Bioaccessibility of arsenic from contaminated soils and alteration of the gut microbiome in an in vitro gastrointestinal model.

Arsenic exposure has been reported to alter the gut microbiome in mice. Activity of the gut microbiome derived from fecal microbiota has been found to affect arsenic bioaccessibility in an in vitro gastrointestinal (GI) model. Only a few studies have explored the relation between arsenic exposure and changes in the composition of the gut microbiome and in arsenic bioaccessibility. Here, we used simulated GI model system (GIMS) containing a stomach, small intestine, colon phases and microorganisms obtained from mouse feces (GIMS-F) and cecal contents (GIMS-C) to assess whether exposure to arsenic-contaminated soils affect the gut microbiome and whether composition of the gut microbiome affects arsenic bioaccessibility. Soils contaminated with arsenic did not alter gut microbiome composition in GIMS-F colon phase. In contrast, arsenic exposure resulted in the decline of bacteria in GIMS-C, including members of Clostridiaceae, Rikenellaceae, and Parabacteroides due to greater diversity and variability in microbial sensitivity to arsenic exposure. Arsenic bioaccessibility was greatest in the acidic stomach phase of GIMS (pH 1.5-1.7); except for GIMS-C colon phase exposed to mining-impacted soil in which greater levels of arsenic solubilized likely due to microbiome effects. Physicochemical properties of different test soils likely influenced variability in arsenic bioaccessibility (GIMS-F bioaccessibility range: 8-37%, GIMS-C bioaccessibility range: 2-18%) observed in this study.

Bioavailable soil Pb minimized by in situ transformation to plumbojarosite.

Exposure to lead (Pb) during early life has persistent adverse health effects. During childhood, ingestion of bioavailable Pb in contaminated soils can be a major route of Pb absorption. Remediation to alter physiochemical properties of soil-borne Pb can reduce Pb bioavailability. Our laboratory-based approach for soil Pb remediation uses addition of iron (Fe) sulfate and application of heat to promote formation of plumbojarosite (PLJ), a sparingly soluble Pb-Fe hydroxysulfate mineral. We treated two soils with anthropogenic Pb contamination and samples of clean topsoil spiked with various Pb compounds (i.e., carbonate, chloride, phosphate [P], or sulfate) to convert native Pb species to PLJ and used a mouse assay to assess relative bioavailability (RBA) of Pb in untreated (U) and remediated soils. Bone and blood Pb levels were significantly lower (P < 0.001, Student's t test) in mice that consumed diets amended with remediated soils than with U soils. Estimated RBA for Pb in both remediated natural soils and Pb-mineral spiked soils were reduced by >90% relative to Pb RBA for U soils, which is substantially more effective than other soil amendments, including P. X-ray absorption spectroscopy showed that >90% of all Pb species in remediated soils were converted to PLJ, and ingested PLJ was not chemically transformed during gastrointestinal tract transit. Post treatment neutralization of soil pH did not affect PLJ stability, indicating the feasibility in field conditions. These results suggest that formation of PLJ in contaminated soils can reduce the RBA of Pb and minimize this medium's role as a source of Pb exposure for young children.

Intra- and Interlaboratory Evaluation of an Assay of Soil Arsenic Relative Bioavailability in Mice.

Hand-to-mouth activity in children can be an important route for ingestion of soil and dust contaminated with inorganic arsenic. Estimating the relative bioavailability of arsenic present in these media is a critical element in assessing the risks associated with aggregate exposure to this toxic metalloid during their early life. Here, we evaluated the performance of a mouse assay for arsenic bioavailability in two laboratories using a suite of 10 soils. This approach allowed us to examine both intralaboratory and interlaboratory variations in assay performance. Use of a single vendor for preparation of all amended test diets and of a single laboratory for arsenic analysis of samples generated in the participating laboratories minimized contributions of these potential sources of variability in assay performance. Intralaboratory assay data showed that food and water intake and cumulative urine and feces production remained stable over several years. The stability of these measurements accounted for the reproducibility of estimates of arsenic bioavailability obtained from repeated intralaboratory assays using sodium arsenate or soils as the test material. Interlaboratory comparisons found that estimates of variables used to evaluate assay performance (recovery and urinary excretion factor) were similar in the two laboratories. For all soils, estimates of arsenic relative bioavailability obtained in the two laboratories were highly correlated (r2 = 0.94 and slope = 0.9) in a linear regression model. Overall, these findings show that this mouse assay for arsenic bioavailability provides reproducible estimates using a variety of test soils. This robust model may be adaptable for use in other laboratory settings.

