Ernesto Burgio

ISDE Scientific Committee

Effetti epigenetici dell’inquinamento

atmosferico

Deaths from urban air pollution in 2000, as estimated by the WHO

World Health Report, 2002

The WHO estimates that air pollution is responsablefor 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 respiratoryillnesses . 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

a

PM10 PM < 1

b

The mechanism for the effect on macrophages may be the increased

oxidative stress from the large surface area of ultrafine particles.

b

d

c2

UP are less well phagocytized

by alveolar macrophages

than larger particles and

inhibit their phagocytic ability

a

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.

2

1

3

4

56

78

Residential exposure to traffic is associated with

coronary atherosclerosis.

Inquinamento e cuore

Hoffmann B et al. Circulation (2007) 116: 489–496

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%

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 Levelsin 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 6

Mortalità per patologie respiratorie

1.3 2.1respiratorie

Mortalità per patologie cardiovascolari

0.9 1.4 12Ricoveri ospedalieriPazienti over 65 anni

0.7

Mortalità per cancro al polmone

? 14

*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 β-

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

interleukin-1β (IL-1β) in olfactory bulb and frontal cortex, as well as neuronal and astrocytic accumulation of the 42 amino acid form of β-

amyloid 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.

PARTE II

Rudolf Jaenisch- Whitehead Institute and

Dept. of Biology, MIT, Cambridge, MA

Everyday levels Everyday levels mattermatter

At truly low levels At truly low levels ……it interferes with gene it interferes with gene

At high levels… arsenic kills people

At moderately low levels… it causes a range of diseases

it interferes with gene it interferes with gene

activationactivation

KaltreiderKaltreider et alet al. 2002. 2002

Many of these substances (Dioxins,

Heavy Metals, Polycyclic aromatic

Hydrocarbons) are dangerous for

humans health at very low-every day-

doses (which are very difficult to be

assessed by the ordinary toxicological

studies)

TCDD

Viruses

EMF

1

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

HERVs

3

2

SYNERGISM !!

“FLUID EPI-GENOME”

4

What’s Epi-Genetics ?

Multiple levels

of packing are

required to fit the

DNA into the cell

nucleus

.. the

Central

Dogma

of

Molecular

Biology

We should remember that..

Biology

depicts

one

direction-

flow

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…

REVERSE

TRANSCRIPTIONGRNetworks

SYSTEMS

GENOMICS

(BIOLOGY)

TRANSPOSABLE ELEMENTS

…we must remember that things are quite

different: information flow is circular

between the genome and the environment

TRANSCRIPTION

FACTORS

(and COFACTORS)

NATURAL GENETIC ENGINEERING

TRANSPOSABLE ELEMENTS

The “Fluid

Genome”

EPIGENETIC

CHANGES

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)

Hormons

Xenobiotics

EMF

VIRUSES

Naesens, M. & Sarwal, M. M Current paradigm of molecular biology Nat. Rev. Nephrol. 6, 614–628 (2010)

in a continuous

dialogue with

the

environment

DNAdoublehelix(2-nmdiameter)

Nucleosome

(10-nm diameter)

Histones

“Beads on

a string”

Euchromatin

Multiple

levels

of packing are

required to fit

the DNA into

the cell

nucleusNuclear DNA is

Metaphase chromosome700nm

Tight helical fiber(30-nm diameter) Supercoil

(200-nm diameter)

Campbell NE et al (Eds):

Biology: Concepts & Connections

4th Edition, 2003

Heterochromatin

nucleusNuclear DNA is

normally tightly

wrapped around

histones

The Epigenetic Players

DNA

Methylation

Histone

modification

Chromatin

Remodeling

machinery

+ON OFF

1

4

32

Euchromatin

Heterochromatin

“The study of heritable changes in gene function that occur without a change in the DNA sequence”

Fraga et al., PNAS. 2005.

4

MicroRNAs

Nuclear DNA is

normally tightly

wrapped around

histones rendering

the DNA inaccessible 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

antisensetranscriptional 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

Histone

Lysine

Acetylation

Histone Deacetylases.

Histone Acetyltransferases;

Histone Methyltransferases

Many toxicants cause

rapid alterations in gene

expression by activating

protein kinase signaling

cascades.

