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1.  Association between length of gestation and cervical DNA methylation of PTGER2 and LINE 1-HS 
Epigenetics  2014;9(8):1083-1091.
Worldwide, more than 1 in 10 infants is born prior to 37 weeks gestation. Preterm birth can lead to increased mortality risk and poor life-long health and neurodevelopmental outcomes. Whether environmental risk factors affect preterm birth through epigenetic phenomena is largely unstudied. We sought to determine whether preterm risk factors, such as smoke exposure and education, were associated with cervical DNA methylation in the prostaglandin E receptor 2 gene (PTGER2) and a repetitive element, long interspersed nuclear element-1 Homo sapiens-specific (LINE 1-HS). Second, we aimed to determine whether mid-pregnancy DNA methylation of these regions in cervical samples could predict the length of gestation. We obtained a cervical swab between 16–19 weeks gestation from 80 women participating in a Mexico City birth cohort, used pyrosequencing to analyze DNA methylation of PTGER2 and LINE 1-HS, and examined associations with maternal covariates. We used accelerated failure time models to analyze associations of DNA methylation with the length of gestation. DNA methylation of both sequences was associated with Pap smear inflammation. LINE 1-HS methylation was associated with smoke exposure, BMI and parity. In adjusted models, gestations were 3.3 days longer (95%CI 0.6, 6.0) for each interquartile range of PTGER2 DNA methylation. Higher LINE 1-HS methylation was associated with shorter gestations (-3.3 days, 95%CI -6.5, -0.2). In conclusion, cervical DNA methylation was associated with risk factors for preterm birth and the length of gestation.
PMCID: PMC4164493  PMID: 24827772
Cervix; DNA methylation; epigenetics; LINE 1; Preterm birth; PTGER2
2.  Pesticide Use and Relative Leukocyte Telomere Length in the Agricultural Health Study 
PLoS ONE  2015;10(7):e0133382.
Some studies suggest that telomere length (TL) may be influenced by environmental exposures, including pesticides. We examined associations between occupational pesticide use reported at three time points and relative telomere length (RTL) in the Agricultural Health Study (AHS), a prospective cohort study of pesticide applicators in Iowa and North Carolina. RTL was measured by qPCR using leukocyte DNA from 568 cancer-free male AHS participants aged 31-94 years with blood samples collected between 2006 and 2008. Self-reported information, including pesticide use, was collected at three time points: enrollment (1993-1997) and two follow-up questionnaires (1998-2003, 2005-2008). For each pesticide, we evaluated cumulative use (using data from all three questionnaires), and more recent use (using data from the last follow-up questionnaire). Multivariable linear regression was used to examine the associations between pesticide use (ever, lifetime days, intensity-weighted lifetime days (lifetime days*intensity score)) and RTL, adjusting for age at blood draw and use of other pesticides. Of the 57 pesticides evaluated with cumulative use, increasing lifetime days of 2,4-D (p-trend=0.001), diazinon (p-trend=0.002), and butylate (p-trend=0.01) were significantly associated with shorter RTL, while increasing lifetime days of alachlor was significantly associated with longer RTL (p-trend=0.03). Only the association with 2,4-D was significant after adjustment for multiple comparisons. Of the 40 pesticides evaluated for recent use, malathion was associated with shorter RTL (p=0.03), and alachlor with longer RTL (p=0.03). Our findings suggest that leukocyte TL may be impacted by cumulative use and recent use of certain pesticides.
PMCID: PMC4510132  PMID: 26196902
3.  GDM Women’s Pre-Pregnancy Overweight/Obesity and Gestational Weight Gain on Offspring Overweight Status 
PLoS ONE  2015;10(6):e0129536.
To examine the association of maternal pre-pregnancy body mass index (BMI) and gestational weight gain (GWG) with anthropometry in the offspring of mothers with gestational diabetes mellitus (GDM).
We performed a retrospective cohort study in 1263 GDM mother-child pairs. General linear models and Logistic regression models were used to assess the single and joint associations of maternal pre-pregnancy BMI (normal weight, overweight, and obesity) and GWG (inadequate, adequate and excessive GWG) with anthropometry and overweight status in the offspring from birth to 1-5 years old.
Maternal pre-pregnancy BMI and GWG were positively associated with birth weight for gestational age Z score and birth weight for length for gestational age Z score at birth, and weight for age Z score, length/height for age Z score, and weight for length/height Z score at of 1-5 years old offspring. Maternal pre-pregnancy overweight, obesity, and excessive GWG were associated with increased risks of large for gestational age [ORs 95% CIs = 1.87 (1.37-2.55), 2.98 (1.89-4.69), and 2.93 (2.07-4.13), respectively] and macrosomia [ORs 95% CIs = 2.06 (1.50-2.84), 2.89 (1.78-4.70), and 2.84 (1.98-4.06), respectively] at birth and childhood overweight at 1-5 years old [ORs 95% CIs = 1.26 (0.92-1.73), 1.96 (1.24-3.09), and 1.59 (1.15-2.21), respectively].
Offspring born to GDM mothers with pre-pregnancy overweight/obesity or excessive GWG were associated with increased risks of large for gestational age and macrosomia at birth, and childhood overweight at 1-5 years old, compared with those born to GDM mothers with pre-pregnancy normal weight and adequate GWG.
