are considered as good bioindicators
of environmental pollution. Chromosome aberration assays,
mutation assays, cytogenetic tests and specific locus
mutation assays were performed Hartl et al. (2006) and
Marcon et al. (1999) using these systems. Advances in
molecular biology have led to the development of a number
of selective and sensitive assays for DNA analysis such as
RAPD; AFLP, SSR and SCAR. The random amplified
polymorphic DNA (RAPD) technique is a semiquantitative
method which has been used for genetic mapping, taxonomy,
phylogeny and detection of various kinds of DNA damage
and mutations (Liu et al., 2005).
The objective of this study was to describe the
impact of Cd treatment on root length, dry weight and total
soluble protein level of okra seedlings, and to evaluate the
application of RAPD as a molecular biomarker to detect
DNA damage in okra seedlings. The genotoxicity indicator
potential of okra (Abelmoschus esculentus L.) seedlings at
different Cd concentrations was also evaluated.
Materials and Methods
Plant material, growth and total soluble protein level : Plant
growth and total soluble protein extractions were performed
as previosly reported by Körpe-Aksoy and Aras (2010).
Okra seeds (Bornova 2003 cultivar from Aegean Agricultural
Research Institute, Izmir, Turkey) were surface sterilized with
Journal of Environmental Biology, November 2013
70% alcohol and 30% sodium hypochlorite solution and
then washed with distilled water. Seeds were germinated to
primary roots of 2 mm long in a petri dish containing two
filter papers at room temperature and then 24 uniformly
germinated seeds were selected and transferred to petri
dishes containing 15 ml test solutions (30, 60 and 120 mg l-1
CdCl2) for three weeks. Petri dishes were incubated at room
temperature with a 16–8 hr day/night photoperiod. Each
treatment was replicated three times. After 21 days of
incubation, the root length, dry weight and total soluble
protein level of okra seedlings were measured. Inhibitory
rate (IR %) was calculated by the following formula: IR = (1x/y) x 100. Where x and y are the average values detected in
the control and each sample treated respectively. After 21
days of incubation, root dry weight was measured, following
treatment at 70ºC for 48 hr. The roots were homogenized
with (1:1, w/v) 0.2 M phosphate buffer (pH 7.0) with a cold
mortar and pestle (Omran, 1980). The homogenate was
centrifuged at 27.000 g for 20 min. The supernatant was
used for assays of total soluble protein content. The total
soluble protein contents of the root extracts were determined
according to Bradford method (Bradford, 1976) using bovine
serum albumin (BSA) as a standard.
DNA extraction and RAPD analysis : After 21 days of
growth, 24 seedlings were collected, ground in liquid
nitrogen, and total genomic DNA was extracted according
to Aras et al. (2003). DNA concentration of each sample
was quantified by NanoDrop ND-1000 Spectrophotometer.
The DNA concentrations were in the range of 969 to 1862
ng µl-1 and 260/280 nm ratios ranged from 1.94 to 2.05.
PCR was performed in a reaction mixture of 20 µl
containing approximately 200 ng of genomic DNA, 0.2 µM
primer, 2.5 µM each of the dNTPs, 2.5 mM MgCl2, 0.5 U of
Taq DNA polymerase (Promega) and 1X reaction buffer. 15
primers were tested and 9 of them gave clear and
reproducible bands. The primers used were of 10 bp in
length. The PCR program had an initial denaturing step of 5
min at 94ºC, followed by 35 cycles of; 94ºC for 30 sec
(denaturation), 50ºC for 60 sec (annealing) and 72ºC for 90
sec (extension) and final extension period of 8 min at 72ºC.
A negative control of PCR mix without any template DNA
was also used to test any other kinds of contamination.
PCR reactions were conducted from each of the three
replicates separately. All amplifications were carried out
twice. PCR products and 100bp DNA ladder (Fermentas)
were resolved electrophoretically in a 1.6 % agarose gels
containing 0.5µl ml-1 ethidium bromide, and run at 60 V for
about 4 hr. Samples were visualized and analyzed under UV
light using the system Gene Genius, Syngene.
Polymorphism in RAPD profiles included
disappearance and appearance of bands in comparison to
control and the average was calculated for each test group
987
Genotoxic effect of Cd in okra seedlings
exposed to different Cd treatments. To compare the
sensitivity of each parameter (root length, root dry weight,
root total soluble protein content), changes in these values
were calculated as a percentage of their control (set to 100%).
were tested by performing one-way analysis of variance
(ANOVA). The Duncan’s test was used to reveal the
statistical differences.
