Glutathione Modulates Recombinant Rat Arsenic (+3 Oxidation

Humans and other species enzymatically convert inorganic arsenic (iAs) into ..... 1 mM AdoMet, 10 μM iAsIII, and 1 mM TCEP that was incubated at 37 Â...
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Chem. Res. Toxicol. 2004, 17, 1621-1629

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Glutathione Modulates Recombinant Rat Arsenic (+3 Oxidation State) Methyltransferase-Catalyzed Formation of Trimethylarsine Oxide and Trimethylarsine Stephen B. Waters,† Vicenta Devesa,‡ Michael W. Fricke,§,| John T. Creed,§ Miroslav Sty´blo,‡,⊥,# and David J. Thomas*,O Curriculum in Toxicology, Center for Environmental Medicine, Asthma, and Lung Biology, Department of Pediatrics, School of Medicine, and Department of Nutrition, School of Public Health, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, Microbiological and Chemical Exposure Assessment Research Division, National Exposure Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, Cincinnati, Ohio 45628, and Pharmacokinetics Branch, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711 Received August 5, 2004

Humans and other species enzymatically convert inorganic arsenic (iAs) into methylated metabolites. Although the major metabolites are mono- and dimethylated arsenicals, trimethylated arsenicals have been detected in urine following exposure to iAs. The AS3MT gene encodes an arsenic (+3 oxidation state) methyltransferase, which catalyzes both the oxidative methylation of trivalent arsenicals and the reduction of pentavalent arsenicals. In reaction mixtures containing recombinant rat AS3MT (rrAS3MT) and radiolabeled arsenite, mono- and dimethylated arsenicals and a third radiolabeled product can be resolved by thin-layer chromatography. Hydride generation atomic absorption spectrometry and electrospray ionization mass spectrometry identified the third reaction product as trimethylarsine oxide. The addition of glutathione to reaction mixtures containing radiolabeled arsenite and rrAS3MT increased the yield of methylated and dimethylated arsenicals but suppressed the formation of trimethylarsine oxide. Although a dimethylarsenic-glutathione complex was rapidly converted to trimethylarsine oxide, the addition of a molar excess of glutathione to dimethylarsenic suppressed the production of trimethylarsine oxide. The nonquantitative recovery of radioarsenic from reaction mixtures suggested that AS3MT catalyzed the formation of a volatile arsenical. This volatile species was identified as trimethylarsine. Thus, AS3MT catalyzes the formation of all products in a reaction sequence leading from an inorganic to a volatile methylated arsenical. The regulation of this pathway by intracellular glutathione may be an important determinant of the pattern and extent of formation of arsenicals.

Introduction Cullen and associates (1) proposed a scheme for the biomethylation of arsenic in which oxidative methylation of trivalent arsenic (AsIII) alternates with the reduction of pentavalent arsenic (AsV) to trivalency. This scheme * To whom correspondence should be addressed. Tel: 919-541-4974. Fax: 919-541-1937. E-mail: [email protected]. † Curriculum in Toxicology, University of North Carolina at Chapel Hill. ‡ Center for Environmental Medicine, Asthma, and Lung Biology, University of North Carolina at Chapel Hill. § Microbiological and Chemical Exposure Assessment Research Division, United States Environmental Protection Agency. | Fellow of the Oak Ridge Institute for Science and Education. ⊥ Department of Pediatrics, School of Medicine, University of North Carolina at Chapel Hill. # Department of Nutrition, School of Public Health, University of North Carolina at Chapel Hill. O Pharmacokinetics Branch, Experimental Toxicology Division, United States Environmental Protection Agency.

AsVO43- + 2e f AsIIIO33- + CH3+ f CH3AsVO32- + 2e f CH3AsIIIO22- + CH3+ f (CH3)2AsVO2- + 2e f (CH3)2AsIIIO- + CH3+ f (CH3)3AsVO links exposure to inorganic As (iAs)1 to the production and excretion of mono-, di-, and trimethylated arsenicals. Reactions for the oxidative methylation of arsenicals and the reduction of pentavalent arsenicals are enzymatically 1 Abbreviations: iAs, inorganic arsenic; AS3MT, arsenic (+3 oxidation state) methyltransferase; rrAS3MT, recombinant rat arsenic (+3 oxidation state) methyltransferase; TCEP, tris(2-carboxylethyl)phosphine; Trx, thioredoxin; TR, thioredoxin reductase; GR, glutathione reductase; GST, glutathione-S-transferase; MAs, methylated arsenicals, DMAs, dimethylated arsenicals; TMAs, trimethylarsine; DMAsIIIGS, a stoichiometric complex of DMAsIII with GSH; ICP-MS, inductively coupled plasma mass spectrometry; ESI-MS, electrospray ionization mass spectrometry.

10.1021/tx0497853 CCC: $27.50 © 2004 American Chemical Society Published on Web 11/12/2004

