Effetti epigenetici dell`inquinamento atmosferico

Effetti epigenetici dell’inquinamento
atmosferico
Ernesto Burgio
ISDE Scientific Committee
Deaths from urban air pollution in 2000, as estimated by the WHO
World Health Report, 2002
The WHO estimates that air pollution is responsable
for 3 million premature deaths each year.
There is consistent evidence that the
levels of fine particulate matter in
the air are associated with the risk of
death from all causes and from
cardiovascular and respiratory
illnesses. These findings strengthen
the rationale for controlling the
levels of respirable particles in
outdoor air. (N Engl J Med
2000;343:1742-9.)
L'associazione tra particolato fine e malattie cardiache emerse negli anni novanta del secolo
scorso, allorché le evidenze epidemiologiche del nesso tra inquinamento dell'aria e ricoveri
ospedalieri per malattie cardiovascolari si andarono accumulando ...
si osservò che l'esposizione annuale media a PM2.5 si associava ad un aumentato rischio di
mortalità da cardiopatia ischemica e aritmie... a dimostrazione che le concentrazioni di particolato
non solo causavano un aggravamento a breve termine della malattia cardiovascolare, ma avevano
un ruolo patogenetico negli eventi coronarici
Standards and monitoring are now being
introduced for PM2.5 particles, termed ‘fine
particles’ and mostly 1,000 to 2,500nm in size,
but there is nothing to cover the much smaller ones
Lack of Standards and Monitoring for UFPs
The current standards are in terms of total mass, yet UFPs are generally around only one percent
of the total mass but present the majority of the surface area that is reactive to human tissues.
If the mass of a single inhaled 2.5 μm particle is divided into typical nanoparticles ~80nm, they would have 1000
times more surface area.
For that reason alone, the mass-based PM standards are far from appropriate for UFPs.
Ultrafine particles have a high specific surface area, which can catalyse
reactions and adsorb high amounts of toxic substances (like PAH..)
A given mass of ultrafine particles has
a much greater surface area than the same
mass of fine, yet respirable, particles and so is
more likely to cause overload,
(causing far more bronchoalveolar chronic
inflammation, fibrosis and tumors).
The adverse effects of ultrafine particles may be mediated in part by their ability to inhibit
phagocytosis.. in part by their pro-inflammatory b
a
PM < 1
PM10
b
d
a
UP are less well phagocytized
by alveolar macrophages
than larger particles and
inhibit their phagocytic ability
c2
The mechanism for the effect on macrophages may be the increased
oxidative stress from the large surface area of ultrafine particles.
UP form complexes with proteins
and biomolecules
which may result in functional
changes of the latter !!
!
L'inquinamento atmosferico consiste in una miscela
eterogenea, complessa di gas, liquidi e particolato.
Studi epidemiologici hanno dimostrato un
consistente aumento di rischio di eventi
cardiovascolari in relazione sia alle esposizioni a
breve che a lungo termine alle concentrazioni di
particolato usuali delle nostre città…
Diversi meccanismi sono stati
descritti, tra cui l'attivazione di
processi trombotici, aritmie,
vasocostrizione arteriosa acuta,
risposta infiammatoria sistemica
(endotelite...) con promozione
dell’aterosclerosi
Nanoparticles could interact with the vascular endothelium or have direct effects on atherosclerotic
plaques and cause local oxidative stress and proinflammatory effects similar to those seen in the lungs.
Increased inflammation could destabilize coronary plaques, which might result in rupture, thrombosis, and
acute coronary syndrome.
1
2
3
4
6
5
7
8
Inquinamento e cuore
Residential exposure to traffic is associated with
coronary atherosclerosis.
4,494 individuals living in close proximity to a major urban road
Increased coronary artery calcium scores
(a marker of coronary atherosclerosis) by 60%
Hoffmann B et al. Circulation (2007) 116: 489–496
Combustion-derived nanoparticulate matter causes
acute and chronic cardiovascular disease
Mills NL et al. Nat Clin Pract Cardiovasc Med doi:10.1038/ncpcardio1399 (2008)
Brook RD et al. Circulation 109: 2655–2671 (2004)
Inquinamento e ictus
Associations of Fine and Ultrafine
Particulate Air Pollution
With Stroke Mortality
in an Area of Low Air Pollution Levels
Jaana Kettunen et al. Stroke 2007;38;918-922
We therefore evaluated the effects of several particle measures including, for the
first time to our knowledge, ultrafine particles (<0.1 µm) on stroke.
