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Preabsorptive Metabolism of Sodium Arsenate by Anaerobic Microbiota of Mouse Cecum Forms a Variety of Methylated and Thiolated Arsenicals Tatyana S. Pinyayev,† Michael J. Kohan,‡ Karen Herbin-Davis,‡ John T. Creed,*,† and David J. Thomas‡ †
Microbiological and Chemical Exposure Assessment Research Division, National Exposure Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268, United States ‡ Integrated System Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, United States
bS Supporting Information ABSTRACT: 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 (MMAV), 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.
H2S-producing organisms in the microbiota11 and the relatively high pH of the distal gastrointestinal tract favor thioarsenical production. Earlier work showed that anaerobic microbiota from mouse cecum or human feces converted iAsIII and iAsV into mono- and dimethylated oxyarsenicals12-14 and DMAV into thiolated metabolites (e.g., DMMTA) and trimethylated metabolites (trimethylarsine oxide, TMAO, and trimethylarsine sulfide, TMAS).15 Here, we have examined the concentration and time dependencies of linkages between the formation of oxy- and thio-arsenicals by the anaerobic microbiota of mouse cecum. Supporting Information summarizes procedures for the preparation of mouse cecal contents for in vitro assays, assay conditions, and sample processing. Procedures for the preparation and use of analytical standards and for the quantitiation of arsenicals are also summarized in Supporting Information. Figure 1 shows the time and concentration dependencies for the appearance of oxy- and thio-arsenicals in supernates from cecal reaction mixtures that initially contained 200, 1000, and 2000 ng iAsV 3 mL-1. Graphs are grouped to reflect the linkage between the standard pathway and thioarsenical production (Table 1). As iAsV concentration decreased with longer incubations, supernates contained increasing concentrations of monothio (AsVS1)-, dithio (AsVS2)-, and trithio (AsVS3)-iAsV, MMAV, monomethyldithioarsonic (MMDTAV), dimethyldithioarsonic
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hronic exposure to inorganic arsenic (iAs) has been linked to increased prevalences of various diseases, including cancers, cardiovascular disease, and diabetes.1 Because some adverse effects associated with exposure to either arsenite (iAsIII) or arsenate (iAsV) are likely mediated by the mono(MMA), di- (DMA), or tri- (TMA) methylated metabolites of iAs,2 elucidating metabolic pathways and the spectrum of metabolites are critical research needs. The traditionally proposed scheme for the production of methylated metabolites of iAs includes alternating steps for the reduction of As from pentavalency to trivalency and for oxidative methylation, as outlined in the top row of Table 1.3 However, the precise metabolic pathway and mechanism for formation of specific toxic metabolites are poorly understood, and there have been reports suggesting direct methylation of the AsIII-glutathione complex to yield MMAIII,4,5 where one of the glutathiones is substituted by a methyl group in the presence of arsenic methyltransferase. Thioarsenicals, structural analogues of oxyarsenicals in which sulfur replaces oxygen, are readily formed by exposure of oxyarsenicals to hydrogen sulfide (H2S).6 Thioarsenicals are detected in tissues and urine after exposure to either iAsV or dimethylarsinic acid (DMAV).7-9 Dimethylthioarsenate (DMMTA) binds to the cysteinyl residue of glutathione, suggesting that AsV-containing thioarsenicals could interact with cysteinyl residues in proteins.10 Preabsorptive metabolism modifies ingested arsenicals before absorption across the gastrointestinal barrier. The prevalence of This article not subject to U.S. Copyright. Published 2011 by the American Chemical Society
Received: January 26, 2011 Published: March 09, 2011 475
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Table 1. Standard Pathway for the Formation of Methylated Oxyarsenicals and Linkages between Oxy- and Thio-Arsenical Metabolism
Figure 2. (A) Time-dependent changes in concentrations of oxy- and thio- arsenicals in supernates from reaction mixtures containing mouse cecal microbiota incubated anaerobically up to 48 h after the addition of 1000 ng of arsenic (as arsenate) 3 mL-1; these time-dependent changes are characteristic of all studied spike levels. The total is the summed concentration of each quantified arsenical. (B) A mass balance of arsenicals in supernatant, combined DDI water extracts, and washed pellet for samples incubated for 48 h initially containing 200 ng of arsenic (as arsenate) 3 mL-1.
