Kevin M. Kubachka

Arsenic metabolism by human gut microbiota upon in vitro digestion of contaminated soils.

Background Speciation analysis is essential when evaluating risks from arsenic (As) exposure. In an oral exposure scenario, the importance of presystemic metabolism by gut microorganisms has been evidenced with in vivo animal models and in vitro experiments with animal microbiota. However, it is unclear whether human microbiota display similar As metabolism, especially when present in a contaminated matrix. Objectives We evaluated the metabolic potency of in vitro cultured human colon microbiota toward inorganic As (iAs) and As-contaminated soils. Methods A colon microbial community was cultured in a dynamic model of the human gut. These colon microbiota were incubated with iAs and with As-contaminated urban soils. We determined As speciation analysis using high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry. Results We found a high degree of methylation for colon digests both of iAs (10 μg methylarsenical/g biomass/hr) and of As-contaminated soils (up to 28 μg/g biomass/hr). Besides the formation of monomethylarsonic acid (MMAV), we detected the highly toxic monomethylarsonous acid (MMAIII). Moreover, this is the first description of microbial thiolation leading to monomethylmonothioarsonic acid (MMMTAV). MMMTAV, the toxicokinetic properties of which are not well known, was in many cases a major metabolite. Conclusions Presystemic As metabolism is a significant process in the human body. Toxicokinetic studies aiming to completely elucidate the As metabolic pathway would therefore benefit from incorporating the metabolic potency of human gut microbiota. This will result in more accurate risk characterization associated with As exposures.

Disruption of the arsenic (+3 oxidation state) methyltransferase gene in the mouse alters the phenotype for methylation of arsenic and affects distribution and retention of orally administered arsenate

The arsenic (+3 oxidation state) methyltransferase (As3mt) gene encodes a 43 kDa protein that catalyzes methylation of inorganic arsenic. Altered expression of AS3MT in cultured human cells controls arsenic methylation phenotypes, suggesting a critical role in arsenic metabolism. Because methylated arsenicals mediate some toxic or carcinogenic effects linked to inorganic arsenic exposure, studies of the fate and effects of arsenicals in mice which cannot methylate arsenic could be instructive. This study compared retention and distribution of arsenic in As3mt knockout mice and in wild-type C57BL/6 mice in which expression of the As3mt gene is normal. Male and female mice of either genotype received an oral dose of 0.5 mg of arsenic as arsenate per kg containing [(73)As]-arsenate. Mice were radioassayed for up to 96 h after dosing; tissues were collected at 2 and 24 h after dosing. At 2 and 24 h after dosing, livers of As3mt knockouts contained a greater proportion of inorganic and monomethylated arsenic than did livers of C57BL/6 mice. A similar predominance of inorganic and monomethylated arsenic was found in the urine of As3mt knockouts. At 24 h after dosing, As3mt knockouts retained significantly higher percentages of arsenic dose in liver, kidneys, urinary bladder, lungs, heart, and carcass than did C57BL/6 mice. Whole body clearance of [(73)As] in As3mt knockouts was substantially slower than in C57BL/6 mice. At 24 h after dosing, As3mt knockouts retained about 50% and C57BL/6 mice about 6% of the dose. After 96 h, As3mt knockouts retained about 20% and C57BL/6 mice retained less than 2% of the dose. These data confirm a central role for As3mt in the metabolism of inorganic arsenic and indicate that phenotypes for arsenic retention and distribution are markedly affected by the null genotype for arsenic methylation, indicating a close linkage between the metabolism and retention of arsenicals.

Exploring the in vitro formation of trimethylarsine sulfide from dimethylthioarsinic acid in anaerobic microflora of mouse cecum using HPLC-ICP-MS and HPLC-ESI-MS

Although metabolism of arsenicals to form methylated oxoarsenical species has been extensively studied, less is known about the formation of thiolated arsenical species that have recently been detected as urinary metabolites. Indeed, their presence suggests that the metabolism of ingested arsenic is more complex than previously thought. Recent reports have shown that thiolated arsenicals can be produced by the anaerobic microflora of the mouse cecum, suggesting that metabolism prior to systemic absorption may be a significant determinant of the pattern and extent of exposure to various arsenic-containing species. Here, we examined the metabolism of 34S labeled dimethylthioarsinic acid (34S-DMTA(V)) by the anaerobic microflora of the mouse cecum using HPLC-ICP-MS and HPLC-ESI-MS/MS to monitor for the presence of various oxo- and thioarsenicals. The use of isotopically enriched 34S-DMTA(V) made it possible to differentiate among potential metabolic pathways for production of the trimethylarsine sulfide (TMAS(V)). Upon in vitro incubation in an assay containing anaerobic microflora of mouse cecum, 34S-DMTA(V) underwent several transformations. Labile 34S was exchanged with more abundant 32S to produce 32S-DMTA(V), a thiol group was added to yield DMDTA(V), and a methyl group was added to yield 34S-TMAS(V). Because incubation of 34S-DMTA(V) resulted in the formation of 34S-TMAS(V), the pathway for its formation must preserve the arsenic-sulfur bond. The alternative metabolic pathway postulated for formation of TMAS(V) from dimethylarsinic acid (DMA(V)) would proceed via a dimethylarsinous acid (DMA(III)) intermediate and would necessitate the loss of 34S label. Structural confirmation of the metabolic product was achieved using HPLC-ESI-MS/MS. The data presented support the direct methylation of DMTA(V) to TMAS(V). Additionally, the detection of isotopically pure 34S-TMAS(V) raises questions about the sulfur exchange properties of TMAS(V) in the cecum material. Therefore, 34S-TMAS(V) was incubated and the exchange was monitored with respect to time. The data suggest that the As-S bond associated with TMAS(V) is less labile than the As-S bond associated with DMTA(V).