Results and Discussion
Estimation of genomic template stability : Genomic template
stability (GTS) values were also calculated according to
results of RAPD analysis. GTS implies a qualitative measure
showing the obvious change to the number of RAPD
profiles generated by the okra seedlings, in relation to
profiles obtained from the control. GTS % was calculated
polymorphic profiles in each sample, and ‘n’ is the number
of total bands in control.
The inhibition of seed germination in response to an
environmental pollutants was evaluated by treating seeds
with different concentrations of Cd solutions (30, 60 and
120 mg l -1). It was observed that root lengths were
substantially decreased with increased Cd concentration
(p<0.05) after 21 days of exposure. The maximum inhibition
was found at 120 mg l-1 Cd concentration. Consequently,
inhibitory rate (IR) of Cd on total soluble protein level, dry
weight and root length were gradually increased with
increased Cd concentrations (Table 3).
Statistical analysis : The SPSS statistical package software
(Windows 13.0) was used to analyze the changes in root
length, dry weight and total soluble protein content. Data
The dry weight and root elongation of okra seedlings
decreased significantly (p < 0.05) in a dose dependent
manner. However, total soluble protein content increased
as GTS
§ a·
¨1 ¸ u 100% , where ‘a’ indicates the RAPD
© n¹
Table 1 : Changes of total bands in control and polymorphic bands in exposed samples in okra seedlings and the polymorphism ratios
of the primers
Primer
OPA 3
OPA 6
OPA 7
OPA 9
OPA 10
OPA 14
OPA 16
OPA 18
OPA 19
a+b
Sequence of
primers (5’t 3’)
Control
AGTCAGCCAC
GGTCCCTGAC
GAAACGGGTG
GGGTAACGCC
GTGATCGCAG
TCTGTGCTGG
AGCCAGCGAA
AGGTGACCGT
CAAACGTCGG
8
7
8
12
10
9
8
12
12
86
a
30 ppm
b
a
60 ppm
b
120 ppm
a
b
0
4
3
2
1
1
3
1
2
1
0
0
1
1
2
2
1
0
1
4
2
0
0
2
2
1
1
2
0
0
1
2
0
0
0
0
1
6
2
1
1
1
2
0
1
25
Total Polymorphic Ratio
band
band
(%)
0
0
0
1
2
0
0
2
2
18
8
7
8
12
10
9
8
12
12
3
6
3
3
5
4
5
3
3
37.5
85.7
37.5
25.0
50.0
44.4
62.5
25.0
25.0
22
a : appearance of new bands, b: dissapearance of control bands, a+b indicates polymorphic bands
Table 2 : Changes of GTS for all primers
Cd Consantra- OPA 3
tions (mg l-1)
%GTS
OPA 6
%GTS
OPA 7
%GTS
OPA 9
%GTS
OPA 10
%GTS
OPA 14
%GTS
OPA 16
%GTS
OPA 18
%GTS
OPA 19
%GTS
Average
%GTS
30
60
120
42.8
42.8
14.3
62.5
75.0
75.0
75.0
91.7
83.3
80.0
80.0
70.0
66.6
77.7
88.8
37.5
75.0
75.0
83.3
91.6
83.3
83.3
91.6
75.0
59.0
76.4
72.5
87.5
62.5
87.5
% GTS: (1- a/n)* 100 a: the average number of polymorphic bands in each treated sample; n : the number of all bands in the control.
Table 3 : Effect of Cd on total protein, dry weight and root elongation of okra seedlings after 21 days of treatment
Cd concentrations (ppm)
Total protein
%IR
Dry weight (mg)
%IR
Control
30
60
120
1.359+0.3
1.410+0.1
1.491+0.2
1.558+0.1
0
3.75
9.71
14.64
0.127+0.1
0.094+0.1
0.070+0.1
0.052+0.1
0
25.9
44.8
59.0
Root elongation (mm)
3.83+0.6
3.16+0.6
3.0+0.6
2.0+0.5
%IR
0
17.5
21.67
47.78
Journal of Environmental Biology, November 2013
988
S.S. Aydin et al.