Figure 1. Time course and concentration dependence for the conversion of arsenate to oxy- and thio-arsenical metabolites in supernates from mouse cecal reaction mixtures [concentration (ppb) as a function of incubation time (h)]. Reaction mixtures containing mouse cecal microbiota were incubated anaerobically up to 48 h after the addition of 200 (gray circle), 1000 (0), or 2000 (2) ng of arsenic (as arsenate) 3 mL-1.
(DMDTAV), and monomethyltrithioarsonic (MMTTA) acids, and of an unidentified arsenical. (Low yield of the latter metabolite precluded identification.) Concentrations of metabolites in supernates increased as a function of initial iAsV concentration and length of incubation, suggesting that metabolic capacity was not exceeded over this range of iAsV concentrations. Notably, the most abundant methylated metabolites in supernates after 48 h were terminal species in which sulfur replaced oxygen, suggesting that in vitro anaerobiosis that mimics conditions in the distal gastrointestinal tract did not favor the oxidation of thioarsenicals to oxyarsenicals.
Time-dependent reduction in recovery of As in supernates (Figure 2A) suggested either that arsenicals were volatilized from reaction mixtures or that arsenic was present in pellets. Therefore, we attempted to recover arsenicals from pellets by extraction with distilled deionized water (DDI). An additional 27% of total As in reaction mixtures was recovered in combined DDI extracts, and an equal percentage of the total As was found in extracted pellets (Figure 2B). High cumulative recovery of total As in supernate, combined DDI extract, and extracted pellet 476
dx.doi.org/10.1021/tx200040w |Chem. Res. Toxicol. 2011, 24, 475–477
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indicate that As was not lost by volatilization but was associated with particulate components of the pellet. Formation of thioarsenicals can be integrated into the standard scheme for iAs methylation (Table 1). Here, arsenicals containing either AsIII or AsV are potential substrates for the conversion from oxy- to thio-arsenicals. In contrast, only AsIIIcontaining oxyarsenicals are substrates for enzymatically catalyzed conversion to methylated species. Linking these two metabolic pathways for As suggests that internal dosimetry for As following the ingestion of iAsV may be more complex than originally thought. Production of thioarsenicals by preabsorptive metabolism may affect both the distribution and toxicity of ingested iAs. For example, tissue distribution patterns for DMMTA and DMDTA differ from that for DMAV.16 AsVS3, iAsIII, and iAsV are about equally cytotoxic to Vibrio fischeri.17 DMMTA is more cytotoxic than iAsIII in human uroepithelial EJ-1 cells18 and more cytotoxic than DMAV to human epidermoid A431 cells.19 Thiolated arsenicals formed in the gastrointestinal tract may also be substrates for further thiolation by H2S produced in mammalian cells by trans-sulfuration pathways of cysteine metabolism.20 Integrating studies of the pre- and postabsorptive metabolism of arsenic to form methylated and thiolated metabolites will provide a better understanding of the role of metabolism in the toxicity of this metalloid.
(3) Challenger, F. (1951) Biological methylation. Adv. Enzymol. 12, 429–491. (4) Hayakawa, T., Kobayashi, Y., Cui, X., and Hirano, S. (2005) A new metabolic pathway of arsenite: arsenic-glutathione complexes are substrates for human arsenic methyltransferase Cyt19. Arch. Toxicol. 183–191. (5) Naranmandura, H., Suzuki, N., and Suzuki, K. T. (2006) Trivalent arsenicals are bound to proteins during reductive methylation. Chem. Res. Toxicol. 19, 1010–1018. (6) Fricke, M. W., Zeller, M., Sun, H., Lai, V. W.-M., Cullen, W. R., Shoemaker, J. A., Witkowski, M. R., and Creed, J. T. (2005) Chromatographic separation and identification of products from the reaction of dimethylarsinic acid with hydrogen sulfide. Chem. Res. Toxicol. 18, 1821–1829. (7) Raml, R., Rumpler, A., Goessler, W., Vahter, M., Li, L., Ochi, T., and Francesconi, K. A. (2007) Thio-dimethylarsinate is a common metabolite in urine samples from arsenic-exposed women in Bangladesh. Toxicol. Appl. Pharmacol. 