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catalyzed. As methyltransferases have been purified and characterized from rabbit and rat liver (2, 3). An arsenic methyltransferase from rat liver, arsenic (+3 oxidation state) methyltransferase (AS3MT),2 has been sequenced, and its gene has been cloned and expressed (4). The rat AS3MT gene is orthologous with mouse and human AS3MT genes, and the protein product of each gene is an As methyltransferase (5). Rat AS3MT appears to function as an AsV reductase, using exogenous reductants, dithiothreitol or tris(2-carboxylethyl)phosphine (TCEP), or endogenous reductants, thioredoxin (Trx), glutaredoxin, and lipoic acid, coupled with thioredoxin reductase (TR) or with glutathione reductase (GR) and glutathione (GSH) and NADPH (6). In addition, AsV can be reduced in reactions catalyzed by AsV reductases (10). Purine nucleoside phosphorylase (7, 8) and glutathioneS-transferase (GST) ω (9) are candidate mammalian AsV reductases. High intracellular concentrations of GSH may also provide a source of reducing equivalents needed to reduce pentavalent arsenicals (10). Although biomethylation of iAs has been commonly regarded as a mechanism for its detoxification, there is abundant evidence that methylated arsenicals (MAs), especially those containing AsIII, exceed iAsIII in potency as cytotoxins, as genotoxins, or as inhibitors of enzyme activity (11). Hence, biomethylation of iAs is an activation process that yields more reactive and toxic intermediates and products. The major products of the biomethylation of iAs are MAs and dimethylated arsenicals (DMAs). However, trimethylated arsenicals have also been identified among the products of these methylation reactions. Trimethylated species have been detected in urine and tissues of humans and other species following treatment with a variety of arsenicals (12-17). Respiratory clearance of trimethylarsine (TMAs) has been demonstrated in hamsters or mice treated with trimethylarsine oxide (TMAO) or TMAs (18-20). Historically, the occurrence of a garliclike odor in expired breath of individuals poisoned with iAs has been attributed to the expiration of TMAs, although it may be due to the expiration of a volatile species of another metalloid (e.g., tellurium) (21). Because the acute toxicities of trimethylated pentavalent arsenicals are relatively low (18-20), little attention has been given to the trimethylation of iAs or to the consequences of exposure to trimethylated arsenicals. However, recent studies have identified adverse effects associated with TMAO exposure. Chronic administration of TMAO to rats increases the yield of preneoplastic GSTplacental form positive foci in the liver (22), and TMAO is a complete liver carcinogen in the rat (23). Furthermore, in in vitro assays, cellular reductants (NADH, NADPH) can reduce in TMAO to TMAs, which is a DNAdamaging species (24). Because trimethylated arsenicals have been detected as metabolites after exposure to a variety of arsenicals, this work investigated the capacity of recombinant rat AS3MT (rrAS3MT) to catalyze the formation of trimethylated arsenicals. The enzyme was found to catalyze 2 We have previously referred to this arsenic methyltransferase and its gene as cyt19. This designation was based on its original identification in GenBank as a methyltransferase of unknown function (GenBank/EBI Data Bank accession number AF393243). In the present work, the recommendations of Human Gene Nomenclature Database (http://www.gene.ucl.ac.uk/cgi-bin/nomenclature/searchgenes.pl) are followed and the protein and its gene are referred to as arsenic (+3 oxidation state) methyltransferase (AS3MT).

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the formation of trimethylated arsenicals in reaction mixtures that contained either iAsIII or DMAsIII as a substrate. On the basis of earlier work (6), it was postulated that this reaction might be modulated by the addition of GSH to reaction mixtures. In fact, GSH sharply reduced the formation of TMAO and increased the production of MAs and DMAs in reaction mixtures that contained rrAS3MT and either iAsIII or DMAsIII. In the absence of GSH, AS3MT also catalyzed the formation of TMAs. Notably, only TMAs was released into the gas phase above aqueous reaction mixtures; no other arsine was detected in the gas phase. The relative concentrations of GSH and glutathione disulfide (GSSG), which reflect the cellular oxidation state, may regulate AS3MT’s activity, affecting the rates of formation of MAs, DMAs, and TMAO. Changing the pattern and amounts of metabolites formed in AS3MT-catalyzed reactions by changing the cellular GSH concentration may be a determinant of the effects of chronic exposure to iAs.

Experimental Procedures Caution: iAs has been classified as a human carcinogen (25) and should be handled accordingly. Arsenicals. [73As]arsenic acid (iAsV) (estimated specific activity of 13.3 Ci/mg As) was obtained from the Isotope Program of the Oak Ridge National Laboratory (Oak Ridge, TN). [73As]arsenous acid (iAsIII) was produced from radiolabeled arsenic acid by reduction with metabisulfite-thiosulfate reagent (26). Iododimethylarsine (DMAsIII) and a complex of DMAsIII with GSH (DMAsIIIGS) were synthesized as previously described (27). rrAS3MT-Catalyzed Methylation of Arsenicals. The cloning of rat AS3MT and the expression and purification of the recombinant protein have been described (4). The rrAS3MTcatalyzed conversion of iAsIII to MAs was monitored by measuring the conversion of [73As]-labeled iAsIII to methylated products or by determination of the conversion of stable arsenicals to methylated products. Reaction mixtures usually contained rrAS3MT, 1 mM AdoMet, a reductant, and an arsenical in 100 mM tris-100 mM phosphate, pH 7.4, buffer. The reductant was 1 mM TCEP or a Trx-regenerating system consisting of 10 µM Escherichia coli Trx, 3 µM rat liver TR, and 300 µM NADPH (6). The effects of addition of up to 5 mM GSH or 5 mM GSSG on the rate of formation of methylated metabolites were tested in this assay system. For most studies, reaction mixtures were incubated in capped 1.5 mL microtubes and were processed for analysis immediately after the period of incubation. Because preliminary analyses found that the recovery of arsenic from reaction mixtures was not quantitative, it was postulated that volatile arsines were formed in and lost from the aqueous phases of reaction mixtures during the course of AS3MT-catalyzed reactions. To assess the loss of volatile metabolites, recoveries of radioarsenic were determined in reaction mixtures from which each component (rrAS3MT, TCEP, AdoMet, or GSH) was individually omitted. After reaction mixtures in capped microtubes were incubated at 37 °C for up to 120 min, they were uncapped at room temperature for 2 min. After they were vented, aliquots of these reaction mixtures were taken for TLC analysis of radioarsenicals. These reaction mixtures were then recapped and quickly frozen at -20 °C. Frozen samples were radiosassayed with a Minaxi 5000 γ counter (Packard, Downers Grove, IL), and their As contents (pmols) were calculated from the specific activity of radiolabeled arsenic used as the substrate. Percent recoveries were calculated as the ratio of counts per minute of [73As]arsenous acid (iAsIII) present in reaction mixtures after incubation and venting to the counts per minute of [73As]arsenous acid (iAsIII) added to reaction mixtures before incubation and venting. In other studies, the effects of the amount of enzyme and substrate concentration on the loss of arsenic by volatilization