C
30ppm
60ppm
120ppm
M
140
120
3000bp
2000bp
100
1500bp
1000bp
800bp
700bp
600bp
500bp
80
40
20
400bp
300bp
200bp
0
Root
elongation
Dry weight
Total soluble
protein content
RAPD
profiling
100bp
Fig 1 : RAPD profiles generated by OPA14 primer from okra seedlings
with increased in Cd treatments.
The effect of Cd treatment on RAPD profile is shown
in Fig. 1. Nine out of 15 primers used for RAPD analysis
produced clear and reproducible bands.
The highest number of appearance and
disappearance of new bands was observed at 30 mg l-1 Cd
treatment with all of the nine primers used. On the other
hand, Cd treatment also displayed an increase in band
appearance and disappearance compared to the control
sample (Fig 1).
To test the reproducibility of the RAPD-PCR,
experiments were repeated at least twice for each primer,
faint bands were ignored and only sharp and reproducible
bands were taken into consideration. Also the reactions
were carried out from each of the three replicates separately.
None of the primers used in this study showed
monomorphic band pattern. As RAPD primers scan almost
the whole genome, it might be suggested that primers used
in this study were able to find DNA regions in which
alterations occurred. Table 1 indicates the number of altered
bands in RAPD analysis as disappearance and/or
appearence. The polymorphism ratios of the primers are
displayed in Table 1. The polymorphic bands observed were
due to the loss and/or gain of the amplified bands in the
treated samples in comparison to the control profiles. The
highest polymorphism was observed in the primer OPA 6
with a ratio of 85.70.
Journal of Environmental Biology, November 2013
Fig 2 : Comparison of root lelongation, dry weight, total soluble
protein content and RAPD profiling in okra seedlings exposed to
different concentrations of Cd
The genomic template stability (GTS, %) values, a
qualitative measure reflecting changes in RAPD profiles
were calculated for each nine primers tested (Table 2). The
lowest GTS value (59.0 %) was obtained in okra seedlings
exposed to 30 mg l-1 Cd, while highest GTS value (76.04 %)
was found at 60 mg l-1 Cd treatment (Table 2).
The genomic template stability was used for
comparing the changes in RAPD profiles and reductions in
root length, root dry weight and total soluble protein content
of okra seedlings. Results showed that root growth
parameters (length, dry weight) reduced with increased Cd
concentrations, while total soluble protein content was
enhanced. Genomic template stability also decreased after
exposure to 30 mg l-1 Cd but stabilized after 60 mg l-1 of Cd
exposure (Fig. 2).
In genetic-ecotoxicology, the effective evaluation
and proper environmental monitoring of potentially
genotoxic pollutants will improve with the development of
sensitive and selective methods to detect toxicant-induced
alterations in the genomes of a wide range of biota
(Theodorakis et al., 2001, 2006; Liu et al., 2007). Molecular
tools, physiological and biochemical paremeters used in
genotoxicology can improve the detection of genotoxic
effects and enable the interpretation of the data at the
molecular level in order to fully understand the effect of a
contaminant on organisms (Liu et al., 2005).
Total soluble protein content, an important indicator
of reversible and irreversible changes in metabolism, is
989
Genotoxic effect of Cd in okra seedlings
known to respond to a wide variety of stressors such as
natural and xenobiotic (Singh and Tewari, 2003). Results
manifested that total soluble protein content in the roots of
okra seedlings enhanced with increased Cd concentration.
Researchers have shown that total soluble protein content
can increase or decrease due to heavy metal contamination,
depending on concentration, formulation and stage of plant
growth (Peixoto et al., 2008). This alteration may be due to
disturbance in protein synthesis, denaturation of proteins
or regulated expression by metal ions like metallothionein.
The results confirmed that Cd is toxic for plants
reported earlier by several authors (Nable et al., 1997;
Shanker et al., 2005; Israr et al. 2006; Broadley et al., 2007).
Inhibitory effect of Cd contamination was evaluated in okra
root growth and was found toxic, causing an inhibition in
seed germination and root growth in okra seedlings even at
low concentrations. The root elongation and dry weight of
root tips decreased at all Cd treatments.