…PM2.5, but also ultrafine particles and carbon monoxide,
are associated with increased risk of fatal stroke…
Effetti sulla salute umana in % per ogni
incremento di 10 µg/m3 di PM10 e PM2.5
Effetti
PM10*
PM10**
PM2.5***
Mortalità generica
0.6
1.3
1.3
2.1
6
Mortalità per patologie
cardiovascolari
0.9
1.4
12
Ricoveri ospedalieri
Pazienti over 65 anni
0.7
?
14
Mortalità per patologie
respiratorie
Mortalità per cancro al
polmone
*Anderson HR WHO Regional Office for Europe 2004
**MISA Meta Analisi Italiana su otto grandi città italiane
*** Pope A.C., Journal American Association 2002
*** Pope Circulation 2004
Exposures to particulate matter and gaseous air pollutants have been associated with respiratory tract inflammation, disruption of the nasal
respiratory and olfactory barriers, systemic inflammation, production of mediators of inflammation capable of reaching the brain and systemic
circulation of particulate matter. Mexico City (MC) residents are exposed to significant amounts of ozone, particulate matter and associated
lipopolysaccharides. MC dogs exhibit brain inflammation and an acceleration of Alzheimer’s-like pathology, suggesting that the brain is
adversely affected by air pollutants. MC children, adolescents and adults have a significant upregulation of cyclooxygenase-2 (COX2) and
interleukin-1β (IL-1β) in olfactory bulb and frontal cortex, as well as neuronal and astrocytic accumulation of the 42 amino acid form of βamyloid peptide (Aβ42), including diffuse amyloid plaques in frontal cortex. The pathogenesis of Alzheimer’s disease (AD) is characterized by
brain inflammation and the accumulation of Aβ42, which precede the appearance of neuritic plaques and neurofibrillary tangles, the
pathological hallmarks of AD. Our findings of nasal barrier disruption, systemic inflammation, and the upregulation of COX2 and IL-1β
expression and Aβ42 accumulation in brain suggests that sustained exposures to significant concentrations of air pollutants such as
particulate matter could be a risk factor for AD and other neurodegenerative diseases.
The frontal cortex of an 11-month-old healthy MC
dog exhibits Aβ42 staining of a diffuse plaque,
surrounded by a microglia-like nucleus
The frontal cortex of a 17-year-old MC boy… shows a diffuse
Aβ42 plaque (red product) and GFAP-negative astrocytes
The frontal cortex of a 36-year-old MC male with an E3/E4 ApoE genotype .. shows
abundant mature and diffuse Aβ42 plaques (red stain) along
with GFAP-positive reactive astrocytosis
PARTE II
Rudolf Jaenisch- Whitehead Institute and
Dept. of Biology, MIT, Cambridge, MA
Everyday levels matter
At high levels… arsenic kills people
At moderately low levels… it causes a range of diseases
At truly low levels …
it interferes with gene
activation
Many of these substances (Dioxins,
Heavy Metals, Polycyclic aromatic
Hydrocarbons) are dangerous for
humans health at very low-every daydoses (which are very difficult to be
assessed by the ordinary toxicological
studies)
Kaltreider et al.
al. 2002
EMF
We may represent the environment as a continuous stream of
information (simple: photons: individual packages of E = M =
Information) or complex (organic molecules, viruses etc)
interacting with our cells [membrane /transmembrane
receptors, signal transduction proteins, nuclear receptors,
genome (DNA + Epigenome)] forcing them to adapt
TCDD
Viruses
1
SYNERGISM !!
3
“FLUID EPI-GENOME”
HERVs
4
2
What’s Epi-Genetics ?
Multiple levels
of packing are
required to fit the
DNA into the cell
nucleus
We should remember that..
