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BACKGROUND: Arsenic exposure through drinking water persists in many regions. Inorganic As (InAs) is methylated to monomethyl-arsenical species (MMAs) and dimethyl-arsenical species (DMAs), facilitating urinary excretion. Arsenic methylation is dependent on one-carbon metabolism, which is influenced by nutritional factors such as folate and creatine. OBJECTIVE: This study investigated the effects of folic acid (FA) and/or creatine supplementation on the proportion of As metabolites in urine. DESIGN: In a 24-wk randomized, double-blinded, placebo-controlled trial, 622 participants were assigned to receive FA (400 or 800 μg per day), 3 g creatine per day, 400 μg FA + 3 g creatine per day, or placebo. The majority of participants were folate sufficient; all received As-removal water filters. From wk 12–24, half of the participants receiving FA received placebo. RESULTS: Among groups receiving FA, the mean decrease in ln(%InAs) and %MMAs and increase in %DMAs exceeded those of the placebo group at wk 6 and 12 (P

Methylation of ingested inorganic arsenic (InAs) to monomethyl- (MMAs) and dimethyl-arsenical species (DMAs) facilitates urinary arsenic elimination. Folate and creatine supplementation influenced arsenic methylation in a randomized controlled trial. Here, we examine if baseline status of one-carbon metabolism nutrients (folate, choline, betaine, and vitamin B12) modified the effects of FA and creatine supplementation on changes in homocysteine, guanidinoacetate (GAA), total blood arsenic, and urinary arsenic metabolite proportions and indices. Study participants (N = 622) received 400 or 800 μg FA, 3 g creatine, 400 μg FA + 3 g creatine, or placebo daily for 12 weeks. Relative to placebo, FA supplementation was associated with greater mean increases in %DMAs among participants with betaine concentrations below the median than those with levels above the median (FDR

Background: Posttranslational histone modifications (PTHMs) are altered by arsenic, an environmental carcinogen. PTHMs are also influenced by nutritional methyl donors involved in one-carbon metabolism (OCM), which may protect against epigenetic dysregulation. Methods: We measured global levels of three PTHMs, which are dysregulated in cancers (H3K36me2, H3K36me3, H3K79me2), in peripheral blood mononuclear cells (PBMC) from 324 participants enrolled in the Folic Acid and Creatine Trial, a randomized trial in arsenic-exposed Bangladeshi adults. Sex-specific associations between several blood OCM indices (folate, vitamin B12, choline, betaine, homocysteine) and PTHMs were examined at baseline using regression models, adjusted for multiple tests by controlling for the false discovery rate (PFDR). We also evaluated the effects of folic acid supplementation (400 μg/d for 12 weeks), compared with placebo, on PTHMs. Results: Associations between choline and H3K36me2 and between vitamin B12 and H3K79me2 differed significantly by sex (Pdiff < 0.01 and Conclusions: OCM indices may influence PTHMs in a sex-dependent manner, and folic acid supplementation, at this dose and duration, does not alter PTHMs in PBMCs. Impact: This is the first study to examine the influences of OCM indices on PTHMs in a population that may have increased susceptibility to cancer development due to widespread exposure to arsenic-contaminated drinking water and a high prevalence of hyperhomocysteinemia. Cancer Epidemiol Biomarkers Prev; 26(2); 261–9. ©2016 AACR.

BACKGROUND Folic acid (FA) supplementation facilitates urinary excretion of arsenic, a human carcinogen. A better understanding of interactions between one-carbon metabolism intermediates may improve the ability to design nutrition interventions that further facilitate arsenic excretion. OBJECTIVE The objective was to determine if FA and/or creatine supplementation increase choline and betaine and decrease dimethylglycine (DMG). METHODS We conducted a secondary analysis of the Folic Acid and Creatine Trial, a randomized trial in arsenic-exposed Bangladeshi adults (n = 605, aged 24-55 y, 50.3% male) who received arsenic-removal water filters. We examined treatment effects of FA and/or creatine supplementation on plasma choline, betaine, and DMG concentrations, measured by LC-tandem mass spectrometry at baseline and at week 12. Group comparisons were between 1) 400 and 800 μg FA/d (FA400 and FA800, respectively) compared with placebo, 2) creatine (3 g/d) compared with placebo, and 3) creatine plus FA400 compared with FA400. RESULTS Choline decreased in the placebo group (-6.6%; 95% CI: -10.2%, -2.9%) but did not change in the FA groups (FA400: 2.5%; 95% CI: -0.9%, 6.1%; FA800: 1.4%; 95% CI: -2.5%, 5.5%; P < 0.05). Betaine did not change in the placebo group (-3.5%; 95% CI: -9.3%, 2.6%) but increased in the FA groups (FA400: 14.1%; 95% CI: 9.4%, 19.0%; FA800: 13.0%; 95% CI: 7.2%, 19.1%; P < 0.01). The decrease in DMG was greater in the FA groups (FA400: -26.7%; 95% CI: -30.9%, -22.2%; FA800: -27.8%; 95% CI: -31.8%, -23.4%) than in the placebo group (-12.3%; 95% CI: -18.1%, -6.2%; P < 0.01). The percentage change in choline, betaine, and DMG did not differ between creatine treatment arms and their respective reference groups. CONCLUSION Supplementation for 12 wk with FA, but not creatine, increases plasma betaine, decreases plasma DMG, and prevents a decrease in plasma choline in arsenic-exposed Bangladeshi adults. This trial was registered at clinicaltrials.gov as NCT01050556.

