Research Arsenic Metabolism by Human Gut Microbiota upon in Vitro Digestion of Contaminated Soils Tom Van de Wiele,1 Christina M. Gallawa,2 Kevin M. Kubachka,2 John T. Creed,2 Nicholas Basta,3 Elizabeth A. Dayton,3 Shane Whitacre,3 Gijs Du Laing,4 and Karen Bradham 5 1Laboratory Microbial Ecology and Technology, Ghent University, Ghent, Belgium; 2Microbiological and Chemical Exposure Assessment Research Division, National Exposure Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Cincinnati, Ohio, USA; 3School of Environment and Natural Resources, The Ohio State University, Columbus, Ohio, USA; 4Laboratory Analytical and Applied Ecochemistry, Ghent University, Ghent, Belgium; 5National Exposure Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, USA 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. o Bjectivs : 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. r esults : 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 monomethyl­ arsonous 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. c onclusi : 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. k ey words : arsenic species, bacteria, colon, gastrointestinal, metalloid, microflora, presystemic metabolism, Simulator of the Human Intestinal Microbial Ecosystem, speciation. Environ Health Perspect 118:1004–1009 (2010). doi:10.1289/ehp.0901794 [Online 26 March 2010] Arsenic (As), a ubiquitous environmental con­ taminant, presents significant human health risks: Chronic exposure is associated with the development of cancer in the bladder, liver, kidney, and lungs (Chen et al. 1992). Regions with a high geogenic As background show an increased risk for elevated exposure by con­ sumption of drinking water and diet. An addi­ tional exposure scenario in urban areas near smelting and mining activities is the ingestion of contaminated soil and dust by children, who display typical hand­ to­ mouth behavior. Although inorganic As (iAs) may be the pre­ dominant form in contaminated soils, As spe­ ciation changes during gastrointestinal transit are not well characterized. The gut represents a highly reducing environment and harbors a complex microbial community, which may contribute to the presystemic biotransfor­ mation of ingested As (systemic metabolism being defined as all metabolic reactions carried out by human cells). Presystemic As specia­ tion analysis must therefore be considered an essential part of the risk evaluation process, especially with respect to toxicity, which is speciation dependent. In short, methylated 1004 trivalent species—monomethylarsonous acid (MMAIII), dimethylarsinous acid (DMAIII), and arsenous acid (iAsIII)—are two orders of magnitude more cytotoxic than is As acid (iAsV) (Naranmandura et al. 2007a). The methylated pentavalent species—monomethyl­ arsonic acid (MMAV) and dimethylarsinic acid (DMAV)—present a 10f­old lower toxic­ ity than iAsV, whereas trimethylarsine oxide (TMAO) is essentially nontoxic (Hirano et al. 2004). In the human body, iAs is sequentially methylated and predominately excreted as DMAV in urine. This methylation process was originally considered a detoxification process, but the formation of reactive intermediates (MMAIII and DMAIII) has forced researchers to reconsider methylation as an activation pro­ cess (Styblo et al. 2002). In addition, a recent study on human urine analysis after iAs expo­ sure revealed new sulfurc­ontaining methylated As metabolites, monomethylmonothioarsonic acid (MMMTAV) and dimethylmonothio­ arsinic acid (DMMTAV) (Naranmandura et al. 2007b; Raml et al. 2007), for which the mechanism of formation and toxicological profile are not yet fully characterized. Given the toxicological importance of As speciation changes, it is clear that a complete risk charac­ terization after As exposure must include the possibility of presystemic metabolism by the microbe­ rich environment of the gastro­ intestinal tract. The colon harbors a vast (1014 bacterial cells) and incredibly diverse (> 1,000 species) microbial community, which has the abil­ ity to metabolize xenobiotics far more exten­ sively than any other part of the body (Sousa et al. 2008). Thus far, the presystemic bio­ transformation of As was primarily studied with gut microbiota from animal models. Rowland and Davies (1981) reported the reduction of iAsV to iAsIII by rat cecal bac­ teria as well as limited formation of MMA and DMA. In another study with rats orally exposed to DMAV Chen et al. (1996) detected demethylated (iAsV, MMAV) and methylated (TMAO) urinary metabolites. Finally, the thiolation of methylated As oxides (DMAV, TMAO) in the cecal contents of a mouse (Kubachka et al. 2009a, 2009b) and the observed thiolation through in vivo experi­ ments (Kuroda et al. 2004; Naranmandura et al. 2007b) have been reported. Presystemic As metabolism in the human body has been less investigated (Hirner et al. 2004). Nevertheless, Michalke et al. (2008) reported that human gut microbes actively volatilize bismuth and other metal­ loids, including As, through methylation and Address correspondence to T. Van de Wiele, Laboratory Microbial Ecology and Technology, Ghent University, 653 Coupure Links, B­ 9000 Gent, Belgium. Telephone: 32­ 9­ 264­ 59­ 76. Fax: 32­9­264­62­48. E­mail: tom.vandewiele@ugent.be Supplemental Material is available online (doi:10. 1289/ehp.0901794 via http://dx.doi.org/). We thank R. Dick, L. Dick, and the Laboratory for Soil Microbiology at Ohio State University for hosting the experimental setup; R. Diaz (University of Duisburg–Essen) for critically reading the manuscript. This study was supported by the special research fund of Ghent University through project 01J05308; the FWO­ Vlaanderen (Flemish Fund for Scientific Research) provided travel grants. T.V.W. is a post­ doctoral research fellow of the FWO­Vlaanderen. The authors declare they have no actual or potential competing financial interests. Received 8 December 2009; accepted 26 March 2010. ovlume 118 | number 7 | July 2010 • Environmental Health Perspectives