Andrea Baccarelli, MD, PhD, MPH

Laboratory of Environmental Epigenetics

Powerful ideas for a healthier world

Novel tools for identifying environmental

programming of aging

Epigenetics & Mitochondriomics

Background on the mitochondrion

• a cellular organelle – in the cytosol of most

nucleated cells

• produces energy – by oxidising organic acids

and fats with oxygen – process of oxidative

phosphorylation

• generates oxygen radicals as a toxic by-product – Reactive Oxygen Species

(ROS)

The power plant of eukaryotic cells

• Like a power plant, the mitochondrion:

– burns fuel (fat and organic acids)

– produces energy (ATP)

– emits pollution (ROS)

Environmental Mitochondriomics

Pollutant sources

Mitochondria

Release pollutants in the environment

Release endogenous ‘pollutants’ within cells

Amplification of exposure effects

Mitochondrial DNA (mtDNA)

• Extranuclear genome – not part of the genetic

code in the nucleus of your cells

• Small DNA molecule – 16,569 bp

• 37 genes – 13 for proteins

(phosphorilation enzymes) [N.B., all other proteins coded in nuclear DNA]

– 22 for tRNAs – 2 for rRNAs (12S, 16S)

7

Unique characteristics of mtDNA

• All the mtDNA in your body came from your mother (sperm has almost no mitochondria)

• Oxidative damage 5 to 10 times higher than nuclear DNA: – direct exposure to endogenous ROS – lacks protective histones – diminished DNA repair capacity

• Damaged mitochondria burn fat and other energy substrates more inefficiently: – less energy – more ROS

Presentation Outline

investigating environmental mitochondriomics

Mitochondrial damage &

dysfunction

Mitochondrial Epigenetics

Mitochondria &

Environmental Disease

Mitochondrial damage & dysfunction

mitochondrial DNA copy number as an environmental biosensor

High copy number of mtDNA genomes

• Hundreds to thousands mitochondria per cell

• 2-10 mtDNA copies per mitochodrion

Mitochondrial damage and copy number

Exposure

Oxidative stress mtDNA damage

Mitochondrial number increases

Increased ROS

production

Damage to nuclear DNA, RNA, proteins,

and lipids

Air Pollution – health effects & sources

• Epidemiology investigations:

– air pollution exposure is associated with increased hospitalization and early death

– Both acute and long-term effects on cardiorespiratory disease, lung cancer, neurological effects

• Traffic is primary source

– traced by air benzene, black carbon

• Proxidant exposure

• Exposed individuals → high levels of oxidative markers

Italy benzene multicity study median personal air benzene, by city and exposure group

0

20

40

60

80

100

120

140

Genoa Milan Cagliari

P<0.001

P<0.001

P<0.001

Carugno et al., Environ Health Perspect 2012

Med

ian

air

be

nze

ne

(µg

/m3)

Relative mtDNA copy number (RmtDNAcn) analysis

• qPCR analysis on 384-well plate format: – Mitochondrial gene (Mt reaction): mtND1 – Single copy nuclear gene (S reaction): β-globin – Mt/S ration reflects MtDNAcn

• Relative mtDNAcn – To avoid plate effects, MtDNAcn is calculated as relative

difference to a standard DNA (run in each plate) – E.g. RmtDNAcn=1.24: the sample’s mtDNAcn is 24% higher

than the standard DNA – CVs of 3-5% on duplicate samples run on different days

• Key features – Easy to measure – Reflects both damage and dysfunction

City Group N RMtDNAcn (Unadjusted) RMtDNAcn (Adjusted*)

Mean (95% CI) p Mean (95% CI) p

Genoa Referents 48 0.75 (0.65-0.86) 0.75 (0.66-0.85)

Bus Drivers 151 0.90 (0.84-0.97) 0.013 0.90 (0.84-0.97) 0.019

Milan Referents 56 0.76 (0.68-0.84) 0.75 (0.69-0.82)