Background: Exposure to inorganic arsenic is associated with numerous adverse health outcomes, with susceptibility differing by sex. Although evidence from in vitro studies suggests that arsenic alters post-translational histone modifications (PTHMs), evidence in humans is limited. Objectives: The objectives were to determine: a) if arsenic exposure is associated with global (percent) levels of PTHMs H3K36me2, H3K36me3, and H3K79me2 in a sex-dependent manner, and b) if %PTHMs are stable when arsenic exposure is reduced. Methods: We examined associations between arsenic, measured in blood and urine, and %PTHMs in peripheral blood mononuclear cells from 317 participants enrolled in the Bangladesh Folic Acid and Creatine Trial (FACT). We also examined the stability of %PTHMs after the use of arsenic-removal water filters (n = 60). Results: Associations between natural log–transformed (ln) urinary arsenic, adjusted for creatinine (uAsCr), and %H3K36me2 differed significantly between men and women (p = 0.01). ln(uAsCr) was positively associated with %H3K36me2 in men [β = 0.12; 95% confidence interval (CI): 0.01, 0.23, p = 0.03] but was negatively associated with %H3K36me2 in women (β = –0.05; 95% CI: –0.12, 0.02, p = 0.19). The patterns of associations with blood arsenic were similar. On average, water filter use was also associated with reductions in %H3K36me2 (p < 0.01), but this did not differ significantly by sex. Arsenic was not significantly associated with %H3K36me3 or %H3K79me2 in men or women. Conclusions: Arsenic exposure was associated with %H3K36me2 in a sex-specific manner but was not associated with %H3K36me3 or %H3K79me2. Additional studies are needed to assess changes in %H3K36me2 after arsenic removal. Citation: Howe CG, Liu X, Hall MN, Slavkovich V, Ilievski V, Parvez F, Siddique AB, Shahriar H, Uddin MN, Islam T, Graziano JH, Costa M, Gamble MV. 2016. Associations between blood and urine arsenic concentrations and global levels of post-translational histone modifications in Bangladeshi men and women. Environ Health Perspect 124:1234–1240; http://dx.doi.org/10.1289/ehp.1510412

Arsenic (As) methylation capacity in epidemiologic studies is typically indicated by the proportions of inorganic As (%InAs), monomethylarsonic acid (%MMA), and dimethylarsinic acid (%DMA) in urine as a fraction of total urinary As. The relationship between renal function and indicators of As methylation capacity has not been thoroughly investigated.Our two aims were to examine (1) associations between estimated glomerular filtration rate (eGFR) and %As metabolites in blood and urine, and (2) whether renal function modifies the relationship of blood %As metabolites with respective urinary %As metabolites.In a cross-sectional study of 375 As-exposed Bangladeshi adults, we measured blood and urinary As metabolites, and calculated eGFR from plasma cystatin C.In covariate-adjusted linear models, a 1 ml/min/1.73 m(2) increase in eGFR was associated with a 0.39% increase in urinary %InAs (p0.0001) and a mean decrease in urinary %DMA of 0.07 (p=0.0005). In the 292 participants with measurable blood As metabolites, the associations of eGFR with increased blood %InAs and decreased blood %DMA did not reach statistical significance. eGFR was not associated with urinary or blood %MMA in covariate-adjusted models. For a given increase in blood %InAs, the increase in urinary %InAs was smaller in those with reduced eGFR, compared to those with normal eGFR (p=0.06); this effect modification was not observed for %MMA or %DMA.Urinary excretion of InAs may be impaired in individuals with reduced renal function. Alternatively, increased As methylation capacity (as indicated by decreased urinary %InAs) may be detrimental to renal function.

Inorganic arsenic (InAs) is metabolized through a series of methylation reactions catalyzed by arsenic(III)-methyltransferase (AS3MT), resulting in the generation of monomethylarsonic (MMAs) and dimethylarsinic acids (DMAs). AS3MT activity requires the presence of the methyl donor S-adenosylmethionine (SAM), a product of folate-dependent one-carbon metabolism, and a reductant. Although glutathione (GSH), the primary endogenous antioxidant, is not required for As methylation, GSH stimulates As methylation rates in vitro. However, the relationship between GSH redox and As methylation capacity in humans is unknown. We wished to test the hypothesis that a more oxidized plasma GSH redox status is associated with decreased As methylation capacity, and examine whether these associations are modified by folate nutritional status. Concentrations of plasma GSH and GSSG, plasma folate, total blood As (bAs), total urinary As (uAs), and uAs metabolites were assessed in a cross-sectional study of n = 376 Bangladeshi adults who were chronically exposed to As in drinking water. We observed that a decreased plasma GSH/GSSG ratio (reflecting a more oxidized redox state) was significantly associated with increased urinary %MMA, decreased urinary %DMA, and increased total bAs in folate-deficient individuals (plasma folate ≤ 9.0 nmol/L). Concentrations of plasma GSH and GSSG were independently associated with increased and decreased As methylation capacity, respectively. No significant associations were observed in folate-sufficient individuals, and interactions by folate status were statistically significant. Our findings suggest that GSH/GSSG redox regulation might contribute to the large interindividual variation in As methylation capacity observed in human populations.