.. the
Central
Dogma
of
Molecular
Biology
depicts
one
directionflow
of genetic
information
..which seems to be a rather simplistic (linear, deterministic) model
If the Central Dogma of Molecular Biology depicted one
direction-flow of genetic information…
…we must remember that things are quite
different: information flow is circular
between the genome and the environment
SYSTEMS
GENOMICS
(BIOLOGY)
REVERSE
TRANSCRIPTION
GRNetworks
TRANSPOSABLE ELEMENTS
NATURAL GENETIC ENGINEERING
TRANSCRIPTION
FACTORS
(and COFACTORS)
EPIGENETIC
CHANGES
The “Fluid
Genome”
ENVIRONMENT
..configuring a new model of genomic molecular network composed by DNA, epigenome, proteome
sequences communicating in cis and trans and, above all, reactive /interactive
(in a continuous dialogue with the environment)
VIRUSES
Hormons
Xenobiotics
EMF
in a continuous
dialogue with
the
environment
Naesens, M. & Sarwal, M. M Current paradigm of molecular biology Nat. Rev. Nephrol. 6, 614–628 (2010)
DNA
double
helix
(2-nm
diameter)
Euchromatin
Histones
“Beads on
a string”
Multiple
levels
of packing are
required to fit
the DNA into
the cell
nucleus
Nucleosome
(10-nm diameter)
Nuclear DNA is
normally tightly
wrapped around
histones
Heterochromatin
Tight helical fiber
(30-nm diameter)
Supercoil
(200-nm diameter)
Metaphase chromosome
700
nm
Campbell NE et al (Eds):
Biology: Concepts & Connections
4th Edition, 2003
The Epigenetic Players
1
Chromatin
Remodeling
machinery
OFF
ON
DNA
Methylation
+
2
Histone
modification
3
4
MicroRNAs
Euchromatin
Heterochromatin
“The study of heritable changes in gene function
that occur without a change in the DNA sequence”
Fraga et al., PNAS. 2005.
Nuclear DNA is
normally tightly
wrapped around
histones rendering
the DNA inaccessible
to the general
transcription
machinery and
hence this tight
association prevents
transcription of DNA
Eterochromatin
The
Histone tails
are a critical
determinant
of chromatin structure
1
Histone Tails
are subject to
a variety of
covalent
modifications
Histone Code”
hypothesis: modifications
of the Histone tails
act as marks read
by other proteins
to control the
expression
or replication of
chromosomal regions
E.g. generally,
Histone Acetylation
is associated with
transcriptionally
active genes
Deacetylation
is associated with
inactive genes
(= gene silencing)
DNA methylation
Covalent modification of the DNA is also important for gene silencing human cells.
Most genes have GC rich areas of DNA in their promoter regions, referred to as CpG islands.
Methylation of the C residues within the CpG islands leads to gene silencing
2
(highly unstable base)
Conclusions: We found decreased
repeated-element methylation after
exposure to traffic particles.
LINE-1 methylation decreased in relation to
higher black carbon and PM2.5 ambient level
with significant associations for all the time
windows evaluated and stronger effects at the
longer time windows (2–7 d).
Alu methylation showed no significant
association with pollutant levels
at any of the time windows evaluated
Hypomethylation of repetitive DNA sequences is expected to lead to the transcriptional activation of those repetitive
sequences that still contain active promoters, potentially resulting in disruption of transcription factor balance, sense or
antisense transcriptional interference, and production of transcripts complementary to endogenous transcripts or to
alterations in genomic organization and stability
3
http://www.gene-quantification.de/micro-rna-1.html#nature
Many toxicants cause
rapid alterations in gene
expression by activating
protein kinase signaling
cascades.
Histone Acetyltransferases;
Histone Methyltransferases
Histone Deacetylases.
Histone
Lysine
Acetylation
P
H3-K9
Nuclear Receptor
DNA Response Element
H3-S10
ATP-dependent Nucleosome
Remodeling Complex
These modifications are believed to be essential
for the full transcriptional response to stressinducing chemicals
The resulting rapid,
defensive alterations in
gene activity require the
transmission of a signal
directly to the histones
present in the chromatin
of stress response genes:
within minutes
of exposure
the phosphorylation of
serine 10 of histone H3
and the
acetylation
of lysines 9 and/or 14
take place
In-vitro, animal, and human investigations have identified
several classes of environmental chemicals that modify epigenetic marks..
including
- metals (cadmium, arsenic, nickel, chromium, CH3-mercury),
- peroxisome proliferators (trichloroethylene,
dichloroacetic acid…),
- Air Pollutants (PM 0,1/2,5/10, black carbon, benzene),
- EDCs - Endocrine-Disrupting/reproductive toxicants
(DES, bisphenol A, persistent organic pollutants, dioxin).
Because these epigenetic changes are small,
potentially cumulative, and they may develop over
time, it may be difficult to establish the cause-effect
relationships among environmental factors,
epigenetic changes, and diseases.