In vitro biotransformation of dimethylarsinic acid and trimethylarsine oxide by anaerobic microflora of mouse cecum analyzed by HPLC-ICP-MS and HPLC-ESI-MS

The capacity of the anaerobic microflora from a mouse cecum to metabolize dimethylarsinic acid (DMAV) and trimethylarsine oxide (TMAO) was examined in an in vitro assay system containing cecal contents in modified VPI buffer. Samples were incubated under anaerobic conditions at 37 °C for up to 24 hours and metabolic products were analyzed by HPLC-ICP-MS and HPLC-ESI-MS/MS. Under these conditions, DMAV was thiolated to dimethylthioarsinic acid (DMTAV) and dimethyldithioarsinic acid (DMDTA). The identities of DMTAV (m/z 154), DMDTA (m/z 170), and trimethylarsine sulfide (TMAS, m/z 152) were confirmed with HPLC-ESI-MS/MS. Three chromatographic separations were utilized to verify the lack of co-elution prior to quantification by ICP-MS. The predominant arsenical in reaction mixtures at 24 hours was DMDTA. The presence of TMAS in reaction mixtures after a six hour incubation implies a metabolism route from DMAV to TMAS possibly via reduction to dimethylarsinous acid, methylation to yield TMAO, and thiolation to TMAS. Addition of TMAO to in vitro assay systems containing cecal contents demonstrated that TMAO was almost quantitatively converted to TMAS within one hour. These combined results indicate that ingested arsenicals can undergo substantial metabolism mediated by the microflora of the gastrointestinal tract. Finally, metabolism of arsenicals occurring before absorption across the gastrointestinal barrier could be a modifier of exposure and dose.

Matrix Extension and Multilaboratory Validation of Arsenic Speciation Method EAM §4.10 to Include Wine

A multilaboratory validation (MLV) was performed to extend the U.S. Food and Drug Administrations (FDA) analytical method Elemental Analysis Manual (EAM) §4.10, High Performance Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometric Determination of Four Arsenic Species in Fruit Juice, to include wine. Several method modifications were examined to optimize the method for the analysis of dimethylarsinic acid, monomethylarsonic acid, arsenate (AsV), and arsenite (AsIII) in various wine matrices with a range of ethanol concentrations by liquid chromatography-inductively coupled plasma-mass spectrometry. The optimized method was used for the analysis of five wines of different classifications (red, white, sparkling, rosé, and fortified) by three laboratories. Additionally, the samples were fortified in duplicate at levels of approximately 5, 10, and 30 μg kg-1 and analyzed by each participating laboratory. The combined average fortification recoveries of dimethylarsinic acid, monomethylarsonic acid, and inorganic arsenic (iAs the sum of AsV and AsIII) in these samples were 101, 100, and 100%, respectively. To further demonstrate the method, 46 additional wine samples were analyzed. The total As levels of all the wines analyzed in this study were between 1.0 and 38.2 μg kg-1. The overall average mass balance based on the sum of the species recovered from the chromatographic separation compared to the total As measured was 89% with a range of 51-135%. In the 51 analyzed samples, iAs accounted for an average of 91% of the sum of the species with a range of 37-100%.

Multilaboratory Validation of First Action Method 2016.04 for Determination of Four Arsenic Species in Fruit Juice by High-Performance Liquid Chromatography–Inductively Coupled Plasma–Mass Spectrometry

Before being designated AOAC First Action Official MethodSM 2016.04, the U.S. Food and Drug Administrations method, EAM 4.10 High Performance Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometric Determination of Four Arsenic Species in Fruit Juice, underwent both a single-laboratory validation and a multilaboratory validation (MLV) study. Three federal and five state regulatory laboratories participated in the MLV study, which is the primary focus of this manuscript. The method was validated for inorganic arsenic (iAs) measured as the sum of the two iAs species arsenite [As(III)] and arsenate [As(V)], dimethylarsinic acid (DMA), and monomethylarsonic acid (MMA) by analyses of 13 juice samples, including three apple juice, three apple juice concentrate, four grape juice, and three pear juice samples. In addition, two water Standard Reference Materials (SRMs) were analyzed. The method LODs and LOQs obtained among the eight laboratories were approximately 0.3 and 2 ng/g, respectively, for each of the analytes and were adequate for the intended purpose of the method. Each laboratory analyzed method blanks, fortified method blanks, reference materials, triplicate portions of each juice sample, and duplicate fortified juice samples (one for each matrix type) at three fortification levels. In general, repeatability and reproducibility of the method was ≤15% RSD for each species present at a concentration >LOQ. The average recovery of fortified analytes for all laboratories ranged from 98 to 104% iAs, DMA, and MMA for all four juice sample matrixes. The average iAs results for SRMs 1640a and 1643e agreed within the range of 96-98% of certified values for total arsenic.