Chronic exposure to inorganic arsenic (InAs) through drinking water is a major problem worldwide. InAs undergoes hepatic methylation to form mono- and dimethyl arsenical species (MMA and DMA, respectively), facilitating arsenic elimination. Both reactions are catalyzed by arsenic (+3 oxidation state) methyltransferase (AS3MT) using S-adenosylmethionine (SAM) as the methyl donor, yielding the methylated product and S-adenosylhomocysteine (SAH), a potent product-inhibitor of AS3MT. SAM biosynthesis depends on folate- and cobalamin-dependent one-carbon metabolism. With the use of samples from 353 participants in the Folate and Oxidative Stress Study, our objective was to test the hypotheses that blood SAM and SAH concentrations are associated with arsenic methylation and that these associations differ by folate and cobalamin nutritional status. Blood SAM and SAH were measured by HPLC. Arsenic metabolites in blood and urine were measured by HPLC coupled to dynamic reaction cell inductively coupled plasma MS. In linear regression analyses, SAH was not associated with any of the arsenic metabolites. However, log(SAM) was negatively associated with log(% urinary InAs) (β: -0.11; 95% CI: -0.19, -0.02; P = 0.01), and folate and cobalamin nutritional status significantly modified associations between SAM and percentage of blood MMA (%bMMA) and percentage of blood DMA (%bDMA) (P = 0.02 and P = 0.01, respectively). In folate- and cobalamin-deficient individuals, log(SAM) was positively associated with %bMMA (β: 6.96; 95% CI: 1.86, 12.05; P < 0.01) and negatively associated with %bDMA (β: -6.19; 95% CI: -12.71, 0.32; P = 0.06). These findings suggest that when exposure to InAs is high, and methyl groups are limiting, SAM is used primarily for MMA synthesis rather than for DMA synthesis, contributing additional evidence that nutritional status may explain some of the interindividual differences in arsenic metabolism and, consequently, susceptibility to arsenic toxicity.

The precise mechanisms by which nickel and arsenic compounds exert their carcinogenic properties are not completely understood. In recent years, alterations of epigenetic mechanisms have been implicated in the carcinogenesis of compounds of these two metals. In vitro exposure to certain nickel or arsenic compounds induces changes in both DNA methylation patterns, as well as, in the levels of posttranslational modifications of histone tails. Changes in DNA methylation patterns have been reported in human subjects exposed to arsenic. Here we review our recent reports on the alterations in global levels of posttranslational histone modifications in peripheral blood mononuclear cells (PBMCs) of subjects with occupational exposure to nickel and subjects exposed to arsenic in their drinking water. Occupational exposure to nickel was associated with an increase in H3K4me3 and decrease in H3K9me2. A global increase in H3K9me2 and decrease in H3K9ac was found in subjects exposed to arsenic. Additionally, exposure to arsenic resulted in opposite changes in a number of histone modifications in males when compared with females in the arsenic population. The results of these two studies suggest that exposure to nickel or arsenic compounds, and possibly other carcinogenic metal compounds, can induce changes in global levels of posttranslational histone modifications in peripheral blood mononuclear cells.

Background Arsenic is a carcinogen to which 35 million people in Bangladesh are chronically exposed. The enzymatic transfer of methyl groups to inorganic As (iAs) generates monomethylarsonic (MMA) and dimethylarsinic acids (DMA) and facilitates urinary As (uAs) elimination. This process is dependent on one-carbon metabolism, a pathway in which folate and cobalamin have essential roles in the recruitment and transfer of methyl groups. Although DMAV is the least toxic metabolite, increasing evidence suggests that MMAIII may be the most cytotoxic and genotoxic As intermediary metabolite. Objective We examined the associations between plasma cobalamin and uAs metabolites. Methods We conducted a cross-sectional study of 778 Bangladeshi adults in which we over-sampled cobalamin-deficient participants. Participants provided blood samples for the measurement of plasma cobalamin and urine specimens for As measurements. Results Cobalamin was inversely associated with the proportion of total uAs excreted as iAs (%iAs) [unstandardized regression coefficient (b) = –0.10; 95% confidence interval (CI), −0.17 to −0.02; p = 0.01] and positively associated with %MMA (b = 0.12; 95% CI, 0.05 to 0.20; p = 0.001). Both of these associations were stronger among folate-sufficient participants (%iAs: b = −0.17; 95% CI, −0.30 to −0.03; p = 0.02. %MMA: b = 0.20; 95% CI, 0.11 to 0.30; p < 0.0001), and the differences by folate status were statistically significant. Conclusions In this group of Bangladeshi adults, cobalamin appeared to facilitate the first As methylation step among folate-sufficient individuals. Given the toxicity of MMAIII, our findings suggest that in contrast to folate, cobalamin may not favorably influence As metabolism.