Police Officers 77 1.14 (1.07-1.22) <0.001 1.10 (1.01-1.19) <0.001

Gas Attendants 76 0.86 (0.79-0.94) 0.037 0.90 (0.83-0.98) 0.005

Cagliari Distant 10 0.94 (0.59-1.48) 0.90 (0.60-1.41)

Close 47 1.24 (1.01-1.52) 0.215 1.25 (1.03-1.51) 0.206

Petrochemical 24 1.64 (1.30-2.07) 0.024 1.63 (1.22-2.18) 0.041

*Geometric mean adjusted for age, sex, smoking habit, number of cigarettes/day

Blood RmtDNAcn, by city and exposure group

Carugno et al., Environ Health Perspect 2012

Blood RmtDNAcn vs. personal air benzene by city and in all subjects

Carugno et al., Environ Health Perspect 2012

RMtDNAcn in the steel worker study

Hou et al, Environmental Health 2010

Paired t-test: p<0.001

Mitochondrial epigenetics

mtDNA methylation as environmental target

Epigenetics

• Programming of gene expression that: – does not depend on the DNA code – (relatively) stable, i.e., replicated through:

• cell mitosis • meiosis, i.e. transgenerational (limited evidence in

humans)

• Characteristics of epigenetic programming – Modifiable (can be reprogrammed)

– Active or poised to be activated: • Potentially associated with current health states or

predict future events

A Symphonic Example

DNA Phenotype

Epigenetics

Epig

en

etic

s &

Mu

sic

Use

th

e

Sam

e M

arki

ngs

Epig

en

etic

s &

Mu

sic

Use

th

e

Sam

e M

arki

ngs

pencil markings (can be erased)

markings in ink (permanent)

DN

A m

eth

ylat

ion

DNA methylation suppresses RNA expression (more accurately: it is usually associated with suppressed RNA)

DNA methylation inactive

DNA demethylation active or poised to be activated

Environmental exposures on nuclear DNA methylation Results from our lab

• Air pollution (PM, foundry PM) – Baccarelli, AJRCCM 2009; – Tarantini, EHP 2009; – Dioni, EHP 2010; – Madrigano, EHP 2011; – Hou, Part Fibre Tox 2011; – Bind, Epidemiol 2012; – Madrigano, AJE 2012 – Sofer, Epigenomics, in press

• Metals – Wright, EHP 2010; – Kile, EHP 2012; – Lambrou, Epidemiology 2012 – Byun, Part Fibre Tox, in press – Guo, under review – Seow, in preparation

• Benzene – Bollati, Cancer Res 2007; – Seow, WH PlosONE 2012; – Fustinoni, Med Lav 2012

• PAHs – Pavanello, Int J Cancer 2009; – Pavanello, Carcinogenesis 2010; – Peluso, Int J Epidemiol; – Alegria, Torres Chemosphere 2012

• POPs and Pesticides – Rusiecki, EHP 2008; – Zhang, Environ Mol Mutagen 2012; – Zhang, Environ Tox Pharmacol 2012; – Villahur, in preparation.

• Phsychosocial stress – Bollati, Chronobiol Int 2010; – Rusiecki, Epigenomics 2012

• Smoking and allergens – Sordillo, Int Arch Aller Immun 2012 – Wan, Hum Mol Gen 2012 – Baccarelli, Epigenomics 2012

Epigenetics of mitochondria

• Methylation of mtDNA has been widely overlooked – total absence of methylation reported in 1973

(Dawid et al, Science)

– subsequent reports showed low methylation levels

• Schock et al., PNAS 2010 – previous studies underestimated the level of cytosine

modification in the mtDNA.

– DNMT1 translocates to the mitochondria • driven by a mitochondrial targeting sequence immediately

upstream of the commonly accepted translational start site.