Polycyclic Aromatic
Hydrocarbons (PAH)
(Ultra)-fine
particles
Heavy Metals
Benzene
Dioxin and
Dioxin-like
molecules
2
3
….particular awareness has aroused a
study that documented the presence
1 of (geno) toxic and mutagenic
substances in all the umbilical
cords tested, demonstrating the
ubiquity of embryo-fetal exposure
Mothers Milk: Record levels of toxic fire
retardants found in American mothers'
breast milk. Washington, DC. Available at
http://www.ewg.org/reports/mothersmilk/
NAS (National Academy of Sciences). 2000.
Scientific Frontiers in Developmental
Toxicology and Risk Assessment.
Committee on Developmental Toxicology.
Washington, DC: National Academies Press;
EPA (U.S. Environmental Protection
Agency). 2003a. America's children and the
environment. Measures of contaminants,
body burdens, and illnesses. Available
online at
http://www.epa.gov/envirohealth/children.
What is the Global Chemical Burden..
Industrial chemicals in mothers and daughters:
the pollution we share and inherit
E’ vero che nel sangue e nei tessuti di tutti gli uomini e le donne che vivono in ambienti urbani
e/o industriali e persino nel sangue cordonale e placentare e nei tessuti fetali sono presenti
questi stessi inquinanti in quantità di anno in anno, di decennio in decennio maggiori ?
Is it true that these pollutants are present in blood and tissues of all men and women living in urban and industrial
environments and even in the cord blood and placental and fetal tissues in more and more significant amounts year
after year ?
E’ vero, in particolare, che metalli, diossine e altri inquinati lipofili accumulati nei tessuti materni
possono passare, anche a distanza di anni dal loro assorbimento, nel sangue e raggiungere il feto ?
DOHAD
XXI secolo: drammatica trasformazione
dell’ambiente  del microambiente uterino
Differentiation occurs numerous times during
the development of a multicellular organism as
the organism changes from
a single zygote to a complex system of tissues
and 200 cell types (genetically identical.. each
with its own epigenetic and morphomorpho-functional1
characteristics)..
Cellular Differentiation: an Epigentic process
Nature 447, 425-432 (24 May 2007)
Differentiation
Fetal Programming
PLASTICITY
2
Gametogenesis. Maturation of germ cells is characterized by an
impressive degree of cellular restructuring and gene regulation that
involves remarkable genomic reorganization.
These events are finely tuned, but are also susceptible to the
introduction of various types of error…
epi-mutations
Napoli C, Infante T, Casamassimi A. Maternal-foetal epigenetic
interactions in the beginning of cardiovascular damage
Cardiovasc Res. 2011 Dec 1;92(3):367-74
Epigenetic modifications during early life
can influence the mature phenotype and
determine sensitivity to later environmental
factors and subsequent risk
of cardiovascular diseases
The active chromatin is
characterized by the presence
of acetyl groups (Ac) on
specific lysine residues of
core histones. CpG sequences
in the promoter regions of
active genes are usually
unmethylated, allowing
the binding of TFs.
Inactive chromatin is
characterized by histone
deacetylation, promoter CpG
methylation, and decreased
binding of TFs.
Post-transcription control can
also be provided by mirRNA
molecules
Examples of epigenetic
machinery enzymes are also
shown
An epigenetically modified precursor during embryogenesis may affect both normal
morphogenesis and post-natal physiological adult progenitor-mediated vascular repair
A•
Generalmente ci si limita a valutare il rischio
legato a singole fonti di inquinamento confrontando
popolazioni più o meno direttamente esposte,
• e trascurando il fatto che oggi l’inquinamento è
B
un fenomeno ubiquitario e capillare e che
l’esposizione ad agenti epi-genotossici
(fisici, (bio)chimici, biologici..) più pericolosi concerne,
e in misura sempre più rilevante,
l’intera popolazione umana,
le generazioni future, l’intera eco/biosfera.
… a causa del bio-accumulo/biomagnificazione degli xeno-biotici
in ambiente, catena alimentare e tessuti degli organismi complessi
e della possibile trasmissione transgenerazionale delle modifiche epigenetiche
CHEMICAL BURDEN-Carico Chimico Globale
Now, 40 years after the original
description of the clinical signs of this
illness by Japanese pediatrician Tomisaku
Kawasaki, we understand that this selflimited vasculitis affords a unique
opportunity to study acute endothelialcell injury, aneurysm formation, and
vascular-wall remodeling in young
children in whom the cumulative effects
of aging and environmental insults have
not yet taken their toll …
La Malattia di Kawasaki è una
(immuno)vasculite sistemica?
Ernesto Burgio
Comitato Scientifico
ISDE Italia
Two generations of
clinicians and researchers
have been stumped
by the riddle of KD..