Background Approximately 35 million people in Bangladesh are chronically exposed to inorganic arsenic (InAs) in drinking water. Methylation of InAs to monomethylarsonic (MMA) and dimethylarsinic acids (DMA) relies on folate-dependent one-carbon metabolism and facilitates urinary arsenic (uAs) elimination. Objectives We examined the relationships between folate, cobalamin, cysteine, total homocysteine (tHcys), and uAs metabolites in a sample of 6-year-old Bangladeshi children (n = 165). Methods Children provided blood samples for measurement of tHcys, folate, cobalamin, and cysteine, and urine specimens for the measurement of total uAs and As metabolites. Results Consistent with our studies in adults, mean tHcys concentrations (7.9 μmol/L) were higher than those reported among children of similar ages in other populations. Nineteen percent of the children had plasma folate concentrations < 9.0 nmol/L. The proportion of total uAs excreted as InAs (%InAs) was inversely correlated with folate (r = −0.20, p = 0.01) and cysteine (r = −0.23, p = 0.003), whereas the correlations between %DMA and both folate (r = 0.12, p = 0.14) and cysteine (r = 0.11, p = 0.15) were positive. Homocysteine was inversely correlated (r = −0.27, p = 0.009) with %MMA in males, and the correlation with %DMA was positive (r = 0.13, p = 0.10). Conclusions These findings suggest that, similar to adults, folate and cysteine facilitate As methylation in children. However, the inverse correlation between tHcys and %MMA, and positive correlation with %DMA, are both opposite to our previous findings in adults. We propose that upregulation of one-carbon metabolism, presumably necessary to meet the considerable demands for DNA and protein biosynthesis during periods of rapid growth, results in both increased tHcys biosynthesis and increased As methylation.

Background: Depletion of global 5-hydroxymethylcytosine (5-hmC) is observed in human cancers and is strongly implicated in skin cancer development. Although arsenic (As)—a class I human carcinogen linked to skin lesion and cancer risk—is known to be associated with changes in global %5-methylcytosine (%5-mC), its influence on 5-hmC has not been widely studied. Methods: We evaluated associations of As in drinking water, urine, and blood with global %5-mC and %5-hmC in two studies of Bangladeshi adults: (i) leukocyte DNA in the Nutritional Influences on Arsenic Toxicity study (n = 196; 49% male, 19–66 years); and (ii) peripheral blood mononuclear cell DNA in the Folate and Oxidative Stress study (n = 375; 49% male, 30–63 years). Results: Overall, As was not associated with global %5-mC or %5-hmC. Sex-specific analyses showed that associations of As exposure with global %5-hmC were positive in males and negative in females (P for interaction < 0.01). Analyses examining interactions by elevated plasma total homocysteine (tHcys), an indicator of B-vitamin deficiency, found that tHcys also modified the association between As and global %5-hmC (P for interaction < 0.10). Conclusion: In two samples, we observed associations between As exposure and global %5-hmC in blood DNA that were modified by sex and tHcys. Impact: Our findings suggest that As induces sex-specific changes in 5-hmC, an epigenetic mark that has been associated with cancer. Future research should explore whether altered %5-hmC is a mechanism underlying the sex-specific influences of As on skin lesion and cancer outcomes. Cancer Epidemiol Biomarkers Prev; 24(11); 1748–57. ©2015 AACR.

Exposure to arsenic (As) in drinking water is a widespread public health problem leading to increased risk for multiple outcomes such as cancer, cardiovascular disease, and possibly renal disease; potential mechanisms include inflammation and oxidative stress. We tested the hypothesis that As exposure is associated with increased inflammation and decreased estimated glomerular filtration rate (eGFR) and examined whether the effects of As were modified by plasma glutathione (GSH), glutathione disulfide (GSSG), or the reduction potential of the GSSG/2GSH pair (EhGSH). In a cross-sectional study of N = 374 Bangladeshi adults having a wide range of As exposure, we measured markers of inflammation (plasma C-reactive protein (CRP), α-1 acid glycoprotein (AGP)), renal function (eGFR), GSH, and GSSG. In covariate-adjusted models, a 10% increase in water As, urinary As adjusted for specific gravity (uAs), or blood As (bAs) was associated with a 0.74% (p = 0.01), 0.90% (p = 0.16), and 1.39% (p = 0.07) increase in CRP, respectively; there was no association with AGP. A 10% increase in uAs or bAs was associated with an average reduction in eGFR of 0.16 (p = 0.12) and 0.21 ml/min/1.73 m2 (p = 0.08), respectively. In stratified analyses, the effect of As exposure on CRP was observed only in participants having EhGSH > median (uAs pWald = 0.03; bAs pWald = 0.05). This was primarily driven by stronger effects of As exposure on CRP in participants with lower plasma GSH. The effects of As exposure on eGFR were not modified significantly by EhGSH, GSH, or GSSG. These data suggest that participants having lower plasma GSH and a more oxidized plasma EhGSH are at increased risk for As-induced inflammation. Future studies should evaluate whether antioxidant treatment lowers plasma EhGSH and reduces risk for As-induced diseases.

