Title: Arsenic, Nutrition, and Metabolic Outcomes
Description: Exposure to arsenic (As) is a major public health concern globally. Inorganic As (InAs) undergoes hepatic methylation to form monomethyl (MMAs)- and dimethyl (DMAs)-arsenical species, facilitating urinary As elimination. MMAsIII is considerably more toxic than either InAsIII or DMAsV, and a higher proportion of MMAs in urine has been associated with risk for a wide range of adverse health outcomes. One-carbon metabolism (OCM) is a biochemical pathway that provides methyl groups for the methylation of As, and is influenced by folate and other micronutrients, such as vitamin B12, choline, betaine and creatine. A growing body of evidence has demonstrated that OCM-related micronutrients play a critical role in As methylation. To analyze the impact of As exposure, it needs to be properly quantitated. Urinary As (uAs) is a biomarker of As exposure. Urinary creatinine (uCr) or specific gravity (SG) are used to correct uAs for urine dilution. However, uCr is correlated with As methylation, whereas SG has limitations in individuals with kidney damage.

Therefore, it is important to determine which urine dilution proxy is appropriate in As-related research. In Chapter 2 we conducted a review that summarized observational epidemiological studies, interventions, and relevant experimental evidence examining the role that OCM-related micronutrients have on As methylation, toxicity of As, and risk for associated adverse health-related outcomes. People with higher relative percentage of MMAs (%MMAs) in urine (inefficient As methylation), have been shown to have a higher risk of cardiovascular disease and several cancers but appear to have a lower risk of diabetes and obesity in populations from the US, Mexico, and Taiwan. It is unknown if this opposite pattern with obesity is present in Bangladesh, a country with lower adiposity and higher As exposure in drinking water. Efficiency of As methylation differs substantially between species, between individuals, and across populations. In Chapter 3, we aimed to evaluate which urine dilution correction methods for uAs most accurately predicted blood As (bAs).

We used data from the Folic Acid and Creatine Trial (FACT; N = 541) and Folate and Oxidative Stress (FOX; N = 343) study in Bangladesh. Three linear regression models were assessed using uAs (1) adjusted for uCr or SG as separate covariates, (2) standardized for uCr or SG, i.e., uAs/uCr, and (3) adjusted for residual corrected uCr or SG following adjustment for age, sex and BMI. Median uAs/bAs for FACT and FOX were 114/8.4 and 140/12.3 µg/L. In FACT, two-fold increases in uAs adjusted for uCr or SG were related to 34% and 22% increases in bAs, respectively, with similar patterns in FOX. In Chapter 4, we investigated the effects of folic acid (FA) and/or creatine supplementation on the concentrations of As species and primary (PMI: MMAs/InAs) and secondary (SMI: DMAs/MMAs) methylation indices in blood in Bangladeshi adults having a wide range of folate status. In a randomized, double-blinded, placebo-controlled trial, 622 participants were assigned to FA (400 or 800 μg/day), 3 g creatine/day, 3 g creatine + 400 μg FA/day, or placebo for 12 weeks.

For the following 12 weeks, half of the FA participants were randomly switched to receive placebo. All participants received As-removal water filters at baseline. Blood As species were measured at baseline, and weeks 1, 12, and 24. In all groups, blood As species concentrations decreased due to filter use. After 1 week, the mean within-person increase in SMI for the creatine + 400FA group was greater than that of the placebo group (p = 0.05). The mean percent decrease (95% CI) in blood concentrations of MMAs (bMMAs) between baseline and week 12 was greater for all treatment groups compared to the placebo group [400FA: -10.3 (-11.9, -8.8); 800FA: -9.5 (-11.1, -8.0); creatine: -5.9 (-8.6, -3.0); creatine + 400FA: -8.4 (-10.0, -6.9); placebo: -2.0 (-4.0, 0.0)], and the percent increase in blood DMAs (bDMAs) concentrations for the FA treated groups all significantly exceeded that of placebo [400 FA: 12.8 (10.5, 15.2); 800 FA: 11.3 (8.90, 13.8); creatine + 400 FA: 7.40 (5.20, 9.70); placebo: -0.10 (-2.80, 2.60)].

The mean decrease in PMI and increase in SMI in all FA groups significantly exceeded placebo (p < 0.05). Data from week 24 showed evidence of a reversal of treatment effects on As species from week 12 in those who switched from 800FA to placebo, with significant decreases in SMI [-9.0% (-3.5, -14.8)] and bDMAs [-5.9% (-1.8, -10.2)] in those who switched from 800FA to placebo, whereas for those who remained on 800FA, PMI and bMMAs concentrations continued to decline [-7.2% (-0.5, -14.3) and -3.1% (-0.1, -6.2), respectively] for those who remained on 800FA supplementation. This trial was registered at https://clinicaltrials.gov as NCT01050556. In Chapter 5, we characterized the association between body mass index (BMI) and As methylation in Bangladeshi adults and adolescents participating in the FACT; FOX; and Metals, Arsenic, and Nutrition in Adolescents Study (MANAS). Arsenic species (InAs, MMAs, DMAs) were measured in urine and blood. Height and weight were measured to calculate BMI. The associations between concurrent BMI with urine and blood As species were analyzed using linear regression models, adjusting for nutrients involved in OCM such as choline.

In FACT, we also evaluated the prospective association between weight change and As species. Mean BMIs were 19.2/20.4, 19.8/21.0, and 17.7/18.7 kg/m2 in males/females in FACT, FOX, and MANAS, respectively. BMI was associated with As species in female but not in male participants. In females, after adjustment for total urine As, age, and plasma folate, the adjusted mean differences (95% confidence) in urinary %MMAs and %DMAs for a 5 kg/m2 difference in BMI were -1.21 (-1.96, -0.45) and 2.47 (1.13, 3.81), respectively in FACT, -0.66 (-1.56, 0.25) and 1.43 (-0.23, 3.09) in FOX, and -0.59 (-1.19, 0.02) and 1.58 (-0.15, 3.30) in MANAS. The associations were attenuated after adjustment for choline. Similar associations were observed with blood As species. In FACT, a 1-kg of weight increase over 2 to 10 (mean 5.4) years in males/ females was prospectively associated with mean %DMAs that was 0.16%/0.19% higher. BMI was negatively associated with %MMAs and positively associated with %DMAs in females but not males in Bangladesh; associations were attenuated after plasma choline adjustment.

In conclusion, we found that FA supplementation lowers bMMAs and increases bDMAs in a sample of primarily folate-replete adults, while creatine supplementation lowers bMMAs. Evidence of the reversal of treatment effects on As species following FA cessation suggests short-term benefits of supplementation and underscores the importance of long-term interventions such as FA fortification. Additionally, there is fairly robust evidence supporting the impact of folate on As methylation, and some evidence from case-control studies indicating that folate nutritional status influences risk for As-induced skin lesions and bladder cancer. However, the potential for folate to be protective for other As-related health outcomes, adverse health risks of high folate/FA levels (particularly in areas where folate supplements are common), and beneficial effects of other OCM-related micronutrients on As methylation and risk for health outcomes are not as well studied and warrant additional research. We also found that the role of body fat on estrogen levels that may influence OCM, e.g. by increasing choline synthesis.

Research is needed to determine whether the associations between BMI and As species are causal and their influence on As-related health outcomes. Finally, we found that in assessing urine dilution correction approaches, models with uCr consistently had lower AIC values than SG across methods. The uAs associations with bAs were stronger after adjustment for uCr vs. SG. Decisions regarding urine dilution methods should consider whether the study outcomes are influenced by factors such as methylation or medical conditions.