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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)
EnvironmentalMitochondriomics
Pollutant sources
Mitochondria
Release pollutants in the environment
Release endogenous ‘pollutants’ within cells
Amplification of exposure effects
Piece by Charles W. SchmidtEnvironmental Health Perspectives, July 2010
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)
Unique characteristics of mtDNA
• 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
Mitochondrialdamage & dysfunction
mitochondrial DNA copy number as an environmental biosensor
Red blood cell Skin Lymphocyte
Heart /brain
X XXX X
Cell type
Cell types, mitochondria and mtDNA
0 few hundred 1,000 5,000 >100,000
Mitochondrion
2 to >10,000
mtDNA
16,5692 to more than 10,000
37435NoNox5
Absent or quite limitedx10
~3,000,000,0002
30,000>28,000,000
YesYesx1Highx1
Size (bp)DNA copies per cell
Genes# of CpGsIntronsHistones
Oxidative stressDNA repair
Mutation rate Byun HM et al., Human Genetics 2014
Mitochondrial and nuclear DNA
mtDNA nDNA
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
Italian multi‐city benzene exposure study
• Benzene is a widespread pollutant associated with vehicular traffic emissions– Low‐level benzene may induce oxidative damage– No mechanistic biomarkers are available to detect biological dysfunction at low doses
• To determine whether low‐level benzene is associated with increased blood mitochondrial DNA copy number (mtDNAcn).
Italy multicity benzene exposure 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): 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
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
DNA methylatio
nDNA methylation suppresses RNA expression(more accurately: it is usually associated with suppressed RNA)
DNA methylationinactive DNA
demethylationactive or poised to be activated
Environmental exposures on nuclear DNA methylation Results from our labs in Milan and Boston• 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, Particle Fib Tox, 2013
mtDNA methylationmodeled dose‐response with PM1
Byun et al, Particle Fib Tox, 2013
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
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, Particle Fib Tox, 2013
41
• Blood buffy coat and buccal cells• mtDNA methylation(D‐loop promoter region and 12s rRNA)• Disease outcomes
2012‐2015
• Cell type specific• Purified mtDNA• Whole mtDNA seq/ bisulfite seq• And more
2015‐onwards
Ongoing plans for mtDNA research at our lab
16S rRNA
NADH Dehydrogenase
subunits
Cytochrome Oxidase subunits ATP Synthase
subunits
Cytochrome Oxidase subunits
NADH Dehydrogenase
subunits
NADH Dehydrogenase
subunits
Cytochrome b
Mitochondrial genome
D-loop
MT-CO1MT-CO2
MT-CO3
MT-ATP6MT-ATP8
MT-ND5
tRNA-leucine MT-TL1
Cardiovascular disease pilot
• 10 patients with atherosclerotic cardiovascular disease
• 15 healthy controls with similar age and sex distributions
• Isolation of mtDNA from platelets
Isolation of mtDNA from human platelets
Why platelets?
“Cell type specific” Single blood cell type
“Technically convenient” Anucleate cell with mitochondria
Disorders in which platelets play a key role: Atherosclerosis, coronary artery
disease, myocardial infarction, cerebrovascular disease, stroke,
asthma etc.