Arsenic contamination of drinking water occurs globally and is associated with numerous diseases including skin, lung and bladder cancers, and cardiovascular disease. Recent research indicates that arsenic may be an endocrine disruptor. This study was conducted to evaluate the nature of gene expression changes among males and females exposed to arsenic contaminated water in Bangladesh at high and low doses. Twenty-nine (55% male) Bangladeshi adults with water arsenic exposure ranging from 50 to 1000 μg/L were selected from the Folic Acid Creatinine Trial. RNA was extracted from peripheral blood mononuclear cells for gene expression profiling using Affymetrix 1.0 ST arrays. Differentially expressed genes were assessed between high and low exposure groups for males and females separately and findings were validated using quantitative real-time PCR. There were 534 and 645 differentially expressed genes (p

Background The World Health Organization estimates that > 140 million people worldwide are exposed to arsenic (As)–contaminated drinking water. As undergoes biologic methylation, which facilitates renal As elimination. In folate-deficient individuals, this process is augmented by folic acid (FA) supplementation, thereby lowering blood As (bAs). Creatinine concentrations in urine are a robust predictor of As methylation patterns. Although the reasons for this are unclear, creatine synthesis is a major consumer of methyl donors, and this synthesis is down-regulated by dietary/supplemental creatine. Objectives Our aim was to determine whether 400 or 800 μg FA and/or creatine supplementation lowers bAs in an As-exposed Bangladeshi population. Methods We conducted a clinical trial in which 622 participants were randomized to receive 400 μg FA, 800 μg FA, 3 g creatine, 3 g creatine+400 μg FA, or placebo daily. All participants received an As-removal filter on enrollment, and were followed for 24 weeks. After the 12th week, half of the two FA groups were switched to placebo to evaluate post-treatment bAs patterns. Results Linear models with repeated measures indicated that the decline in ln(bAs) from baseline in the 800-μg FA group exceeded that of the placebo group (weeks 1–12: β= –0.09, 95% CI: –0.18, –0.01; weeks 13–24: FA continued: β= –0.12, 95% CI: –0.24, –0.00; FA switched to placebo: β= –0.14, 95% CI: –0.26, –0.02). There was no rebound in bAs related to cessation of FA supplementation. Declines in bAs observed in the remaining treatment arms were not significantly different from those of the placebo group. Conclusions In this mixed folate-deficient/replete study population, 12- and 24-week treatment with 800 μg (but not 400 μg) FA lowered bAs to a greater extent than placebo; this was sustained 12 weeks after FA cessation. In future studies, we will evaluate whether FA and/or creatine altered As methylation profiles. Citation Peters BA, Hall MN, Liu X, Parvez F, Sanchez TR, van Geen A, Mey JL, Siddique AB, Shahriar H, Uddin MN, Islam T, Balac O, Ilievski V, Factor-Litvak P, Graziano JH, Gamble MV. 2015. Folic acid and creatine as therapeutic approaches to lower blood arsenic: a randomized controlled trial. Environ Health Perspect 123:1294–1301; http://dx.doi.org/10.1289/ehp.1409396

Background Arsenic is a major environmental toxin that is detoxified in the liver by biochemical mechanisms that are still under study. In the traditional metabolic pathway, arsenic undergoes two methylation reactions, each followed by a reduction, after which it is exported and released in the urine. Recent experiments show that glutathione plays an important role in arsenic detoxification and an alternative biochemical pathway has been proposed in which arsenic is first conjugated by glutathione after which the conjugates are methylated. In addition, in rats arsenic-glutathione conjugates can be exported into the plasma and removed by the liver in the bile. Methods We have developed a mathematical model for arsenic biochemistry that includes three mechanisms by which glutathione affects arsenic methylation: glutathione increases the speed of the reduction steps; glutathione affects the activity of arsenic methyltranferase; glutathione sequesters inorganic arsenic and its methylated downstream products. The model is based as much as possible on the known biochemistry of arsenic methylation derived from cellular and experimental studies. Results We show that the model predicts and helps explain recent experimental data on the effects of glutathione on arsenic methylation. We explain why the experimental data imply that monomethyl arsonic acid inhibits the second methylation step. The model predicts time course data from recent experimental studies. We explain why increasing glutathione when it is low increases arsenic methylation and that at very high concentrations increasing glutathione decreases methylation. We explain why the possible temporal variation of the glutathione concentration affects the interpretation of experimental studies that last hours. Conclusions The mathematical model aids in the interpretation of data from recent experimental studies and shows that the Challenger pathway of arsenic methylation, supplemented by the glutathione effects described above, is sufficient to understand and predict recent experimental data. More experimental studies are needed to explicate the detailed mechanisms of action of glutathione on arsenic methylation. Recent experimental work on the effects of glutathione on arsenic methylation and our modeling study suggest that supplements that increase hepatic glutathione production should be considered as strategies to reduce adverse health effects in affected populations.