RAPD technique is a reliable, sensitive and
reproducible assay (Atienzar and Jha, 2006) used to detect
a wide range of DNA damage (e.g. DNA adducts, DNA
breakage) and mutation (point mutations and large
rearrangements) in organisms exposed to potentially
genotoxic agents (Liu et al., 2005). In the current study,
RAPD assay gave information about DNA alterations as
disappearance of normal bands and/or appearance of new
bands in Cd treated okra seedlings. Most of the appearances
and disappearances of new bands were observed at 30 mg
l-1 of Cd treatment which might have resulted due to
inhibition in replication machinery or a change in some of
the oligonucleotide priming sites due to heavy metal
contamination (Liu et al., 2005). A dose-dependent changes
in the number of RAPD polymorphic bands has been
detected in other organisms exposed to cadmium, lead and
benzo (a) pyrene (Jones and Kortenkamp, 2000; Castan˜o
and Becerril, 2004; Liu et al., 2005; Cenkci et al., 2010; Aras
et al., 2010).
The changes in RAPD profiles due to genotoxic
effect of heavy metals are reflected as genomic template
stability values (GTS) which is a qualitative method used
for detection of genotoxic effect of heavy metals and related
to the level of DNA damage, the efficiency of DNA repair
and replication. Therefore, a high level DNA damage does
not necessarily decrease the genomic template stability (in
comparison to a low level of DNA alterations), because
DNA repair and replication are inhibited by high frequency
of DNA damage (Liu et al., 2005). Increase in Cd
concentration is responsible for the reduction in root growth
and also genomic DNA template stability. The result
suggests that DNA damage reflected as a decrease in GTS
might also be responsible for the reduction of root tip growth
in okra seedlings. In the current study, a complex proportion
was found between metal concentration and GTS values.
Results of the current research suggests that lower
concentration of Cd treatment may induce high DNA
damage and mutation than higher concentration of Cd
contamination on okra seedlings. Higher GTS values
obtained at 60 and 120 mg l-1 Cd concentrations, might be an
indication of introduction of an effective repair system or
any other kind of cellular adaptation and/or defense sytem.
According to Liu et al. (2005) this situation was explained
by the plateau effect which was ascribed to multiple changes
in RAPD profiles (appearance of new, disappearance of the
existing bands), which tend to counterbalance each other
(Liu et al., 2005).
Therefore it is concluded that Cd may pose genotoxic
effect and induce DNA damages and mutations. These can
be successfully detected in conjunction with RAPD method
and population parameters.
Acknowledgment
The authors would like to thank Biotechnology
Institute of Ankara University, Ankara for providing
equipments via the projects number 8, 61 and 171.
References
Ahmed, Md. K., E. Parvin, M. Arif, M.S. Akter, M.S. Khan and Md.
M. Islam: Measurements of genotoxic potential of cadmium in
different tissues of fresh water climbing perch Anabas
testudineus (Bloch) using comet assay. Environ. Toxicol.
Pharmacol., 30, 80-84 (2010).
Amat, A., A. Pfohl-Leszkowicz and M. Castegnaro: Genotoxic
activity of thiophenes on liver human cell line (hepg2).
Polycycl. Aromat. Comp., 24, 733-742 (2010).
Aras, S., A. Duran and G. Yenilmez: Isolation of DNA for RAPD
analysis from dry leaf material of some Hesperis L. specimens.
Plant Mol. Biol. Rep., 21, 461a-461f (2003).
Aras, S., Ç. Kanlitepe, D. Cansaran-Duman, M.G. Halici and T.
Beyaztas: Assesment of air pollution genotoxicity by molecular
markers in the exposed samples of Pseudevernia furfuracea
(L.) Zopf in the Province of Kayseri (Central Anatolia). J.
Environ. Monit., 12, 536-543 (2010).
Atienzar, A.F. and A.N. Jha: The random amplified polymorphic
DNA (RAPD) assay and related techniques applied to
genotoxicity and carcinogenesis studies: A critical review. Mutat.
Res., 613, 76-102 (2006).
Bradford, M.M.: A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of
protein-dye binding. Anal. Biochem., 72, 254-284 (1976).
Broadley, M.R., P.J. White, J.P. Hammond, I. Zelko and A. Lux: Zinc
in plants. New Phytol., 173, 677-702 (2007).
Carreras, H.A., D.W. Eduardo and M.L. Pignata: Assessment of human
health risk related to metals by the use of biomonitors in the
province of Cordoba, Argentina. Environ. Pollut., 157, 117122 (2009).
Castan˜o, A. and C. Becerril: In vitro assessment of DNA damage
after short-and long-term exposure to benzo(a)pyrene using
RAPD and the RTG-2 fish cellline. Mutat. Res., 552, 141-151
(2004).