Glutathione and Arsenic Methyltransferase were quantified in reaction mixtures containing 5 or 50 µg of rrAS3MT and 1 or 10 µM [73As]-labeled iAsIII in the presence of 1 mM AdoMet and 1 mM TCEP that were incubated at 37 °C for 120 min in capped microtubes. At the end of the incubation period, microtubes were uncapped at room temperature for 2 min. Aliquots of these reactions mixtures were then taken for TLC analysis of radioarsenicals. The remainders of these reaction mixtures were recapped and snap frozen at -20 °C and radiosassayed with a Minaxi 5000 γ counter. The picomoles of As remaining in the reaction mixtures after incubation and venting were calculated from the specific activity of 73As-labeled substrate. To identify volatile arsenicals formed during rrAS3MTcatalyzed methylation of iAsIII, reaction mixtures containing 100 µg of rrAS3MT, 10 µM iAsIII, 1 mM TCEP, and 1 mM AdoMet in 100 mM tris-100 mM NaPO4, pH 7.4, buffer (0.2 mL final volume) were incubated in sealed 3 mL conical reaction vials (Ace Glass, Vineland, NJ) for 2 h at 37 °C. To test the effect of GSH on the formation of volatile arsenicals, some reaction mixtures also contained 5 mM GSH. Reaction vials were purged with He immediately before the beginning of the incubation period. At the end of the incubation, 60 mL of He was slowly injected into each sealed vial to displace the gas phase (about 2.8 mL in volume) above the aqueous phase of the reaction mixture. Purging He was directed through a U-tube filled with Chromosorb WAW-dimethyldichlorosilane 46/60 (Supelco, Inc., Bellefonte, PA) positioned in a Dewar flask that contained liquid N2. After the gas phase was purged, the cooled U-tube was attached to the inlet of a custom-designed quartz atomization cuvette aligned in the light path of a model 5100 atomic absorption spectrophotometer (Perkin-Elmer, Norwalk, CT) equipped with a model EDL II electrodeless discharge lamp (Perkin-Elmer). The U-tube was then removed from the Dewar flask and warmed to thermally separate arsines. Conditions for the detection of arsines by atomic absorption spectrometry have been described (28, 29). A reaction mixture from which AS3MT was omitted was incubated and sampled under identical conditions. Following purging of the gas phase, aqueous phases of reaction mixtures were analyzed for iAs and its methylated metabolites by hydride generation atomic absorption spectrometry (HG-AAS) as described below. TLC and Quantitation of Radiolabeled Arsenicals. Radiolabeled standards and aliquots of reaction mixtures were oxidized by treatment with 10% H2O2 before TLC on PEI-F cellulose TLC plates (Baker, Phillipsburg, NJ) with a 2-propanol: water:acetic acid (10:2.5:1) solvent system (30, 31). Radiolabeled arsenicals separated by TLC were quantified with a model FLA2000 fluorescent image analyzer (Fujifilm, Stamford, CT) equipped with Image Gauge (version 3.0). HG-AAS. Stable arsenicals in reaction mixtures were separated and quantified by HG-AAS using a Perkin-Elmer model 5100 atomic absorption spectrometer (28, 29). This method uses pH selective generation of arsines from arsenicals containing trivalent or pentavalent arsenic to determine the oxidation state of arsenic in inorganic and methylated species. Recent modifications of this method permit the reliable separation and quantitation of TMAO (29). Calibration curves for five concentrations of each arsenical (0.5, 2.5, 10, 20, and 80 ng As) based on peak areas were used for quantification in these studies. Ion Chromatography. Both anion and cation chromatography were performed on a Hewlett-Packard 1100 system (Agilent, Bellvue, WA). Anion chromatography used a PRP-X100 column (Hamilton, Reno, NV) with a mobile phase of 20 mM (NH4)2CO3, pH 9. This mobile phase was prepared from (NH4)2CO3 (ACS reagent, Aldrich, Milwaukee, WI) with NH4OH [trace metal grade, Fisher Scientific (Pittsburgh, PA)] used for pH adjustment. Anion chromatography easily resolved iAsIII, iAsV, MAs, and DMAs. Cation chromatography on an Ionosphere C column (Varian, Palo Alto, CA) separated and quantified TMAO. The mobile phase for cation chromatography was 10 mM pyridinium formate, pH 2.7. This mobile phase was prepared from pyridine and formic acid (ACS reagent grade, Fisher). For

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Figure 1. Time course for the appearance of radiolabeled metabolites. Aliquots of reaction mixtures that contained 5 µg of rrAS3MT, 1 mM AdoMet, 1 µM [73As]iAsIII, and a reductant (1 mM TCEP or a Trx-regenerating system consisting of 10 µM E. coli Trx, 3 µM rat liver TR, and 300 µM NADPH in 100 mM tris-100 mM phosphate, pH 7.4) were incubated at 37 °C for up to 120 min. Aliquots of the reaction mixtures were chromatographed in a 2-propanol:water:acetic acid (10:2.5:1) solvent system. Known species (iAs, MAs, and DMAs) and unknown arsenical are labeled, and the direction of chromatography is indicated by an arrow. anion or cation chromatography, the flow rate for the mobile phase was 1 mL/min. Samples containing anionic and cationic arsenic species were diluted 1:4 with eluent before injection. The injection volume for either anion or cation chromatography was 100 µL. Separation by cation chromatography was monitored by either inductively coupled plasma mass spectrometry (ICP-MS) or electrospray ionization mass spectrometry (ESIMS). Peak identification was based on a retention time match with a verified standard. For these analyses, verified standards were iAsIII (Spex, Metuchen, NJ), iAsV (Spex), MAsV (ChemService, West Chester, PA), DMAsV (ChemService), and TMAO (generously provided by Professor William R. Cullen, University of British Columbia, Vancouver). Standard Reference Material 1640-Trace Elements in Natural Water (National Institute of Standards and Technology, Gaithersburg, MD) was used to validate the concentrations of arsenic in these verified standards. Mobile phases and arsenical standards were prepared in deionized distilled water (18 MΩ; Millipore, Bedford, MA). ICP-MS. Studies reported here used a Hewlett-Packard 4500 ICP-MS (Palo Alto, CA) operated with the following instrumental parameters: rf power ) 1210 W, carrier gas flow ) 1.26 L/min, and plasma gas flow ) 15 L/min. The spray chamber temperature was 5 °C. Ion chromatography and ICP-MS were fully integrated for unattended operation. Quantitation was performed using integrated signals from the ion chromatography ICP-MS for m/z ) 75. ESI-MS. A Finnigan LCQ Deca (ThermoQuest, San Jose, CA) ion trap mass spectrometer was used as a secondary means for peak identification of TMAO. The presence of TMAO was confirmed by ion chromatography ESI-MS and a retention time match to an authentic standard using the molecular ion (M + 1 ) 137). Operational parameters for this instrument were as follows: sheath gas flow at 80 units, auxiliary gas flow at 25 units, spray voltage at 4.3 kV, capillary temperature of 250 °C, and capillary voltage at 5.9 V.

