Title: Chronic arsenic exposure in Bangladesh and the United States
Description: Background: Chronic arsenic (As) exposure in a global public health concern. Arsenic exposure through drinking water affects over 140 million people in at least 70 countries, including 40 million people in Bangladesh. In the United States (US), 2.4 million people rely on private wells or public water systems with As levels above the US maximum contaminant level. Ingested inorganic arsenic (InAs) is methylated to monomethyl (MMAs)- and dimethyl (DMAs)-arsenical species using the methyl donor S-adenosylmethionine (SAM). Full methylation of InAs to DMAs decreases As toxicity and facilitates urinary As excretion. Arsenic methylation capacity is influenced by nutrients involved in one-carbon metabolism (OCM), the biochemical pathway that synthesizes SAM. Folate recruits one-carbon units for the remethylation of homocysteine and the synthesis of SAM. The availability of one-carbon units is also impacted by nutrients including the alternative methyl donor betaine, its precursor choline, and possibly the cofactor vitamin B12.

In addition, As methylation capacity may also be influenced by creatine; an estimated 50% of SAM is consumed by the final step of endogenous creatine synthesis. The adverse health outcomes associated with chronic As exposure include impaired intellectual function, cardiovascular disease, diabetes, inflammation, and cancers of the bladder, lung, kidney, liver, and skin. In utero As exposure is associated with adverse birth outcomes include decreased birth weight and gestational age. Elevated health risks persist after exposure has been reduced or ended, leading to the hypothesis that epigenetic dysregulation, including changes in DNA methylation, may be a biological mechanism linking As exposure to health outcomes. Objectives: This research has three main objectives: (1) to investigate the influence of OCM nutritional factors on As methylation by evaluating effects of folic acid (FA) and creatine supplementation on As methylation capacity, and effect modification by baseline status of OCM-related nutrients; (2) to examine associations between As exposure and loci-specific DNA methylation in an epigenome-wide association study (EWAS); and (3) to assess mediation of the association between in utero As exposure and birth outcomes (i.e., gestational age and birth weight) by DNA methylation of target genes identified in an EWAS, as well as the candidate gene DNA methyltransferase 3 alpha (DNMT3A), a protein-coding gene involved in de novo DNA methylation.

Methods: This research used data from three studies of As-exposed individuals. To address the first objective, we used data from the Folic Acid and Creatine Trial (FACT), a 24-week randomized clinical trial of FA (400 or 800 μg/day) and/or creatine supplementation (3 g/day or 3 g creatine and 400 μg FA/day) among As-exposed adults in Bangladesh recruited independent of folate status (N = 622). We investigated overall FA and creatine treatment effects on mean within-person changes in As metabolite proportions in urine compared to the placebo group (weeks 0 to 12). Rebound of As methylation capacity following the cessation of FA supplementation was assessed from weeks 12 to 24. We also assessed effect modification by baseline choline, betaine, vitamin B12, and plasma folate of treatment effects on changes in homocysteine, guanidinoacetate (GAA) (biomarkers of OCM and endogenous creatine synthesis, respectively), total blood As, and urinary As metabolite proportions and indices. To address the second objective, we used data from the Strong Heart Study (SHS), a population-based prospective cohort of American Indians with low-moderate levels of As exposure.

DNA methylation was measured in 2,325 participants using the Illumina MethylationEPIC array, which interrogates > 850,000 loci. We tested for differentially methylated positions (DMPs) and regions (DMRs), and conducted gene ontology (GO) enrichment analysis to understand functions of genes containing differential methylation. To address the third objective, we used data from a prospective birth cohort in Bangladesh. In a discovery phase, an EWAS was conducted to identify CpGs with methylation measured in cord blood that are associated with maternal water As levels and birth outcomes (N = 44). In a validation phase, DNA methylation in cord blood was measured using bisulfite pyrosequencing at three target CpGs annotated to miR124-3, MCC, and GNAL (N = 569). We applied structural equation models (SEMs) to assess mediation of the association between in utero As exposure and gestational age by DNA methylation. In addition, mediation of the association between in utero As exposure and birth outcomes by DNA methylation of the candidate gene DNA methyltransferase alpha (DNMT3A) was assessed.

