Urinary Sulfur-Containing Metabolite Produced by Intestinal Bacteria

Aug 14, 2003 - Division of Customer Training Group Application Center, Yokogawa .... Analytical Systems), was performed under the following condi-...
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Chem. Res. Toxicol. 2003, 16, 1124-1129

Urinary Sulfur-Containing Metabolite Produced by Intestinal Bacteria Following Oral Administration of Dimethylarsinic Acid to Rats Kaoru Yoshida,*,† Koichi Kuroda,† Xu Zhou,† Yoshinori Inoue,† Yukiko Date,§ Hideki Wanibuchi,‡ Shoji Fukushima,‡ and Ginji Endo† Department of Preventive Medicine and Environmental Health and First Department of Pathology, Osaka City University Medical School, 1-4-3, Asahi-machi, Abeno-ku, Osaka, 545-8585, Japan, and Division of Customer Training Group Application Center, Yokogawa Analytical Systems, Inc., 2-11-13, Naka-cho, Musashino-shi, Tokyo, 180-0006, Japan Received February 11, 2003

Our long-term oral administration of dimethylarsinic acid (DMAV) in rats revealed that three unidentified metabolites, M-1, M-2, and M-3, were detected in urine and feces. DMAV and trimethylarsine oxide (TMAO) were converted to M-2 and M-3 and M-1 by Escherichia coli strain A3-6 isolated from the ceca of DMAV-administered rats, respectively. In this study, we report on the mechanism of production and the chemical properties of these unknown metabolites. To investigate the pattern of conversion of DMAV or TMAO by A3-6 in the presence of cysteine (Cys), arsenic metabolites of DMAV or TMAO in medium after incubation with A3-6 and Cys were analyzed by liquid chromatography with inductively coupled plasma mass spectrometry (LC-ICP-MS). DMAV was reduced to dimethylarsinous acid (DMAIII) to form M-2 in the presence of Cys and A3-6, and M-2 was further converted to M-3. TMAO was rapidly converted to M-1 by A3-6. The cytotoxicity of the unidentified metabolites was investigated. M-2 was more cytotoxic than DMAV, M-1, and M-3 in V79 cells. The cytotoxicity of M-2 in HL-60 cells was decreased by the addition of superoxide dismutase, suggesting that the cytotoxicity of M-2 might be due to the production of reactive oxygen species. In addition, we examined the chemical properties of M-2 by LC-ICP-MS and LC-MS. M-2 was oxidized to DMAV by hydrogen peroxide, suggesting that M-2 may be a reduced form of DMAV. M-2 was consistent with the reactant of DMAV with metabisulfite-thiosulfate reagent but not DMAIII by analyses of LC-ICP-MS and LC-MS. The molecular weight of M-2 was 154, and M-2 was a sulfurcontaining metabolite.

Introduction Epidemiological studies have demonstrated that longterm ingestion or inhalation of inorganic arsenic can increase the risk of developing skin, lung, bladder, and liver cancers (1, 2). It is still a question which arsenic species are the ultimate carcinogens in such cancers. Inorganic arsenic is methylated to the organic pentavalent arsenicals, MMAV1 and DMAV, in humans. DMAV is the major metabolite formed after exposure to inorganic arsenic in many, but not all, mammalian species (3). Methylation of inorganic arsenic occurs via alternating reduction of pentavalent to trivalent arsenic followed * To whom correspondence should be addressed. Tel: (81)6-66453751. Fax: (81)6-6646-0722. E-mail: [email protected]. † Department of Preventive Medicine and Environmental Health, Osaka City University Medical School. ‡ First Department of Pathology, Osaka City University Medical School. § Division of Customer Training Group Application Center, Yokogawa Analytical Systems, Inc.. 1 Abbreviations: MMAV, monomethylarsonic acid; DMAV, dimethylarsinic acid; GSH, glutathione; MMAIII, monomethylarsonous acid; DMAIII, dimethylarsinous acid; AsIII, arsenite; AsV, arsenate; TMAO, trimethylarsine oxide; TeMA, tetramethylarsonium; Cys, cysteine; AsBe, arsenobetaine; LC-ICP-MS, liquid chromatography with inductively coupled plasma mass spectrometry; SOD, superoxide dismutase; PBN, R-phenyl-N-tert-butyl nitrone; ROS, reactive oxygen species; 8-OHdG, 8-hydroxy-2′-deoxyguanosine.