Cenkci, S., I.H. Cigerci, M. Yildiz, C. Ozay, A. Bozdag and H. Terzi:
Lead contamination reduces chlorophyll biosynthesis and
Journal of Environmental Biology, November 2013
990
genomic template stability in Brassica rapa L. Environ. Exp.
Bot., 67, 467-473 (2010).
Citterio, S., R. Aina, M. Labra, A. Ghiani, P. Fumagalli, S. Sgorbati
and A. Santagostino: Soil genotoxicity: A new strategy based
on biomolecular tools and plants bioindicators. Environ. Sci.
Tech., 36, 2748-2753 (2002).
Collin-Hansen, C., R.A. Andersen and E. Steinnes: Damage to DNA
and lipids in Boletus edulis exposed to heavy metals. Mycol.
Res., 109, 1386-1396 (2005).
Duquesnay, I., G.M. Champeau, G. Evray, G. Ledoigt and A. PiquetPissaloux: Enzymatic adaptations to arsenic-induced oxidative
stress in Zea mays and genotoxic effect of arsenic in root tips
of Vicia faba and Zea mays. CR Biol., 333, 814-824 (2010).
Fiskesjo, G.: Allium test for screening chemicals; evaluation of
cytological parameters. In: Plants for Environmental Studies.
Lewis Publisher. Boca Raton, New York, pp. 307-333 (1997).
Fomin, A., A. Paschke and U. Arndt: Assesment of the genotoxicity
of mine-dump material using the Tradescantia stamen hair
(trad-shm) and the Tradescantia-micronucleus (trad-shm)
bioassay. Mutat. Res., 426, 173-181 (1999).
Hamon, S. and D.H. Sloten van: Characterization and evaluation of
okra. In: The Use of Plant Genetic Resources. Cambridge
University Press, Cambridge, pp. 173-196 (1989).
Hartl, M.G.H., M. Kilemade, B.M. Coughlan, J. O’Halloran, F.V.
Pelt, D. Sheehan, C. Mothersill and N.M. O’Brien: A twospecies biomarker model for the assessment of sediment toxicity
in the marine and estuarine environment using the comet assay.
J. Environ. Sci. Hlth., 41, 939-953 (2006).
Israr, M., S. Sahi, R. Data and D. Sarkar: Bioaccumulation and
physiological effects of mercury in Sesbania drummonii.
Chemosphere, 65, 591-598 (2006).
Jones, C. and A. Kortenkamp: RAPD library fingerprinting of
bacterial and human DNA: Applications in mutation detection.
Teratogen. Carcinogen. Mutagen., 20, 49-63 (2000).
Khudsar, T., M. Iqbal and R.K. Sairam: Zinc induced changes in
morpho-physiological and biochemical parameters in Artemisia
annua. Biol. Plantar., 48, 255-260 (2004).
Koppen, G. and L. Verschaeve: The alkaline comet test on plant
cells: A new genotoxicity test for DNA strand breaks in Vicia
faba root cells. Mutat. Res., 360, 193-200 (1996).
Körpe-Aksoy, D. and S. Aras: Evaluation of copper stress on eggplant
(Solanum melongena L.) seedlings at molecular and population
levels using various biomarkers. Mutat. Res., 719, 29-34 (2010).
Liu, W., P. Li, X.M. Qi, Q. Zhou, L. Zheng, T. Sun and Y. Yang: DNA
changes in barley (Hordeum vulgare) seedlings induced by
cadmium pollution using RAPD analysis. Chemosphere, 61,
158-167 (2005).
Liu, W., Y. Yang, Q. Zhou, L. Xie, P. Li and T. Sun: Impact assessment
of cadmium contamination on rice (Oryza sativa L.) seedlings
at molecular and population levels using multiple biomarkers.
Chemosphere, 67, 1155-1163 (2007).
Ma, X.L, D.L. Cowles and R.L. Carter: Effect of pollution on genetic
diversity in the Bay Mussel Mytilus galloprovincialis and the
Acorn Barnacles Balanus glandula. Mar. Environ. Res., 50,
559-563 (2000).
Mahajan, R.K., I.S. Bisht, R.C. Agrawal and R.S. Rana: Studies on
South Asian characterization data. Genet. Resour. Crop Ev.,
43, 249-255 (1996).
