Metabolomic Effects of Folic Acid Supplementation in Adults: Evidence from the FACT Trial.

BACKGROUND: Folic acid (FA) is the oxidized form of folate found in supplements and FA-fortified foods. Most FA is reduced by dihydrofolate reductase to 5-methyltetrahydrofolate (5mTHF); the latter is the form of folate naturally found in foods. Ingestion of FA increases the plasma levels of both 5mTHF and unmetabolized FA (UMFA). Limited information is available on the downstream metabolic effects of FA supplementation, including potential effects associated with UMFA. OBJECTIVE: We aimed to assess the metabolic effects of FA-supplementation, and the associations of plasma 5mTHF and UMFA with the metabolome in FA-naïve Bangladeshi adults. METHODS: Sixty participants were selected from the Folic Acid and Creatine Trial; half received 800 µg FA/day for 12 weeks and half placebo. Plasma metabolome profiles were measured by high-resolution mass spectrometry, including 170 identified metabolites and 26,541 metabolic features. Penalized regression methods were used to assess the associations of targeted metabolites with FA-supplementation, plasma 5mTHF, and plasma UMFA. Pathway analyses were conducted using Mummichog. RESULTS: In penalized models of identified metabolites, FA-supplementation was associated with higher choline. Changes in 5mTHF concentrations were positively associated with metabolites involved in amino acid metabolism (5-hydroxyindoleacetic acid, acetylmethionine, creatinine, guanidinoacetate, hydroxyproline/n-acetylalanine) and 2 fatty acids (docosahexaenoic acid and linoleic acid). Changes in 5mTHF concentrations were negatively associated with acetylglutamate, acetyllysine, carnitine, propionyl carnitine, cinnamic acid, homogentisate, arachidonic acid, and nicotine. UMFA concentrations were associated with lower levels of arachidonic acid. Together, metabolites selected across all models were related to lipids, aromatic amino acid metabolism, and the urea cycle. Analyses of nontargeted metabolic features identified additional pathways associated with FA supplementation. CONCLUSION: In addition to the recapitulation of several expected metabolic changes associated with 5mTHF, we observed additional metabolites/pathways associated with FA-supplementation and UMFA. Further studies are needed to confirm these associations and assess their potential implications for human health. TRIAL REGISTRATION NUMBER: This trial was registered at https://clinicaltrials.gov as NCT01050556.

Effects of e-liquid flavor, nicotine content, and puff duration on metal emissions from electronic cigarettes.

Vaping is the action of inhaling and exhaling aerosols from electronic cigarettes. The aerosols contain various amounts of toxic chemicals, including metals. The purpose of this study was to evaluate factors that can influence metal levels, including flavor and nicotine content in the e-liquid, and puff duration. Aerosols were collected from both closed-system (cartridge-based) and open-system e-cigarettes using e-liquids with different flavors (fruit, tobacco, and menthol), nicotine content (0, 6, 24, and 59 mg/mL), and different puff durations (1, 2, and 4 s). The concentrations of 14 metals in the collected aerosols were measured using inductively coupled plasma mass spectroscopy. Aerosol concentrations of As, Fe, and Mn varied significantly among fruit, tobacco, and menthol flavors in both closed-system and open-system devices. Concentrations of Al, Fe, Sn, and U were significantly higher in tobacco or menthol flavored aerosols compared to fruit flavors in closed-system devices. Aerosol W levels were significantly higher in tobacco flavored aerosols compared to fruit flavors in open-system devices. Concentrations of As, Fe, and Mn were higher in tobacco flavored aerosols compared to menthol flavors in both types of devices. The median Pb concentration decreased significantly from 15.8 to 0.88 µg/kg when nicotine content increased from 0 to 59 mg/mL, and median Ni concentration was 9.60 times higher in aerosols with nicotine of 59 mg/mL compared to 24 mg/mL (11.9 vs. 1.24 µg/kg) for closed-system devices. No significant differences were observed in aerosol metal concentrations for different puff durations. Aerosol metal concentrations varied widely between different flavors and nicotine content but not by puff duration. Flavor and nicotine content of the e-liquid could be potential factors in metal emissions. Some elements showed higher concentrations under certain conditions, highlighting the urgent need of developing strict product regulations, especially on e-liquid composition and nicotine content to inform e-cigarette users about metal exposure through vaping.

Mixed metals exposure and cognitive function in Bangladeshi adolescents.