Results Figure 1 shows the time course for the appearance of radiolabeled metabolites formed in reactions containing 5 µg of rrAS3MT, 1 mM AdoMet, 1 µM [73As]iAsIII, and a reductant (1 mM TCEP or a Trx/TR/NADPH-coupled system). With either reductant, 1 µM iAsIII was converted to three radiolabeled species that were separated by TLC with a 2-propanol:water:acetic acid (10:2.5:1) solvent

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Figure 2. Analysis of reaction mixtures using HG-AAS with arsine generation at pH 6. (A) Pattern of chromatography for a mixture containing 20 ng each of authentic iAs, MAs, DMAs, and TMAO. (B) Pattern of chromatography for an aliquot of a reaction mixture containing 5 µg of rrAS3MT, 1 mM AdoMet, 1 µM [73As]iAsIII, and 1 mM TCEP in 100 mM tris-100 mM phosphate, pH 7.4, that was incubated 37 °C for 60 min.

system. Although both MAsV and DMAsV were identified by their cochromatography with authentic [14C]-labeled MAsV or DMAsV, the most rapidly migrating metabolite had not been identified in previous studies. Because this third metabolite appeared at later time points than did either MAs or DMAs, its formation was postulated to depend on prior formation of these methylated metabolites. Further information on the identity of the third metabolite was sought by analysis of reaction mixtures using HG-AAS. In reaction mixtures containing 5 µg of rrAS3MT, 1 mM AdoMet, 1 µM [73As]iAsIII, and 1 mM TCEP, the third product of metabolism cochromatographed with TMAs generated from authentic TMAO (Figure 2). The identity of the third metabolite as TMAO

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was confirmed by cation chromatography ESI-MS of an aliquot of a reaction mixture that contained 50 µg of rrAS3MT, 1 mM AdoMet, 10 µM iAsIII, and 1 mM TCEP that was incubated at 37 °C for 16 h. Figure 3 shows the identification of TMAO in the reaction mixture by selective ion monitoring for the molecular ion (M + 1 ) 137). The retention time was identical to that of an authentic standard of TMAO. The pattern of metabolism of iAsIII in rrAS3MTcatalyzed reactions was further examined in reaction mixtures that contained 5 or 50 µg of rrAS3MT, 1 mM AdoMet, 10 µM [73As]-labeled iAsIII, and 1 mM TCEP that were incubated at 37 °C for 16 h. The pattern of metabolites in these reaction mixtures was determined by anion chromatography ICP-MS. These studies demonstrated that the recovery of arsenic from reaction mixtures was nonquantitative. For triplicate assays initially containing 1000 pmol of iAsIII, the average recovery of arsenic from reaction mixtures with 5 µg of rrAS3MT was 72.5%; from reaction mixtures with 50 µg of rrAS3MT, the average recovery was 58.7%. As shown in Figure 4, the fractional contribution of TMAO to the total As recovered from reaction mixtures increased with an increasing amount of AS3MT in the reaction mixture. The effects of concentration of GSH in reaction mixture on the pattern of metabolites formed in AS3MT-catalyzed reactions were examined in assays in which up to 5 mM GSH was added to reaction mixtures that contained 5 µg of rrAS3MT, 1 mM AdoMet, 1 µM [73As]iAsIII, and 1 mM TCEP that were incubated at 37 °C for 60 min. Increasing the concentration of GSH in the reaction mixture increased the fraction of iAsIII converted to methylated metabolites (Figure 5). However, as the concentration of GSH increased, the yield of TMAO decreased. The effect of GSH on the production of TMAO in rrAS3MT-catalyzed reactions was examined by determining the effect of GSH on the conversion of DMAsIII to TMAO. For these studies, DMAsIII was added as the substrate in reaction mixtures that contained rrAS3MT, 1 mM AdoMet, and 1 mM TCEP that were incubated at 37 °C for up to 120 min. Substrates for these reactions included 1 µM DMAsIII, 1 µM DMAsIIIGS, or 1 µM DMAsIII and 5 mM GSH. Both DMAsIII and DMAsIII GS were substrates for conversion to TMAO; however, a competing oxidation reaction that produced DMAsV reduced the concentration of these trivalent arsenicals in reaction mixtures (Figure 6). In contrast, rrAS3MT did not catalyze the formation of TMAO when 1 µM DMAsIII and 5 mM GSH were added to the reaction

Figure 3. Identification of the third metabolite as TMAO by cation chromatography ESI-MS. An aliquot of a reaction mixture that contained 50 µg of rrAS3MT, 1 mM AdoMet, 10 µM [73As]iAsIII, and 1 mM TCEP in 100 mM tris-100 mM phosphate, pH 7.4, that was incubated at 37 °C for 16 h was analyzed by cation chromatography ESI-MS. TMAO in the reaction mixture was identified by a retention time match (7.44 min) to authentic TMAO in 100 mM tris-100 mM phosphate, pH 7.4, using the molecular ion (M + 1 ) 137). Cation chromatography was performed on an Ionosphere C column using a mobile phase of 10 mM pyridinium formate, pH 2.7, at 1 mL per minute.

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Figure 4. Effect of the concentration of rrAS3MT on the formation of TMAO. Reaction mixtures that contained 5 or 50 µg of rrAS3MT, 1 mM AdoMet, 10 µM [73As]iAsIII, and 1 mM TCEP in 100 mM tris-100 mM phosphate, pH 7.4, that were incubated at 37 °C for 16 h were analyzed for iAs (black), MAs (white), DMAs (gray), and TMAO (striped) by anion chromatography ICP-MS. Results expressed as pmols of arsenic are present as these arsenical species (means and SDs; n ) 3).