Kidney disease is emerging as an arsenic (As)-linked disease outcome, however further evidence of this association is warranted. Our first objective for this paper was to examine the potential renal toxicity of As exposure in Bangladesh. Our second objective relates to examining whether the previously reported positive association between urinary creatinine (uCrn) and As methylation may be explained by renal function. We had hypothesized that these associations relate to supply and demand for s-adenosylmethionine, the methyl donor for both creatine synthesis and As methylation. Alternatively, renal function could influence both As and creatinine excretion, or the As metabolites may influence renal function, which in turn influences uCrn. We conducted a cross-sectional study (N = 478) of adults, composed of a sample recruited in 2001 and a sample recruited in 2003. We assessed renal function using plasma cystatin C, and calculated the estimated glomerular filtration rate (eGFR). Consistent with renal toxicity of As, log-uAs had a marginal inverse association with eGFR in the 2003 sample (b = -5.6, p = 0.07), however this association was not significant in the 2001 sample (b = -1.9, p = 0.24). Adjustment for eGFR did not alter the associations between uCrn and the %uAs metabolites, indicating that GFR does not explain these associations. Increased eGFR was associated with increased odds of having %uInAs >12.2% (2001: OR = 1.01, 95%CI (1.00,1.03); 2003: OR = 1.04, 95%CI (1.01,1.07)). In the 2003 sample only, there was a negative association between eGFR and %uDMA (b = -0.08, p = 0.02). These results may indicate differential effects of renal function on excretion of InAs and DMA. Alternatively, a certain methylation pattern, involving decreased %InAs and increased %DMA, may reduce renal function. Given that these studies were cross-sectional, we cannot distinguish between these two possibilities. Discrepancies between the samples may be due to the higher As exposure, poorer nutrition, and lower As methylation capacity in the 2003 sample.

Background: Several studies employing cell culture and animal models have suggested that arsenic (As) exposure induces global DNA hypomethylation. However, As has been associated with global DNA hypermethylation in human study populations. We hypothesized that this discrepancy may reflect a nonlinear relationship between As dose and DNA methylation. Objective: The objective of this study was to examine the dose–response relationship between As and global methylation of peripheral blood mononuclear cell (PBMC) DNA in apparently healthy Bangladeshi adults chronically exposed to a wide range of As concentrations in drinking water. Methods: Global PBMC DNA methylation, plasma folate, blood S-adenosylmethionine (SAM), and concentrations of As in drinking water, blood, and urine were measured in 320 adults. DNA methylation was measured using the [3H]-methyl incorporation assay, which provides disintegration-per-minute (DPM) values that are negatively associated with global DNA methylation. Results: Water, blood, and urinary As were positively correlated with global PBMC DNA methylation (p < 0.05). In multivariable-adjusted models, 1-μg/L increases in water and urinary As were associated with 27.6-unit (95% CI: 6.3, 49.0) and 22.1-unit (95% CI: 0.5, 43.8) decreases in DPM per microgram DNA, respectively. Categorical models indicated that estimated mean levels of PBMC DNA methylation were highest in participants with the highest As exposures. Conclusions: These results suggest that As is positively associated with global methylation of PBMC DNA over a wide range of drinking water As concentrations. Further research is necessary to elucidate underlying mechanisms and physiologic implications. Citation: Niedzwiecki MM, Hall MN, Liu X, Oka J, Harper KN, Slavkovich V, Ilievski V, Levy D, van Geen A, Mey JL, Alam S, Siddique AB, Parvez F, Graziano JH, Gamble MV. 2013. A dose–response study of arsenic exposure and global methylation of peripheral blood mononuclear cell DNA in Bangladeshi adults. Environ Health Perspect 121:1306–1312; http://dx.doi.org/10.1289/ehp.1206421

Background: In vitro and rodent studies have shown that arsenic (As) exposure can deplete glutathione (GSH) and induce oxidative stress. GSH is the primary intracellular antioxidant; it donates an electron to reactive oxygen species, thus producing glutathione disulfide (GSSG). Cysteine (Cys) and cystine (CySS) are the predominant thiol/disulfide redox couple found in human plasma. Arsenic, GSH, and Cys are linked in several ways: a) GSH is synthesized via the transsulfuration pathway, and Cys is the rate-limiting substrate; b) intermediates of the methionine cycle regulate both the transsulfuration pathway and As methylation; c) GSH serves as the electron donor for reduction of arsenate to arsenite; and d) As has a high affinity for sulfhydryl groups and therefore binds to GSH and Cys. Objectives: We tested the hypothesis that As exposure is associated with decreases in GSH and Cys and increases in GSSG and CySS (i.e., a more oxidized environment). Methods: For this cross-sectional study, the Folate and Oxidative Stress Study, we recruited a total of 378 participants from each of five water As concentration categories: < 10 (n = 76), 10–100 (n = 104), 101–200 (n = 86), 201–300 (n = 67), and > 300 µg/L (n = 45). Concentrations of GSH, GSSG, Cys, and CySS were measured using HPLC. Results: An interquartile range (IQR) increase in water As was negatively associated with blood GSH (mean change, –25.4 µmol/L; 95% CI: –45.3, –5.31) and plasma CySS (mean change, –3.00 µmol/L; 95% CI: –4.61, –1.40). We observed similar associations with urine and blood As. There were no significant associations between As exposure and blood GSSG or plasma Cys. Conclusions: The observed associations are consistent with the hypothesis that As may influence concentrations of GSH and other nonprotein sulfhydryls through binding and irreversible loss in bile and/or possibly in urine. Citation: Hall MN, Niedzwiecki M, Liu X, Harper KN, Alam S, Slavkovich V, Ilievski V, Levy D, Siddique AB, Parvez F, Mey JL, van Geen A, Graziano J, Gamble MV. 2013. Chronic arsenic exposure and blood glutathione and glutathione disulfide concentrations in Bangladeshi adults. Environ Health Perspect 121:1068–1074; http://dx.doi.org/10.1289/ehp.1205727