BACKGROUND: Over 57 million people in Bangladesh have been chronically exposed to arsenic-contaminated drinking water. They also face environmental exposure to elevated levels of cadmium (Cd), manganese (Mn), and lead (Pb), all of which have been previously observed in environmental and biological samples for this population. These metals have been linked to adverse neurocognitive outcomes in adults and children, though their effects on adolescents are not yet fully characterized. Additionally, previous studies have linked selenium (Se) to protective effects against the toxicity of these other metals. OBJECTIVES: To examine the associations between mixed metals exposure and cognitive function in Bangladeshi adolescents. METHODS: The Metals, Arsenic, & Nutrition in Adolescents study (MANAs) is a cross-sectional study of 572 Bangladeshi adolescents aged 14-16 years, whose parents were enrolled in the Health Effects of Arsenic Longitudinal Study (HEALS). Biosamples were collected from these adolescents for measurement of whole blood metalloid/metal levels of As, Cd, Mn, Pb, and Se. Participants also completed an abbreviated version of The Cambridge Neuropsychological Test Automated Battery (CANTAB), a cognitive function test designed to measure performance across several aspects of executive function. Linear regression was used to examine associations for each metal while controlling for the other metals. Bayesian Kernel Machine Regression (BKMR) assessed the overall mixture effect in addition to confirming the effects of individual metal components observed via linear regression. RESULTS: Linear regression revealed negative associations for Spatial Working Memory and both As and Mn (As B=-2.40, Mn B=-5.31, p < 0.05). We also observed negative associations between Cd and Spatial Recognition Memory (B=-2.77, p < 0.05), and Pb and Delayed Match to Sample, a measure of visual recognition and memory (B=-3.67, p < 0.05). Finally, we saw a positive association for Se and Spatial Span Length (B=0.92, p < 0.05). BKMR results were largely consistent with the regression analysis, showing meaningful associations for individual metals and CANTAB subtests, but no overall mixture effect. Via BKMR, we observed negative associations between Pb and Delayed Match to Sample, and Cd and Spatial Recognition Memory; this analysis also showed positive associations for Se and the Planning, Reaction Time, and Spatial Span subtests. BKMR posterior inclusion probability consistently reported that Se, the only component of the mixture to show a positive association with cognition, was the most important member of the mixture. CONCLUSIONS: Overall, we found Se to be positively associated with cognition, while Mn and As were linked to poorer working memory, and Cd and Pb were associated with poorer visual recognition and memory. Our observations are consistent with previous reports on the effects of these metal exposures in adults and children. Our findings also suggest agreement between linear regression and BKMR methods for analyzing metal mixture exposures. Additional studies are needed to evaluate the impact of mixed metals exposure on adverse health and poorer cognition later in life for those exposed during adolescence. Findings also suggest that metal exposure mitigation efforts aimed at adolescents might influence lifelong cognitive outcomes in regions where environmental exposure to metals is endemic.

Betaine and choline status modify the effects of folic acid and creatine supplementation on arsenic methylation in a randomized controlled trial of Bangladeshi adults.

PURPOSE: 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. METHODS: 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. RESULTS: 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 < 0.05). 400 µg FA/day was associated with a greater decrease in homocysteine among participants with plasma folate concentrations below, compared with those above, the median (FDR < 0.03). Creatine treatment was associated with a significant decrease in %MMAs among participants with choline concentrations below the median (P = 0.04), but not among participants above the median (P = 0.94); this effect did not significantly differ between strata (P = 0.10). CONCLUSIONS: Effects of FA and creatine supplementation on arsenic methylation capacity were greater among individuals with low betaine and choline status, respectively. The efficacy of FA and creatine interventions to facilitate arsenic methylation may be modified by choline and betaine nutritional status. CLINICAL TRIAL REGISTRATION: Clinical Trial Registry Identifier: NCT01050556, U.S. National Library of Medicine, https://clinicaltrials.gov ; registered January 15, 2010.

Nutrition, one-carbon metabolism and arsenic methylation in Bangladeshi adolescents.