PMCID: PMC4476720  PMID: 26098307
4.  IARC Monographs: 40 Years of Evaluating Carcinogenic Hazards to Humans 
Pearce, Neil | Blair, Aaron | Vineis, Paolo | Ahrens, Wolfgang | Andersen, Aage | Anto, Josep M. | Armstrong, Bruce K. | Baccarelli, Andrea A. | Beland, Frederick A. | Berrington, Amy | Bertazzi, Pier Alberto | Birnbaum, Linda S. | Brownson, Ross C. | Bucher, John R. | Cantor, Kenneth P. | Cardis, Elisabeth | Cherrie, John W. | Christiani, David C. | Cocco, Pierluigi | Coggon, David | Comba, Pietro | Demers, Paul A. | Dement, John M. | Douwes, Jeroen | Eisen, Ellen A. | Engel, Lawrence S. | Fenske, Richard A. | Fleming, Lora E. | Fletcher, Tony | Fontham, Elizabeth | Forastiere, Francesco | Frentzel-Beyme, Rainer | Fritschi, Lin | Gerin, Michel | Goldberg, Marcel | Grandjean, Philippe | Grimsrud, Tom K. | Gustavsson, Per | Haines, Andy | Hartge, Patricia | Hansen, Johnni | Hauptmann, Michael | Heederik, Dick | Hemminki, Kari | Hemon, Denis | Hertz-Picciotto, Irva | Hoppin, Jane A. | Huff, James | Jarvholm, Bengt | Kang, Daehee | Karagas, Margaret R. | Kjaerheim, Kristina | Kjuus, Helge | Kogevinas, Manolis | Kriebel, David | Kristensen, Petter | Kromhout, Hans | Laden, Francine | Lebailly, Pierre | LeMasters, Grace | Lubin, Jay H. | Lynch, Charles F. | Lynge, Elsebeth | ‘t Mannetje, Andrea | McMichael, Anthony J. | McLaughlin, John R. | Marrett, Loraine | Martuzzi, Marco | Merchant, James A. | Merler, Enzo | Merletti, Franco | Miller, Anthony | Mirer, Franklin E. | Monson, Richard | Nordby, Karl-Cristian | Olshan, Andrew F. | Parent, Marie-Elise | Perera, Frederica P. | Perry, Melissa J. | Pesatori, Angela Cecilia | Pirastu, Roberta | Porta, Miquel | Pukkala, Eero | Rice, Carol | Richardson, David B. | Ritter, Leonard | Ritz, Beate | Ronckers, Cecile M. | Rushton, Lesley | Rusiecki, Jennifer A. | Rusyn, Ivan | Samet, Jonathan M. | Sandler, Dale P. | de Sanjose, Silvia | Schernhammer, Eva | Costantini, Adele Seniori | Seixas, Noah | Shy, Carl | Siemiatycki, Jack | Silverman, Debra T. | Simonato, Lorenzo | Smith, Allan H. | Smith, Martyn T. | Spinelli, John J. | Spitz, Margaret R. | Stallones, Lorann | Stayner, Leslie T. | Steenland, Kyle | Stenzel, Mark | Stewart, Bernard W. | Stewart, Patricia A. | Symanski, Elaine | Terracini, Benedetto | Tolbert, Paige E. | Vainio, Harri | Vena, John | Vermeulen, Roel | Victora, Cesar G. | Ward, Elizabeth M. | Weinberg, Clarice R. | Weisenburger, Dennis | Wesseling, Catharina | Weiderpass, Elisabete | Zahm, Shelia Hoar
Environmental Health Perspectives  2015;123(6):507-514.
Background: Recently, the International Agency for Research on Cancer (IARC) Programme for the Evaluation of Carcinogenic Risks to Humans has been criticized for several of its evaluations, and also for the approach used to perform these evaluations. Some critics have claimed that failures of IARC Working Groups to recognize study weaknesses and biases of Working Group members have led to inappropriate classification of a number of agents as carcinogenic to humans.
Objectives: The authors of this Commentary are scientists from various disciplines relevant to the identification and hazard evaluation of human carcinogens. We examined criticisms of the IARC classification process to determine the validity of these concerns. Here, we present the results of that examination, review the history of IARC evaluations, and describe how the IARC evaluations are performed.
Discussion: We concluded that these recent criticisms are unconvincing. The procedures employed by IARC to assemble Working Groups of scientists from the various disciplines and the techniques followed to review the literature and perform hazard assessment of various agents provide a balanced evaluation and an appropriate indication of the weight of the evidence. Some disagreement by individual scientists to some evaluations is not evidence of process failure. The review process has been modified over time and will undoubtedly be altered in the future to improve the process. Any process can in theory be improved, and we would support continued review and improvement of the IARC processes. This does not mean, however, that the current procedures are flawed.
Conclusions: The IARC Monographs have made, and continue to make, major contributions to the scientific underpinning for societal actions to improve the public’s health.
Citation: Pearce N, Blair A, Vineis P, Ahrens W, Andersen A, Anto JM, Armstrong BK, Baccarelli AA, Beland FA, Berrington A, Bertazzi PA, Birnbaum LS, Brownson RC, Bucher JR, Cantor KP, Cardis E, Cherrie JW, Christiani DC, Cocco P, Coggon D, Comba P, Demers PA, Dement JM, Douwes J, Eisen EA, Engel LS, Fenske RA, Fleming LE, Fletcher T, Fontham E, Forastiere F, Frentzel-Beyme R, Fritschi L, Gerin M, Goldberg M, Grandjean P, Grimsrud TK, Gustavsson P, Haines A, Hartge P, Hansen J, Hauptmann M, Heederik D, Hemminki K, Hemon D, Hertz-Picciotto I, Hoppin JA, Huff J, Jarvholm B, Kang D, Karagas MR, Kjaerheim K, Kjuus H, Kogevinas M, Kriebel D, Kristensen P, Kromhout H, Laden F, Lebailly P, LeMasters G, Lubin JH, Lynch CF, Lynge E, ‘t Mannetje A, McMichael AJ, McLaughlin JR, Marrett L, Martuzzi M, Merchant JA, Merler E, Merletti F, Miller A, Mirer FE, Monson R, Nordby KC, Olshan AF, Parent ME, Perera FP, Perry MJ, Pesatori AC, Pirastu R, Porta M, Pukkala E, Rice C, Richardson DB, Ritter L, Ritz B, Ronckers CM, Rushton L, Rusiecki JA, Rusyn I, Samet JM, Sandler DP, de Sanjose S, Schernhammer E, Seniori Costantini A, Seixas N, Shy C, Siemiatycki J, Silverman DT, Simonato L, Smith AH, Smith MT, Spinelli JJ, Spitz MR, Stallones L, Stayner LT, Steenland K, Stenzel M, Stewart BW, Stewart PA, Symanski E, Terracini B, Tolbert PE, Vainio H, Vena J, Vermeulen R, Victora CG, Ward EM, Weinberg CR, Weisenburger D, Wesseling C, Weiderpass E, Zahm SH. 2015. IARC Monographs: 40 years of evaluating carcinogenic hazards to humans. Environ Health Perspect 123:507–514;
PMCID: PMC4455595  PMID: 25712798
5.  Global Methylation and Hydroxymethylation in DNA from Blood and Saliva in Healthy Volunteers 
BioMed Research International  2015;2015:845041.
