Carrie V. Breton

Prenatal tobacco smoke exposure affects global and gene-specific DNA methylation.

RATIONALE Prenatal exposure to tobacco smoke increases the risk for diseases later in the childs life that may be mediated through alterations in DNA methylation. OBJECTIVES To demonstrate that differences in DNA methylation patterns occur in children exposed to tobacco smoke and that variation in detoxification genes may alter these associations. METHODS Methylation of DNA repetitive elements, LINE1 and AluYb8, was measured using bisulfite conversion and pyrosequencing in buccal cells of 348 children participating in the Childrens Health Study. Gene-specific CpG methylation differences associated with smoke exposure were screened in 272 participants in the Childrens Health Study children using an Illumina GoldenGate panel. CpG loci that demonstrated a statistically significant difference in methylation were validated by pyrosequencing. Estimates were standardized across loci using a Z score to enable cross-comparison of results. MEASUREMENTS AND MAIN RESULTS DNA methylation patterns were associated with in utero exposure to maternal smoking. Exposed children had significantly lower methylation of AluYb8 (beta, -0.31; P = 0.03). Differences in smoking-related effects on LINE1 methylation were observed in children with the common GSTM1 null genotype. Differential methylation of CpG loci in eight genes was identified through the screen. Two genes, AXL and PTPRO, were validated by pyrosequencing and showed significant increases in methylation of 0.37 (P = 0.005) and 0.34 (P = 0.02) in exposed children. The associations with maternal smoking varied by a common GSTP1 haplotype. CONCLUSIONS Life-long effects of in utero exposures may be mediated through alterations in DNA methylation. Variants in detoxification genes may modulate the effects of in utero exposure through epigenetic mechanisms.

Environmental epigenetics: prospects for studying epigenetic mediation of exposure–response relationships

Changes in epigenetic marks such as DNA methylation and histone acetylation are associated with a broad range of disease traits, including cancer, asthma, metabolic disorders, and various reproductive conditions. It seems plausible that changes in epigenetic state may be induced by environmental exposures such as malnutrition, tobacco smoke, air pollutants, metals, organic chemicals, other sources of oxidative stress, and the microbiome, particularly if the exposure occurs during key periods of development. Thus, epigenetic changes could represent an important pathway by which environmental factors influence disease risks, both within individuals and across generations. We discuss some of the challenges in studying epigenetic mediation of pathogenesis and describe some unique opportunities for exploring these phenomena.

Dietary Arsenic Exposure in Bangladesh

Background Millions of people in Bangladesh are at risk of chronic arsenic toxicity from drinking contaminated groundwater, but little is known about diet as an additional source of As exposure. Methods We employed a duplicate diet survey to quantify daily As intake in 47 women residing in Pabna, Bangladesh. All samples were analyzed for total As, and a subset of 35 samples were measured for inorganic arsenic (iAs) using inductively coupled plasma mass spectrometry equipped with a dynamic reaction cell. Results Median daily total As intake was 48 μg As/day [interquartile range (IQR), 33–67) from food and 4 μg As/day (IQR, 2–152) from drinking water. On average, iAs comprised 82% of the total As detected in dietary samples. After adjusting for the estimated inorganic fraction, 34% [95% confidence interval (CI), 21–49%] of all participants exceeded the World Health Organization’s provisional tolerable daily intake (PTDI) of 2.1 μg As/kg-day. Two of the 33 women who used a well with < 50 μg As/L exceeded this recommendation. Conclusions When drinking water concentrations exceeded the Bangladesh drinking water standard of 50 μg As/L, ingested water was the dominant source of exposure. However, as drinking water As concentrations decrease, the relative contribution of dietary As sources becomes more important to ingested dose. The combined intake from both diet and drinking water can cause some individuals to exceed the PTDI in spite of using a tube well that contains < 50 μg As/L.