BACKGROUND: Over 57 million people in Bangladesh are chronically exposed to arsenic-contaminated drinking water. Ingested inorganic arsenic (InAs) undergoes hepatic methylation generating monomethyl- (MMAs) and dimethyl- (DMAs) arsenic species in a process that facilitates urinary As (uAs) elimination. One-carbon metabolism (OCM), a biochemical pathway that is influenced by folate and vitamin B12, facilitates the methylation of As. OCM also supports nucleotide and amino acid synthesis, particularly during periods of rapid growth such as adolescence. While folate supplementation increases As methylation and lowers blood As (bAs) in adults, little data is available for adolescents. OBJECTIVES: To examine the associations between OCM-related micronutrients and As methylation in Bangladeshi adolescents chronically exposed to As-contaminated drinking water. METHODS: We conducted a cross-sectional study of 679 Bangladeshi adolescents, including 320 boys and 359 girls aged 14-16 years. Nutritional status was assessed by red blood cell (RBC) folate, plasma folate, plasma B12 and homocysteine (Hcys). Arsenic-related outcomes included blood arsenic (bAs), urinary arsenic (uAs), and urinary arsenic metabolites expressed as a percentage of total urinary As: %InAs, %MMAs, %DMAs. RESULTS: Boys had significantly lower B12, higher Hcys, higher bAs, higher uAs, higher %MMAs, and a trend toward lower RBC folate compared to girls. Therefore, regression analyses controlling for water As and BMI were sex stratified. Among girls, RBC folate was inversely associated with bAs, plasma B12 was inversely associated with uAs, and plasma Hcys was inversely associated with %MMA. Among boys, plasma folate was inversely associated with %InAs and positively associated with %DMA, RBC folate was inversely associated with %InAs and positively associated with %MMA, while Hcys was positively associated with %InAs. CONCLUSIONS: These findings suggest that associations between OCM nutritional status, bAs, and distribution of As metabolites in adolescents are similar to previously reported observations in adults and in children. The As methylation findings are statistically significant among boys but not among girls; this may be related to estrogen which more strongly influences OCM in females. The inverse association between Hcys and %MMA in girls is somewhat unexpected given that Hcys is known to be an indicator of impaired OCM and low folate/B12 in adults. Overall, these results indicate that the associations between OCM-related micronutrients and arsenic methylation in adolescents are generally similar to prior findings in adults, though these associations may differ by sex. Additionally, these findings suggest that more investigation into the role of Hcys in adolescent physiology is needed, perhaps particularly for girls. Additional studies are needed to evaluate the impact of OCM and As methylation on As-related adverse health outcomes (such as cancer and cardiovascular disease) in people exposed to As during adolescence.

Exposure to e-cigarette aerosol over two months induces accumulation of neurotoxic metals and alteration of essential metals in mouse brain.

Despite a recent increase in e-cigarette use, the adverse human health effects of exposure to e-cigarette aerosol, especially on the central nervous system (CNS), remain unclear. Multiple neurotoxic metals have been identified in e-cigarette aerosol. However, it is unknown whether those metals accumulate in the CNS at biologically meaningful levels. To answer this question, two groups of mice were whole-body exposed twice a day, 5 days a week, for two months, to either a dose of e-cigarette aerosol equivalent to human secondhand exposure, or a 5-fold higher dose. After the last exposure, the olfactory bulb, anterior and posterior frontal cortex, striatum, ventral midbrain, cerebellum, brainstem, remaining brain tissue and spinal cord were collected for metal quantification by inductively coupled plasma mass spectrometry and compared to tissues from unexposed control mice. The two-month exposure caused significant accumulation of several neurotoxic metals in various brain areas - for some metals even at the low exposure dose. The most striking increases were measured in the striatum. For several metals, including Cr, Cu, Fe, Mn, and Pb, similar accumulations are known to be neurotoxic in mice. Decreases in some essential metals were observed across the CNS. Our findings suggest that chronic exposure to e-cigarette aerosol could lead to CNS neurotoxic metal deposition and endogenous metal dyshomeostasis, including potential neurotoxicity. We conclude that e-cigarette-mediated metal neurotoxicity may pose long-term neurotoxic and neurodegenerative risks for e-cigarette users and bystanders.

Exposure to arsenic at different life-stages and DNA methylation meta-analysis in buccal cells and leukocytes.