Dietary Lead and Phosphate Interactions Affect Oral Bioavailability of Soil Lead in the Mouse.

Effects of dietary P level on the oral bioavailability of Pb present in soil were examined in a mouse model. Adult female C57BL/6 mice had free access to AIN-93G purified rodent diet amended with Pb as a soluble salt, Pb acetate, or in a soil matrix (NIST SRM 2710a). In these studies, the basal diet contained P at a nutritionally sufficient level (0.3% w/w) and the modified diets contained P at a lower (0.15%) or a higher (1.2%) level. For either dietary Pb source (Pb acetate or NIST SRM 2710a), low dietary P level markedly increased accumulation of Pb in bone, blood, and kidney. Tissue Pb levels in mice fed a high P in diet were not different from mice fed the basal P diet. Dietary P and Pb interacted to affect body weight change and feed efficiency in mice. The relative contribution of different Pb species in diet and feces was also affected by dietary P level. Differences in Pb species between diet and feces indicated that transformation of Pb species can occur during gastrointestinal tract transit. These interactions between Pb and P that alter Pb speciation may be important determinants of the bioavailability of Pb ingested in soil.

An in vitro assessment of bioaccessibility of arsenicals in rice and the use of this estimate within a probabilistic exposure model.

In this study, an in vitro synthetic gastrointestinal extraction protocol was used to estimate bioaccessibility of different arsenicals present in 17 rice samples of various grain types that were collected across the United States. The across matrix average for total arsenic was 209 ng/g±153 (\[xmacr]±2σ). The bioaccessibility estimate produced an across matrix average of 61%±19 (\[xmacr]±2σ). The across matrix average concentrations of inorganic arsenic (iAs) and dimethylarsinic acid (DMA) were 81 ng/g±67.7 and 41 ng/g±58.1 (\[xmacr]±2σ), respectively. This distribution of iAs concentrations in rice was combined with the distribution of consumption patterns (from WWEIA) in a Stochastic Human Exposure and Dose Simulator model to estimate population-based exposures. The mean consumption rate for the population as a whole was 15.7 g per day resulting in a 0.98 µg iAs per day exposure. The mean consumption rate for children 1-2 years old was 7 g per day resulting in a 0.48 µg iAs per day exposure. Presystemic biotransformation of DMA in rice was examined using an in vitro assay containing the anaerobic microbiota of mouse cecum. This assay indicated that DMA extracted from the rice was converted to dimethylthioarsinic acid, although a second oxygen-sulfur exchange to produce DMDTA was not observed.

Effect of sodium arsenite dose administered in the drinking water on the urinary bladder epithelium of female arsenic (+3 oxidation state) methyltransferase knockout mice.

The enzyme arsenic (+3 oxidation state) methyltransferase (As3mt) catalyzes reactions converting inorganic arsenic to methylated metabolites, some of which are highly cytotoxic. In a previous study, female As3mt knockout (KO) mice treated with diet containing 100 or 150 ppm arsenic as arsenite showed systemic toxicity and significant effects on the urothelium. In the present study, we showed that the cytotoxic and proliferative effects of arsenite administration on the urothelium are dose dependent. Female wild-type C57BL/6 mice and As3mt KO mice were divided into five groups (n = 7) with free access to drinking water containing 0, 1, 10, 25, or 50 ppm arsenic as arsenite for 4 weeks. At sacrifice, urinary bladders of both As3mt KO and wild-type mice showed hyperplasia by light microscopy; however, the hyperplasia was more severe in the As3mt KO mice. Intracytoplasmic granules were detected in the urothelium of As3mt KO and wild-type mice at arsenic doses ≥ 10 ppm but were more numerous, more extensive, and larger in the KO mice. A no effect level for urothelial effects was identified at 1 ppm arsenic in the wild-type and As3mt KO mice. In As3mt KO mice, livers showed mild acute inflammation and kidneys showed hydronephrosis. The present study shows a dose-response for the effects of orally administered arsenite on the bladder urothelium of wild-type and As3mt KO mice, with greater effects in the KO strain but with a no effect level of 1 ppm for both.