Genetic and epigenetic variations in inducible nitric oxide synthase promoter, particulate pollution, and exhaled nitric oxide levels in children

BACKGROUND Inducible nitric oxide synthase (iNOS; encoded by nitric oxide synthase isoform 2 [NOS2]) is the major enzyme for nitric oxide synthesis in airways. As such, measurement of fractional concentration of exhaled nitric oxide (Feno) provides an in vivo assessment of iNOS activity. Short-term exposure to air pollution, haplotypes, and DNA methylation in the NOS2 promoter has been associated independently with iNOS expression, Feno levels, or both. OBJECTIVE We aimed to examine the effects of ambient air pollutants, NOS2 promoter haplotypes, and NOS2 promoter methylation on Feno levels in children. METHODS We selected 940 participants in the Childrens Health Study who provided buccal samples and had undergone Feno measurement on the same day. DNA methylation was measured with a bisulfite-PCR Pyrosequencing assay. Seven single nucleotide polymorphisms captured the haplotype diversity in the NOS2 promoter. Average particulate matter with an aerodynamic diameter of 2.5 μm or less (PM(2.5)) and 10 μm (PM(10)) or less and ozone and nitrogen dioxide levels 7 days before Feno measurement were estimated based on air pollution data obtained at central monitoring sites. RESULTS We found interrelated effects of PM(2.5), NOS2 promoter haplotypes, and iNOS methylation on Feno levels. Increased 7-day average PM(2.5) exposure was associated with lower iNOS methylation (P = .01). NOS2 promoter haplotypes were globally associated with NOS2 promoter methylation (P = 6.2 × 10(-8)). There was interaction among 1 common promoter haplotype, iNOS methylation level, and PM(2.5) exposure on Feno levels (P(interaction) = .00007). CONCLUSION Promoter variants in NOS2 and short-term PM(2.5) exposure affect iNOS methylation. This is one of the first studies showing contributions of genetic and epigenetic variations in air pollution-mediated phenotype expression.

Maternal arsenic exposure associated with low birth weight in Bangladesh.

Objective: To characterize the effects of maternal arsenic exposure on birth weight. Methods: Hair, toenail, and drinking water samples were collected from pregnant women (n = 52) at multiple time points during pregnancy and from their newborns after birth. Total arsenic was measured using inductively coupled plasma-mass spectrometry. The association between arsenic and birth weight was investigated using linear and logistic regression models. Results: Maternal hair arsenic measured early in pregnancy was associated with decreased birth weight (&bgr; = −193.5 ± 90.0 g, P = 0.04). Maternal hair and drinking water arsenic levels measured at first prenatal visit were significantly correlated with newborn hair arsenic level (&rgr; = 0.32, P = 0.04 and &rgr; = 0.31, P = 0.04). Conclusions: Results suggest that maternal arsenic exposure early in pregnancy negatively affects newborn birth weight and that maternal hair provides the best integrated measure of arsenic exposure.

Prenatal Tobacco Smoke Exposure Is Associated with Childhood DNA CpG Methylation

Background Smoking while pregnant is associated with a myriad of negative health outcomes in the child. Some of the detrimental effects may be due to epigenetic modifications, although few studies have investigated this hypothesis in detail. Objectives To characterize site-specific epigenetic modifications conferred by prenatal smoking exposure within asthmatic children. Methods Using Illumina HumanMethylation27 microarrays, we estimated the degree of methylation at 27,578 distinct DNA sequences located primarily in gene promoters using whole blood DNA samples from the Childhood Asthma Management Program (CAMP) subset of Asthma BRIDGE childhood asthmatics (n = 527) ages 5–12 with prenatal smoking exposure data available. Using beta-regression, we screened loci for differential methylation related to prenatal smoke exposure, adjusting for gender, age and clinical site, and accounting for multiple comparisons by FDR. Results Of 27,578 loci evaluated, 22,131 (80%) passed quality control assessment and were analyzed. Sixty-five children (12%) had a history of prenatal smoke exposure. At an FDR of 0.05, we identified 19 CpG loci significantly associated with prenatal smoke, of which two replicated in two independent populations. Exposure was associated with a 2% increase in mean CpG methylation in FRMD4A (p = 0.01) and Cllorf52 (p = 0.001) compared to no exposure. Four additional genes, XPNPEP1, PPEF2, SMPD3 and CRYGN, were nominally associated in at least one replication group. Conclusions These data suggest that prenatal exposure to tobacco smoke is associated with reproducible epigenetic changes that persist well into childhood. However, the biological significance of these altered loci remains unknown.