BACKGROUND: Arsenic (As) exposure through drinking water is a global public health concern. Epigenetic dysregulation including changes in DNA methylation (DNAm), may be involved in arsenic toxicity. Epigenome-wide association studies (EWAS) of arsenic exposure have been restricted to single populations and comparison across EWAS has been limited by methodological differences. Leveraging data from epidemiological studies conducted in Chile and Bangladesh, we use a harmonized data processing and analysis pipeline and meta-analysis to combine results from four EWAS. METHODS: DNAm was measured among adults in Chile with and without prenatal and early-life As exposure in PBMCs and buccal cells (N = 40, 850K array) and among men in Bangladesh with high and low As exposure in PBMCs (N = 32, 850K array; N = 48, 450K array). Linear models were used to identify differentially methylated positions (DMPs) and differentially variable positions (DVPs) adjusting for age, smoking, cell type, and sex in the Chile cohort. Probes common across EWAS were meta-analyzed using METAL, and differentially methylated and variable regions (DMRs and DVRs, respectively) were identified using comb-p. KEGG pathway analysis was used to understand biological functions of DMPs and DVPs. RESULTS: In a meta-analysis restricted to PBMCs, we identified one DMP and 23 DVPs associated with arsenic exposure; including buccal cells, we identified 3 DMPs and 19 DVPs (FDR < 0.05). Using meta-analyzed results, we identified 11 DMRs and 11 DVRs in PBMC samples, and 16 DMRs and 19 DVRs in PBMC and buccal cell samples. One region annotated to LRRC27 was identified as a DMR and DVR. Arsenic-associated KEGG pathways included lysosome, autophagy, and mTOR signaling, AMPK signaling, and one carbon pool by folate. CONCLUSIONS: Using a two-step process of (1) harmonized data processing and analysis and (2) meta-analysis, we leverage four DNAm datasets from two continents of individuals exposed to high levels of As prenatally and during adulthood to identify DMPs and DVPs associated with arsenic exposure. Our approach suggests that standardizing analytical pipelines can aid in identifying biological meaningful signals.

Metal concentrations in electronic cigarette aerosol: Effect of open-system and closed-system devices and power settings.

BACKGROUND: Electronic cigarettes (E-cigarettes) generate aerosol containing metal contaminants. Our goals were to quantify aerosol metal concentrations and to compare the effects of power setting and device type (closed-system vs. open-system) on metal release. METHODS: Aerosol samples were collected from two closed-system devices (a cigalike and pod) and two open-system devices (mods). Each open-system device was operated at three different power settings to examine the effect of device power on metal release. Concentrations of 14 metals in e-cigarette aerosol collected via droplet deposition were measured using inductively coupled plasma mass spectroscopy. Aerosol metal concentrations were reported as mass fractions (µg/kg) in the e-liquid. RESULTS: For open-system device 1 (OD1), median arsenic (As), chromium (Cr), copper (Cu), iron (Fe), manganese (Mn), nickel (Ni), lead (Pb), antimony (Sb), tin (Sn), and zinc (Zn) concentrations increased 14, 54, 17, 30, 41, 96, 14, 81, 631, and 7-fold when the device power was increased from low (20 W) to intermediate (40 W) setting. When the power was further increased from intermediate (40 W) to high (80 W) setting, concentrations of As, Cr, Cu, Mn, Ni, and Sb did not change significantly. For open-system device 2 (OD2), Cr and Mn concentrations increased significantly when device power was increased from low (40 W) to intermediate (120 W) setting, and then decreased significantly when power was further increased from intermediate (120 W) to high (200 W) setting. Among the four devices, aerosol metal concentrations were higher for the open-system than the closed-system devices, except for aluminum (Al) and uranium (U). For Cr, median (interquartile range) concentrations (µg/kg) from the open-system devices were 2.51 (1.55, 4.23) and 15.6 (7.88, 54.5) vs. 0.39 (0.05, 0.72) and 0.41 (0.34, 0.57) for the closed-system devices. For Ni, concentrations (µg/kg) from the open-system devices were 793 (508, 1169) and 2148 (851, 3397) vs. 1.32 (0.39, 3.35) and 11.9 (10.7, 22.7) from the closed-system devices. Inhalation of 0% and 100% of samples from OD1, 7.4% and 88.9% from OD2 by typical e-cigarette users would exceed chronic minimum risk levels (MRL) of Mn and Ni, respectively. No MRL exceedance was predicted for the closed-system devices. A large fraction of users of OD1 (100%) and OD2 (77.8%) would be exposed to Ni levels higher than those from reference tobacco cigarette 3R4F. CONCLUSIONS: Our findings suggest that power setting and device type affect metal release from devices to aerosol which would subsequently be inhaled by users. Metal concentrations from open-system devices first increased with device power, and then leveled off for most metals. Open-system devices generate aerosol with higher metal concentrations than closed-system devices. These findings inform tobacco regulatory science, policy makers and health professionals on potential metal health risks associated with e-cigarette use, design and manufacturing.

A custom-built low-cost chamber for exposing rodents to e-cigarette aerosol: practical considerations.