Basu et al. (2011) reported the associations of both dietary and blood nutrient measures, as well as urinary creatinine (uCr), with arsenic (As) methylation capacity, as assessed by the proportions of urinary inorganic, monomethyl, and dimethyl As metabolites. One finding was that uCr was the strongest predictor of As methylation; participants with higher uCr concentrations had a higher percentage of total urinary As as dimethylarsinic acid (DMA) compared to those with lower uCr. This is consistent with what we have previously reported in Bangladeshi adults and children (Gamble et al. 2005; Ahsan et al. 2007; Hall et al. 2009), and is an interesting and potentially very important observation. Approximately 40% of S-adenosylmethionine (SAM)-derived methyl groups are devoted to the biosynthesis of creatine, the precursor of creatinine (Brosnan et al. 2011; Mudd and Poole 1975). At high levels of As exposure (500–1,000 µg/L), based on one-carbon kinetics (Schalinske and Steele 1989), we estimated that methylation of 80% of a daily dose of inorganic As (InAs) to DMA would require approximately 50 µmol SAM, thus consuming approximately 2–4% of the SAM normally turning over in a well-nourished adult per day. Low dietary creatine intake associated with low-protein or vegetarian diets places an increased demand for SAM for creatine biosynthesis (Brosnan 2011). This could potentially reduce the availability of SAM for As methylation, providing a plausible mechanism underlying this highly reproducible observation. This assumes that uCr reflects, to some extent, dietary creatine intake, as we have observed (Gamble M, unpublished data). Conversely, dietary creatine intake and/or creatine supplementation down-regulates endogenous creatine biosynthesis, potentially sparing SAM for methylation of other substrates such as As. We are currently testing this hypothesis in a randomized controlled trial of creatine supplementation. In addition, as Basu et al. (2011) noted, and as we have previously reported (Gamble and Liu 2005), one implication of the observed association between uCr and As methylation capacity is that urinary As should not be expressed per gram creatinine to correct for urine concentration. Rather, uCr should be included as a covariate in regression models. One concerning aspect of the study by Basu et al. (2011) is the handling of blood samples used for nutrient measurements. As noted by Basu et al. and in a previous publication on these same participants (Chung et al. 2006), the blood samples were stored in an ice chest in the field for up to 24 hr before processing. This 24-hr delay can be problematic for some nutrients, especially folate, which is extremely sensitive to oxidative degradation (Drammeh et al. 2008). Basu et al. (2011) reported that in univariate analyses, they observed higher urinary percentages of InAs in individuals with higher serum folate concentrations. This finding is contrary to our previous findings that folate facilitates As methylation (Gamble et al. 2005, 2006, 2007; Hall et al. 2007, 2009). This discrepancy might be explained by differences in sample processing. Basu et al. (2011) also reported associations between dietary intake of several nutrients (assessed using a modified 24-hr recall) and As methylation capacity. One of the most critical and widely discussed issues in nutritional epidemiology is the method used to adjust for total energy intake (TEI) (Willett et al. 1997). The main reasons to adjust for TEI are to a) adjust for potential confounding by TEI, b) remove extraneous variation in nutrient intakes that is due only to their correlation with TEI, and c) simulate a dietary intervention. What is often most relevant is diet composition, or nutrient intake in relation to TEI (Willett et al. 1997). Several methods are available to adjust for TEI, and the best approach can vary depending on the nutrient and question of interest. Basu et al. (2011) adjusted for TEI by dividing each nutrient intake by TEI (nutrient density method). While this approach is appealing because of its simplicity, in reality it can create a complex variable (Willett and Stampfer 1998). For example, when TEI is related to the outcome of interest, the use of nutrient densities can actually induce confounding in the opposite direction. Although we cannot determine from Basu et al.’s article whether TEI measured by the 24-hr recall was associated with As methylation, in theory, an association seems plausible. Also, because their statistical analysis tested for associations between multiple nutrients and urinary As metabolites, it is best to acknowledge that some of the statistically significant associations might be due to chance alone.

Exposure to arsenic (As) through drinking water is a substantial problem worldwide. The methylation of As, a reactive metalloid, generates monomethyl- (MMA) and dimethyl-arsenical (DMA) species. The biochemical pathway that catalyzes these reactions, one-carbon metabolism, is regulated by folate and other micronutrients. Arsenic methylation exerts a critical influence on both its urinary elimination and chemical reactivity. Mice having the As methyltransferase null genotype show reduced urinary As excretion, increased As retention, and severe systemic toxicity. The most toxic As metabolite in vitro is MMA(III), an intermediate in the generation of DMA(V), a much less toxic metabolite. These findings have raised the question of whether As methylation is a detoxification or bioactivation pathway. Results of population-based studies suggest that complete methylation of inorganic As to DMA is associated with reduced risk for As-induced health outcomes, and that nutrients involved in one-carbon metabolism, such as folate, can facilitate As methylation and elimination.

Background and Aims: An emerging body of evidence indicates that early-life arsenic (As) exposure may influence the trajectory of health outcomes later in life; however, the mechanisms underlying t...