Preabsorptive metabolism of sodium arsenate by anaerobic microbiota of mouse cecum forms a variety of methylated and thiolated arsenicals.

The conventional scheme for arsenic methylation accounts for methylated oxyarsenical production but not for thioarsenical formation. Here, we report that in vitro anaerobic microbiota of mouse cecum converts arsenate into oxy- and thio- arsenicals. Besides methylarsonic acid (MMA(V)), arsenate was transformed into six unique metabolites: mono-, di-, and trithio-arsenic acid, monomethyldithio- and monomethyltrithio-arsonic acid, and dimethyldithioarsonic acid. Thioarsenicals were found in soluble and particulate fractions of reaction mixtures, suggesting interactions with anaerobic microbiota. Metabolism of ingested arsenate to oxy- and thio-arsenicals before absorption across the gastrointestinal barrier could affect bioavailability, systemic distribution, and resulting toxicity.

Arsenic (+3 oxidation state) methyltransferase genotype affects steady-state distribution and clearance of arsenic in arsenate-treated mice.

Arsenic (+3 oxidation state) methyltransferase (As3mt) catalyzes formation of mono-, di-, and tri-methylated metabolites of inorganic arsenic. Distribution and retention of arsenic were compared in adult female As3mt knockout mice and wild-type C57BL/6 mice using a regimen in which mice received daily oral doses of 0.5mg of arsenic as arsenate per kilogram of body weight. Regardless of genotype, arsenic body burdens attained steady state after 10 daily doses. At steady state, arsenic body burdens in As3mt knockout mice were 16 to 20 times greater than in wild-type mice. During the post dosing clearance period, arsenic body burdens declined in As3mt knockout mice to ~35% and in wild-type mice to ~10% of steady-state levels. Urinary concentration of arsenic was significantly lower in As3mt knockout mice than in wild-type mice. At steady state, As3mt knockout mice had significantly higher fractions of the body burden of arsenic in liver, kidney, and urinary bladder than did wild-type mice. These organs and lung had significantly higher arsenic concentrations than did corresponding organs from wild-type mice. Inorganic arsenic was the predominant species in tissues of As3mt knockout mice; tissues from wild-type mice contained mixtures of inorganic arsenic and its methylated metabolites. Diminished capacity for arsenic methylation in As3mt knockout mice prolongs retention of inorganic arsenic in tissues and affects whole body clearance of arsenic. Altered retention and tissue tropism of arsenic in As3mt knockout mice could affect the toxic or carcinogenic effects associated with exposure to this metalloid or its methylated metabolites.

Severe systemic toxicity and urinary bladder cytotoxicity and regenerative hyperplasia induced by arsenite in arsenic (+3 oxidation state) methyltransferase knockout mice. A preliminary report.

