Title: Nutritional influences on arsenic toxicity in Bangladeshi men and women
Description: Background: In Bangladesh, more than 57 million individuals are exposed to arsenic-contaminated drinking water at concentrations that exceed the World Health Organization guideline for safe drinking water, which is 10 μg/L. Arsenic is a human carcinogen, which has also been associated with numerous non cancer outcomes, including cardiovascular disease. For many arsenic-related health outcomes, susceptibility differs by sex, with some outcomes preferentially afflicting males and others females. Although reducing exposure to arsenic-contaminated drinking water is the primary strategy for preventing arsenic toxicity, cancer risks remain elevated decades after arsenic exposure has been reduced. Therefore, public health approaches which complement arsenic remediation efforts are needed. One potential set of strategies includes nutritional interventions. Deficiencies in one-carbon metabolism (OCM nutrients can cause hyperhomocysteinemia (HHcys), which has been associated with adverse health outcomes, including cancers and cardiovascular disease.

In Bangladesh, the prevalence of HHcys is quite high and differs by sex (63% among men, 26% among women). Nutrients involved in the OCM pathway may also protect against arsenic toxicity. Two potential mechanisms include: 1) by enhancing arsenic metabolism and 2) by preventing/reversing arsenic-induced epigenetic dysregulation. Arsenic metabolism facilitates urinary arsenic elimination and depends on two sequential S-adenosylmethionine (SAM)-dependent methylation steps, which yield the mono- and dimethyl arsenical species (MMA and DMA, respectively and S-adenosylhomocysteine (SAH), a potent inhibitor of most methyltransferases. SAM is synthesized via OCM, a pathway with many nutritional influences, including folate and cobalamin. There is substantial evidence from experimental studies that the OCM pathway is important for facilitating arsenic metabolism and elimination. However, the relationships between SAM, SAH, and arsenic methylation may be particularly complex in populations exposed continuously to arsenic, because 1) the arsenic metabolites compete for methylation, since each methylation step is catalyzed by the arsenic (+3) methyltransferase and requires a methyl group from SAM, and 2) folate and cobalamin nutritional status may vary between individuals.

Although the mechanisms mediating arsenic toxicity remain largely unclear and are likely multifactorial, there is increasing evidence that arsenic induces epigenetic dysregulation, including alterations in both DNA methylation and posttranslational histone modifications (PTHMs), and these effects may differ by sex. Arsenic has also been shown to alter gene expression in a sex dependent manner. However, the sex-specific effects of arsenic on PTHMs and gene expression have not been confirmed in a large epidemiological study. Since many of the enzymes involved in epigenetic regulation, including DNA methyltransferases and lysine histone methyltransferases, depend on SAM, epigenetic modifications are also influenced by OCM. Previous studies have demonstrated that nutritional methyl donors involved in the OCM pathway buffer against/modify toxicant-induced alterations in DNA methylation. This may also be true for arsenic-induced alterations in PTHMs. However, the relationships between OCM indices and PTHMs have not been characterized in arsenic-exposed populations.

Objectives: We had five main objectives: 1) to examine the relationships between SAM, SAH, and arsenic methylation capacity, and potential effect modification by folate and cobalamin nutritional status; 2) to characterize a specific cleavage product of histone H3, which we identified in human peripheral blood mononuclear cells (PBMCs) in our early analyses of PTHMs; 3) to evaluate the effects of arsenic exposure and arsenic removal on three candidate PTHMs (di- and tri-methylation at lysine 36 of histone H3 (H3K36me2 and H3K36me3, respectively) and di-methylation at lysine 79 of histone H3 (H3K79me2)), which were selected because they are dysregulated in cancers and are altered by arsenic and/or nutritional methyl donors in vitro; 4) to examine associations between arsenic exposure and gene-specific DNA methylation and mRNA expression, particularly for genes involved in pathways implicated in arsenic toxicity; and 5) to characterize the relationships between OCM indices and our three candidate PTHMs, and the effect of folic acid (FA) supplementation on these same PTHMs.

For objectives 3-5, we also examined potential differences by sex. Methods: To address these objectives, we used data from three epidemiological studies of arsenic-exposed Bangladeshi adults: 1) the Folate and Oxidative Stress (FOX) study, a cross-sectional study of healthy individuals; 2) the Folic Acid and Creatine Trial (FACT), a randomized placebo-controlled trial (duration 24 weeks) in which healthy participants received an arsenic-removal water filter at baseline and were also randomized to one of five nutrition intervention arms: placebo, 400 μg FA/day (FA400), 800 μg FA/day (FA800), 3 g creatine/day (Creatine), and Creatine + FA400; and 3) the Bangladesh Vitamin E and Selenium Trial (BEST), a randomized placebo controlled trial (duration 6 years) in which individuals with arsenicosis were randomized to one of four nutrition intervention arms: placebo, vitamin E (alphatocopheral, 100 mg/day), selenium (L-selenomethionine, 200 μg/day), or a combination of vitamin E and selenium. In Chapter 3, we examined associations between blood SAM and SAH and the proportion (%) of each arsenic metabolite, measured in blood and urine, among FOX participants.