Background We utilized data from the Health Effects of Arsenic Longitudinal Study (HEALS) in Araihazar, Bangladesh, to evaluate the association of steamed rice consumption with urinary total arsenic concentration and arsenical skin lesions in the overall study cohort (N=18,470) and in a subset with available urinary arsenic metabolite data (N=4,517). Methods General linear models with standardized beta coefficients were used to estimate associations between steamed rice consumption and urinary total arsenic concentration and urinary arsenic metabolites. Logistic regression models were used to estimate prevalence odds ratios (ORs) and their 95% confidence intervals (CIs) for the associations between rice intake and prevalent skin lesions at baseline. Discrete time hazard models were used to estimate discrete time (HRs) ratios and their 95% CIs for the associations between rice intake and incident skin lesions. Results Steamed rice consumption was positively associated with creatinine-adjusted urinary total arsenic (β=0.041, 95% CI: 0.032-0.051) and urinary total arsenic with statistical adjustment for creatinine in the model (β=0.043, 95% CI: 0.032-0.053). Additionally, we observed a significant trend in skin lesion prevalence (P-trend=0.007) and a moderate trend in skin lesion incidence (P-trend=0.07) associated with increased intake of steamed rice. Conclusions This study suggests that rice intake may be a source of arsenic exposure beyond drinking water.

Background Methyltransferase (MT) reactions, in which methyl groups are attached to substrates, are fundamental to many aspects of cell biology and human physiology. The universal methyl donor for these reactions is S-adenosylmethionine (SAM) and this presents the cell with an important regulatory problem. If the flux along one pathway is changed then the SAM concentration will change affecting all the other MT pathways, so it is difficult for the cell to regulate the pathways independently. Methods We created a mathematical model, based on the known biochemistry of the folate and methionine cycles, to study the regulatory mechanisms that enable the cell to overcome this difficulty. Some of the primary mechanisms are long-range allosteric interactions by which substrates in one part of the biochemical network affect the activity of enzymes at distant locations in the network (not distant in the cell). Because of these long-range allosteric interactions, the dynamic behavior of the network is very complicated, and so mathematical modeling is a useful tool for investigating the effects of the regulatory mechanisms and understanding the complicated underlying biochemistry and cell biology. Results We study the allosteric binding of 5-methyltetrahydrofolate (5mTHF) to glycine-N-methyltransferase (GNMT) and explain why data in the literature implies that when one molecule binds, GNMT retains half its activity. Using the model, we quantify the effects of different regulatory mechanisms and show how cell processes would be different if the regulatory mechanisms were eliminated. In addition, we use the model to interpret and understand data from studies in the literature. Finally, we explain why a full understanding of how competing MTs are regulated is important for designing intervention strategies to improve human health. Conclusions We give strong computational evidence that once bound GNMT retains half its activity. The long-range allosteric interactions enable the cell to regulate the MT reactions somewhat independently. The low Km values of many MTs also play a role because the reactions then run near saturation and changes in SAM have little effect. Finally, the inhibition of the MTs by the product S-adenosylhomocysteine also stabilizes reaction rates against changes in SAM. Electronic supplementary material The online version of this article (doi:10.1186/s12918-015-0215-6) contains supplementary material, which is available to authorized users.

Background Arsenic in drinking water, a major health hazard to millions of people in South and East Asia and in other parts of the world, is ingested primarily as trivalent inorganic arsenic (iAs), which then undergoes hepatic methylation to methylarsonic acid (MMAs) and a second methylation to dimethylarsinic acid (DMAs). Although MMAs and DMAs are also known to be toxic, DMAs is more easily excreted in the urine and therefore methylation has generally been considered a detoxification pathway. A collaborative modeling project between epidemiologists, biologists, and mathematicians has the purpose of explaining existing data on methylation in human studies in Bangladesh and also testing, by mathematical modeling, effects of nutritional supplements that could increase As methylation. Methods We develop a whole body mathematical model of arsenic metabolism including arsenic absorption, storage, methylation, and excretion. The parameters for arsenic methylation in the liver were taken from the biochemical literature. The transport parameters between compartments are largely unknown, so we adjust them so that the model accurately predicts the urine excretion rates of time for the iAs, MMAs, and DMAs in single dose experiments on human subjects. Results We test the model by showing that, with no changes in parameters, it predicts accurately the time courses of urinary excretion in mutiple dose experiments conducted on human subjects. Our main purpose is to use the model to study and interpret the data on the effects of folate supplementation on arsenic methylation and excretion in clinical trials in Bangladesh. Folate supplementation of folate-deficient individuals resulted in a 14% decrease in arsenicals in the blood. This is confirmed by the model and the model predicts that arsenicals in the liver will decrease by 19% and arsenicals in other body stores by 26% in these same individuals. In addition, the model predicts that arsenic methyltransferase has been upregulated by a factor of two in this population. Finally, we also show that a modification of the model gives excellent fits to the data on arsenic metabolism in human cultured hepatocytes. Conclusions The analysis of the Bangladesh data using the model suggests that folate supplementation may be more effective at reducing whole body arsenic than previously expected. There is almost no data on the upregulation of arsenic methyltransferase in populations chronically exposed to arsenic. Our model predicts upregulation by a factor of two in the Bangladesh population studied. This prediction should be verified since it could have important public health consequences both for treatment strategies and for setting appropriate limits on arsenic in drinking water. Our model has compartments for the binding of arsenicals to proteins inside of cells and we show that these comparments are necessary to obtain good fits to data. Protein-binding of arsenicals should be explored in future biochemical studies.