Objectives: To (1) design and build a low-cost exposure chamber system for whole-body exposure of rodents to electronic cigarette aerosol, (2) provide detailed instructions with particular focus on automated e-cigarette activation, and (3) develop a simple mathematical model for aerosol levels in the exposure chamber.Methods: We built the system with standard laboratory equipment and an open-source electronics platform (Arduino) for e-cigarette activation. Arduino is used to control a solenoid, which pushes the activation button of so-called "Mod" e-cigarettes, and a pump to move the aerosol from the mouthpiece of the e-cigarette into the chamber. For "Pods" and "Cigalikes," the solenoid is not used as they are activated by the vacuum created by the pump. Aerosol concentrations were measured with a light-scattering laser photometer.Results: The system allows varying the air exchange rate, monitoring aerosol levels, and programing arbitrary puff topography. Aerosol concentrations observed for different chamber operating conditions (puff time and period, e-cigarette power output, air exchange rate) were consistent with the mathematical model.Conclusions: Our low-cost exposure chamber can be used in animal experimental studies of the health effects of e-cigarettes. Our model allows estimating design parameters such as chamber volume, air exchange rate, and puff period.

Supplementation with Folic Acid, but Not Creatine, Increases Plasma Betaine, Decreases Plasma Dimethylglycine, and Prevents a Decrease in Plasma Choline in Arsenic-Exposed Bangladeshi Adults.

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.

Low-Dose Creatine Supplementation Lowers Plasma Guanidinoacetate, but Not Plasma Homocysteine, in a Double-Blind, Randomized, Placebo-Controlled Trial.

BACKGROUND: Creatine synthesis from guanidinoacetate consumes ~50% of s-adenosylmethionine (SAM)-derived methyl groups, accounting for an equivalent proportion of s-adenosylhomocysteine (SAH) and total homocysteine (tHcys) synthesis. Dietary creatine inhibits the synthesis of guanidinoacetate, thereby lowering plasma tHcys in rats. OBJECTIVE: We tested the hypotheses that creatine supplementation lowers plasma guanidinoacetate, increases blood SAM, lowers blood SAH, and lowers plasma tHcys. METHODS: Bangladeshi adults were randomly assigned to receive 1 of 4 treatments for 12 wk: placebo (n = 101), 3 g/d creatine (Cr; n = 101), 400 µg/d folic acid (FA; n = 153), or 3 g/d creatine plus 400 µg/d folic acid (Cr+FA; n = 103). The outcomes of plasma guanidinoacetate and tHcys, as well as whole blood SAM and SAH, were analyzed at baseline and week 12 by HPLC. Treatment effects of creatine supplementation were examined with the use of the group comparisons of Cr vs. placebo and Cr+FA vs. FA. RESULTS: Plasma guanidinoacetate declined by 10.6% (95% CI: 4.9, 15.9) in the Cr group while increasing nonsignificantly in the placebo group (3.7%; 95% CI: -0.8, 8.5) (Pgroup difference = 0.0002). Similarly, plasma guanidinoacetate declined by 9.0% (95% CI: 3.4, 14.2) in the Cr+FA group while increasing in the FA group (7.0%; 95% CI: 2.0, 12.2) (Pgroup difference < 0.0001). Plasma tHcys declined by 23.4% (95% CI: 19.5, 27.1) and 21.0% (95% CI: 16.4, 25.2) in the FA and Cr+FA groups, respectively (Pgroup difference = 0.41), with no significant changes in the placebo or Cr groups (Pgroup difference = 0.35). A decrease in guanidinoacetate over time was associated with a decrease in tHcys over time in the Cr+FA group (ß = 0.30; 95% CI: 0.17, 0.43; P < 0.0001). CONCLUSIONS: Our findings indicate that whereas creatine supplementation downregulates endogenous creatine synthesis, this may not on average lower plasma tHcys in humans. However, tHcys did decrease in those participants who experienced a decline in plasma guanidinoacetate while receiving creatine plus folic acid supplementation. This trial was registered at clinicaltrials.gov as NCT01050556.

Renal function is associated with indicators of arsenic methylation capacity in Bangladeshi adults.

BACKGROUND: 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. OBJECTIVES: 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. METHODS: 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. RESULTS: 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 (p<0.0001) 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. CONCLUSIONS: 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.

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) (ß: -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 (ß: 6.96; 95% CI: 1.86, 12.05; P < 0.01) and negatively associated with %bDMA (ß: -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.

Contribution of arsenic and uranium in private wells and community water systems to urinary biomarkers in US adults: The Strong Heart Study and the Multi-Ethnic Study of Atherosclerosis.