Aims. We describe a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify and compare simultaneously global methylation and hydroxymethylation in human DNA of different tissues. Materials and Methods. Blood and saliva DNA from fourteen volunteers was processed for epigenetic endpoints using LC-MS/MS and PCR-pyrosequencing technology. Results. Global DNA methylation was significantly lower in saliva (mean 4.61% ±  0.80%), compared to blood samples (5.70% ± 0.22%). In contrast, saliva (0.036% ± 0.011%) revealed significantly higher hydroxymethylation compared to blood samples (mean 0.027% ± 0.004%). Whereas we did not find significant correlations for both epigenetic measures between the tissues, a significant association was observed between global methylation and global hydroxymethylation in saliva DNA. Neither LINE-1 nor Alu elements of blood and saliva correlated, nor were they correlated with the DNA hydroxymethylation of blood or saliva, respectively. Conclusion. Global DNA methylation and hydroxymethylation of cytosine can be quantified simultaneously by LC-MS/MS. Saliva DNA cannot be considered as a surrogate for blood DNA to study epigenetic endpoints.
PMCID: PMC4450276  PMID: 26090450
6.  Effects of Short-term Exposure to Inhalable Particulate Matter on DNA Methylation of Tandem Repeats 
There is compelling evidence that particulate matter (PM) increases lung cancer risk by triggering systemic inflammation, and leukocyte DNA hypomethylation. However, previous investigations focused on repeated element sequences from LINE-1 and Alu families. Tandem repeats, which display a greater propensity to mutate, and are often hypomethylated in cancer patients, have never been investigated in individuals exposed to PM. We measured methylation of three tandem repeats (SATα, NBL2, D4Z4) by polymerase chain reaction–pyrosequencing on blood samples from truck drivers and office workers (60 per group) in Beijing, China. We used lightweight monitors to measure personal PM2.5 (PM with aerodynamic diameter ≤2.5 µm) and elemental carbon (EC, a tracer of PM from vehicular traffic). Ambient PM10 data were obtained from air quality measuring stations. Overall, an interquartile increase in personal PM2.5 and ambient PM10 levels was associated with a significant covariate-adjusted decrease in SATα methylation (−1.35% 5-methyl cytosine [5mC], P = 0.01; and −1.33%5mC; P = 0.01, respectively). Effects from personal PM2.5 and ambient PM10 on SATα methylation were stronger in truck drivers (−2.34%5mC, P = 0.02; −1.44%5mC, P = 0.06) than office workers (−0.95%5mC, P = 0.26; −1.25%5mC, P = 0.12, respectively). Ambient PM10 was negatively correlated with NBL2 methylation in truck drivers (−1.38%5mC, P = 0.03) but not in office workers (1.04%5mC, P = 0.13). Our result suggests that PM exposure is associated with hypomethylation of selected tandem repeats. Measuring tandem-repeat hypomethylation in easy-to-obtain blood specimens might identify individuals with biological effects and potential cancer risk from PM exposure.
PMCID: PMC4426495  PMID: 24436168
Air pollution; epigenetics; epidemiology
7.  Effects of airborne fine particles (PM2.5) on Deep Vein Thrombosis Admissions in North Eastern United States 
Literature relating air pollution exposure to DVT and pulmonary embolism (PE), in spite of biological plausibility, is sparse. No comprehensive study examining associations between both short and long term exposure to Particulate matter (PM)2.5 and DVT or PE has been published to date. Using a novel PM2.5 prediction model we study whether long and short term PM2.5 exposure is associated with DVT and PE admissions among elderly across the northeastern USA.
We estimated daily exposure of PM2.5 in each zipcode. We investigated long and short-term effects of PM2.5 on DVT and PE hospital admissions. There were 453,413 DVT and 151,829 PE admissions in the study. For short term exposure, we performed a case crossover analysis matching on month and year and defined the hazard period as lag 01 (exposure of day of admission and previous day). For the long term association, we used a Poisson regression.
A 10-µg/m3 increase in short term exposure was associated with a 0.63 % increase in DVT admissions (95% CI = 0.03 to 1.25) and a 6.98 % (95% CI = 5.65 to 8.33) increase in long term exposure admissions. For PE, the associated risks were 0.38 (95% CI = −0.68 to 1.25) and 2.67 % (95% CI = 5.65 to 8.33). These results persisted when analyses were restricted to location-periods meeting the current EPA annual standard of 12-µg/m3.
Our findings showed that PM2.5 exposure was associated with DVT and PE hospital admissions, and that current standards are not protective of this result.
PMCID: PMC4424156  PMID: 25678264
Air Pollution; Public health; Epidemiology; Environment; Venous Thrombosis; Deep-Venous Thrombosis
8.  β-2 adrenergic receptor gene methylation is associated with decreased asthma severity in Inner-City School Children 
Genetic variation in the β-2 adrenergic receptor gene (ADRB2) has been implicated in asthma severity and control with conflicting results. Epigenetic variation in the β-2 adrenergic receptor gene (ADRB2) may play an important role in asthma phenotype.
We aimed to evaluate whether DNA methylation of ADRB2 is associated with asthma phenotypes in inner city school aged children.
Multiple CpG sites in the promoter region of ADRB2 gene were analyzed in 177 children enrolled in the School Inner-City Asthma Study (SICAS). Blood or saliva-derived DNA was measured by bisulfite-polymerase chain reaction pyrosequencing assay. Average percent DNA methylation across the sites was evaluated for association with asthma severity (report of dyspnea, nighttime symptoms, rescue medication use, and baseline spirometry) and morbidity (school absences and unscheduled healthcare visits). Three clades composed of highly correlated methylation sites within the methylated segment of ADRB2 were further analyzed.
