Vesna Ilievski

Folate, homocysteine, and arsenic metabolism in arsenic-exposed individuals in Bangladesh.

Chronic exposure to arsenic is occurring throughout South and East Asia due to groundwater contamination of well water. Variability in susceptibility to arsenic toxicity may be related to nutritional status. Arsenic is methylated to monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) via one-carbon metabolism, a biochemical pathway that is dependent on folate. The majority of one-carbon metabolism methylation reactions are devoted to biosynthesis of creatine, the precursor of creatinine. Our objectives of this cross-sectional study were to characterize the relationships among folate, cobalamin, homocysteine, and arsenic metabolism in Bangladeshi adults. Water arsenic, urinary arsenic, urinary creatinine, plasma folate, cobalamin, and homocysteine were assessed in 1,650 adults; urinary arsenic metabolites were analyzed for a subset of 300 individuals. The percentage of DMA in urine was positively associated with plasma folate (r = 0.14, p = 0.02) and negatively associated with total homocysteine (tHcys; r = −0.14, p = 0.01). Conversely, percent MMA was negatively associated with folate (r = −0.12, p = 0.04) and positively associated with tHcys (r = 0.21, p = 0.0002); percent inorganic arsenic (InAs) was negatively associated with folate (r = −0.12, p = 0.03). Urinary creatinine was positively correlated with percent DMA (r = 0.40 for males, p < 0.0001; 0.25 for females, p = 0.001), and with percent InAs (r = −0.45 for males, p < 0.0001; −0.20 for females, p = 0.01). Collectively, these data suggest that folate, tHcys, and other factors involved in one-carbon metabolism influence arsenic methylation. This may be particularly relevant in Bangladesh, where the prevalence of hyperhomocysteinemia is extremely high.

Folate Deficiency, Hyperhomocysteinemia, Low Urinary Creatinine, and Hypomethylation of Leukocyte DNA Are Risk Factors for Arsenic-Induced Skin Lesions

Background Arsenic methylation relies on folate-dependent one-carbon metabolism and facilitates urinary As elimination. Clinical manifestations of As toxicity vary considerably among individuals and populations, and poor methylation capacity is thought to confer greater susceptibility. Objective After determining that folate deficiency, hyperhomocysteinemia, and low urinary creatinine are associated with reduced As methylation, and that As exposure is associated with increased genomic methylation of leukocyte DNA, we asked whether these factors are associated with As-induced skin lesion risk among Bangladeshi adults. Methods We conducted a nested case–control study of 274 cases who developed lesions 2 years after recruitment, and 274 controls matched to cases for sex, age, and water As. Results The odds ratios and 95% confidence intervals (CIs) for development of skin lesions for participants who had low folate (< 9 nmol/L), hyperhomocysteinemia (men, > 11.4 μmol/L; women, > 10.4 μmol/L), or hypomethylated leukocyte DNA at recruitment (< median) were 1.8 (95% CI, 1.1–2.9), 1.7 (95% CI, 1.1–2.6), and 1.8 (95% CI, 1.2–2.8), respectively. Compared with the subjects in the first quartile, those in the third and fourth quartiles for urinary creatinine had a 0.4-fold decrease in the odds of skin lesions (p < 0.01). Conclusions These results suggest that folate deficiency, hyperhomocysteinemia, and low urinary creatinine, each associated with decreased As methylation, are risk factors for As-induced skin lesions. The increased DNA methylation associated with As exposure previously observed, and confirmed among controls in this study, may be an adaptive change because hypomethylation of leukocyte DNA is associated with increased risk for skin lesions.