BACKGROUND: Chronic exposure to inorganic arsenic (As) and uranium (U) in the United States (US) occurs from unregulated private wells and federally regulated community water systems (CWSs). The contribution of water to total exposure is assumed to be low when water As and U concentrations are low. OBJECTIVE: We examined the contribution of water As and U to urinary biomarkers in the Strong Heart Family Study (SHFS), a prospective study of American Indian communities, and the Multi-Ethnic Study of Atherosclerosis (MESA), a prospective study of racially/ethnically diverse urban U.S. communities. METHODS: We assigned residential zip code-level estimates in CWSs (µg/L) and private wells (90th percentile probability of As >10 µg/L) to up to 1485 and 6722 participants with dietary information and urinary biomarkers in the SHFS (2001-2003) and MESA (2000-2002; 2010-2011), respectively. Urine As was estimated as the sum of inorganic and methylated species, and urine U was total uranium. We used linear mixed-effects models to account for participant clustering and removed the effect of dietary sources via regression adjustment. RESULTS: The median (interquartile range) urine As was 5.32 (3.29, 8.53) and 6.32 (3.34, 12.48) µg/L for SHFS and MESA, respectively, and urine U was 0.037 (0.014, 0.071) and 0.007 (0.003, 0.018) µg/L. In a meta-analysis across both studies, urine As was 11% (95% CI: 3, 20%) higher and urine U was 35% (5, 73%) higher per twofold higher CWS As and U, respectively. In the SHFS, zip-code level factors such as private well and CWS As contributed 46% of variation in urine As, while in MESA, zip-code level factors, e.g., CWS As and U, contribute 30 and 49% of variation in urine As and U, respectively. IMPACT STATEMENT: We found that water from unregulated private wells and regulated CWSs is a major contributor to urinary As and U (an estimated measure of internal dose) in both rural, American Indian populations and urban, racially/ethnically diverse populations nationwide, even at levels below the current regulatory standard. Our findings indicate that additional drinking water interventions, regulations, and policies can have a major impact on reducing total exposures to As and U, which are linked to adverse health effects even at low levels.

Influence of folic acid and vitamin B12 supplementation on arsenic methylation: A double-blinded, placebo-controlled trial in Bangladeshi children.

BACKGROUND: Inorganic arsenic is metabolized to monomethyl- (MMAs) and dimethyl- (DMAs) species via one-carbon metabolism (OCM); this facilitates urinary arsenic elimination. OCM is influenced by folate and vitamin B12 and previous randomized control trials (RCTs) showed that folic acid (FA) supplementation increases arsenic methylation in adults. This RCT investigated the effects of FA + B12 supplementation on arsenic methylation in children, a key developmental stage where OCM supports growth. METHODS: A total of 240 participants (8-11 years, 53 % female) drinking from wells with arsenic concentrations > 50 µg/L, were encouraged to switch to low arsenic wells and were randomized to receive 400 µg FA + 5 µg B12 or placebo daily for 12-weeks. Urine and blood samples were collected at baseline, week 1 (only urine) and week 12. Generalized estimated equation (GEE) models were used to assess treatment effects on arsenic species in blood and urine. RESULTS: At baseline, the mean ± SD total blood and urinary arsenic were 5.3 ± 2.9 µg/L and 91.2 ± 89.5 µg/L. Overall, total blood and urine arsenic decreased by 11.7% and 17.6%, respectively, at the end of follow up. Compared to placebo, the supplementation group experienced a significant increase in the concentration of blood DMAs by 14.0% (95% CI 5.0, 25.0) and blood secondary methylation index (DMAs/MMAs) by 0.19 (95% CI: 0.09, 0.35) at 12 weeks. Similarly, there was a 1.62% (95% CI: 0.43, 20.83) significantly higher urinary %DMAs and -1.10% (95% CI: -1.73, -0.48) significantly lower urinary %MMAs in the supplementatio group compared to the placebo group after 1 week. The direction of the changes in the urinary %iAs, %MMAs, and %DMAs at week 12 were consistent with those at week 1, though estimates were not significant. Treatment effects were stronger among participants with higher baseline blood arsenic concentrations. Results were consistent across males and females, and participants with higher and lower folate and B12 status at baseline. CONCLUSION: This RCT confirms that FA + B12 supplementation increases arsenic methylation in children as reflected by decreased MMAs and increased DMAs in blood and urine. Nutritional interventions may improve arsenic methylation and elimination in children, potentially reducing arsenic toxicity while also improving nutritional status.

Metabolomic changes associated with chronic arsenic exposure in a Bangladeshi population.