Methylation of individual sites generally ranged from 0 – 6% with average percent methylation across sites of 2.4%. Univariate analyses strongly favored the association of higher percent methylation with lower asthma severity measured by report of dyspnea. Furthermore, there was a non-significant trend toward less rescue medication use, nighttime symptoms, school absences, activity limitation due to asthma, and improved lung function measurements with increased methylation. Multivariate analysis demonstrated methylation of ADRB2 gene significantly associated with less dyspnea (odds ratio (OR) 0.2, 95%confidence interval (CI), 0.1 – 0.6, P = 0.002). Each of the 3 clades of methylation sites showed a strong, but not statistically significant, effect on decreased dyspnea.
Conclusions and Clinical Relevance
DNA methylation in the ADRB2 gene is associated with decreased asthma symptom severity, suggesting a role for methylation in asthma phenotypes.
PMCID: PMC3989375  PMID: 24131275
9.  Effect of prenatal arsenic exposure on DNA methylation and leukocyte subpopulations in cord blood 
Epigenetics  2014;9(5):774-782.
Prenatal arsenic exposure is associated with increased risk of disease in adulthood. This has led to considerable interest in arsenic’s ability to disrupt fetal programming. Many studies report that arsenic exposure alters DNA methylation in whole blood but these studies did not adjust for cell mixture. In this study, we examined the relationship between arsenic in maternal drinking water collected ≤ 16 weeks gestational age and DNA methylation in cord blood (n = 44) adjusting for leukocyte-tagged differentially methylated regions. DNA methylation was quantified using the Infinium HumanMethylation 450 BeadChip array. Recursively partitioned mixture modeling examined the relationship between arsenic and methylation at 473,844 CpG sites. Median arsenic concentration in water was 12 µg/L (range < 1- 510 µg/L). Log10 arsenic was associated with altered DNA methylation across the epigenome (P = 0.002); however, adjusting for leukocyte distributions attenuated this association (P = 0.013). We also observed that arsenic had a strong effect on the distribution of leukocytes in cord blood. In adjusted models, every log10 increase in maternal drinking water arsenic exposure was estimated to increase CD8+ T cells by 7.4% (P = 0.0004) and decrease in CD4+ T cells by 9.2% (P = 0.0002). These results show that prenatal exposure to arsenic had an exposure-dependent effect on specific T cell subpopulations in cord blood and altered DNA methylation in cord blood. Future research is needed to determine if these small changes in DNA methylation alter gene expression or are associated with adverse health effects.
PMCID: PMC4063836  PMID: 24525453
arsenic; DNA methylation; cord blood; immune function; leukocytes; developmental programming
10.  Platelet mitochondrial DNA methylation: a potential new marker of cardiovascular disease 
Clinical Epigenetics  2015;7(1):44.
Platelets are critical in the etiology of cardiovascular disease (CVD), and the mitochondria in these cells serve as an energy source for platelet function. Epigenetic factors, especially DNA methylation, have been employed as markers of CVD. Unlike nuclear DNA methylation, mitochondrial DNA (mtDNA) methylation has not been widely studied, in part, due to debate about its existence and role. In this study, we examined platelet mtDNA methylation in relation to CVD.
We measured mtDNA methylation in platelets by bisulfite-PCR pyrosequencing and examined associations of CVD with methylation in mitochondrial genes; cytochrome c oxidase (MT-CO1, MT-CO2, and MT-CO3); tRNA leucine 1 (MT-TL1); ATP synthase (MT-ATP6 and MT-ATP8); and NADH dehydrogenase (MT-MD5). We report that CVD patients have significantly higher mtDNA methylation than healthy controls in MT-CO1 (18.53%, P < 0.0001), MT-CO2 (3.33%, P = 0.0001), MT-CO3 (0.92%, P < 0.0001), and MT-TL1 (1.67%, P = 0.0001), which are involved in ATP synthesis. Platelet mtDNA methylation was not related with age, BMI, and race in this study.
Our results suggest that platelet mtDNA methylation, which could serve as non-invasive and easy-to-obtain markers, may be implicated in the etiology of CVD.
Electronic supplementary material
The online version of this article (doi:10.1186/s13148-015-0078-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4404685  PMID: 25901189
Mitochondrial epigenetics; DNA methylation; Platelet; Cardiovascular disease
11.  Pessimistic orientation in relation to telomere length in older men: the VA Normative Aging Study 
Psychoneuroendocrinology  2014;42:68-76.
Recent research suggests pessimistic orientation is associated with shorter leukocyte telomere length (LTL). However, this is the first study to look not only at effects of pessimistic orientation on average LTL at multiple time points, but also at effects on the rate of change in LTL over time.
Participants were older men from the VA Normative Aging Study (n=490). The Life Orientation Test (LOT) was used to measure optimistic and pessimistic orientations at study baseline, and relative LTL by telomere to single copy gene ratio (T:S ratio) was obtained repeatedly over the course of the study (1999-2008). A total of 1,010 observations were included in the analysis. Linear mixed effect models with a random subject intercept were used to estimate associations.
Higher pessimistic orientation scores were associated with shorter average LTL (percent difference by 1-SD increase in pessimistic orientation (95% CI): -3.08 (-5.62, -0.46)), and the finding was maintained after adjusting for the higher likelihood that healthier individuals return for follow-up visits (-3.44 (-5.95,-0.86)). However, pessimistic orientation scores were not associated with rate of change in LTL over time. No associations were found between overall optimism and optimistic orientation subscale scores and LTL.
Higher pessimistic orientation scores were associated with shorter LTL in older men. While there was no evidence that pessimistic orientation was associated with rate of change in LTL over time, higher levels of pessimistic orientation were associated with shorter LTL at baseline and this association persisted over time.
PMCID: PMC4070424  PMID: 24636503
Optimism; Pessimism; Telomere length
12.  Epigenetic effects of low perinatal doses of flame retardant BDE-47 on mitochondrial and nuclear genes in rat offspring 
Toxicology  2014;328:152-159.
