Van de Wiele et al. (23 µg/L) as detected methylarsenicals. The methylation percentage for colon digests of soil 2 (22.8%) and soil 3 (21.2%) was higher, with soil 2 displaying MMAV (111 µg/L), MMAIII (9 µg/L), and MMMTAV (158 µg/L) and soil 3 displaying only MMAV (28 µg/L) and MMMTAV (68 µg/L). Finally, no meth­ ylated As species were detected in the colon digests of slag soil 4. Summarizing the in vitro As speciation changes by human gut microorganisms, we calculated the specific production rates of methylated arsenicals by taking into account the initial microbial biomass and As con­ centrations. We obtained a methylation rate of 10 µg methylarsenicals/g biomass/hr for the colon digest of iAsV (Table 3). Although no methylarsenicals were detected in colon digests of the slag soil 4, the presence of the other soil matrices did not necessarily lower the above­mentioned methylation rate. We obtained methylation rates of 4, 29, and 10 µg/g/hr for colon digests of soils 1, 2, and 3, respectively (Table 3). Discussion The present study demonstrates that human colon microorganisms have the potency to actively metabolize As into methylated arsenicals and thioarsenicals, which indicates that presystemic As metabolism may not be neglected when assessing risks from oral As exposure. We observed this upon colon incubation of both iAs and As­ contaminated soils. These findings parallel those from studies with animal gut microbiota (Hall et al. 1997; Rowland and Davies 1981) and suggest the existence of a presystemic As metabolism in the human body. The most important result was the detection of signif­ icant levels of MMMTAV in colon digests of both iAsV (25% of bioaccessible As) and of As­ contaminated soils (up to 20% of bio­ accessible As). To our knowledge, this is the first time that MMMTAV production from iAsV by human colon microbiota has been described. MMMTAV production from this source resembles the methylation and thiola­ tion of DMAV into trimethylarsine sulfide by mouse cecal microbiota (Kubachka et al. 2009a) and the production of methylated thioarsenicals from DMAV by rat intestinal microbiota (Kuroda et al. 2004; Yoshida et al. 2001). Yet, mammalian cells also have the ability to form methylated thioarsenicals. Kuroda et al. (2004) described rapid detection (5 min) of DMMTAV and dimethyldithioars­ inic acid (DMDTAV) after injection of of rats with DMAIII, and Naranmandura and Suzuki (2008) reported that DMAIII was converted to DMDTAV by human red blood cells. The finding of presystemic MMMTAV formation by human gut microorganisms raises questions about its toxicological 1008 importance. Although the absorption kinetics of MMMTAV and other thiolated arsenicals across the epithelium are unknown, there is evidence that some methylated thioarsenicals elicit a higher toxicity than iAsV because of their more efficient absorption by mam­ malian cells (Naranmandura et al. 2007a). Preliminary cytotoxicity (Naranmandura et al. 2007a; Yoshida et al. 2003) and genotoxicity (Kuroda et al. 2004) data for DMMTAV show levels of toxicity similar to those of trivalent As species. Our observa­ tions in the present study emphasize the need to investigate the behavior of MMMTAV in the gut lumen and the absorption rate across the intestinal epithelium. In addition, the mechanism behind the microbial production pathway needs to be elucidated. Interestingly, MMMTAV levels in the colon digests cor­ related with those of MMAV (R2 = 0.76), whereas the correlation with levels of MMAIII was much lower (R2 = 0.42). This seems to indicate that MMMTAV in the colon digests arises from the thiolation of MMAV, which would correspond with earlier observations describing the interconversion between oxide and sulfide forms of MMAV, DMAV, and TMAO (Conklin et al. 2008). The sulfide source may originate from microbial sulfate reduction to hydrogen sulfide, which is a common process in the colon environment (Deplancke et al. 2000), and can trigger the formation of thioarsenosugars upon the incubation of arsenosugars with mouse cecal contents (Conklin et al. 2006). The role of sulfate­ reducing microorganisms in the pre­ systemic production pathway of MMMTAV must therefore be studied further. The significant formation of MMAV and MMAIII after incubation of iAsV with colon microorganisms was not unexpected. Arsenic methylation by rodent gut microbes (Hall et al. 1997; Rowland and Davies 1981) and human gut microbes (Diaz­ Bone and Van de Wiele 2009; Meyer et al. 2008) has been described previously. Taking into account the initial biomass concentration, we observed specific methylation rates of 10 µg methyl­ arsenicals/hr/g biomass. This roughly corre­ sponds to 130 pmol/hr/mg biomass, which is > 16 pmol/hr/mg obtained with rat cecal microbiota (Hall et al. 1997). Interestingly, the presence of a soil matrix did not neces­ sarily result in lower As methylation rates, yet soil­dependent parameters may have affected the methylation rate. First, comparison of the mineralogy from soils 1, 2, and 3 with that of slag (soil 4) showed an important difference in reactive iron oxide content (Table 1), which is highly efficient in sorbing As (Beak et al. 2006). The reactive iron oxide content in slag soil 4 was particularly high (18,759 µg/kg; Table 1), presumably leading to much lower As availability to colon microorganisms (0.3% bioaccessibility) and thus also limiting methylation. This observation may confirm earlier observations of slag soil mineralogy sig­ nificantly decreasing As bioavailability (Davis et al. 1996). A second element in the soil­ dependent As methylation may be the differ­ ence in toxic elements. Compared with the first three soils, slag (soil 4) contained high amounts of cadmium, chromium, copper, molybdenum, lead, and zinc (Table 1), which may be toxic to intestinal microorganisms. Our finding of a 70% lower fermentation activity in soil 4 colon digests versus colon digests of the other soils (data not shown) may support the assumption of slag­soil–induced toxicity. The actual relationship between gut microbial As metabolism and soil characteris­ tics therefore needs further study. A final aspect of our study concerns the metabolic potency of fecal microbes toward high levels of iAsV (90 mg iAsV/L) and the influence of cofactors. Nonamended colon digests of iAsV resulted in the efficient reduc­ tion to iAsIII and the production of mTA. Similar to the finding of MMMTAV, the for­ mation of mTA may result from an oxygen­ for­sulfur exchange in iAs V because of the availability of sulfide, originating from the abovem­entioned microbial sulfate reduction. The absence of mTA in glutathionea­mended samples may be explained by the complete reduction of iAsV into iAsIII by glutathione as reducing agent. We also evaluated the effect of methyl group donors. In contrast to methi­ onine, methylcobalamin may be an effective methyl group donor, resulting in the efficient methylation (19%) of iAsV into MMAV and MMAIII (Table 2). The methylation efficiency increased to 25% upon cosupplementation of methylcobalamin and glutathione. We attributed this to the increased reduction of MMAV into MMAIII by glutathione as reduc­ ing agent. In contrast to the colon digests with low levels of iAsV (225 µg/L), no MMMTAV was detected in the fecal digests. A probable explanation is the difference in experimental setup, the difference in microbial community composition and activity, or a difference in sulfide availability. These observations confirm a previous report that addition of cofactors may increase As methylation by enteric micro­ organisms, yet it is not a prerequisite for the methylation of low levels of As (micrograms per liter range) (Hall et al. 1997). The present study provides evidence for the existence of significant presystemic As metabolism by human gut microorgan­ isms, but the relevance for the total risk of oral As exposure is not yet clear. So far, the in vitro approach for assessing the risks from oral contaminant exposure mainly involved the use of models that focus on gastric and intestinal processes. Methylation of As by intestinal microorganisms was thought ovlume 118 | number 7 | July 2010 • Environmental Health Perspectives