Chronic exposure to arsenic (As) remains a global public health concern and our understanding of the biological mechanisms underlying the adverse effects of As exposure remains incomplete. Here, we used a high-resolution metabolomics approach to examine how As affects metabolic pathways in humans. We selected 60 non-smoking adults from the Folic Acid and Creatine Trial (FACT). Inorganic (AsIII, AsV) and organic (monomethylarsonous acid [MMAs], dimethylarsinous Acid [DMAs]) As species were measured in blood and urine collected at baseline and at 12 weeks. Plasma metabolome profiles were measured using untargeted high-resolution mass spectrometry. Associations of blood and urinary As with 170 confirmed metabolites and >26,000 untargeted spectral features were modeled using a metabolome-wide association study (MWAS) approach. Models were adjusted for age, sex, visit, and BMI and corrected for false discovery rate (FDR). In the MWAS screening of confirmed metabolites, 17 were associated with ≥1 blood As species (FDR<0.05), including fatty acids, neurotransmitter metabolites, and amino acids. These results were consistent across blood As species and between blood and urine As. Untargeted MWAS identified 423 spectral features associated with ≥1 blood As species. Unlike the confirmed metabolites, untargeted model results were not consistent across As species, with AsV and DMAs showing distinct association patterns. Mummichog pathway analysis revealed 12 enriched metabolic pathways that overlapped with the 17 identified metabolites, including one carbon metabolism, tricarboxylic acid cycle, fatty acid metabolism, and purine metabolism. Exposure to As may affect numerous essential pathways that underlie the well-characterized associations of As with multiple chronic diseases.

The Folic Acid and Creatine Trial: Treatment Effects of Supplementation on Arsenic Methylation Indices and Metabolite Concentrations in Blood in a Bangladeshi Population.

BACKGROUND: Chronic arsenic (As) exposure is a global environmental health issue. Inorganic As (InAs) undergoes methylation to monomethyl (MMAs) and dimethyl-arsenical species (DMAs); full methylation to DMAs facilitates urinary excretion and is associated with reduced risk for As-related health outcomes. Nutritional factors, including folate and creatine, influence one-carbon metabolism, the biochemical pathway that provides methyl groups for As methylation. OBJECTIVE: Our aim was to investigate the effects of supplementation with folic acid (FA), creatine, or the two combined on the concentrations of As metabolites and the primary methylation index (PMI: MMAs/InAs) and secondary methylation index (SMI: DMAs/MMAs) in blood in Bangladeshi adults having a wide range of folate status. METHODS: In a randomized, double-blinded, placebo (PBO)-controlled trial, 622 participants were recruited independent of folate status and assigned to one of five treatment arms: a) PBO (n=102), b) 400µg FA/d (400FA; n=153), c) 800µg FA/d (800FA; n=151), d) 3g creatine/d (creatine; n=101), or e) 3g creatine+400µg of FA/d (creatine+400FA; n=103) for 12 wk. For the following 12 wk, half of the FA participants were randomly switched to the PBO while the other half continued FA supplementation. All participants received As-removal water filters at baseline. Blood As (bAs) metabolites were measured at weeks 0, 1, 12, and 24. RESULTS: At baseline, 80.3% (n=489) of participants were folate sufficient (≥9 nmol/L in plasma). In all groups, bAs metabolite concentrations decreased, likely due to filter use; for example, in the PBO group, blood concentrations of MMAs (bMMAs) (geometric mean±geometric standard deviation) decreased from 3.55±1.89µg/L at baseline to 2.73±1.74 at week 1. After 1 wk, the mean within-person increase in SMI for the creatine+400FA group was greater than that of the PBO group (p=0.05). The mean percentage decrease in bMMAs between baseline and week 12 was greater for all treatment groups compared with the PBO group [400FA: -10.4 (95% CI: -11.9, -8.75), 800FA: -9.54 (95% CI: -11.1, -7.97), creatine: -5.85 (95% CI: -8.59, -3.03), creatine+400FA: -8.44 (95% CI: -9.95, -6.90), PBO: -2.02 (95% CI: -4.03, 0.04)], and the percentage increase in blood DMAs (bDMAs) concentrations for the FA-treated groups significantly exceeded that of PBO [400FA: 12.8 (95% CI: 10.5, 15.2), 800FA: 11.3 (95% CI: 8.95, 13.8), creatine+400FA: 7.45 (95% CI: 5.23, 9.71), PBO: -0.15 (95% CI: -2.85, 2.63)]. The mean decrease in PMI and increase in SMI in all FA groups significantly exceeded PBO (p<0.05). Data from week 24 showed evidence of a reversal of treatment effects on As metabolites from week 12 in those who switched from 800FA to PBO, with significant decreases in SMI [-9.0% (95% CI: -3.5, -14.8)] and bDMAs [-5.9% (95% CI: -1.8, -10.2)], whereas PMI and bMMAs concentrations continued to decline [-7.16% (95% CI: -0.48, -14.3) and -3.1% (95% CI: -0.1, -6.2), respectively] for those who remained on 800FA supplementation. CONCLUSIONS: FA supplementation lowered bMMAs and increased bDMAs in a sample of primarily folate-replete adults, whereas creatine supplementation lowered bMMAs. Evidence of the reversal of treatment effects on As metabolites following FA cessation suggests short-term benefits of supplementation and underscores the importance of long-term interventions, such as FA fortification. https://doi.org/10.1289/EHP11270.