Polybrominated diphenyl ethers (PBDEs) are known endocrine disrupting chemicals used commonly as flame retardants in everything from electronics to furniture. Exposure to PBDEs during early development has been linked to neurodevelopmental delays. Despite mounting evidence of neurological harm from PBDE exposure, the molecular mechanisms underlying these effects on brain function remain unknown. We examined the effects of perinatal exposure to BDE-47, the most biologically active and prevalent BDE congener in North America, on epigenetic patterns in the frontal lobe of Wistar rats. Dams were gavaged with BDE-47 (0.002 and 0.2 mg/kg body weight) at gestation days 9 and 16, and postnatal days 1, 8, and 15. Frontal lobes from offspring at postnatal day 41 were collected to measure 5-methylcytosine (5mC) in mitochondrial cytochrome c oxidase genes (Mt-co1, Mt-co2, and Mt-co3), global nuclear 5-hydroxymethylcytosine (5hmC) content, 5mC in repetitive elements L1Rn, and 5mC in nuclear genes (Bdnf, Crhr1, Mc2r, Nr3c1, and Snca) related to behavioral and brain functions in the nuclear genome. We observed a significant decrease in %5mC in Mt-co2 (difference from control= −0.68%, p=0.01 at the 0.2 mg/kg BDE-47). 5mC in repetitive elements L1Rn decreased at 0.002 mg/kg BDE-47 (difference= −1.23%, p=0.02). Decreased nuclear 5mC was observed in Bdnf and Nr3c1 in BDE-47 exposed rats. However, we did not observe significant effects of PBDE toxicity on DNA methylation patterns for the majority of genes in the brain.
PMCID: PMC4353575  PMID: 25533936
BDE-47; PBDE; Endocrine Disruptors; Epigenetics; DNA methylation; Brain
13.  Environmental exposure and mitochondrial epigenetics: study design and analytical challenges 
Human genetics  2014;133(3):247-257.
The environment can influence human health and disease in many harmful ways. Many epidemiological studies have been conducted with the aim of elucidating the association between environmental exposure and human disease at the molecular and pathological levels, and such associations can often be through induced epigenetic changes. One such mechanism for this is through environmental factors increasing oxidative stress in the cell, and this stress can subsequently lead to alterations in DNA molecules. The two cellular organelles that contain DNA are the nucleus and mitochondria, and the latter are particularly sensitive to oxidative stress, with mitochondrial functions often disrupted by increased stress. There has been a substantial increase over the past decade in the number of epigenetic studies investigating the impact of environmental exposures upon genomic DNA, but to date there has been insufficient attention paid to the impact upon mitochondrial epigenetics in studying human disease with exposure to environment.
Here, in this review, we will discuss mitochondrial epigenetics with regards to epidemiological studies, with particular consideration given to study design and analytical challenges. Furthermore, we suggest future directions and perspectives in the field of mitochondrial epigenetic epidemiological studies.
PMCID: PMC4351743  PMID: 24402053
Mitochondria; epigenetics; epidemiology; DNA methylation; disease
14.  Air pollution and gene-specific methylation in the Normative Aging Study 
Epigenetics  2014;9(3):448-458.
The mechanisms by which air pollution has multiple systemic effects in humans are not fully elucidated, but appear to include inflammation and thrombosis. This study examines whether concentrations of ozone and components of fine particle mass are associated with changes in methylation on tissue factor (F3), interferon gamma (IFN-γ), interleukin 6 (IL-6), toll-like receptor 2 (TLR-2), and intercellular adhesion molecule 1 (ICAM-1). We investigated associations between air pollution exposure and gene-specific methylation in 777 elderly men participating in the Normative Aging Study (1999–2009). We repeatedly measured methylation at multiple CpG sites within each gene’s promoter region and calculated the mean of the position-specific measurements. We examined intermediate-term associations between primary and secondary air pollutants and mean methylation and methylation at each position with distributed-lag models. Increase in air pollutants concentrations was significantly associated with F3, ICAM-1, and TLR-2 hypomethylation, and IFN-γ and IL-6 hypermethylation. An interquartile range increase in black carbon concentration averaged over the four weeks prior to assessment was associated with a 12% reduction in F3 methylation (95% CI: -17% to -6%). For some genes, the change in methylation was observed only at specific locations within the promoter region. DNA methylation may reflect biological impact of air pollution. We found some significant mediated effects of black carbon on fibrinogen through a decrease in F3 methylation, and of sulfate and ozone on ICAM-1 protein through a decrease in ICAM-1 methylation.
PMCID: PMC4053463  PMID: 24385016
air pollution; traffic; gene-specific DNA methylation; effect modification; mediation analysis; elderly
15.  Maternal Glucose during Pregnancy and after Delivery in Women with Gestational Diabetes Mellitus on Overweight Status of Their Children 
BioMed Research International  2015;2015:543038.
Objective. To examine the association of maternal glycemia during pregnancy and after delivery with anthropometry in the offspring of mothers with gestational diabetes mellitus (GDM). Methods. A total of 1,263 GDM mothers and their children finished the health survey at 1–5 years after delivery. Results. Offspring of GDM mothers who were diagnosed with diabetes during pregnancy had higher prevalence of overweight, higher mean weight for height Z scores, and higher mean BMI for age Z scores at 1–5 years old than the offspring of GDM mothers who were diagnosed with impaired glucose tolerance (IGT) during pregnancy. Offspring of GDM mothers who developed diabetes 1–5 years after delivery had higher mean values of Z scores for weight for height and BMI for age at 1–5 years old than the offspring of GDM mothers who had normal glucose or prediabetes after delivery. Conclusions. Offspring of GDM mothers who were diagnosed with diabetes during pregnancy or after delivery had an increased risk of childhood overweight or weight gain at 1–5 years old compared with children of GDM mothers with IGT during pregnancy or with normal glucose or prediabetes after delivery.
PMCID: PMC4352735  PMID: 25802854
16.  DNA methylation age of blood predicts all-cause mortality in later life 
Genome Biology  2015;16(1):25.
DNA methylation levels change with age. Recent studies have identified biomarkers of chronological age based on DNA methylation levels. It is not yet known whether DNA methylation age captures aspects of biological age.
Here we test whether differences between people’s chronological ages and estimated ages, DNA methylation age, predict all-cause mortality in later life. The difference between DNA methylation age and chronological age (Δage) was calculated in four longitudinal cohorts of older people. Meta-analysis of proportional hazards models from the four cohorts was used to determine the association between Δage and mortality. A 5-year higher Δage is associated with a 21% higher mortality risk, adjusting for age and sex. After further adjustments for childhood IQ, education, social class, hypertension, diabetes, cardiovascular disease, and APOE e4 status, there is a 16% increased mortality risk for those with a 5-year higher Δage. A pedigree-based heritability analysis of Δage was conducted in a separate cohort. The heritability of Δage was 0.43.