Figure 5. Effect of glutathione concentration on the pattern and extent of AS3MT-catalyzed methylation of iAs. Effect of addition of up to 5 mM GSH to reaction mixtures that contained 5 µg of rrAS3MT, 1 mM AdoMet, 1 µM [73As]iAsIII, and 1 mM TCEP in 100 mM tris-100 mM phosphate, pH 7.4, that were incubated at 37 °C for 60 min. Aliquots of reaction mixtures were chromatographed in a 2-propanol:water:acetic acid (10: 2.5:1) solvent system. Amounts (pmols) of MAs (black), DMAs (white), and TMAO (gray) are shown (means and SDs; n ) 3).

mixture. Hence, a molar excess of GSH effectively limits the formation of TMAO from DMAs. The extent of loss of volatile arsenic from reaction mixtures was quantified using radiolabeled and stable arsenicals as substrates for AS3MT-catalyzed reactions. Omission of 5 mM GSH from reaction mixtures was associated with the production of TMAO and the loss of [73As] (Figure 7A,B). After 120 min at 37 °C, reaction mixtures lacking GSH had lost an average of 4.6% of the radiolabeled arsenic that was initially present. This loss was statistically significant by a two-tailed student’s test (P ) 0.026). Loss of radioarsenic did not occur in reaction mixtures from which other components (rrAS3MT, TCEP, and AdoMet) were individually omitted. The volatilization of arsenic was also evaluated in reaction mixtures in which the amounts of both rrAS3MT and substrate concentration were varied. Here, reaction mixtures lacking GSH were incubated for 120 min at 37 °C. For either substrate concentration (1 or 10 µM iAsIII), increasing the amount of rrAS3MT increased the amount of radioarsenic lost from the reaction mixture (Figure 7C). At the higher concentration of rrAS3MT, nearly 30% of the radioarsenic was lost from reaction mixtures. The identity of the volatilized arsenic formed in reaction mixtures was determined by sampling the gas phase of reaction mixtures, which were incubated in sealed reaction vials for

Figure 6. Effect of glutathione on the pattern and extent of AS3MT-catalyzed methylation of iododimethylarsine. Reaction mixtures containing 5 µg of rrAS3MT, 1 mM AdoMet, and 1 mM TCEP with (A) 1 µM DMAsIII, (B) 1 µM DMAsIII GS, or (C) 1 µM DMAsIII and 5 mM GSH were incubated at 37 °C for up to 120 min. The oxidation state of arsenicals in reaction mixtures was determined by pH selective HG-AAS. The percentage of arsenic present as DMAsIII (black), DMAsV (white), or TMAO (gray) is shown.

120 min at 37 °C. These experiments also examined the effect of GSH on the formation of volatile arsenic in rrAS3MT-catalyzed reactions. Gas phases of reaction mixtures containing volatile arsines were collected in a liquid N2-cooled U-tube filled with Chromosorb WAWdimethyldichlorosilane 46/60. Because no reductant (NaBH4) was added to reaction mixtures before the gas phases were sampled, only volatile arsines were collected by this procedure. For reaction mixtures that contained 5 mM GSH, no volatile arsenic was detected in the gas phase; essentially all of the arsenic recovered in the aqueous phase was present as DMAs (Figure 8A,B). For reaction mixtures that lacked GSH, TMAs was detected in the gas phase of the reaction mixture and iAs, DMAs, and TMAO were detected in the aqueous phase (Figure 8C,D). On the basis of the results of two experiments, TMAs in the gas phase of the reaction mixtures accounted for an average of 3.5% of the total As recovered from reaction mixtures.

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Figure 7. Recovery of radioarsenic from AS3MT-catalyzed reaction mixtures. (A) Pattern of metabolites in reaction mixtures that contained 5 µg of rrAS3MT, 1 mM AdoMet, 1 µM [73As]iAsIII, and 1 mM TCEP in 100 mM tris-100 mM phosphate, pH 7.4, that were incubated at 37 °C for up to 120 min. Identities of arsenicals shown and direction of chromatography in a 2-propanol:water:acetic acid (10:2.5:1) solvent system are indicated by an arrow. (B) Effect of omission of components on the recovery of radioarsenic from reaction mixtures. A complete reaction mixture contained 5 µg of rrAS3MT, 1 mM AdoMet, 1 mM TCEP, 5 mM GSH, and 1 µM [73As]-labeled arsenite in 100 mM tris-100 mM Na phosphate, pH 7.4, buffer (O). Effect of omission of 5 mM GSH (+), 5 µg of rrAS3MT (2), 1 mM AdoMet (1), or 1 mM TCEP (9) on recovery of radioarsenic from reaction mixtures. Reaction mixtures with and without GSH were incubated from 0 to 120 min at 37 °C. Other reaction mixtures were incubated for 120 min at 37 °C. Reaction mixtures initially contained 50 pmol of arsenic. Aliquots of reaction mixtures were chromatographed in a 2-propanol:water:acetic acid (10:2.5:1) solvent system. Means and SDs are shown; n ) 3. (C) Effect of the amount of enzyme and substrate concentration on the recovery of radioarsenic from reaction mixtures. The reaction mixtures contained 5 or 50 µg of rrAS3MT, 1 mM AdoMet, 1 mM TCEP, and 5 mM GSH, with 1 (left panel) or 10 (right panel) µM [73As]-labeled arsenite in 100 mM tris-100 mM Na phosphate, pH 7.4, buffer. The reaction mixtures were incubated for 120 min at 37 °C before radioassay. The reaction mixtures initially contained 50 pmol of arsenic (1 µM arsenite) or 500 pmol of arsenic (10 µM arsenite). Means and standard deviations are shown; n ) 3.

Discussion The solvent system for TLC commonly used in earlier studies (acetone:water:acetic acid) resolved two 73Aslabeled products (MAs and DMAs) that were formed in AS3MT-catalyzed reactions (4, 6, 31). However, use of a 2-propanol:water:acetic acid (10:2.5:1) solvent system for TLC demonstrated that a third radiolabeled metabolite was formed in reaction mixtures that contained rrAS3MT, AdoMet, [73As]iAsIII, and a reductant. The appearance of the third metabolite lagged behind the appearance of MAs and DMAs in AS3MT-catalyzed reactions, suggesting that its formation depended on the formation of these MAs. Because earlier studies of the metabolism of iAs in organisms indicated that trimethylated arsenicals might be formed in the course of metabolism, we postulated the third radiolabeled product in AS3MT-catalyzed reaction mixtures to be a trimethylated species. TMAO was identified in the aqueous phase of reaction mixtures by HG-AAS and by mass spectral analysis. Additional studies showed that the volatile arsine TMAs was also a product of a reaction catalyzed by AS3MT. Hence, AS3MT catalyzes all steps of a sequence of reactions that leads from iAsIII to TMAs. GSH was shown to exert two distinct effects of GSH on the catalytic activity of AS3MT. We have previously shown that in reaction mixtures that contained 5 mM GSH and no other reductant, rrAS3MT only slowly catalyzed the formation of MAs from iAsIII. However, in reactions with either TCEP or Trx as the reductant, the addition of GSH markedly stimulated the rate of methylation by AS3MT (6). The present work shows that this stimulatory effect of GSH on the rate of MAs and DMAs

