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 Release pollutants in the environment Mitochondria Release endogenous ‘pollutants’ within cells Amplification of exposure effects Piece by Charles W. Schmidt Environmental 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 (phosphorilation enzymes) [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 Mitochondrial Mitochondrial Mitochondria damage & & dysfunction Epigenetics Environmental Disease investigating environmental mitochondriomics Mitochondrial damage & dysfunction mitochondrial DNA copy number as an environmental biosensor Cell types, mitochondria and mtDNA Cell type Mitochondrion mtDNA X X X XX Red blood cell 0 Skin few hundred Lymphocyte 1,000 2 to >10,000 5,000 Heart /brain >100,000 Mitochondrial and nuclear DNA mtDNA nDNA Size (bp) 16,569 ~3,000,000,000 DNA copies per cell 2 to more than 10,000 2 Genes 37 30,000 # of CpGs 435 >28,000,000 Introns No Yes Histones No Yes Oxidative stress x5 x1 DNA repair Absent or quite limited High Mutation rate x10 x1 Byun HM et al., Human Genetics 2014 Mitochondrial damage and copy number Exposure Damage to nuclear DNA, RNA, proteins, and lipids Oxidative stress mtDNA Increased damage ROS production Mitochondrial number increases 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 oxidave 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 study median personal air benzene, by city and exposure group ) 3 140 P<0.001 120 (µg/m 100 80 P<0.001 benzene 60 air P<0.001 40 20 Median 0 Genoa Milan Cagliari Carugno et al., Environ Health Perspect 2012 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 Blood RmtDNAcn, by city and exposure group 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 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 methylation suppresses RNA expression (more accurately: it is usually associated with suppressed RNA) DNA methylation inactive DNA methylation demethylation active or poised to be activated DNA Environmental exposures on nuclear DNA methylation Results from our labs in Milan and Boston • Air pollution (PM, foundry PM) • PAHs – Baccarelli, AJRCCM 2009; – Pavanello, Int J Cancer 2009; – Tarantini, EHP 2009; – Pavanello, Carcinogenesis 2010; – Dioni, EHP 2010; – Peluso, Int J Epidemiol; – Madrigano, EHP 2011; – Alegria, Torres Chemosphere 2012 – Hou, Part Fibre Tox 2011; • POPs and Pesticides – Bind, Epidemiol 2012; – Rusiecki, EHP 2008; – Madrigano, AJE 2012 – Zhang, Environ Mol Mutagen 2012; – Sofer, Epigenomics, in press – Zhang, Environ Tox Pharmacol 2012; • Metals – Villahur, in preparation. – Wright, EHP 2010; • Phsychosocial stress – Kile, EHP 2012; – Bollati, Chronobiol Int 2010; – Lambrou, Epidemiology 2012 – Rusiecki, Epigenomics 2012 – Byun, Part Fibre Tox, in press • Smoking and allergens – Guo, under review – Sordillo, Int Arch Aller Immun 2012 – Seow, in preparation – Wan, Hum Mol Gen 2012 • Benzene – Baccarelli, Epigenomics 2012 – Bollati, Cancer Res 2007; – Seow, WH PlosONE 2012; – Fustinoni, Med Lav 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 mtDNA methylation in steel workers exposed to metal‐rich air particles (PM1) P=0.002 Methylation (%) MT-TF & MT-RNR1 & MT-RNR1 MT-TF Controls High-exposed (n=20) steel workers (n=20) Byun et al, Particle Fib Tox, 2013 mtDNA methylation modeled dose‐response with PM1 P=0.02 for linear effect 1 (%) RNR1 in ‐ MT 0 & Change TF ‐ -1 MT-TF & MT-RNR1 & MT-RNR1 MT-TF Methylation % Methylation MT -2 0.5 1.0 1.5 2.0 2.5 Log (PM1 exposure level) Byun et al, Particle Fib Tox, 2013 mtDNAcn copy number and mtDNA methylation 3.0 r=0.36 P=0.02 2.5 2.0 1.5 1.0 0.5 Relative mitochondrial copy number 0.0 0.0 2.0 4.0 6.0 8.0 10.0 12.0 MT-TF & MT-RNR1 % Methylation Byun et al, Particle Fib Tox, 2013 Ongoing plans for mtDNA research at our lab 2012‐2015 • Blood buffy coat and buccal cells • mtDNA methylation (D‐loop promoter region and 12s rRNA) • Disease outcomes 2015‐onwards • Cell type specific • Purified mtDNA • Whole mtDNA seq/ bisulfite seq • And more 41 Cardiovascular disease pilot • 10 patients with atherosclerotic D-loop Cytochrome b 16S rRNA NADH MT-ND5 cardiovascular disease Dehydrogenase subunits NADH • 15 healthy controls Dehydrogenase Mitochondrial genome tRNA-leucine MT-TL1 subunits NADH Dehydrogenase with similar age and subunits Cytochrome Cytochrome Oxidase Oxidase MT-CO3 sex distributions subunits ATP Synthasesubunits MT-CO1 subunits MT-CO2 • Isolation of mtDNA MT-ATP6 MT-ATP8 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 related” disease, myocardial infarction, cerebrovascular disease, stroke, asthma etc. Isolation of mtDNA from human platelets platelets 200 μl Plasma Centrifuge Buffy coat Erythrocytes Lysis buffer AL Centrifuge DNase I (3hr) (Qiagen) mtDNA Isolating mtDNA from nuclear DNA Total Cellular DNA Mitochondrial DNA X Nuclear DNA XX XX Nuclear DNA + Numts Nuclear DNA sequences of Mitochondrial origin (Numts) Purity of isolated mtDNA by Real Time‐PCR