“Disease related”
Plasma
Buffy coat
Erythrocytes
200 μl
Centrifuge DNase I (3hr)Lysis buffer AL (Qiagen)
Centrifuge
mtDNA
platelets
Isolation of mtDNA from human platelets
Isolating mtDNA from nuclear DNA
XX
XX X
Total Cellular DNA
Nuclear DNA + Numts
Nuclear DNA sequences of Mitochondrial origin (Numts)
Nuclear DNA
Mitochondrial DNA
Purity of isolated mtDNA by Real Time‐PCR
1. Positive control ‘chrM:3313-3322’ 2. Positive control HBB3. Test sample ‘chrM:3313-3322’4. Test sample HBB
1 2 3
4
Byun HM et al. Methods Mol Biol submitted 2015
Healthy CVD0
5
10
15
20
25
30
% m
tDN
A m
ethy
latio
n
MT-CO1
Healthy CVD012345678
% m
tDN
A m
ethy
latio
n
MT-CO2
Healthy CVD0.0
0.5
1.0
1.5
2.0
2.5
% m
tDN
A m
ethy
latio
n
MT-CO3
Healthy CVD0
1
2
3
4
5
6
% m
tDN
A m
ethy
latio
n
MT-TL1a) b) c) d)**** *** **** ***
Healthy CVD0
1
2
3
4
5
% m
tDN
A m
ethy
latio
n
MT-ATP6
(n=12) (n=10) (n=12) (n=10) (n=15) (n=10) (n=10) (n=9)
e)
Healthy CVD0
1
2
3
4
5
% m
tDN
A m
ethy
latio
n
MT-ATP8f)
Healthy CVD0
2
4
6
8
10
12%
mtD
NA
met
hyla
tion
MT-ND5g)
(n=9) (n=9) (n=12) (n=10) (n=9) (n=10)
CVD patients have different mtDNA methylation
0 10 20 30 40 50 60 70 80 90 1000
102030405060708090
100
100% - Specificity%
Sens
itivi
ty%
MT-CO2
0 10 20 30 40 50 60 70 80 90 1000
102030405060708090
100
100% - Specificity%
Sens
itivi
ty%
MT-CO3
0 10 20 30 40 50 60 70 80 90 1000
102030405060708090
100
100% - Specificity%
Sens
itivi
ty%
MT-TL1
0 10 20 30 40 50 60 70 80 90 1000
102030405060708090
100
100% - Specificity%
Sens
itivi
ty%
MT-CO1
a) b) c) d)
AUROC: 0.99Sensitivity: 100%Specificity: 90%PPV%: 92%NPV%: 100%
AUROC: 0.94Sensitivity: 100%Specificity: 70%PPV%: 80%NPV%: 100%
AUROC: 0.96Sensitivity: 100%Specificity: 70%PPV%: 82%NPV%: 100%
AUROC: 0.97Sensitivity: 100%Specificity: 78%PPV%: 83%NPV%: 100%
Predictive value of mtDNA methylation
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 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
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 18.0haplogroup 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
0.50
1.00
2.00
4.00
8.00
All subjects Cluster 1 Cluster 2 Cluster 3 Cluster 4
OR (95%
CI)
Estimated Risk of low MMSE due to traffic PM
Colicino et al., Environmental Health, 2014
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
Summary of our findings
• Effects of air pollution exposure on:– mtDNA copy number– mtDNA methylation
• Opportunities for disease investigations– Cardiovascular disease– Air pollution acceleration of cognitive aging
• Questions and future directions– Relevance of mtDNA copy numbers and mtDNA 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• Golareh Agha• Kasey Brennan• Juan Carmona• Akin Cayir• Elena Colicino• Deep Deb• Alexandra Dereix• Hanine Haji• Allan Just• Oskar Karlsson• Hannah Laue• Rosie Martinez• Jamaji Nwanaji‐Enwerem• Cheng Peng• Diddier Prada• Rodos Rodostensis• Marco Guerra Sanchez• Letizia Trevisi• Yan Zhao• Jia Zhong
Andrea Baccarelli ‐ Harvard School of Public Health
Collaborators on mtDNA researcg• Pier Bertazzi, Milan• Michele Carugno, Milan• Miriam Hoxha, Milan• Valentina Bollati, Milan• Valeria Motta, Harvard/Milan• Laura Dioni, Milan• Mindy Powers, Johns Hopkins• Marc Weisskopf, Harvard• Joel Schwartz, Harvard• Pan Vokonas, VA Boston• Hyang‐Min Byun, Newcastle
Funding
• R01ES021733Molecular and Epigenetic Mitochondriomics of Air Particles, Lead and Cognition
Environmental Epigenetics [email protected]