E-cigarette aerosol collection using converging and straight tubing Sections: Physical mechanisms.

HYPOTHESIS: Identification and quantification of harmful chemicals in e-cigarette aerosol requires collecting the aerosolized e-liquid for chemical analysis. In 2016, Olmedo at al. empirically developed a simple method for aerosol collection by directing the aerosol through a sequence of alternating straight and converging tubing sections, which drain the recovered e-liquid into a collection vial. The tubing system geometry and flow conditions promote inertial impaction of aerosolized e-liquid on tube walls, where it deposits and flows into the collection vial. EXPERIMENTS: We use high-speed optical imaging to visualize aerosol transport in proxies of the collection system. We also determined collection efficiencies of various configurations of the collection system. FINDINGS: A turbulent jet emerges from converging conical sections and impinges onto the wall of downstream tubing sections, resulting in inertial impaction and deposition of the aerosol. For inertial impaction to occur the tip radius of the converging section must be small enough for a jet to be formed and the sequence of tubing sections must be curved in a polygon-like manner such that the jet emerging from a converging section impinges on the downstream tube wall. The collection efficiency is significantly smaller without such curvature.

Identification of newly formed toxic chemicals in E-cigarette aerosols with Orbitrap mass spectrometry and implications on E-cigarette control.

The increasing use of electronic nicotine delivery systems (ENDS) is of concern due to multiple emerging adverse health effects. Most analyses of the harmful chemicals of ENDS have targeted metals or carbonyls generated by thermal decomposition of carrier liquids such as propylene glycol. However, new complex compounds not routinely identified and with unknown health consequences could be formed. ENDS aerosol samples were collected by the direct aerosol droplet deposition method. Untargeted analysis was performed using Orbitrap mass spectrometry with high mass accuracy. We identified more than 30 "features" in the aerosol characterized by pairs of the mass-to-charge ratio "m/z" of the compound and the retention time. We identified several compounds containing nicotine and propylene glycol (NIC-PG), whose abundance relative to nicotine increased along with vaping power used. On the basis of the prediction by the Environmental Protection Agency Toxicity Estimation Software Tool, these compounds exert developmental toxicity. In addition, a nitrogen-containing compound, likely tributylamine (a known lung irritant), was identified based on the molecular weight. This compound has not been previously identified in ENDS e-liquids and aerosols. ENDS produce not only small toxic compounds such as aldehydes, but also large complex toxic compounds such as NIC-PG. Predicted development toxicity for NIC-PG is concerning for fetal development in pregnant women who use ENDS, children exposed to secondhand or thirdhand ENDS aerosols, and teenage ENDS users whose brains are still developing. The strong positive association between NIC-PG levels and ENDS power output supports regulating high-powered ENDS.

Association between body mass index and arsenic methylation in three studies of Bangladeshi adults and adolescents.

BACKGROUND: Water-borne arsenic (As) exposure is a global health problem. Once ingested, inorganic As (iAs) is methylated to mono-methyl (MMA) and dimethyl (DMA) arsenicals via one-carbon metabolism (OCM). People with higher relative percentage of MMA (MMA%) 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. OBJECTIVE: To characterize the association between body mass index (BMI) and As methylation in Bangladeshi adults and adolescents participating in the Folic Acid and Creatine Trial (FACT); Folate and Oxidative Stress (FOX) study; and Metals, Arsenic, and Nutrition in Adolescents Study (MANAS). METHODS: Arsenic species (iAs, MMA, DMA) 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. RESULTS: 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 MMA% and DMA% 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 DMA% that was 0.16%/0.19% higher. DISCUSSION: BMI was negatively associated with MMA% and positively associated with %DMA in females but not males in Bangladesh; associations were attenuated after plasma choline adjustment. These findings may be related to the role of body fat on estrogen levels that can influence one-carbon metabolism, 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.