222, 374–380. (8) Hansen, H. R., Raab, A., Jaspars, M., Milne, B. F., and Feldmann, J. (2004) Sulfur-containing arsenical mistaken for dimethylarsinous acid [DMA(III)] and identified as a natural metabolite in urine: Major implications for studies on arsenic metabolism and toxicity. Chem. Res. Toxicol. 17, 1086–1091. (9) Hansen, H. R., Pickford, R., Thomas-Oates, J., Jaspars, M., and Feldmann, J. (2004) 2-Dimethylarsinothioyl acetic acid identified in a biological sample: The first occurrence of a mammalian arsinothioyl metabolite. Angew. Chem., Int. Ed. 43, 337–340. (10) Raab, A., Wright, S., Jaspars, M., Meharg, A., and Feldmann, J. (2007) Pentavalent arsenic can bind to biomolecules. Angew. Chem., Int. Ed. 119, 2648–2651. (11) Deplancke, B., Hristova, K. R., Oakley, H. A., McCracken, V. J., Aminov, R., Mackie, R. I., and Gaskins, H. R. (2000) Molecular ecological analysis of the succession and diversity of sulfate-reducing bacteria in the mouse gastrointestinal tract. Appl. Environ. Microbiol. 66, 2166–2174. (12) Hall, L. L., George, S. E., Kohan, M. J., Styblo, M., and Thomas, D. J. (1997) In vitro methylation of inorganic arsenic in mouse intestinal cecum. Toxicol. Appl. Pharmacol. 147, 101–109. (13) Rowland, I. R., and Davies, M. J. (1981) In vitro metabolism of inorganic arsenic by the gastro-intestinal microflora of the rat. J. Appl. Toxicol. 1, 278–283. (14) Van de Wiele, T., Gallawa, C. M., Kubachka, K. M., Creed, J. T., Basta, N., Dayton, E. A., Whitacre, S., Du Laing, G., and Bradham, K. (2010) Arsenic metabolism by human gut microbiota upon in vitro digestion of contaminated soils. Environ. Health Perspect. 118, 1004–1009. (15) Kubachka, K. M., Kohan, M. C., Herbin-Davis, K., Creed, J. T., and Thomas, D. J. (2009) 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. Toxicol. Appl. Pharmacol. 239, 137–143. (16) Naranmandura, H., Ogra, Y., Iwata, K., Lee, J., Suzuki, K. T., Weinfeld, M., and Le, X. C. (2009) Evidence for toxicity differences between inorganic arsenite and thioarsenicals in human bladder cancer cells. Toxicol. Appl. Pharmacol. 238, 133–140. (17) Planer-Friedrich, B., Franke, D., Merkel, B., and Wallschlager, D. (2008) Acute toxicity of thioarsenates to Vibrio fischeri. Environ. Toxicol. Chem. 27, 2027–2035. (18) Naranmandura, H., Ibata, K., and Suzuki, K. T. (2007) Toxicity of dimethylmonothioarsinic acid toward human epidermoid carcinoma A431 cells. Chem. Res. Toxicol. 20, 1120–1125. (19) Suzuki, K. T., Iwata, K., Naranmandura, H., and Suzuki, N. (2007) Metabolic differences between two dimethylthioarsenicals in rats. Toxicol. Appl. Pharmacol. 218, 166–173. (20) Kamoun, P. (2004) Endogenous production of hydrogen sulfide in mammals. Amino Acids 26, 243–254.
’ ASSOCIATED CONTENT
bS
Supporting Information. Further details of experimental conditions, chemical syntheses and characterizations, and analytical methods. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Tel: þ1-513-569-7617. Fax: þ1-513-569-7757. E-mail: creed.
[email protected]. Funding Sources
This research was funded and managed by The Office of Research and Development, United States Environmental Protection Agency, and this manuscript was subjected to US EPA’s administrative review and approved for publication.
’ DISCLOSURE Mention of trade names or commercial products does not constitute endorsement or recommendation for use. ’ ACKNOWLEDGMENT We thank Drs. Kevin Kubachka and Michael Fricke for valuable advice. Professor William R. Cullen, Department of Chemistry, University of British Columbia, generously provided trimethylarsine oxide.
’ REFERENCES (1) Schuhmacher-Wolz, U., Dieter, H., Klein, D., and Schneider, K. (2009) Oral exposure to inorganic arsenic: evaluation of its carcinogenic and non-carcinogenic effects. Crit. Rev. Toxicol. 39, 271–298. (2) Drobna, Z., Styblo, M., and Thomas, D. J. (2009) An overview of arsenic metabolism and toxicity. Curr. Protoc. Toxicol. 42:4.31, 1–6. 477
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