Marcon, F., A. Zijno, R. Crebelli, A. Carere, T. Veidebaum, K. Peltonen,
Journal of Environmental Biology, November 2013
S.S. Aydin et al.
R. Parks, M. Schuler and D.A. Eastmond: Chromosome damage
and aneuploidy detected by multicolour FISH in benzene
exposed shale oil workers. Mutant. Res., 445, 155-166 (1999).
Moraga, D., E. Mdelgi-Lasram, M.S. Romdhane, E. El Abed, I. Boutet,
A. Tanguy and M. Auffret: Genetic responses to metal
contamination in two clams: Ruditapes decussates and Ruditapes
philippinarum. Mar. Environ. Res., 54, 521-525 (2002).
Nable, R.O., G.S. Banuelos and J.G. Paull: Boron toxicity. Plant Soil,
193, 181-198 (1997).
Omran, R.G.: Peroxide levels and the activities of catalase, peroxidase,
and indolacetic acid oxidase during and after chilling cucumber
seedlings. Plant Physiol., 65, 407-408 (1980).
Ouzounidou, G., M. Giamparova, M. Moustakas and S. Karataglis:
Responses of maize (Zea mays L.) plants to copper stress. Int.
Environ. Exp. Bot., 35, 167-176 (1995).
Peixoto, F.P, J. Gomes-Laranjo, J.A. Vicente and V.M.C. Madeira:
Comparative effects of the herbicides dicamba, 2, 4-D and
paraquat on non-green potato tuber calli. J. Plant Physiol.,
165, 1125-1133 (2008).
Ross, K., N. Cooper, J.R. Bidwell and J. Elder: Genetic diversity and
metal tolerance of two marine species: A comparison between
populations from contaminated and reference sites. Mar. Pollut.
Bull., 44, 671-679 (2002).
Schützendübel, A., P. Schwanz, T. Teichmann, K. Gross, R. LangenfeldHeyser, D.L. Godbold and A. Polle: Cadmium-induced changes
in antioxidative systems, hydrogen peroxide content, and
differentiation in Scots pine roots. Plant Physiol., 127, 887898 (2001).
Sharma, R.K. and S.B. Agrawal: Responses of Abelmoschus esculentus
L. (lady’s finger) to elevated levels of Zn and Cd. J. Trop.
Ecol., 51, 389-396 (2010).
Shanker, A.K., C. Cervantes, H. Loza-Tavera and S. Avudainayagam:
Chromium toxicity in plants. Environ. Int., 31, 739-753 (2005).
Singh, P.K. and S.K. Tewari: Cadmium toxicity induced changes in
plant-water relations and oxidative metabolism of Brassica
juncea L. plants. J. Environ. Biol., 24, 107-117 (2003).
Sokolowski, A., D. Fichet, P. Garcia-Meunier, G. Rafenac, M.
Wolowicz and G. Blanchard: The relationship between metal
concentrations and phenotypes in the Baltic clam Macoma
balthica (L.) from the Gulf of Gdansk, Southern Baltic.
Chemosphere, 47, 475-484 (2002).
Theodorakis, C.W., J.W. Bickham, T. Lamb, P.A. Medica and T.B.
Lyne: Integration of genotoxicity and population genetic
analyses in kangaroo rats (Dipodomys merriami) exposed to
radionuclide contamination at the Nevada Test Site, USA.
Environ. Toxicol. Chem., 20, 317-326 (2001).
Theodorakis, C.W., R. Pantino, E. Snyder and A. Albers: Perclorate
effects in fish. In: (Eds.: R.J. Kendall and P.N. Smith), In
Perclorate Ecotoxicology. Soc. Environ. Toxicol. Chem.
(SETAC), Pensacola, FL, 155-186 (2006).
Waldemar, M.: Signaling responses in plants to heavy metal stress.
Acta Physiol. Plant., 29, 177-187 (2007).
Yap, C.K., S.G. Tan, A. Ismail and H. Omar: Allozyme polymorphisms
and heavy metal levels in the green-lipped mussel Perna viridis
(Linnaeus) collected from contaminated and uncontaminated
sites in Malaysia. Environ. Int., 30, 39-46 (2004).
Yuan, M., X. Li, J. Xiao and S. Wang: Molecular and functional
analyses of COPT/Ctr-type copper transporter-like gene family
in rice. BMC Plant Biol., 11, 69 (2011).

Similar documents