DNA Methylation in the Arginase–Nitric Oxide Synthase Pathway Is Associated with Exhaled Nitric Oxide in Children with Asthma

RATIONALE Genetic variation in arginase (ARG) and nitric oxide synthase (NOS) has been associated with exhaled nitric oxide (FeNO) levels in children. Little is known about whether epigenetic variation in these genes modulates FeNO. OBJECTIVES To evaluate whether DNA methylation in ARG and NOS genes is associated with FeNO. METHODS A subset of 940 participants in the Childrens Health Study were selected for this study. Children were eligible if they had FeNO measurements and buccal cells collected on the same day. CpG loci located in the promoter regions of NOS1, NOS2A, NOS3, ARG1, and ARG2 genes were analyzed. Multiple loci in each gene were evaluated individually and averaged together. DNA methylation was measured using a bisulfite-polymerase chain reaction pyrosequencing assay. Linear regression models were used to investigate the association between DNA methylation and FeNO and whether associations differed by asthma status. MEASUREMENTS AND MAIN RESULTS DNA methylation in ARG2 was significantly associated with FeNO. A 1% increase in average DNA methylation of ARG2 was associated with a 2.3% decrease in FeNO (95% confidence interval, -4 to -0.6). This association was significantly larger in children with asthma (%diff = -8.7%) than in children with no asthma (%diff = -1.6%; p(int) = 0.01). Differences in FeNO by asthma status were also observed for ARG1 (%diff(asthma) = -4.4%; %diff(non-asthma) = 0.3%; p(int) = 0.02). DNA methylation in NOS genes was not associated with FeNO. CONCLUSIONS DNA methylation in ARG1 and ARG2 is associated with FeNO in children with asthma and suggests a possible role for epigenetic regulation of nitric oxide production.

Particulate Matter, DNA Methylation in Nitric Oxide Synthase, and Childhood Respiratory Disease

Background: Air pollutants have been associated with childhood asthma and wheeze. Epigenetic regulation of nitric oxide synthase—the gene responsible for nitric oxide production—may be affected by air pollutants and contribute to the pathogenesis of asthma and wheeze. Objective: Our goal was to investigate the association between air pollutants, DNA methylation, and respiratory outcomes in children. Methods: Given residential address and buccal sample collection date, we estimated 7-day, 1-month, 6-month, and 1-year cumulative average PM2.5 and PM10 (particulate matter ≤ 2.5 and ≤ 10 µm aerodynamic diameter, respectively) exposures for 940 participants in the Children’s Health Study. Methylation of 12 CpG sites in three NOS (nitric oxide synthase) genes was measured using a bisulfite-polymerase chain reaction Pyrosequencing assay. Beta regression models were used to estimate associations between air pollutants, percent DNA methylation, and respiratory outcomes. Results: A 5-µg/m3 increase in PM2.5 was associated with a 0.20% [95% confidence interval (CI): –0.32, –0.07] to 1.0% (95% CI: –1.61, –0.56) lower DNA methylation at NOS2A position 1, 0.06% (95% CI: –0.18, 0.06) to 0.58% (95% CI: –1.13, –0.02) lower methylation at position 2, and 0.34% (95% CI: –0.57, –0.11) to 0.89% (95% CI: –1.57, –0.21) lower methylation at position 3, depending on the length of exposure and CpG locus. One-year PM2.5 exposure was associated with 0.33% (95% CI: 0.01, 0.65) higher in average DNA methylation of 4 loci in the NOS2A CpG island. A 5-µg/m3 increase in 7-day and 1-year PM2.5 was associated with 0.6% (95% CI: 0.13, 0.99) and 2.8% (95% CI: 1.77, 3.75) higher NOS3 DNA methylation. No associations were observed for NOS1. PM10 showed similar but weaker associations with DNA methylation in these genes. Conclusions: PM2.5 exposure was associated with percent DNA methylation of several CpG loci in NOS genes, suggesting an epigenetic mechanism through which these pollutants may alter production of nitric oxide.