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:

H3-K9 H3-S10

P

Nuclear Receptor

DNA Response Element

ATP-dependent Nucleosome

Remodeling Complex

within minutes

of exposure

the phosphorylation of

serine 10 of histone H3

and the

acetylation

of lysines 9 and/or 14

take place

H3-K9 H3-S10

These modifications are believed to be essential

for the full transcriptional response to stress-

inducing chemicals

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 - 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.

….particular awareness has aroused a study that documented the presence 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

Dioxin and Dioxin-like molecules

(Ultra)-fine particles

Heavy Metals

Polycyclic Aromatic Hydrocarbons (PAH)

Benzene

1

body burdens, and illnesses. Available online at http://www.epa.gov/envirohealth/children.

2

3

E’ vero che nel sangue e nei tessuti di tutti gli uomini e le donne che vivono in ambienti urbanie/o industriali e persino nel sangue cordonale e placentare e nei tessuti fetali sono presentiquesti stessi inquinanti in quantità di anno in anno, di decennio in decennio maggiori ?

What is the Global Chemical Burden..

Industrial chemicals in mothers and daughters:

the pollution we share and inherit

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 ?

DOHAD

XXI secolo: drammatica trasformazione

dell’ambiente del microambiente uterino

Fetal Programming

Differentiation

Cellular Differentiation: an Epigentic process 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 morphoown epigenetic and morpho--functionalfunctional

characteristics)..

1

Nature 447, 425-432 (24 May 2007)

Fetal Programming

epi-mutations

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…

2

PLASTICITY

Epigenetic modifications during early life

can influence the mature phenotype and

determine sensitivity to later environmental

Napoli C, Infante T, Casamassimi A. Maternal-foetal epigeneticinteractions in the beginning of cardiovascular damageCardiovasc Res. 2011 Dec 1;92(3):367-74

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 histonecharacterized 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

• 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 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,

A

B

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 self-limited vasculitis affords a uniqueopportunity 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 …

Ernesto Burgio

Comitato Scientifico

ISDE Italia

La Malattia di Kawasaki è una

(immuno)vasculite sistemica?

How can an illness look like an

infectious disease but not have a

recoverable agent..

1

Two generations of

clinicians and researchers

have been stumped

by the riddle of KD..

.. look like an immune-mediated vasculitis

but not be easily treated with corticosteroids

2

3

..look like a benign, self-limited illness but be the leading cause

of acquired heart disease in children?

3

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.

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.

The earliest changes are observed in the

endothelium of the musculoelastic arteries

..more advanced lesions show edema and

inflammatory-cell infiltrate in the

subendothelial space

1

2

These changes progress to include

destruction of the media 3

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

and then replacement of the intima and media with fibrous

connective tissue, thinning of the media with aneurysm

formation, scarring, and stenosis

4

Coronary-artery rupture is extremely rare but

can occur during the subacute phase

5

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.

• [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

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.

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 ?

• 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)

Microbes

Viruses

IsIs KD a “KD a “NEWNEW” DISEASE ?” DISEASE ?

“changing

environment”

Biosphere

(Coeliac Disease)

Kawasaki Disease(systemic endothelitis)

Immuno-systems

Hypersensitivity

Systemic (micro)vascular

phlogosis

“Neurodegenerative” diseases

Fluid

GenosphereAutism (?)

Atherosclerosis type I

type II

type IIItype IVtype 0Genetic

background

(systemic endothelitis)

Natural Immunity TLRs

Adaptive Immunity

Microbes

Gut Ecosystem

tolerance

La Rivoluzione Epidemica

del XX Secolo

The Barker Hypothesis (1989)

Insulinoresistance

Metabolic Syndrome

Diabetes

Cardiovascular Diseases

(1989)

Asthma - Allergy

Autoimmune Diseases

Salsomaggiore 18/20-04-2008

ISDE International Conference

L’incremento delle patologie cronico-degenerative

[immunomediate, neuro-degenerative, endocrine, neoplastiche, cardiocircolatorie]

è il prodotto di una drammatica trasformazione ambientalee di una (conseguente) alterazione del programming embrio-fetale ?

Ernesto Burgio

(ISDE Italy)

THANK YOU