Chronic arsenic exposure in Bangladesh and the United States: from nutritional influences on arsenic methylation to arsenic-induced epigenetic dysregulation

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

Nutritional Influences on One-Carbon Metabolism: Effects on Arsenic Methylation and Toxicity

Exposure to inorganic arsenic (InAs) via drinking water and/or food is a considerable worldwide problem. Methylation of InAs generates monomethyl (MMAsIII+V)- and dimethyl (DMAsIII+V)-arsenical species in a process that facilitates urinary As elimination; however, MMAs is considerably more toxic than either InAs or DMAs. Emerging evidence suggests that incomplete methylation of As to DMAs, resulting in increased MMAs, is associated with increased risk for a host of As-related health outcomes. The biochemical pathway that provides methyl groups for As methylation, one-carbon metabolism (OCM), is influenced by folate and other micronutrients, including choline and betaine. Individuals and species differ widely in their ability to methylate As. A growing body of research, including cell-culture, animal-model, and epidemiological studies, has demonstrated the role of OCM-related micronutrients in As methylation. This review examines the evidence that nutritional status and nutritional interventions can influence the metabolism and toxicity of As, with a primary focus on folate

Frequent Users of Services in New York City: Investigating Antecedent Factors, Life Experiences, and Triggers Associated with Cycles of Homelessness and Incarceration

This research investigates the antecedent factors and triggers of frequent episodes of homelessness and incarceration among individuals in New York City. Prior studies have shown a high rate of homelessness among formerly incarcerated individuals and, conversely, a high rate of incarceration among homeless individuals. In New York City, as many as one in five inmates was homeless immediately prior to incarceration and many return to homelessness upon discharge. Individuals who cycle between homeless shelters and jails and prisons have distinct social and health service needs. Improved knowledge of the factors and experiences associated with entrance into the cycle of homelessness and incarceration may improve the delivery and efficacy of services to help prevent and break the cycle. This research consists of a secondary analysis of data derived from the Housing and Services Evaluation (HASE) study of frequent users of New York City homeless shelters and jails. Qualitative and quantitative analysis was used to address the following aims: The first aim was to examine risk factors antecedent to homelessness and incarceration by investigating individual-level characteristics including demographics, clinical conditions, human capital, and social ties. The second was to investigate negative life experiences that appear to increase vulnerability of experiencing frequent homelessness and incarceration. The third aim was to examine proximal events that trigger the initial episode of homelessness. Results showed high rates of limited human capital, limited family networks, chronic health conditions, mental illness, substance abuse, and negative experiences in childhood and adolescence. Analysis of narrative responses identified events that commonly trigger homelessness, including involvement with the criminal justice system, interpersonal conflict, change in household composition, being "kicked out" of shared housing, and employment or other economic problems

Cadmium, Smoking, and Human Blood DNA Methylation Profiles in Adults from the Strong Heart Study.

The epigenetic effects of individual environmental toxicants in tobacco remain largely unexplored. Cadmium (Cd) has been associated with smoking-related health effects, and its concentration in tobacco smoke is higher in comparison with other metals. We studied the association of Cd and smoking exposures with human blood DNA methylation (DNAm) profiles. We also evaluated the implication of findings to relevant methylation pathways and the potential contribution of Cd exposure from smoking to explain the association between smoking and site-specific DNAm. We conducted an epigenome-wide association study of urine Cd and self-reported smoking (current and former vs. never, and cumulative smoking dose) with blood DNAm in 790,026 CpGs (methylation sites) measured with the Illumina Infinium Human MethylationEPIC (Illumina Inc.) platform in 2,325 adults 45-74 years of age who participated in the Strong Heart Study in 1989-1991. In a mediation analysis, we estimated the amount of change in DNAm associated with smoking that can be independently attributed to increases in urine Cd concentrations from smoking. We also conducted enrichment analyses and in silico protein-protein interaction networks to explore the biological relevance of the findings. At a false discovery rate (FDR)-corrected level of 0.05, we found 6 differentially methylated positions (DMPs) for Cd; 288 and 17, respectively, for current and former smoking status; and 77 for cigarette pack-years. Enrichment analyses of these DMPs displayed enrichment of 58 and 6 Gene Ontology and Kyoto Encyclopedia of Genes and Genomes gene sets, respectively, including biological pathways for cancer and cardiovascular disease. In in silico protein-to-protein networks, we observed key proteins in DNAm pathways directly and indirectly connected to Cd- and smoking-DMPs. Among DMPs that were significant for both Cd and current smoking (annotated to PRSS23, AHRR, F2RL3, RARA, and 2q37.1), we found statistically significant contributions of Cd to smoking-related DNAm. Beyond replicating well-known smoking epigenetic signatures, we found novel DMPs related to smoking. Moreover, increases in smoking-related Cd exposure were associated with differential DNAm. Our integrative analysis supports a biological link for Cd and smoking-associated health effects, including the possibility that Cd is partly responsible for smoking toxicity through epigenetic changes. https://doi.org/10.1289/EHP6345.This work was supported by grants by the National Heart, Lung, and Blood Institute (NHLBI) (under contract numbers 75N92019D00027, 75N92019D00028, 75N92019D00029, & 75N92019D00030) and previous grants (R01HL090863, R01HL109315, R01HL109301, R01HL109284, R01HL109282, and R01HL109319 and cooperative agreements U01HL41642, U01HL41652, U01HL41654, U01HL65520, and U01HL65521), by the National Institute of Health Sciences (R01ES021367, R01ES025216, P42ES010349, P30ES009089), by the Spanish Funds for Research In Health Sciences, Carlos III Health Institute, co-funded by European Regional Development Fund (CP12/03080 and PI15/00071), by Chilean CONICYT/FONDECYT-POSTDOCTORADO Nº3180486 (A.L.R.-C) and a fellowship from “La Caixa” Foundation (ID 100010434). The fellowship code is “LCF/BQ/DR19/11740016.”S