– mitochondrial DNMT1 • is upregulated in response to hypoxia

• affects mtDNA gene expression

mtDNA methylation in foundry workers

• Foundry workers are exposed to metal-rich air particles (PM)

• mtDNA methylation analysis of a sequence ajdjacent two genes key to mitochondrial protein translation – MT-RNR1 : protein that facilitates formation of RNA

secondary structures, assembly of the mitochondrial ribosome, and mitochondrial translation

– MT-TF gene: a mitochondrion-specific transfer RNA

• Blood DNA from 20 foundry workers with high PM exposure vs. 20 controls

CpG sites in mtDNA

The outer ring (in black) shows the relative position of each of the 435 predicted CpGs

Chinnery et al, Int J Epidemiol 2012

MT-T

F &

MT-R

NR

1

Meth

yla

tion (

%)

P=0.002

Controls

(n=20)

High-exposed

steel workers

(n=20)

mtDNA methylation in steel workers exposed to metal-rich air particles (PM1)

Byun et al, Particle Fib Tox, 2013

mtDNA methylation modeled dose-response with PM1

Byun et al, Particle Fib Tox, 2013

MT-T

F &

MT-R

NR

1

% M

eth

yla

tio

n

0.5

Log (PM1 exposure level) 1.0 1.5 2.0 2.5

1

0

-1

-2

Ch

ange

in

MT-

TF &

MT-

RN

R1

M

eth

ylat

ion

(%

)

P=0.02 for linear effect

mtDNAcn copy number and mtDNA methylation

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0.0 2.0 4.0 6.0 8.0 10.0 12.0

Rela

tive m

itochondria

l copy n

um

be

r

MT-TF & MT-RNR1 % Methylation

r=0.36

P=0.02

Byun et al, Particle Fib Tox, 2013

mitochondria & environmental disease

Mitochondrial haplogroup clusters and cognitive aging

Air pollution and age-related cognitive decline

• >10% of individuals >65 years and 50% of those ≥85 years have some form of cognitive impairment

• Environmental exposures that augment systemic oxidative stress have been shown to hasten cognitive aging by as much as 5 years – PM from vehicular traffic associated with lower mini–

mental state examination (MMSE) in the Normative Aging Study (Powers, EHP 2012)

– Results consistent with data from the NHANES (Chen, Neurotoxicology 2009), China (Zheng AJPH 2010) and Germany (Rantf Environ Res 2008)

• Rare mitochondrial DNA mutations/deletion produce neurocognitive phenotypes

Air pollution, age related cognitive loss and mitochondria in the NAS

• The Normative Aging Study – ongoing longitudinal cohort study of ~700 elderly men

– followed up every 3-5 years from 1996 to date

– mean age 73 years (range 55-100)

• Haplogroups measured for most of the individuas.

• Exposure to Black Carbon – a tracer of particulate air pollution from traffic

– validated spatio-temporal land-use regression model

– 1-year average the participant’s address prior to the date of the first cognitive assessment

40

mtDNA mutations/deletions are associated with neurological phenotypes

• Leber’s Hereditary Optic Neuropathy

• Mitochondrial encephalopathy, Lactic Acidosis and Stroke-like apisodes (MELAS)

• Kearns-Sayre syndrome

• Progressive encephalopathy

Super-haplogroup clusters in the Normative Aging Study (n=616)

Haplotypes N %

Cluster 1 (J or T) 111 18.0

haplogroup J 52 8.4

haplogroup T 59 9.6

Cluster 2 (H or V) 314 51.0

haplogroup H 53 8.6

haplogroup V 261 42.4

Cluster 3 (K or U) 126 20.5

haplogroup K 60 9.7

haplogroup U 66 10.7

Cluster 4 (I, W or X) 65 10.6

haplogroup I 32 5.2

haplogroup W 10 1.6

haplogroup X 23 3.7

Cluster 1

Cluster 2

Cluster 3

Cluster 4

Clusters of haplogroups created using phylogenetic network and evolutionary tree

AD

Fronto-temporal degeneration

Mini-mental state examination (MMSE)

Category Possible points

Description

Orientation to time

5 From broadest to most narrow. Orientation to time has been

correlated with future decline. Orientation to

place 5

From broadest to most narrow. This is sometimes narrowed down to streets, and sometimes to floor.