production from iAsIII is concentration-dependent. By comparison, the activity of another iAsIII methyltransferase purified from rabbit liver is also markedly stimulated by the addition of GSH to assays (2, 32). The stimulatory effect of GSH on the activity of AS3MT could be related to a glutathionylation reaction (33) involving an interaction between one of the 13 cys residues of rat AS3MT and GSSG, which is present in GSH as a contaminant. However, addition of GSSG (5 mM) to reaction mixtures containing a TR/Trx/NAPDH-coupled system decreased rrAS3MT-catalyzed formation of MAs and DMAs from iAsIII (data not shown). This suggests that GSSG does not function as a reductant, which stimulates this enzyme’s activity. GSH could induce conformational changes in AS3MT by interactions that do not involve cys residues in the either the tripeptide or the protein. For example, interactions between the γ-glutamyl and the glycyl residues of GSH and residues in the carboxy terminus of human GSH transferase A1-1 induce R helix formation and increase the turnover rate for the enzyme (34). A similar interaction between GSH and AS3MT could underlie the effects of the tripeptide on the catalytic function of the enzyme. In the present study, it was also found that the addition of GSH to reaction mixtures suppressed the formation of TMAO as the formation of MAs and DMAs was stimulated. The inhibitory effect to GSH appeared to be exerted at the trimethylation reaction catalyzed by rrAS3MT. A molar excess of GSH to DMAsIII in the reaction mixture blocked formation of TMAO. In contrast, DMAsIIIGS, an equimolar complex of GSH and DMAsIII, was a substrate for conversion to TMAO. This suggests that GSH does not

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Figure 8. Identification of volatile arsine formed in AS3MT-catalyzed reaction mixtures. Reaction mixtures containing 100 µg of rrAS3MT, 10 µM iAsIII, 1 mM TCEP, and 1 mM AdoMet in 100 mM tris-100 mM NaPO4, pH 7.4, with or without 5 mM GSH were incubated in sealed 3 mL conical reaction vials for 2 h at 37 °C. Gas and liquid phases of reaction mixtures were sampled to identify arsenicals present by HG-AAS at pH 6. (A) Chromatogram of the liquid phase of the reaction mixture with GSH (positions for elution of MAs, DMAs, and TMAO are noted). (B) Chromatogram of the gas phase of the reaction mixture with GSH. (C) Chromatogram of the liquid phase of the reaction mixture without GSH (positions for elution of MAs, DMAs, and TMAO are noted). (D) Chromatogram of the gas phase of the reaction mixture without GSH.

inhibit the production of TMAO by means of complexation of DMAsIII but rather affects some interaction between DMAsIII and AS3MT or between AS3MT and another component of the reaction mixture that is necessary for catalysis. Notably, interpretation of studies involving DMAsIIIGS may be complicated by the uncertainties about the stability of this complex under conditions used for assays of AS3MT’s activity. DMAsIIIGS is rapidly decomposed in aqueous solutions at pH 8.3 and 37 °C (35), and the integrity of the complex in reaction mixtures was not measured in the current study. The capacity of GSH to affect the methylation of arsenicals has been demonstrated in other experimental systems. In in vitro assays systems using rat liver cytosol or in rat liver slices, GSH has been shown to stimulate the methylation of iAsIII (36-38). Studies in hamsters and rats have also shown that metabolism, retention, and toxicity of iAsIII are affected by depletion of tissue GSH (39, 40). It is possible that an interaction between AS3MT and GSH underlies the effects of this thiol compound on the methylation of arsenicals that has been reported at each level of biological complexity. The novel observation that AS3MT catalyzes the formation of TMAO and of TMAs suggests that the production, fate, and effects of these arsenicals should also be considered in evaluating the consequences of exposure to iAsIII. By comparison, little is known of the mammalian enzymes that catalyze the formation of volatile hydrides of other metalloids (selenium, tellurium), which can be detected in expired breath (41).

The relation between AS3MT, a mammalian arsenic methyltransferase, which catalyzes the formation of TMAO and TMAs and the prokaryotic enzymes that catalyze the formation of these metabolites, is uncertain. A wide variety of microorganisms can produce methylated arsenicals, including arsines, from iAs (42). Recent work has identified the arsM gene in Halobacterium sp. strain NRC-1, which encodes a protein homologous to mammalian AS3MT (43). These investigators suggested that the methyltransferase encoded by arsM could catalyze the production of methylated arsines and that the diffusion of these volatile arsines from the cell might confer resistance to arsenite. In mammals, AS3MTcatalyzed formation of TMAs could result in the rapid loss of this volatile species in expired air. Notably, the inhibition of TMAO and TMAs production in rrAS3MTcatalyzed reactions containing GSH did not result in the appearance of other arsines in the gas phase of reaction mixtures. The presence of TMAs in the gas phase may reflect its high vapor pressure (498 Torr at 37 °C, 44) and relatively slow rate of oxidation (10-6 M-1 s-1, 45). Thus, AS3MT may be less efficient catalyzing the formation of arsine, methylarsine, or dimethylarsine than of TMAs or these products may be lost through rapid oxidation. Although GSH suppresses AS3MT-catalyzed TMAO production, formation of TMAO does occur in GSH-containing reaction mixtures, albeit at a relatively low rate. We (data not shown) and others (1) have found that TMAO is efficiently reduced to TMAs by GSH. Hence, TMAs may be a slowly evolved terminal product

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as a terminal product of iAs methylation even under cellular conditions in which GSH is present at a millimolar concentration. TMAs generated from TMAO in an in vitro assay can induce single strand breaks in plasmid or phage DNA (24). Because TMAs is about 100-fold more potent than DMAsIII as an inducer of single strand breaks in DNA in in vitro assays, its formation in a AS3MTcatalyzed reaction should be considered a pathway for metabolic activation of arsenicals. Modulation by GSH of AS3MT-catalyzed formation of TMAO and TMAs may affect the extent of exposure to these MAs. Given new information on the genotoxicity and carcinogenicity of TMAO and TMAs, control of their synthesis could be an important determinant of the risk associated with chronic exposure to arsenicals.