Results: In FACT, the mean within-person decreases %InAs and %MMAs and increase in %DMAs were greater among all groups receiving FA supplementation at weeks 6 and 12 compared to placebo (P < 0.05) (Chapter 3). Stratified by median choline and betaine concentrations at baseline, we observed a trend towards greater FA treatment effects among participants with levels below the median of both nutrients compared to participants above the median (Chapter 4). Among participants who discontinued FA supplementation, at week 24, %InAs and %DMAs were not significantly different than baseline levels, suggesting a rebound in As methylation capacity with cessation of FA supplementation. We observed a significantly greater mean within-person decreases in %MMAs with creatine supplementation compared to placebo at weeks 1, 6, and 12; mean within-person changes in %InAs and %DMAs did not differ significantly between the creatine and placebo groups (Chapter 3). The mean within-person decrease in urinary %MMAs at week 12 with creatine treatment was significantly greater than placebo among participants with baseline choline concentrations below the median, but did not differ from placebo among participants with choline concentrations above the median (Chapter 4).

In an EWAS conducted in SHS, we identified 20 DMPs associated with urinary As levels at FDR < 0.05; five DMPs were significant at PBonferroni < 0.05 (Chapter 5). The top significant CpG, cg06690548, was located in solute carrier family 7 member 11 (SLC7A11 ), part of the amino-acid transporter cystine:glutamate antiporter system xc-, which is involved in biosynthesis of the endogenous antioxidant glutathione (GSH). Additional Bonferroni-significant CpGs were located in ANKS3, LINGO3, CSNK1D, and ADAMTSL4. We identified one FDR-significant DMR (chr11:2,322,050-2,323,247) including the open reading frame C11orf21 and tetraspanin 32 (TSPAN32 ). Mediation of the association between in utero As exposure and birth outcomes by cord blood DNA methylation was assessed in a Bangladeshi birth cohort. In the discovery phase (N = 44), the association between maternal water As levels and gestational age was fully mediated by DNA methylation of the top 10 CpGs associated with both variables. In a discovery phase (N = 569), there were significant indirect effects of maternal water As levels on gestational age through DNA methylation of miR124-3 and MCC ; the indirect effect through DNA methylation of GNAL was not significant (Chapter 6).

In an adjusted SEM including miR124-3 and MCC, mediation of the association between in utero As exposure and gestational age by DNA methylation of miR124-3 was borderline significant (P = 0.06); DNA methylation of MCC did not act as a mediator. We also assessed mediation by DNA methylation of DNMT3A (Chapter 7). In an adjusted SEM including birth weight and gestational age, there was a significant indirect effect of maternal toenail As levels on gestational age through DNMT3A methylation, the indirect effect on birth weight was borderline significant (P = 0.082). However, the indirect effects of maternal toenail As levels on birth weight through all pathways including gestational age were statistically significant. A doubling in maternal toenail As concentrations had a total effect of a decrease in gestational age of 2.1 days and a decrease in birth weight of 28.9 g. Conclusions: Results from FACT (Chapters 3 and 4) provide evidence of the associations between OCM-related nutrients and As methylation capacity.

Specifically, FA and creatine supplementation may increase As methylation capacity by increasing the availability of SAM, and treatment effects may be greater among individuals with low betaine and choline status, respectively. In addition, results reported in Chapters 5-7 support the hypotheses that chronic As exposure is associated with epigenetic dysregulation, and that changes in the epigenome may mediate the association between As exposure and adverse health effects. Findings from the research presented here may help inform public health interventions to reduce the adverse health effects of chronic As exposure. However, further research is needed to fully understand the biological mechanism that influence As methylation and that underlie the associations between chronic As exposure and adverse health outcomes.