Registration 3 Repeating named prompts

Attention and calculation

5 Serial sevens, or spelling "world" backwards It has been

suggested that serial sevens may be more appropriate in a population where English is not the first language.

Recall 3 Registration recall Language 2 Naming a pencil and a watch Repetition 1 Speaking back a phrase Complex

commands 6 Varies. Can involve drawing figure shown.

• 30-point questionnaire test of cognitive function • Commonly used to screen for dementia. • Score<25 → low MMSE

0.00

1.00

2.00

3.00

4.00

5.00

6.00

All subjects Cluster 1 Cluster 2 Cluster 3 Cluster 4

OR

(9

5%

CI)

Estimated Risk of low MMSE due to traffic PM

Colicino et al., submitted

P=0.01 for interaction between exposure and clusters

Adjusted for education, alcohol, physical activity, diabetes, dark fish consumption, computer experience, first language, non-white census tract percentage, college degree census tract percentage, first cognitive assessment, part time residents, matrilineal ethnicity.

Risk for a doubling in 1-yr black carbon at baseline

Molecular and epigenetic mitochondriomics of air particles, lead and cognition (R01ES021733)

Inflammation & oxidative stress

Air Particulate

Matter Soluble constituents

Lead

Inhalation

Gastro Intestinalabsorption

Healthy mitochondriain blood

Aged, enlarged, damagedmitochondria in blood

Lead

Mitochondrial DNA

oxidation

heteroplasmy

abundancy

Increased systemicoxidation

Nuclear DNA genes coding for mitochondrial proteins

Altered DNAmethylation

Bone accumulation

Use forrisk prediction(biomarkers)

Cognitive decline

Blood

Longitudinal studies with multiple biospecimens and data collections over up to 20 yrs: -700 elderly participants in the Normative Aging Study (Discovery cohort) -600 elderly participants in MOBILIZE (Replication cohort)

Effects of Air Pollution and Lead on DNA methylation of nuclear –encoded methylation genes

Mitochondrial Protein

Synthesis

Respiratory Chain

Mitochondrial Signaling

Black Carbon

MMSE decline

8 genes p=0.03

9 genes p=0.02

13 genes p=0.005

Bone Lead

Pilot data from 145 elderly participants in the Normative Aging Study Data from genome-wide nuclear methylation arrays

Summary

• Effects of air pollution exposure on: – mtDNA copy number – mtDNA methylation – nDNA methylation of genes encoding for mitochondrial proteins

• Age-related cognitive loss – Hastened by traffic related air pollution – Stronger effects in superhaplogroup clusters 1 & 4

• Questions and future directions – Relevance of mtDNA copy numbers and mtDNA/nDNA

methylation to human disease? – Novel techniques, e.g., deep sequencing – Need for longitudinal prospective studies linking past

exposures→mtDNA markers →phenotypes

Acknowledgments

Harvard Environmental Epigenetics Lab • Yamna Anwar • Kasey Brennan • Hyang-Min Byun • Juan Carmona • Akin Cayir • Elena Colicino • Hanine Haji • Abby Jensen • Allan Just • Kaylene Lin • Pia Pafundi • Ivan Pantic • Cheng Peng • Diddier Prada • Rodos Rodostensis • Marco Guerra Sanchez • Paolo Testa • Jing Zhang • Yan Zhao • Jia Zhong

Collaborators • Pier Bertazzi, Milan • Michele Carugno, Milan • Joel Schwartz, Harvard • Marc Weisskopf, Harvard • Valentina Bollati, Milan • Pan Vokonas, VA Boston

Funding • Multiple NIEHS grants • NIH Epigenomics Roadmap • NHLBI • American Heart Association • Harvard Catalyst • NIEHS-HSPH Center • HSPH Dean’s fund

Andrea Baccarelli - Harvard School of Public Health

Environmental Epigenetics Lab

abaccare@hsph.harvard.edu Thanks!