Prenatal metal exposure, cord blood DNA methylation and persistence in childhood: an epigenome-wide association study of 12 metals

Background Prenatal exposure to essential and non-essential metals impacts birth and child health, including fetal growth and neurodevelopment. DNA methylation (DNAm) may be involved in pathways linking prenatal metal exposure and health. In the Project Viva cohort, we analyzed the extent to which metals (As, Ba, Cd, Cr, Cs, Cu, Hg, Mg, Mn, Pb, Se, and Zn) measured in maternal erythrocytes were associated with differentially methylated positions (DMPs) and regions (DMRs) in cord blood and tested if associations persisted in blood collected in mid-childhood. We measured metal concentrations in first-trimester maternal erythrocytes, and DNAm in cord blood (N = 361) and mid-childhood blood (N = 333, 6–10 years) with the Illumina HumanMethylation450 BeadChip. For each metal individually, we tested for DMPs using linear models (considered significant at FDR < 0.05), and for DMRs using comb-p (Sidak p < 0.05). Covariates included biologically relevant variables and estimated cell-type composition. We also performed sex-stratified analyses. Results Pb was associated with decreased methylation of cg20608990 (CASP8) (FDR = 0.04), and Mn was associated with increased methylation of cg02042823 (A2BP1) in cord blood (FDR = 9.73 × 10–6). Both associations remained significant but attenuated in blood DNAm collected at mid-childhood (p < 0.01). Two and nine Mn-associated DMPs were identified in male and female infants, respectively (FDR < 0.05), with two and six persisting in mid-childhood (p < 0.05). All metals except Ba and Pb were associated with ≥ 1 DMR among all infants (Sidak p < 0.05). Overlapping DMRs annotated to genes in the human leukocyte antigen (HLA) region were identified for Cr, Cs, Cu, Hg, Mg, and Mn. Conclusions Prenatal metal exposure is associated with DNAm, including DMRs annotated to genes involved in neurodevelopment. Future research is needed to determine if DNAm partially explains the relationship between prenatal metal exposures and health outcomes

DNA methylation in cord blood as mediator of the association between prenatal arsenic exposure and gestational age

Prenatal arsenic exposure is associated with adverse birth outcomes and disease risk later in life, which could be mediated through epigenetic dysregulation. We evaluated the association between arsenic and gestational age (GA) that was mediated through DNA methylation (DNAm) using data from a Bangladeshi birth cohort. Arsenic exposure was measured in maternal drinking water at ≤16 weeks GA and maternal toenails collected ≤1 month postpartum. Cord blood DNAm was measured using Infinium HumanMethylation450 arrays (n = 44, discovery phase). Top loci identified in the discovery phase were then pyrosequenced in a second group (n = 569, validation phase). Structural equation models (SEM) evaluated the direct and indirect effects of arsenic and DNAm on GA. In the discovery phase, arsenic was associated with differential DNAm of 139 loci that were associated with GA (P 0.10). Each doubling in water arsenic concentration decreased GA by 2 days, which was fully mediated through the main principal component of the top-ten CpGs (P < 0.001). In the validation phase, there were direct and indirect effects of miR214-3 and MCC DNAm on GA. In an adjusted SEM model, mediation of the association between arsenic and GA by miR124-3 was borderline significant (P = 0.061). This study therefore identified DNAm at specific loci in cord blood that mediated the effect of arsenic exposure on GA. Specifically, prenatal arsenic exposure was associated with lower methylation of miR124-3 that mediated the exposure-response of arsenic on GA. Future research should evaluate if these epigenetic changes are persistent and associated with disease risk

Associations between infant sex and DNA methylation across umbilical cord blood, artery, and placenta samples

DNA methylation (DNAm) is vulnerable to dysregulation by environmental exposures during epigenetic reprogramming that occurs in embryogenesis. Sexual dimorphism in environmentally induced DNAm dysregulation has been identified and therefore it is important to understand sex-specific DNAm patterns. DNAm at several autosomal sites has been consistently associated with sex in cord blood and placental foetal tissues. However, there is limited research comparing sex-specific DNAm across tissues, particularly differentially methylated regions (DMRs). This study leverages DNAm data measured using the Illumina HumanMethylation450 BeadChip in cord blood (N = 179), placenta (N = 229), and umbilical artery samples (N = 229) in the PRogramming of Intergenerational Stress Mechanisms (PRISM) cohort to identify autosomal DMRs and differentially methylated positions (DMPs). A replication analyses was conducted in an independent cohort (GEO Accession GSE129841). We identified 183, 257, and 419 DMRs and 2119, 2281, and 3405 DMPs (pBonferroni < 0.05) in cord blood, placenta, and artery samples, respectively. Thirty-nine DMRs overlapped in all three tissues, overlapping with genes involved in spermatogenesis (NKAPL, PIWIL2 and AURKC) and X–inactivation (LRIF1). In replication analysis, 85% of DMRs overlapped with those identified in PRISM. Overall, DMRs and DMPs had higher methylation levels among females in cord blood and artery samples, but higher methylation levels among males in placenta samples. Further research is necessary to understand biological mechanisms that contribute to differences in sex-specific DNAm signatures across tissues, as well as to determine if sexual dimorphism in the epigenome impacts response to environmental stressors