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Andrea Baccarelli, MD, PhD, MPHLaboratory of Environmental Epigenetics
Mitochondrial Genetics and Epigenetics
Powerful ideas for a healthier world
Novel paths linking air pollution and human disease
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 (phosphorilationenzymes)[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
MitochondrialEpigenetics
Mitochondria &
Environmental Disease
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 stressmtDNA 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 oxida ve markers
Italy benzene multicity studymedian 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 b
enzene
(µg/m
3 )
Relative mtDNA copy number (RmtDNAcn) analysis
• qPCR analysis on 384‐well plate format:– Mitochondrial gene (Mt reaction)– Single copy nuclear gene (S reaction)– 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% longer 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
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-
TF &
MT-
RN
R1
Met
hyla
tion
(%) P=0.002
Controls(n=20)
High-exposed steel workers
(n=20)
mtDNA methylationin steel workers exposed to metal‐rich air particles (PM1)
Byun et al, under review
MT-
TF &
MT-
RN
R1
% M
ethy
latio
n
0.5
Log (PM1 exposure level)1.0 1.5 2.0 2.5
1
0
-1
-2
mtDNA methylationmodeled dose‐response with PM1
Byun et al, under review
Change in
MT‐TF & M
T‐RN
R1
Methylatio
n (%
)
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
Rel
ativ
e m
itoch
ondr
ial c
opy
num
ber
MT-TF & MT-RNR1 % Methylation
r=0.36P=0.02
Byun et al, under review
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 lossand 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
31
Super‐haplogroup clusters in the Normative Aging Study (n=616)
Haplotypes N %
Cluster 1 (J or T) 111 18.0haplogroup J 52 8.4haplogroup T 59 9.6
Cluster 2 (H or V) 314 51.0haplogroup H 53 8.6haplogroup V 261 42.4
Cluster 3 (K or U) 126 20.5haplogroup K 60 9.7haplogroup U 66 10.7
Cluster 4 (I, W or X) 65 10.6haplogroup I 32 5.2haplogroup W 10 1.6haplogroup 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 watchRepetition 1 Speaking back a phraseComplex 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
Estimated Risk of low MMSE due to traffic PM
0.5
1
2
4
Cluster 1 Cluster 2 Cluster 3 Cluster 4
Odd
s Ra
tio
Colicino et al., in preparation
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.
Risk for a doubling in 1‐yr black carbon at baseline
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?– Relationships with mtDNA haplogroups?– Need for longitudinal prospective studies linking past
exposures→mtDNA markers →phenotypes
Acknowledgments
Harvard Environmental Epigenetics lab• Andrea Riganti• Cheng Peng• Elena Colicino• Francesco Nordio• Giulia Giuliano• Katy Monaco• Jia Zhong• Jitendra Barupal• Juan Carmona• Ivan Pantic• Hyang‐Min Byun• Liqiong Guo• Maria Chiara Frisardi• Marco Guerra Sanchez• Nadia Villahur• Rodos Rodostensis• Octavio Jimenez Garza• Valeria Motta
Normative Aging Study Team• Melinda Power• Marc Weisskopf• Lifang Hou• Neil Sondheimer• Avron Spiro• Pantel Vokonas• Joel Schwartz
Multicity Benzene Study• Michele Carugno• Angela Pesatori• Silvia Fustinoni• Pierluigi Cocco• Franco Merlo
Additional funding from the NIH Epigenomics Roadmap, NIEHS, NINR, NHLBI, HSPH‐NIEHS center, and Harvard Catalyst.
NIEHS R01 ES021733Molecular and Epigenetic Mitochondriomics of Air Particles, Lead and Cognition
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