John T. Creed

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.

Speciation and preservation of inorganic arsenic in drinking water sources using EDTA with IC separation and ICP-MS detection

The native distribution of As(III) and As(v) in drinking water supplies can influence the treatment removal strategy. The stability of As(III) and As(v) in iron-rich drinking waters can be affected by the formation of Fe precipitates (Fe oxides and/or hydroxides designated by FeOOH). These precipitates (ppts) can form during the transport of the sample to the laboratory for arsenic speciation analysis. The analysis of the ppt indicates considerable loss of the aqueous arsenic species (As(aq)) to the solid phase FeOOH ppt. Studies of laboratory reagent water containing both As(III) and Fe(III) indicate that the resulting FeOOH ppt contained a mixture of As(III) and As(v) with near quantitative removal of the As(aq) in 18 hr. The corresponding aqueous fraction after filtration through a 0.45 microm filter was composed primarily of As(v). The formation of FeOOH ppt and the loss of As(aq) to the ppt can be virtually eliminated by the use of EDTA, which sequesters the FeIII). Reagent water fortified with Fe(III), As(III) and EDTA produced less than a 1 ppb change in the As(III)aq concentration over 16 d. The EDTA treatment was also tested on three well waters with different native As(III )/As(v) ratios. The native distribution of As(III)/As(v) was stabilized over a period of 10 d with a worst case conversion of As(III) to As(v) of 2 ppb over a 30 d period. All well waters not treated with EDTA had dramatic losses (a factor of 2-5) of As(aq) in less than 1 d. These results indicated that EDTA preservation treatment can be used to preserve As(aq) in waters where the predominant species is the reduced form [As(III)] or in waters which the predominant species is the oxidized form [As(v)]. This preliminary investigation of EDTA to preserve As species in Fe-rich waters indicates stability can be achieved for greater than 14 d.

Determination of bromate in drinking waters by ion chromatography with inductively coupled plasma mass spectrometric detection

Abstract Bromate is a disinfection by-product in drinking water, formed during the ozonation of source water containing bromide. An inductively coupled plasma mass spectrometer is combined with an ion chromatograph for the analysis of bromate in drinking waters. Three chromatographic columns are evaluated in terms of detection limits, analysis time and tolerance to potentially interfering inorganic anions. The detection limits for all columns are in the 1–2 μg/l range for the direct analysis of bromate. A 5-min analysis time was achieved using a Dionex AG10 column and 100 mM NaOH as the eluent. Recoveries for bromate in fortified samples containing chloride (1000 ppm) or nitrate (50 ppm) were 96–107%. Recoveries for bromate in fortified samples containing sulfate (1000 ppm) were 91–124%. The R.S.D. values for drinking water analyses are in the 2–6% range. A 1.8-ml sample was preconcentrated on a Dionex AG10 column. This system produced bromate detection limits in the 0.1–0.2 μg/l range. Coupling the AG10 preconcentrator column with an ultrasonic nebulizer produced a detection limit of 50 ppt for bromate. The precision for samples which are preconcentrated is degraded due to an adjacent peak interfering with integration of the bromate peak.

An investigation of the chemical stability of arsenosugars in simulated gastric juice and acidic environments using IC-ICP-MS and IC-ESI-MS/MS

A more quantitative extraction of arsenic-containing compounds from seafood matrices is essential in developing better dietary exposure estimates. More quantitative extraction often implies a more chemically aggressive set of extraction conditions. However, these conditions may result in undesirable chemical changes in the native arsenicals which may further complicate the toxicological risk assessment. This balance between quantitative extraction and species-specific integrity may be best addressed by using simulated gastric juice as an extraction solvent to mimic bioavailability. This, conceptually, should extract the bioavailable fraction and induce any chemical changes that would occur because of ingestion. The most chemically labile species associated with seafood are thought to be the arsenosugars and for this reason their chemical stability is investigated in this study. Four arsenosugars (3-[5-deoxy-5-(dimethylarsinoyl)-beta-ribofuranosyloxy]-2-hydroxypropylene glycol, As(328); 3-[5-deoxy-5-(dimethylarsinoyl)-beta-ribofuranosyloxy]-2-hydroxypropanesulfonic acid, As(392); 3-[5-deoxy-5-(dimethylarsinoyl)-beta-ribofuranosyloxyl-2-hydroxypropyl hydrogen sulfate, As(408); and 3-[5-deoxy-5-(dimethylarsinoyl)-beta-ribofuranosyloxy]-2-hydroxypropyl-2,3-hydroxypropyl phosphate, As(482)) were isolated from seaweed extracts and subjected to simulated gastric juice and acidic conditions which mimic the stomachs pH of 1.1. Three acid solutions were used to test the chemical stability of the arsenosugars: simulated gastric juice, 78 mM nitric acid and 78 mM hydrochloric acid. The composition of the solutions was monitored over time (up to 48 h) using IC-ICP-MS for detection. The arsenosugars were found to degrade at the rate of 1.4% per h at 38 degrees C and 12.2% per h at 60 degrees C. The plots of percent conversion versus time were found to be independent of the starting arsenosugar and all had r2 values of greater than 0.97. A single common degradation product was observed in all the stability studies. A mass balance between the starting arsenosugar (As(392), As(408) and As(482)) and the degradation product was conducted with each set of experiments. This mass balance indicated that the degradation process did not produce any unchromatographable species. This degradation product was tentatively identified as As(254) as determined by ESI-MS/MS spectral data. An acid hydrolysis mechanism was proposed for the formation of As(254) from each of the native arsenosugars by hydrolysis at the C-1 carbon on the ribose ring.