1
How can an illness look like an
infectious disease but not have a
recoverable agent..
2
.. look like an immune-mediated vasculitis
but not be easily treated with corticosteroids
3
..look like a benign, self-limited illness but be the leading cause
of acquired heart disease in children?
The answers to the riddle continue to elude us…
The hallmark of the arteritis is the presence of
lesions in various stages of progression within
the same patient at any given time.
The earliest changes are observed in the
endothelium of the musculoelastic arteries
1
2
3
..more advanced lesions show edema and
inflammatory-cell infiltrate in the
subendothelial space
These changes progress to include
destruction of the media
4 and then replacement of the intima and media with fibrous
connective tissue, thinning of the media with aneurysm
formation, scarring, and stenosis
Coronary-artery rupture is extremely rare but
can occur during the subacute phase
5
Myointimal proliferation and stenosis occur over a
period of months to years, and these lesions may
remain silent until the moment of acute thrombotic
occlusion, often decades after the acute illness
Un po’ come per la malattia
reumatica (Lasègue, 1816-83) si
può affermare che anche la KD
sfiori cute, articolazioni ecc.. e
colpisca cuore/vasi.
Endothelial Dysfunction
Now, 40 years after the original description of the clinical signs of this illness by Tomisaku Kawasaki, we understand that this
self-limited vasculitis affords a unique opportunity to study acute endothelial-cell injury, aneurysm formation, and
vascular-wall remodeling in young children in whom the cumulative effects of aging and environmental insults have not
yet taken their toll. These lessons.. have relevance for the study of chronic conditions involving vascular injury, including
atherosclerosis and abdominal aortic aneurysm.
•
•
•
•
•
This study revealed that the adult
patients with a history of KD had
systemic vascular endothelial
dysfunction, and also suggested
that a history of KD was possibly
one of the risk factors for early
onset of atherosclerosis.
[Kawasaki disease and atherosclerosis]
Niboshi A, Ozawa S, Hamaoka K.
Nippon Rinsho. 2008 Feb;66(2):387-92
Department of Paediatric Cardiology and Nephrology, Kyoto Prefectural
University of Medicine Graduate School of Medical Science.
The existence of endothelial damage in impaired coronary arteries,
both aneurysms and regressed aneurysms, even during the chronic
stage of Kawasaki disease (KD) has been reported by morphologic and
functional studies.
In addition, based on our results of endothelial-dependent dilatation in
systemic arteries or the pathological findings in weaning rabbits in the
chronic stage of KD-like allergic coronary arteritis by administering a
hypercholesterol diet, it is suggested that vascular endothelial
dysfunction, not only in coronary arteries but also in systemic
arteries, could exist in the KD patients even in a chronic stage, and
also suggested that a history of KD was possible to be one of the risk
factor for the early onset of atherosclerosis.
Is KD a “NEW” DISEASE ?
Is KD a ““NEW
NEW”” DISEASE ?
• G. Fanconi “ Non vi sono malattie
nuove.. nuovo è il modo di
interpretarle”
(Burgio-Notarangelo
Malattie Maestre, 2002
pag. 170)
• M. Grmek: da Vesalio e Sydenham
malattia
come quadro sintomatologico ben definito =
nuova ontologia

pathocenosis (cfr. biocenosis)
Viruses
Microbes
Systemic (micro)vascular
phlogosis
Kawasaki Disease
(systemic endothelitis)
TLRs
“changing
environment”
Biosphere
tolerance
Natural Immunity
Gut Ecosystem
Immuno-systems
Adaptive Immunity
Hypersensitivity
Atherosclerosis
(Coeliac Disease)
(systemic endothelitis)
Fluid
Genosphere
Autism (?)
“Neurodegenerative” diseases
Genetic
background
type 0
type I
type II
type III
type IV
Salsomaggiore 18/20-04-2008
ISDE International Conference
(1989)
La Rivoluzione Epidemica
del XX Secolo
The Barker Hypothesis (1989)
Insulinoresistance
Metabolic Syndrome
Diabetes
Cardiovascular Diseases
Asthma - Allergy
Autoimmune Diseases
Ernesto Burgio
(ISDE Italy)
L’incremento delle patologie cronico-degenerative
[immunomediate, neuro-degenerative, endocrine,
neoplastiche, cardiocircolatorie]
è il prodotto di una drammatica trasformazione ambientale
e di una (conseguente) alterazione del programming embrio-fetale ?
THANK YOU