DNA methylation-derived measures of accelerated aging are heritable traits that predict mortality independently of health status, lifestyle factors, and known genetic factors.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-015-0584-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4350614  PMID: 25633388
17.  Microvesicle-associated microRNA expression is altered upon particulate matter exposure in healthy workers and in A549 cells 
Journal of Applied Toxicology  2014;35(1):59-67.
Cardiovascular disease risk has been consistently linked with particulate matter (PM) exposure. Cell-derived microvesicles (MVs) are released into plasma and transfer microRNAs (miRNAs) between tissues. MVs can be produced by the respiratory system in response to proinflammatory triggers, enter the circulatory system and remotely modify gene expression in cardiovascular tissues. However, whether PM affects MV signaling has never been investigated. In this study, we evaluated expression of microRNAs contained within plasma MVs upon PM exposure both in vivo and in vitro. In the in vivo study, we isolated plasma MVs from healthy steel plant workers before and after workplace PM exposure. We measured the expression of 88 MV-associated miRNAs by real-time polymerase chain reaction. To assess a possible source of the MV miRNAs identified in vivo, we measured their miRNA expression in PM-treated A549 pulmonary cell lines in vitro. MiRNA profiling of plasma MVs showed 5.62- and 13.95-fold increased expression of miR-128 and miR-302c, respectively, after 3 days of workplace PM exposure (P < 0.001). According to Ingenuity Pathway Analysis, miR-128 is part of coronary artery disease pathways, and miR-302c is part of coronary artery disease, cardiac hypertrophy and heart failure pathways. In vitro experiments confirmed a dose-dependent expression of miR-128 in MVs released from A549 cells after 6 h of PM treatment (P = 0.030). MiR-302c was expressed neither from A549 cells nor in reference lung RNA. These results suggest novel PM-activated molecular mechanisms that may mediate the effects of air pollution and could lead to the identification of new diagnostic and therapeutic interventions. Copyright © 2014 The Authors. Journal of Applied Toxicology Published by John Wiley & Sons Ltd.
Cell-derived microvesicles (MVs) are found in plasma and may transfer signals between tissues. In this article, we report in-vivo and in-vitro studies demonstrating that Particulate Matter (PM) affects systemic MV signaling by inducing MV release from alveolar cells into plasma. In-vivo microRNA screening showed increased miR-128 level in plasma MVs after PM exposure. In-vitro experiments confirmed PM-induced release of miR-128 in MVs from A549 alveolar cells. Future studies are warranted to determine the roles of MVs in mediating PM effects.
PMCID: PMC4125569  PMID: 24515752
Particulate matter; microRNAs; microvesicles; steel plant workers; A549 cells
18.  Aberrant Methylation of Hypermethylated-in-Cancer-1 and Exocyclic DNA Adducts in Tobacco Smokers 
Toxicological Sciences  2013;137(1):47-54.
Tobacco smoke has been shown to produce both DNA damage and epigenetic alterations. However, the potential role of DNA damage in generating epigenetic changes is largely underinvestigated in human studies. We examined the effects of smoking on the levels of DNA methylation in genes for tumor protein p53, cyclin-dependent kinase inhibitor2A, hypermethylated-in-cancer-1 (HIC1), interleukin-6, Long Interspersed Nuclear Element type1, and Alu retrotransposons in blood of 177 residents in Thailand using bisulfite-PCR andpyrosequencing. Then, we analyzed the relationship of this methylation with the oxidative DNA adduct, M1dG (a malondialdehyde adduct), measured by 32P-postlabeling. Multivariate statistical analyses showed that HIC1 methylation levels were significantly increased in smokers compared with nonsmokers (p ≤ .05). A dose response was observed, with the highest HIC1 methylation levels in smokers of ≥ 10 cigarettes/day relative to nonsmokers and intermediate values in smokers of 1–9 cigarettes/day (p for trend ≤ .001). No additional relationships were observed. We also evaluated correlations between M1dG and the methylation changes at each HIC1 CpG site individually. The levels of this adduct in smokers showed a significant linear correlation with methylation at one of the 3 CpGs evaluated in HIC1: hypermethylation at position 1904864340 was significantly correlated with the adduct M1dG (covariate-adjusted regression coefficient (β) = .224 ± .101 [SE], p ≤ .05). No other correlations were detected. Our study extends prior work by others associating hypermethylation of HIC1 with smoking; shows that a very specific hypermethylation event can arise from smoking; and encourages future studies that explore a possible role for M1dG in connecting smoking to this latter hypermethylation.
PMCID: PMC3871933  PMID: 24154486
tobacco smoking; overall and site specific methylation; HIC1; oxidative DNA damage; M1dG.
19.  Effects of Temperature and Relative Humidity on DNA Methylation 
Epidemiology (Cambridge, Mass.)  2014;25(4):561-569.
Previous studies have found relationships between DNA methylation and various environmental contaminant exposures. Associations with weather have not been examined. Because temperature and humidity are related to mortality even on non-extreme days, we hypothesized that temperature and relative humidity may affect methylation.
We repeatedly measured methylation on long interspersed nuclear elements (LINE-1), Alu, and 9 candidate genes in blood samples from 777 elderly men participating in the normative aging Study (1999–2009). We assessed whether ambient temperature and relative humidity are related to methylation on LINE-1 and Alu, as well as on genes controlling coagulation, inflammation, cortisol, DNA repair, and metabolic pathway. We examined intermediate-term associations of temperature, relative humidity, and their interaction with methylation, using distributed lag models.
Temperature or relative humidity levels were associated with methylation on tissue factor (F3), intercellular adhesion molecule 1 (ICAM-1), toll-like receptor 2 (TRL-2), carnitine O-acetyltransferase (CRAT), interferon gamma (IFN-γ), inducible nitric oxide synthase (iNOS), and glucocorticoid receptor, LINE-1, and Alu. For instance, a 5°c increase in 3-week average temperature in ICAM-1 methylation was associated with a 9% increase (95% confidence interval: 3% to 15%), whereas a 10% increase in 3-week average relative humidity was associated with a 5% decrease (−8% to −1%). The relative humidity association with ICAM-1 methylation was stronger on hot days than mild days.