Arsenic (+3 oxidation state) methyltransferase (As3mt) catalyzes reactions which convert inorganic arsenic to methylated metabolites. This study determined whether the As3mt null genotype in the mouse modifies cytotoxic and proliferative effects seen in urinary bladders of wild type mice after exposure to inorganic arsenic. Female wild type C57BL/6 mice and As3mt KO mice were divided into 3 groups each (n=8) with free access to a diet containing 0, 100 or 150 ppm of arsenic as arsenite (As(III)). During the first week of As(III) exposure, As3mt KO mice exhibited severe and lethal systemic toxicity. At termination, urinary bladders of both As3mt KO and wild type mice showed hyperplasia by light microscopy. As expected, arsenic-containing granules were found in the superficial urothelial layer of wild type mice. In As3mt KO mice these granules were present in all layers of the bladder epithelium and were more abundant and larger than in wild type mice. Scanning electron microscopy of the bladder urothelium of As3mt KO mice treated with 100 ppm As(III) showed extensive superficial necrosis and hyperplastic changes. In As3mt KO mice, livers showed severe acute inflammatory changes and spleen size and lymphoid areas were decreased compared with wild type mice. Thus, diminished arsenic methylation in As3mt KO mice exacerbates systemic toxicity and the effects of As(III) on the bladder epithelium, showing that altered kinetic and dynamic behavior of arsenic can affect its toxicity.

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 an arsenosugar by mouse anaerobic cecal microflora and cecal tissue as examined using IC-ICP-MS and LC-ESI-MS/MS.

This investigation examined chemical and microbiological transformations of an arsenosugar by mouse cecum. To mimic the low oxygen environment in the mammalian gastrointestinal tract, reaction mixtures were incubated under anaerobic conditions. An arsenosugar extracted from ribbon kelp, 3-[5'-deoxy-5-(dimethylarsinoyl)-beta-ribofuranosyloxy]-2-hydroxypropanesulfonic acid, As392, was added to reaction mixtures that contained either cecal microflora or cecal tissue homogenate. These reaction mixtures were incubated at 0 or 37 degrees C for up to 48 hours to monitor biotransformation of the arsenosugar. Analysis of the reaction mixtures by IC-ICP-MS and LC-ESI-MS/MS indicated that the arsenosugar was converted primarily (95%) to its sulfur analog in less than 1 h at 37 degrees C. Conversion of As392 to its sulfur analog was much slower at 0 degrees C (21% conversion after 48 h). In reaction mixtures with cecal tissue homogenate, conversion of As392 to its sulfur analog was slower (77% conversion after 48 h at 37 degrees C). A good mass balance was found in all reaction mixtures between the amount of arsenosugar added and the sum of all detected arsenic-containing products. LC-ESI-MS/MS spectra of the sulfur-containing arsenosugar formed in all reaction mixtures containing cecal microflora compared well with those of a synthetic standard. These results suggest that the anaerobic microflora of the gastrointestinal tract can rapidly convert ingested arsenosugars to sulfur analogs. This biotransformation may affect the subsequent absorption, metabolism, and disposition of arsenic present in arsenosugars.

A novel S-adenosyl-L-methionine:arsenic(III) methyltransferase from rat liver cytosol.

S-Adenosyl-l-methionine (AdoMet):arsenic(III) methyltransferase, purified from liver cytosol of adult male Fischer 344 rats, catalyzes transfer of a methyl group from AdoMet to trivalent arsenicals producing methylated and dimethylated arsenicals. The kinetics of production of methylated arsenicals in reaction mixtures containing enzyme, AdoMet, dithiothreitol, glutathione (GSH), and arsenite are consistent with a scheme in which monomethylated arsenical produced from arsenite is the substrate for a second methylation reaction that yields dimethylated arsenical. The mRNA for this protein predicts a 369-amino acid residue protein (molecular mass 41056) that contains common methyltransferase sequence motifs. Its sequence is similar to Cyt19, a putative methyltransferase, expressed in human and mouse tissues. Reverse transcription-polymerase chain reaction detects S-adenosyl-l-methionine:arsenic(III) methyltransferase mRNA in rat tissues and in HepG2 cells, a human cell line that methylates arsenite and methylarsonous acid. S-Adenosyl-l-methionine:arsenic(III) methyltransferase mRNA is not detected in UROtsa cells, an immortalized human urothelial cell line that does not methylate arsenite. Because methylation of arsenic is a critical feature of its metabolism, characterization of this enzyme will improve our understanding of this metalloid's metabolism and its actions as a toxin and a carcinogen.