We further examined if these associations differed within strata of folate and/or cobalamin nutritional status. In Chapter 4, we characterized a specific cleavage product of histone H3, which we identified in human PBMCs from a subset of FACT participants (n = 32). We also determined the prevalence of H3 cleavage in these samples and the impact of H3 cleavage on the measurement of downstream PTHMs. In Chapter 5, we presented sex-specific associations between pre-intervention measures of blood arsenic and creatinine-adjusted urinary arsenic (uAsCr) and PTHMs, measured in PBMCs collected from FACT participants (n = 317). We also evaluated whether PTHMs were stable for the 12 week duration after FACT participants received arsenic-removal water filters (n = 60 from placebo group). In Chapter 6, we presented associations between pre-intervention uAsCr and gene-specific DNA methylation (whole blood, n = 400) and mRNA expression (PBMCs, n = 1799) for 47 candidate genes involved in arsenic metabolism, OCM, epigenetic regulation, DNA repair, or tumor suppression/oncogenesis, using baseline-collected samples from BEST participants.

We also evaluated these associations separately by sex. In Chapter 6, we examined sex-specific associations between baseline circulating concentrations of OCM indices, including folate, cobalamin, choline, betaine, and homocysteine, and PTHMs measured in PBMCs collected from FACT participants (n = 324). We also evaluated whether FA400 (n = 106), compared with placebo (n = 60), for a duration of 12 weeks increased global levels of PTHMs. Results: We observed that folate and cobalamin nutritional status significantly modified associations between SAM and the % arsenic metabolites, as hypothesized (Chapter 3). Among folate and cobalamin deficient individuals, SAM was positively associated with the %MMA, and negatively associated with the %DMA, in blood. In Chapter 4, we determined that H3 cleavage was evident in one third of the FACT PBMC samples examined. We further demonstrated that H3 cleavage impacts the measurement of certain PTHMs. In Chapter 5, we reported that biomarkers of arsenic exposure were associated with H3K36me2 in a sex-dependent manner.

In particular, uAsCr was positively associated with H3K36me2 among men, but not women. Furthermore, the use of arsenic-removal water filters was associated with significant reductions in H3K36me2 over a 12 week period, but this did not differ by sex. We also observed that uAsCr was associated with the methylation and expression of several genes involved in OCM, epigenetic regulation, DNA repair, and tumor suppression, and many of these associations differed by sex (Chapter 6). The associations between several OCM indices and PTHMs were also sex-dependent (Chapter 7). Specifically, choline was positively associated with H3K36me2 among men only, while cobalamin was positively associated with H3K79me2 among women only. However, FA400 for 12 weeks did not alter global levels of the PTHMs examined. Conclusions: Given that cancer risks remain elevated decades after arsenic exposure has ceased, public health interventions which complement arsenic remediation efforts are needed. Nutritional interventions may be one promising approach.

Previous studies have observed that a higher %MMA, and a lower DMA, in urine is associated with an increased risk of developing adverse health outcomes. Our finding that SAM was positively associated with %MMA, and negatively associated with %DMA, among individuals deficient for folate and cobalamin contributes additional evidence that nutritional status may explain some of the inter-individual differences in arsenic methylation capacity and, consequently, in susceptibility to arsenic toxicity. Our observation that arsenic exposure was positively associated with global levels of H3K36me2 among men, but not women, and that arsenic was associated with gene specific DNA methylation and mRNA expression in a sex-dependent manner, adds to a growing literature that arsenic induces epigenetic dysregulation differentially by sex. Furthermore, these findings suggest that this may have functional consequences, such as alterations in mRNA expression, including for genes involved in pathways implicated in arsenic toxicity.

While it is tempting to speculate that this may explain some of the sex differences in susceptibility to arsenic toxicity, the clinical implications of our findings will require additional study. We also provided the first evidence from an arsenic exposed population that choline and cobalamin are associated with PTHMs(H3K36me2 and H3K79me2, respectively) in a sex-dependent manner, and that 12 weeks’ supplementation with FA, at a dose based on the recommended dietary allowance for folate, does not significantly alter global levels of H3K36me2, H3K36me3, or H3K79me2 in human PBMCs. Previous studies have shown that nutrients in the OCM pathway protect against toxicant induced alterations in DNA methylation. Our findings suggest that some OCM nutrients, particularly choline and cobalamin, may also influence PTHMs in human PBMCs. These findings lay the groundwork for future studies which further examine whether these nutrients can protect against or modify arsenic induced alterations in PTHMs.