Influence of Prenatal Arsenic Exposure and Newborn Sex on Global Methylation of Cord Blood DNA

Background 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 these observations are unknown. Objective The objective of this study was to investigate the influence of prenatal As exposure on global methylation of cord blood DNA in a study of mother/newborn pairs in Matlab, Bangladesh. Design Maternal and cord blood DNA were available from a convenience sample of 101 mother/newborn pairs. Measures of As exposure included maternal urinary As (uAs), maternal blood As (mbAs) and cord blood As (cbAs). Several measures of global DNA methylation were assessed, including the [3H]-methyl-incorporation assay and three Pyrosequencing assays: Alu, LINE-1 and LUMA. Results In the total sample, increasing quartiles of maternal uAs were associated with an increase in covariate-adjusted means of newborn global DNA methylation as measured by the [3H]-methyl-incorporation assay (quartile 1 (Q1) and Q2 vs. Q4; p = 0.06 and 0.04, respectively). Sex-specific linear regression analyses, while not reaching significance level of 0.05, indicated that the associations between As exposures and Alu, LINE-1 and LUMA were positive among male newborns (N = 58) but negative among female newborns (N = 43); tests for sex differences were borderline significant for the association of cbAs and mbAs with Alu (p = 0.05 and 0.09, respectively) and for the association between maternal uAs and LINE-1 (p = 0.07). Sex-specific correlations between maternal urinary creatinine and newborn methyl-incorporation, Alu and LINE-1 were also evident (p<0.05). Conclusions These results suggest that prenatal As exposure is associated with global DNA methylation in cord blood DNA, possibly in a sex-specific manner. Arsenic-induced epigenetic modifications in utero may potentially influence disease outcomes later in life. Additional studies are needed to confirm these findings and to examine the persistence of DNA methylation marks over time.

Associations of Plasma Selenium with Arsenic and Genomic Methylation of Leukocyte DNA in Bangladesh

Background Global hypomethylation of DNA is thought to constitute an early event in some cancers and occurs in response to arsenic (As) exposure and/or selenium (Se) deficiency in both in vitro and animal models. In addition, antagonism between As and Se, whereby each reduces toxicity of the other, has been well documented in animal models. Se status may therefore modify the health effects of As in As-exposed populations. Objective The primary objectives of our study were to test the hypothesis that Se deficiency is associated with genomic hypomethylation of lymphocyte DNA and to determine whether Se levels are associated with blood As (bAs) and urinary As (uAs) concentrations in adults exposed to As-contaminated groundwater in Bangladesh. A secondary objective was to explore the relationships between plasma Se and As metabolites. Design We assessed plasma Se concentrations, As metabolite profiles in blood and urine, and genomic methylation of leukocyte DNA in a cross-sectional study of 287 adults. Results After adjustment for potential confounders, we observed an inverse association between Se (micrograms per liter) and genomic DNA methylation (disintegrations per minute per 1-μg/L increase in Se): β = 345.6; 95% confidence interval (CI), 59–632. Se concentrations were inversely associated with total As concentrations (micrograms per liter) in blood (β = −0.04; 95% CI, −0.08 to −0.01) and urine (β = −20.1; 95% CI, −29.3 to −10.9). Se levels were negatively associated with the percentage of monomethylarsinic acid (β = −0.59; 95% CI, −1.04 to −0.13) and positively associated with the percentage of dimethylarsinic acid (β = 0.53; 95% CI, 0.04 to 1.01) in blood. Conclusions Our results suggest that Se is inversely associated with genomic DNA methylation. The underlying mechanisms and implications of this observation are unclear and warrant further investigation. In addition, Se may influence bAs and uAs concentrations, as well as relative proportions of As metabolites in blood.

Folate, Cobalamin, Cysteine, Homocysteine, and Arsenic Metabolism among Children in Bangladesh

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.

Chronic Arsenic Exposure and Blood Glutathione and Glutathione Disulfide Concentrations in Bangladeshi Adults

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

Folic Acid and Creatine as Therapeutic Approaches to Lower Blood Arsenic: A Randomized Controlled Trial

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

Folate and Cobalamin Modify Associations between S-adenosylmethionine and Methylated Arsenic Metabolites in Arsenic-Exposed Bangladeshi Adults

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.

A dose-response study of arsenic exposure and global methylation of peripheral blood mononuclear cell DNA in Bangladeshi adults.

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

Creatinine, arsenic metabolism, and renal function in an arsenic-exposed population in bangladesh.

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.