Acknowledgment. S.B.W. is a postdoctoral fellow in the Curriculum in Toxicology, University of North Carolina at Chapel Hill, and is supported by Training Grant T901915 of the U.S. Environmental Protection Agency-University of North Carolina Toxicology Research Program. V.D. is supported by a MECD-Fulbright Fellowship from the Department of Education, Culture, and Sport of Spain. M.S. is supported by NIH Grant ES010845, a Clinical Nutrition Research Center Grant DK 56350, and U.S. EPA collaborative agreement CR829522. We thank Professor William R. Cullen, Department of Chemistry, University of British Columbia, Vancouver, for the generous gift of the arsenicals used in this research. This manuscript has been reviewed in accordance with the policy of the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

References (1) Cullen, W. R., McBride, B. C., and Reglinski, J. (1984) The reduction of trimethylarsine oxide to trimethylarsine by thiols: A mechanistic model for the biological reduction of arsenicals. J. Inorg. Biochem. 21, 45-60. (2) Zakharyan, R., Wu, Y., Bogdan, G. M., and Aposhian, H. V. (1995) Enzymatic methylation of arsenic compounds: Assay, partial purification, and properties of arsenite methyltransferase and monomethylarsonic acid methyltransferase from rabbit liver. Chem. Res. Toxicol. 8, 1029-1038. (3) Lin, S., Shi, Q., Nix, F. B., Styblo, M., Beck, M. A., Herbin-Davis, K. M., Hall, L. L., Simeonsson, J. B., and Thomas, D. J. (2002) A novel S-adenosyl-L-methionine: Arsenic(III) methyltransferase from rat liver cytosol. J. Biol. Chem. 277, 10795-10803. (4) Walton, F. S., Waters, S. B., Jolley, S. L., LeCluyse, E. L., Thomas, D. J., and Styblo, M. (2003) Selenium compounds modulate activity of recombinant rat AsIII methyltransferase and methylation of arsenite by rat and human hepatocytes. Chem. Res. Toxicol. 16, 261-265. (5) Thomas, D. J., Waters, S. B., and Styblo, M. (2004) Elucidating the pathway for arsenic methylation. Toxicol. Appl. Pharmacol. 198, 319-326. (6) Waters, S. B., Styblo, M., and Thomas, D. J. (2004) Endogenous reductants support the catalytic function of recombinant rat cyt19, an arsenic methyltransferase. Chem. Res. Toxicol. 17, 404-409. (7) Gregus, Z., and Nemeti, B. (2002) Purine nucleoside phosphorylase as a cytosolic arsenate reductase. Toxicol. Sci. 70, 1319. (8) Radabaugh, T. R., Sampayo-Reyes, A., Zakharyan, R. A., and Aposhian, H. V. (2002) Arsenate reductase II. Purine nucleoside phosphorylase in the presence of dihydrolipoic acid is a route for reduction of arsenate to arsenite in mammalian systems. Chem. Res. Toxicol. 15, 692-698. (9) Zakharyan, R. A., Sampayo-Reyes, A., Healy, S. M., Tsaprailis, G., Board, P. G., Liebler, D. C., and Aposhian, H. V. (2001) Human

Waters et al.

(10)

(11)

(12)

(13)

(14)

(15)

(16)

(17)

(18)

(19)

(20)

(21) (22)

(23)

(24)

(25)

(26)

(27)

(28)

(29)

monomethylarsonic acid (MMA(V)) reductase is a member of the glutathione-S-transferase superfamily. Chem. Res. Toxicol. 14, 1051-1057. Delnomdedieu, M., Basti, M. M., Styblo, M., Otvos, J. D., and Thomas, D. J. (1994) Complexation of arsenic species in rabbit erythrocytes. Chem. Res. Toxicol. 7, 621-627. Thomas, D. J., Styblo, M., and Lin, S. (2001). The cellular metabolism and systemic toxicity of arsenic. Toxicol. Appl. Pharmacol. 176, 127-144. Marafante, E., Vahter, M., Norin, H., Envall, J., Sandstro¨m, M., Christakopoulos, A., and Ryhage, R. (1987) Biotransformation of dimethylarsinic acid in mouse, hamster and man. J. Appl. Toxicol. 7, 111-117. Yoshida, K., Chen, H., Inoue, Y., Wanibuchi, H., Fukushima, S., Kuroda, K., and Endo, G. (1997) The urinary excretion of arsenic metabolites after a single oral administration of dimethylarsinic acid to rats. Arch. Environ. Contam. Toxicol. 32, 416-421. Yoshida, K., Inoue, Y., Kuroda, K., Chen, H., Wanibuchi, H., Fukushima, S., and Endo, G. (1998) Urinary excretion of arsenic metabolites after long-term oral administration of various arsenic compounds to rats. J. Toxicol. Environ. Health A 54, 179-192. Cohen, S. M., Arnold, L. L., Uzvolgyi, E., Cano, M., St. John, M., Yamamoto, S., Lu, X., and Le, X. C. (2002) Possible role of dimethylarsinous acid in dimethylarsinic acid-induced urothelial toxicity and regenerationn in the rat. Chem. Res. Toxicol. 15, 1150-1157. Lu, X., Arnold, L. L., Cohen, S. M., Cullen, W. R., and Le, X. C. (2003) Speciation of dimethylarsinous acid and trimethylarsine oxide in urine from rats fed with dimethylarsinic acid and dimercaptopropane sulfonate. Anal. Chem. 75, 6463-6438. Hughes, M. F., Kenyon, E. M., Edwards, B. C., Mitchell, C. T., Del Razo, L. M., and Thomas, D. J. (2003) Accumulation and metabolism of arsenic in mice after repeated oral administration of arsenate. Toxicol. Appl. Pharmacol. 191, 202-210. Yamauchi, H., Takahashi, K., Yamamura, Y., and Kaise, T. (1989) Metabolism and excretion of orally and intraperitoneally administered trimethylarsine oxide in the hamster. Toxicol. Environ. Chem. 22, 69-76. Kaise, T., Yamauchi, H., Horiguchi, Y., Tarai, T., Watanabe, S., Hirayama, T., and Fukui, S. (1989) A comparative study on acute toxicity of methylarsonic acid, dimethylarsinic acid and trimethylarsine oxide in mice. Appl. Organomet. Chem. 3, 273-277. Yamauchi, H., Kaise, T., Takahashi, K., and Yamamura, Y. (1990) Toxicity and metabolism of trimethylarsine oxide in mice and hamster. Fundam. Appl. Toxicol. 14, 399-407. Pinto, S. S., and McGill, C. M. (1953) Arsenic trioxide exposure in industry. Ind. Med. Surg. 22, 281-287. Nishikawa, T., Wanibuchi, H., Ogawa, M., Kinoshita, A., Morimura, K., Hiroi, T., Funae, Y., Kishida, H., Nakae, D., and Fukushima, S. (2002) Promoting effects of monomethylarsonic acid, dimethylarsinic acid and trimethylarsine oxide on induction of rat liver preneoplastic glutathione S-transferase placental form positive foci: A possible reactive oxygen species mechanism. Int. J. Cancer 100, 136-139. Shen, J., Wanibuchi, H., Salim, E. I., Wei, M., Kinoshita, A., Yoshida, K., Endo, G., and Fukushima, S. (2003) Liver tumorigenicity of trimethylarsine oxide in male Fischer 344 ratss Association with oxidative DNA damage and enhanced cell proliferation. Carcinogenesis 24, 1827-1835. Andrewes, P., Kitchin, K. T., and Wallace, K. (2003) Dimethylarsine and trimethylarsine are potent genotoxins in vitro. Chem. Res. Toxicol. 16, 994-1003. International Agency for Research on Cancer (1987) Arsenic and arsenic compounds. IARC Monograph on the Evaluation of Carcinogenic Risks to HumanssOverall Evaluations of Carcinogenicity: An Update of IARC Monographs 1 to 42, Suppl. 7, p 100, IARC, Lyon. Reay, P. F., and Asher, C. J. (1977) Preparation and purification of 74As-labeled arsenate and arsenite for use in biological experiments. Anal. Biochem. 78, 557-560. Styblo, M., Serves, S. V., Cullen, W. R., and Thomas, D. J. (1997) Comparative inhibition of yeast glutathione reductase by arsenicals and arsenothiols. Chem. Res. Toxicol. 10, 27-33. Del Razo, L. M., Styblo, M., Cullen, W. R., and Thomas, D. J. (2001) Determination of trivalent methylated arsenicals in biological matrices. Toxicol. Appl. Pharmacol. 174, 282-293. Devesa, V., Del Razo, L. M., Adair, B., Drobna, Z., Waters, S. B., Hughes, M. F, Sty´blo, M., and Thomas, D. J. (2004) Comprehensive analysis of arsenic metabolites by pH-specific hydride generation atomic absorption spectrometry. J. Anal. At. Spectrom. 19, 1460-1467.