A Pathway-based Analysis of Urinary Arsenic Metabolites and Skin Lesions

Inorganic arsenic is metabolized to monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA). Limited evidence suggests that the ability to fully metabolize arsenic into DMA influences susceptibility to disease. To determine whether percentage of MMA was predictive of disease, the authors used data from a case-control study conducted in Bangladesh (2001-2003). Persons who were diagnosed with keratosis, melanosis, Bowens disease, or squamous cell carcinoma were matched on age, sex, and village to persons without these conditions. This analysis was restricted to persons who had no missing data on covariates (859 cases, 868 controls). A path analysis was used to evaluate simultaneously the association between the percentage of all urinary arsenic metabolites and the odds of skin lesions using PROC CALIS in SAS, version 9.1 (SAS Institute, Inc., Cary, North Carolina) and Mplus, version 6.1 (Muthén & Muthén, Los Angeles, California). The odds of skin lesions were significantly associated with log(10) percentage of MMA (adjusted odds ratio (OR(adj)) = 1.56, 95% confidence interval (CI): 1.15, 2.12) but not log(10) percentage of inorganic arsenic (OR(adj) = 1.06, 95% CI: 0.75, 1.50) or log(10) percentage of DMA (OR(adj) = 1.07, 95% CI: 0.33, 3.46). This novel analysis confirmed that persons who excrete a higher proportion of MMA have a greater risk of skin lesions after data are adequately controlled for urinary arsenic metabolites, current arsenic exposure, and other risk factors.

Epigenome-Wide Meta-Analysis of Methylation in Children Related to Prenatal NO2 Air Pollution Exposure

Background: Prenatal exposure to air pollution is considered to be associated with adverse effects on child health. This may partly be mediated by mechanisms related to DNA methylation. Objectives: We investigated associations between exposure to air pollution, using nitrogen dioxide (NO2) as marker, and epigenome-wide cord blood DNA methylation. Methods: We meta-analyzed the associations between NO2 exposure at residential addresses during pregnancy and cord blood DNA methylation (Illumina 450K) in four European and North American studies (n = 1,508) with subsequent look-up analyses in children ages 4 (n = 733) and 8 (n = 786) years. Additionally, we applied a literature-based candidate approach for antioxidant and anti-inflammatory genes. To assess influence of exposure at the transcriptomics level, we related mRNA expression in blood cells to NO2 exposure in 4- (n = 111) and 16-year-olds (n = 239). Results: We found epigenome-wide significant associations [false discovery rate (FDR) p < 0.05] between maternal NO2 exposure during pregnancy and DNA methylation in newborns for 3 CpG sites in mitochondria-related genes: cg12283362 (LONP1), cg24172570 (3.8 kbp upstream of HIBADH), and cg08973675 (SLC25A28). The associations with cg08973675 methylation were also significant in the older children. Further analysis of antioxidant and anti-inflammatory genes revealed differentially methylated CpGs in CAT and TPO in newborns (FDR p < 0.05). NO2 exposure at the time of biosampling in childhood had a significant impact on CAT and TPO expression. Conclusions: NO2 exposure during pregnancy was associated with differential offspring DNA methylation in mitochondria-related genes. Exposure to NO2 was also linked to differential methylation as well as expression of genes involved in antioxidant defense pathways. Citation: Gruzieva O, Xu CJ, Breton CV, Annesi-Maesano I, Antó JM, Auffray C, Ballereau S, Bellander T, Bousquet J, Bustamante M, Charles MA, de Kluizenaar Y, den Dekker HT, Duijts L, Felix JF, Gehring U, Guxens M, Jaddoe VV, Jankipersadsing SA, Merid SK, Kere J, Kumar A, Lemonnier N, Lepeule J, Nystad W, Page CM, Panasevich S, Postma D, Slama R, Sunyer J, Söderhäll C, Yao J, London SJ, Pershagen G, Koppelman GH, Melén E. 2017. Epigenome-wide meta-analysis of methylation in children related to prenatal NO2 air pollution exposure. Environ Health Perspect 125:104–110; http://dx.doi.org/10.1289/EHP36