hydrogenation. Moreover, Meyer et al. (2008) postulated that gut methanogens play a crucial role in metalloid volatilization, thereby exert­ ing toxic effects to the human body—not only by direct interaction with the host but also by disturbing the endogenous gut microbiota composition and metabolism. Finally, a thor­ ough in vitro exploration with the Simulator of the Human Intestinal Microbial Ecosystem (SHIME), a dynamic human gastrointestinal simulator, revealed a high microbial meta­ bolic potency toward metal(loid)s (Diaz­ Bone and Van de Wiele 2009). This was demon­ strated by the finding of significant volatiliza­ tion of As, selenium, bismuth, tellurium (Te), and antimony; the formation of highly toxic AsH3 (arsine) and (CH3)2Te (dimethyl tellu­ ride); and the discovery of two new As–sulfur metabolites. These data indicate the need for more studies with human gut microorganisms, which can confirm the presystemic metabo­ lism as observed with animal gut microbiota. Therefore, in the present study we investigated the metabolic potency of human gut micro­ organisms toward iAs and As from contami­ nated urban soils, assessing the importance of presystemic As biotransformation upon an oral exposure scenario and the actual specia­ tion of As that enters the bloodstream upon gastrointestinal digestion. Materials and Methods Chemicals and media. We used degassed, ultrapure 18 mΩ water (DDI; Millipore, Bedford, MA, USA) to prepare the chromato­ graphic mobile phase and the standard stock solutions. American Chemical Society–grade ammonium nitrate and ammonium dihydro­ gen phosphate (Fisher Scientific, Pittsburgh, PA, USA) and technical­ grade EDTA, tet­ rasodium salt dehydrate (Fisher Scientific, Fair Lawn, NJ, USA) were used in the chro­ matographic mobile phase. We obtained stock solutions of iAs (AsIII and AsV) from Spex Industries (Metuchen, NJ, USA) and certi­ fied stock solutions of MMAV and DMAV from Chem Service (West Chester, PA). W.R. Cullen (Department of Chemistry, University of British Columbia, Vancouver, BC, Canada) provided tetramethyl­cyclo­tetraarsaoxane [cyclo­(CH 3AsO)4] crystals that were syn­ thesized and characterized as described else­ where (Cullen et al. 1989); these crystals were stored at –21°C and were hydrolyzed by degassed, deionized water at the time of analy­ sis to obtain a stock solution of a MMAIII and MMAV mixture (Cullen et al. 1989). We pur­ chased sodium arsenate (Na2HAsO4·7H2O), methionine, methylcobalamine, and gluta­ thione from Sigma­Aldrich (St. Louis, MO, USA). Arsenate stock solutions were prepared in deionized water at 4,500 mg As/L and 45 mg As/L. Soils. The U.S. Environmental Protection Agency kindly provided four Asc­ontaminated soils that originated from urban areas around former smelting sites. We sieved all soils at 250 µm before in vitro gastrointestinal incu­ bation; this sieving reflects the size of particles that most likely sticks to the hands of exposed humans (Kelly et al. 2002). Soil specifications are reported in Table 1. Production and characterization of colon microbiota for SHIME. The in vitro colon microbial community used in this study was cultured and maintained in a modified SHIME, which consisted of four compart­ ments simulating the stomach, small intes­ tine, and both proximal and distal colon. A detailed description of the SHIME, the carbohydrateb­ased medium , and the in vitro colon microbiota has been described previ­ ously (Van de Wiele et al. 2004). Briefly, fecal microbiota previously obtained from a 29­ year­ old male volunteer (who had no his­ tory of antibiotic treatment in the 6 months before the study) were inoculated in the dif­ ferent colon compartments. The SHIME reactor was fed carbohydrate­based medium three times per day to provide digested nutri­ tion for the colon microbiota. After 3 weeks of adaptation, a stable microbial community was obtained in the respective colon com­ partments. We found microbial fermentation activity of the distal colon (short­ chain fatty acid production and ammonium production) and community composition to be consistent with that of previous SHIME runs and the in vivo situation (Molly et al. 1994; Van de Wiele et al. 2004) [see Supplemental Material, Table 1 (doi:10.1289/ehp.0901794)]. Noncontinuous incubation studies. Metabolic potency of fecal microbial inoculum. The first experiment constituted a screening phase to test whether the fecal microbial community from human ori­ gin actively metabolized As. The microbial Characteristic Organic carbon (%) pHa Electric conductivity (dS/m) Fe (mg/kg) Mn (mg/kg) Reactive Feb Percent bioaccessibility with IVGc Toxic trace element content (mg/kg) As Cadmium Chromium Copper Molybdenum Nickel Lead Selenium Zinc Soil 1 5.7 6.1 0.3 14,800 429 3,489 58.5 990 9.7 32.0 51.9 2.1 12.5 885 330 562 Arsenic metabolism by human gut microbiota community was isolated from a fecal sample as previously described by Molly et al. (1994). Thirty milliliters of microbial fecal suspension was sampled, placed in 60­ mL serum bot­ tles, and incubated with NaH2AsO4·7H2O (iAsV; 90 mg iAsV/L), similar to the method of Herbel et al. (2002). Serum bottles were capped with butyl rubber stoppers that are impervious to O2 and subsequently made anaerobic by flushing with N2 gas for 30 min. Samples were then incubated at 37°C on a rotary shaker (150 rpm) for 48 hr. We compared the effect of specific methyl group donors toward microbial As methy­ lation by comparing methionine­amended (5 mmol/L) and methylcobalamin­amended (5 mmol/L) samples with control samples (incubation of the sample in the presence of heat­sterilized fecal microbiota). The effect of glutathione as a reducing agent was eval­ uated by comparing glutathione­ amended samples (10 mmol/L) with control samples. Duplicate incubations were performed on two different days to evaluate the reproduc­ ibility. A scheme of the experimental setup is presented in Supplemental Material, Figure 1 (doi:10.1289/ehp.0901794). Metabolic potency of colon microbiota toward As from contaminated soils. The objec­ tive of the second experiment was to screen for microbial speciation changes of iAsV at more relevant concentrations (i.e., 50–500 µg/L) by mimicking conditions of oral exposure to environmental samples. In addition, four As­contaminated soil samples (one slag soil and three from urban sites) were subjected to a gastrointestinal digestion procedure. To better mimic in vivo conditions, all gastrointestinal stages—gastric, small intestine, and colon— were simulated. We combined the in vitro gastrointestinal method (IVG) from Ohio State University with the SHIME to subsequently simulate stomach and small intestine (IVG) and colon (SHIME) conditions, respectively. The Soil 2 4.0 6.3 0.5 15,250 525 3,592 62.4 829 5.5 31.4 60.1 2.4 10.9 602 430 803 Soil 3 3.1 5.0 0.9 12,100 207 1,634 47.7 379 1.5 28.7 22.2 1.5 9.3 172 127 151 Soil 4 (slag) 2.2 7.2 0.5 202,000 1,640 18,759 1.0 837 28.9 187 1,520 73.9 10.4 8,702 294 12,500 Table 1. Characteristics and elemental composition of the four As-contaminated soils. aSoil pH was determined in a 1:10 (wt/vol) aqueous soil slurry. bIron dissolved by acid ammonium oxalate extraction. cData from Whitacre (2009). Environmental Health Perspectives • volume 118 | number 7 | July 2010 1005