DNA methylation in blood cells may reflect biological effects of temperature and relative humidity. Temperature and relative humidity may also interact to produce stronger effects.
PMCID: PMC4224120  PMID: 24809956
20.  Cardiac Autonomic Dysfunction: Particulate Air Pollution Effects Are Modulated by Epigenetic Immunoregulation of Toll‐like Receptor 2 and Dietary Flavonoid Intake 
Short‐term fine particles (PM2.5) exposure is associated with reduced heart rate variability, a strong predictor of cardiac mortality among older people. Identifying modifiable factors that confer susceptibility is essential for intervention. We evaluated whether Toll‐like receptor 2 (TLR2) methylation, a reversible immune‐epigenetic process, and its dietary modulation by flavonoids and methyl nutrients, modify susceptibility to heart rate variability effects following PM2.5 exposure.
Methods and Results
We measured heart rate variability and PM2.5 repeatedly over 11 years (1275 total observations) among 573 elderly men from the Normative Aging Study. Blood TLR2 methylation was analyzed using pyrosequencing. Daily flavonoid and methyl nutrients intakes were assessed through the Food Frequency Questionnaire (FFQ). Every 10 μg/m3 increase in 48‐hour PM2.5 moving average was associated with 7.74% (95% CI: −1.21% to 15.90%; P=0.09), 7.46% (95% CI: 0.99% to 13.50%; P=0.02), 14.18% (95% CI: 1.14% to 25.49%; P=0.03), and 12.94% (95% CI: −2.36% to 25.96%; P=0.09) reductions in root mean square of successive differences, standard deviation of normal‐to‐normal intervals, low‐frequency power, and high‐frequency power, respectively. Higher TLR2 methylation exacerbated the root mean square of successive differences, standard deviation of normal‐to‐normal intervals, low‐frequency, and high‐frequency reductions associated with heightened PM2.5 (Pinteraction=0.006, 0.03, 0.05, 0.04, respectively). Every interquartile‐range increase in flavonoid intake was associated with 5.09% reduction in mean TLR2 methylation (95% CI: 0.12% to 10.06%; P=0.05) and counteracted the effects of PM2.5 on low frequency (Pinteraction=0.05). No significant effect of methyl nutrients on TLR2 methylation was observed.
Higher TLR2 methylation may confer susceptibility to adverse cardiac autonomic effects of PM2.5 exposure in older individuals. Higher flavonoid intake may attenuate these effects, possibly by decreasing TLR2 methylation.
PMCID: PMC4330067  PMID: 25628407
epidemiology; epigenetics; heart rate variability; inflammation; nutrition
21.  Heart rate variability and DNA methylation levels are altered after short-term metal fume exposure among occupational welders: a repeated-measures panel study 
BMC Public Health  2014;14:1279.
In occupational settings, boilermakers are exposed to high levels of metallic fine particulate matter (PM2.5) generated during the welding process. The effect of welding PM2.5 on heart rate variability (HRV) has been described, but the relationship between PM2.5, DNA methylation, and HRV is not known.
In this repeated-measures panel study, we recorded resting HRV and measured DNA methylation levels in transposable elements Alu and long interspersed nuclear element-1 (LINE-1) in peripheral blood leukocytes under ambient conditions (pre-shift) and right after a welding task (post-shift) among 66 welders. We also monitored personal PM2.5 level in the ambient environment and during the welding procedure.
The concentration of welding PM2.5 was significantly higher than background levels in the union hall (0.43 mg/m3 vs. 0.11 mg/m3, p < 0.0001). The natural log of transformed power in the high frequency range (ln HF) had a significantly negative association with PM2.5 exposure (β = -0.76, p = 0.035). pNN10 and pNN20 also had a negative association with PM2.5 exposure (β = -0.16%, p = 0.006 and β = -0.13%, p = 0.030, respectively). PM2.5 was positively associated with LINE-1 methylation [β = 0.79%, 5-methylcytosince (%mC), p = 0.013]; adjusted for covariates. LINE-1 methylation did not show an independent association with HRV.
Acute decline of HRV was observed following exposure to welding PM2.5 and evidence for an epigenetic response of transposable elements to short-term exposure to high-level metal-rich particulates was reported.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2458-14-1279) contains supplementary material, which is available to authorized users.
PMCID: PMC4302115  PMID: 25512264
22.  Mitochondria, Energetics, Epigenetics, and Cellular Responses to Stress 
Environmental Health Perspectives  2014;122(12):1271-1278.
Background: Cells respond to environmental stressors through several key pathways, including response to reactive oxygen species (ROS), nutrient and ATP sensing, DNA damage response (DDR), and epigenetic alterations. Mitochondria play a central role in these pathways not only through energetics and ATP production but also through metabolites generated in the tricarboxylic acid cycle, as well as mitochondria–nuclear signaling related to mitochondria morphology, biogenesis, fission/fusion, mitophagy, apoptosis, and epigenetic regulation.
Objectives: We investigated the concept of bidirectional interactions between mitochondria and cellular pathways in response to environmental stress with a focus on epigenetic regulation, and we examined DNA repair and DDR pathways as examples of biological processes that respond to exogenous insults through changes in homeostasis and altered mitochondrial function.
Methods: The National Institute of Environmental Health Sciences sponsored the Workshop on Mitochondria, Energetics, Epigenetics, Environment, and DNA Damage Response on 25–26 March 2013. Here, we summarize key points and ideas emerging from this meeting.
Discussion: A more comprehensive understanding of signaling mechanisms (cross-talk) between the mitochondria and nucleus is central to elucidating the integration of mitochondrial functions with other cellular response pathways in modulating the effects of environmental agents. Recent studies have highlighted the importance of mitochondrial functions in epigenetic regulation and DDR with environmental stress. Development and application of novel technologies, enhanced experimental models, and a systems-type research approach will help to discern how environmentally induced mitochondrial dysfunction affects key mechanistic pathways.
Conclusions: Understanding mitochondria–cell signaling will provide insight into individual responses to environmental hazards, improving prediction of hazard and susceptibility to environmental stressors.