Glutathione and Arsenic Methyltransferase (30) Styblo, M., Delnomdedieu, M., Hughes, M. F., and Thomas, D. J. (1995) Identification of methylated metabolites of inorganic arsenic by thin-layer chromatography. J. Chromatogr. B 668, 2129. (31) Styblo, M., Hughes, M. F., and Thomas, D. J. (1996) Liberation and analysis of protein-bound arsenicals. J. Chromatogr. B 677, 161-166. (32) Zakharyan, R. A., Ayala-Fierro, F., Cullen, W. R., Carter, D. M., and Aposhian, H. V. (1999) Enzymatic methylation of arsenic compounds. VII. Monomethylarsonous acid (MMAIII) is the substrate for MMA methyltransferase of rabbit liver and human hepatocytes. Toxicol. Appl. Pharmacol. 158, 9-15. (33) Fratelli, M., Demol, H., Puype, M., Casagrande, S., Eberini, I., Salmona, M., Bonetto, V., Mengozzi, M., Duffieux, F., Miclet, E., Bachi, A., Vandekerckhove, J., Gianazza, E., and Ghezzi, P. (2002) Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes. Proc. Natl. Acad. Sci. U.S.A. 99, 3505-3510. (34) Zhan, Y., and Rule, G. S. (2004) Glutathione induces helical formation in the carboxy terminus of human glutathione transferase A1-1. Biochemistry 43, 7244-7254. (35) Raab, A., Meharg, A. A., Jaspars, M., Genney, D. R., and Feldmann, J. (2004) Arsenic-glutathione complexessTheir stability in solution and during separation by different HPLC modes. J. Anal. At. Spectrom. 19, 183-190. (36) Buchet, J. P., and Lauwerys, R. (1988) Role of thiols in the in vitro methylation of inorganic arsenic by rat liver cytosol. Biochem. Pharmacol. 37, 3149-3153. (37) Georis, B., Cardenas, A., Buchet, J. P., and Lauwerys, R. (1990) Inorganic arsenic methylation by rat tissue slices. Toxicology 63, 73-84.

Chem. Res. Toxicol., Vol. 17, No. 12, 2004 1629 (38) Styblo, M., Delnomdedieu, M., and Thomas, D. J. (1996) Monoand dimethylation of arsenic in rat liver cytosol in vitro. Chem.Biol. Interact. 99, 147-164. (39) Hirata, M., Tanaka, A., Hisanaga, A., and Ishinishi, N. (1990) Effects of glutathione depletion on the acute nephrotoxic potential of arsenite and on arsenic metabolism in hamsters. Toxicol. Appl. Pharmacol. 106, 469-481. (40) Csanaky, I., Nemeti, B., and Gregus, Z. (2003) Dose-dependent biotransformation of arsenite in rats B not S-adenosylmethionine depletion impairs arsenic methylation at high dose. Toxicology 183, 77-91. (41) Feldmann, J., Riechmann, T., and Hirner, A. V. (1996) Determination of organometallics in intra-oral air by LT-GC/ICP-MS. Fresenius J. Anal. Chem. 354, 620-623. (42) Bentley, R., and Chasteen, T. G. (2002) Microbial methylation of metalloids: Arsenic, antimony, and bismuth. Microbiol. Mol. Biol. Rev. 66, 250-271. (43) Wang, G., Kennedy, S. P., Fasiludeen, S., Rensing, C., and DasSarma, S. (2004) Arsenic resistance in Halobacterium sp. strain NRC-1 examined by using an improved gene knockout system. J. Bacteriol. 186, 3187-3194. (44) Rosenbaum, E. J., and Sandberg, C. R. (1940) Vapor pressure of trimethylphosphine, trimethylarsine and trimethylstibine. J. Am. Chem. Soc. 62, 1622-1623. (45) Parris, G. E., and Brinckman, F. E. (1976) Reactions which relate to environmental mobility of arsenic and antimony. II. Oxidation of trimethylarsine and trimethylstibine. Environ. Sci. Technol. 10, 1128-1134.

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