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).

A comparison of urinary arsenic speciation via direct nebulization and on-line photo-oxidation–hydride generation with IC separation and ICP-MS detection

Urinary arsenic speciation provides information on recent arsenic exposure. The literature reported analysis of NIST SRM 2670 Freeze-dried Urine indicates considerable discrepancy in species specific concentration. In this study, two complementary sample introduction pathways, direct nebulization (DN) and hydride generation (HG), were utilized and compared for urinary arsenic speciation via ion chromatography (IC)-ICP-MS. The retention characteristics of arsenobetaine (AsB), arsenite [As(III)], dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), arsenate [As(V)] and Cl− were systematically evaluated with respect to column temperature and the (NH4)2CO3 eluent molarity using the DN method. This characterization indicated that three early eluters [AsB, As(III) and DMA] were best separated at a higher column temperature and lower eluent molarity, whereas MMA, As(V) and Cl− were best separated at a lower column temperature and higher eluent molarity. From these observations, a gradient elution program was developed using 40 and 70xa0mM (NH4)2CO3 (pHxa010.5) at 60u2006°C. This gradient condition produced satisfactory resolution for all five arsenic species with a Cl− tolerance up to 0.3% w/w. In the membrane hydride generation (HG) configuration, a photo-reactor interface was installed between the column and the HG device to facilitate the detection of non-hydride active arsenic species. Isocratic elution using 40xa0mM (NH4)2CO3 was adequate in resolving all five arsenic species while the chloride interference was removed by a gas–liquid separator. NIST SRM 2670 Freeze-dried Urine was analyzed using the DN and HG methods and the sum of the arsenical concentrations was 77.7 ± 3.5 and 71.1 ± 2.8xa0ngxa0mL−1, respectively.

Extraction and detection of a new arsine sulfide containing arsenosugar in molluscs by IC-ICP-MS and IC-ESI-MS/MS

Using IC-ICP-MS and IC-ESI-MS/MS, an unknown arsenical compound in molluscs has been identified as a new arsine sulfide containing analog of a known arsenosugar and is referred to as As(498). This species has been observed in four separate shellfish species following a mild methanol–water extraction. As(498) is unstable, especially in acid, and converts to the arsine oxide containing arsenosugar As(482) over time. Chromatographic retention of As(498) was observed on an anion exchanger ION-120 column but the species did not elute as a well defined peak from a PRP-X100. Mass spectrometric analysis of As(498) at pH 9.0 produced an [M–H]− species at a mass to charge of 497 in the negative-ion mode. A synthetic standard of As(498) was made by bubbling hydrogen sulfide into a stock solution of arsenosugar As(482). The retention time and ESI-MS/MS data were identical for the synthetic standard of As(498) and the unknown arsenical in shellfish extracts.

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.

An investigation of the chemical stability of arsenosugars in basic environments using IC-ICP-MS and IC-ESI-MS/MS.

This paper evaluates the chemical stability of four arsenosugars using tetramethylammonium hydroxide (TMAOH) as an extraction solvent. This solvent was chosen because of the near quantitative removal of these arsenicals from difficult to extract seafood (oysters and shellfish). Four arsenosugars (3-[5-deoxy-5-(dimethylarsinoyl)-beta-ribofuranosyloxy]-2-hydroxypropylene glycol--As(328), 3-5-deoxy-5-(dimethylarsinoyl)-beta-ribofuranosyloxy]-2-hydroxypropanesulfonic acid--As(392), 3-[5-deoxy-5-(dimethylarsinoyl)-beta-ribofuranosyloxy]-2-hydroxypropyl hydrogen sulfate--As(408), and 3-[5-deoxy-5-(dimethylarsinoyl)-beta-ribofuranosyloxy]-2-hydroxypropyl-2,3-hydroxypropyl phosphate--As(482)) were evaluated. The stability of these four arsenosugars were studied independently in a solution of 2.5% TMAOH at 60 degrees C over a period of up to 8 h. Two arsenosugars, As(328) and As(392), were found to be relatively stable in this solution for up to 8 h. However, As(408) and As(482) formed detectable quantities of dimethylarsinic acid (DMAA) and As(328) within 0.5 and 2 h, respectively. It was found that 97% of As(408) degrades after 8 h of treatment producing 3.4 times as much DMAA as As(328). This is contrary to As(482), which produces 13 times as much As(328) as DMAA and only 37% of the As(482) was converted by the 8 h treatment at 60 degrees C. These degradation products led to the investigation of weaker TMAOH extraction solvents. Three different concentrations (2.5%, 0.83% and 0.25%) were used to determine the effect of TMAOH concentration on the degradation rate of As(408). By reducing the TMAOH concentration to 0.83%, the conversion of the arsenosugar to As(328) and DMAA is nearly eliminated (less than 5% loss). Arsenosugars, As(408) and As(482), were also studied in 253 mM NaOH to verify the degradation products. The NaOH experiments were conducted to investigate a possible hydroxide based reaction mechanism. Similar degradation plots were found for each arsenosugar when compared to the 2.5% TMAOH data. A mechanism has been proposed for the formation of As(328) from As(408) and As(482) in base via an SN2 reaction (hydroxide attack) at the side chain carbon adjacent to the inorganic ester. The formation of DMAA is observed in all arsenosugars after prolonged exposure. This probably occurs via an SN2 attack at the arsenic atom.