Citation: Shaughnessy DT, McAllister K, Worth L, Haugen AC, Meyer JN, Domann FE, Van Houten B, Mostoslavsky R, Bultman SJ, Baccarelli AA, Begley TJ, Sobol RW, Hirschey MD, Ideker T, Santos JH, Copeland WC, Tice RR, Balshaw DM, Tyson FL. 2014. Mitochondria, energetics, epigenetics, and cellular responses to stress. Environ Health Perspect 122:1271–1278;
PMCID: PMC4256704  PMID: 25127496
23.  A-clustering: a novel method for the detection of co-regulated methylation regions, and regions associated with exposure 
Bioinformatics  2013;29(22):2884-2891.
Motivation: DNA methylation is a heritable modifiable chemical process that affects gene transcription and is associated with other molecular markers (e.g. gene expression) and biomarkers (e.g. cancer or other diseases). Current technology measures methylation in hundred of thousands, or millions of CpG sites throughout the genome. It is evident that neighboring CpG sites are often highly correlated with each other, and current literature suggests that clusters of adjacent CpG sites are co-regulated.
Results: We develop the Adjacent Site Clustering (A-clustering) algorithm to detect sets of neighboring CpG sites that are correlated with each other. To detect methylation regions associated with exposure, we propose an analysis pipeline for high-dimensional methylation data in which CpG sites within regions identified by A-clustering are modeled as multivariate responses to environmental exposure using a generalized estimating equation approach that assumes exposure equally affects all sites in the cluster. We develop a correlation preserving simulation scheme, and study the proposed methodology via simulations. We study the clusters detected by the algorithm on high dimensional dataset of peripheral blood methylation of pesticide applicators.
Availability: We provide the R package Aclust that efficiently implements the A-clustering and the analysis pipeline, and produces analysis reports. The package is found on
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC3810849  PMID: 23990415
24.  Short-term airborne particulate matter exposure alters the epigenetic landscape of human genes associated with the mitogen-activated protein kinase network: a cross-sectional study 
Environmental Health  2014;13:94.
Exposure to air particulate matter is known to elevate blood biomarkers of inflammation and to increase cardiopulmonary morbidity and mortality. Major components of airborne particulate matter typically include black carbon from traffic and sulfates from coal-burning power plants. DNA methylation is thought to be sensitive to these environmental toxins and possibly mediate environmental effects on clinical outcomes via regulation of gene networks. The underlying mechanisms may include epigenetic modulation of major inflammatory pathways, yet the details remain unclear.
We sought to elucidate how short-term exposure to air pollution components, singly and/or in combination, alter blood DNA methylation in certain inflammation-associated gene networks, MAPK and NF-κB, which may transmit the environmental signal(s) and influence the inflammatory pathway in vivo. To this end, we utilized a custom-integrated workflow—molecular processing, pollution surveillance, biostatical analysis, and bioinformatic visualization—to map novel human (epi)gene pathway-environment interactions.
Specifically, out of 84 MAPK pathway genes considered, we identified 11 whose DNA methylation status was highly associated with black carbon exposure, after adjusting for potential confounders—age, sulfate exposure, smoking, blood cell composition, and blood pressure. Moreover, after adjusting for these confounders, multi-pollutant analysis of synergistic DNA methylations significantly associated with sulfate and BC exposures yielded 14 MAPK genes. No associations were found with the NF-κB pathway.
Exposure to short-term air pollution components thus resulted in quantifiable epigenetic changes in the promoter areas of MAPK pathway genes. Bioinformatic mapping of single- vs. multi-exposure-associated epigenetic changes suggests that these alterations might affect biological pathways in nuanced ways that are not simply additive or fully predictable via individual-level exposure assessments.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-069X-13-94) contains supplementary material, which is available to authorized users.
PMCID: PMC4273424  PMID: 25395096
25.  Air pollution exposure and lung function in highly exposed subjects in Beijing, China: a repeated-measure study 
Exposure to ambient particulate matter (PM) has been associated with reduced lung function. Elemental components of PM have been suggested to have critical roles in PM toxicity, but their contribution to respiratory effects remains under-investigated. We evaluated the effects of traffic-related PM2.5 and its elemental components on lung function in two highly exposed groups of healthy adults in Beijing, China.
The Beijing Truck Driver Air Pollution Study (BTDAS) included 60 truck drivers and 60 office workers evaluated in 2008. On two days separated by 1-2 weeks, we measured lung function at the end of the work day, personal PM2.5, and nine elemental components of PM2.5 during eight hours of work, i.e., elemental carbon (EC), potassium (K), sulfur (S), iron (Fe), silicon (Si), aluminum (Al), zinc (Zn), calcium (Ca), and titanium (Ti). We used covariate-adjusted mixed-effects models including PM2.5 as a covariate to estimate the percentage change in lung function associated with an inter-quartile range (IQR) exposure increase.
The two groups had high and overlapping exposure distributions with mean personal PM2.5 of 94.6 μg/m3 (IQR: 48.5-126.6) in office workers and 126.8 μg/m3 (IQR: 73.9-160.5) in truck drivers. The distributions of the nine elements showed group-specific profiles and generally higher levels in truck drivers. In all subjects combined, forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) did not significantly correlate with PM2.5. However, FEV1 showed negative associations with concentrations of four elements: Si (-3.07%, 95% CI: -5.00; -1.11, IQR: 1.54), Al (-2.88%, 95% CI: -4.91; -0.81, IQR: 0.86), Ca (-1.86%, 95% CI: -2.95; -0.76, IQR: 1.33), and Ti (-2.58%, 95% CI: -4.44; -0.68, IQR: 0.03), and FVC showed negative associations with concentrations of three elements: Si (-3.23%, 95% CI: -5.61; -0.79), Al (-3.26%, 95% CI: -5.73; -0.72), and Ca (-1.86%, 95% CI: -3.23; -0.47). In stratified analysis, Si, Al, Ca, and Ti showed associations with lung function only among truck drivers, and no significant association among office workers.
Selected elemental components of PM2.5 showed effects on lung function that were not found in analyses of particle levels alone.
Electronic supplementary material
The online version of this article (doi:10.1186/s12989-014-0051-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4192276  PMID: 25272992
Lung function; Metals; Particulate matter; Traffic exposure; FEV1; FVC

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