by addition of a methyl group. GSH (4) plays an important role in this reduction. AsV reductase (5) and MMAV reductase (6) catalyze the reduction in a reaction. The methylation pathway of inorganic arsenic has generally been considered to be a detoxification process, because methylated pentavalent arsenic compounds have lower toxicity and lower affinity for tissue constituents (7). However, recent findings suggest that methylation also converts inorganic arsenicals to metabolites with acute toxic or carcinogenic effects. Methylated arsenicals have unique biological effects. DMAV is a tumor promoter and a complete carcinogen of the bladder in rats (8, 9). DMAV is also both fetotoxic and teratogenic in rats and mice and genotoxic in mammalian and human cells (10). Trivalent methylated arsenicals, MMAIII and DMAIII, which are proposed to be intermediates in the methylation pathway, are more cytotoxic (11, 12) and genotoxic (13) than inorganic arsenicals. The presence of MMAIII or DMAIII has recently been confirmed in the urine of humans chronically consuming arsenic-contaminated water (14, 15), in the urine of DMAV-treated rats (12), in the bile of rats injected with AsIII or AsV (16), and in hamster tissue after administration of AsV (17). Studies in the past decade have indicated that DMAV has a multiorgan tumor-promoting activity in rats and that it is a complete carcinogen in the urinary bladder

10.1021/tx030008x CCC: $25.00 © 2003 American Chemical Society Published on Web 08/14/2003

Sulfur-Containing Metabolite in DMAV-Treated Rats

of rats. Although excretion and accumulation of arsenic in rats have been reported to differ from that in many other species because of high affinity of DMA to rat hemoglobin (3), the biotransformation of arsenic in the rat is similar to other animals (18). The proportion of urinary arsenic species is considered a reliable indicator of arsenic metabolism in mammals (19). Thus, to better understand the mechanism of urinary bladder carcinogenecity of DMAV, it is important to investigate the urinary metabolites excreted by rats chronically exposed to DMAV. Unidentified metabolites, M-1 and M-2, were detected in urine after long-term oral administration of DMAV, MMAV, AsIII, or TMAO in our previous study (20). The amounts of excretion of M-1 and M-2 were higher in rats administered DMAV than in other arsenictreated rats. A further unidentified metabolite, M-3, has been detected in feces as a metabolite of DMAV after 20 weeks of exposure to DMAV (21). Marafante et al. (22) reported that an unidentified DMA complex was detected in urine and feces after oral administration of DMAV to mice and hamsters. Hughes and Kenyon (23) also reported an unstable metabolite that was easily oxidized to DMAV in the urine of mice after intravenous administration of DMAV. They suggested that these unknown compounds might be DMA-thiol complexes since DMAV easily react with thiols. Furthermore, they suggested that such DMA complexes might be intermediates in further methylation to TMAO in the liver. However, we have reported that there was no difference in the amounts of production of M-1 or M-2 between L-buthionine-SRsulfoximine-pretreated rats and controls and that the amounts of excretion of M-1 and M-2 after intraperitoneal administration were less than those after oral administration. These results suggested that M-1 and M-2 might be produced not in the liver but in the intestinal tract (21). We have also reported that the unidentified metabolites M-1, M-2, and M-3 and unmetabolized DMAV were excreted mainly as fecal metabolites after chronic oral administration of DMAV (21). This finding also suggested that M-1, M-2, and M-3 might be produced in the intestinal tract. An Escherichia coli strain A3-6, which metabolized DMAV to M-2 and M-3, was isolated from the cecum of rats administered 100 mg/L of DMAV via drinking water for 9 months (24). The E. coli A3-6 converted DMAV to M-2 and M-3 in GAM medium for anaerobes and converted TMAO to M-1 (24). Kuroda et al. (24) reported that the conversion of DMAV to M-2 and M-3 by E. coli A3-6 in bouillon medium required Cys, suggesting that Cys was involved in the conversion of DMAV to M-2 or M-3. The production of methylated trivalent arsenicals or their complexes is an attractive mechanism for triggering carcinogenic effects of DMAV in rats. In the present study, the mechanisms of production of the unknown metabolites in rats or E. coli A3-6 were examined. The cytotoxicities and chemical properties of the unknown metabolites were also examined.

Materials and Methods Reagents. Sodium AsIII, sodium AsV, MMAV, DMAV, TMAO, TeMA iodide, and AsBe, with purities of at least 99.99%, were obtained from Tri Chemical Lab (Yamanashi, Japan). Iododimethylarsine was obtained from Dr. Cullen (University of British Columbia, Vancouver, Canada) and was used to prepare a standard solution for LC-ICP-MS of DMAIII. Bouillon medium was purchased from Nissui (Tokyo, Japan). Other

Chem. Res. Toxicol., Vol. 16, No. 9, 2003 1125 chemicals (analytical grade) were obtained from Wako Pure Chemical Industry (Osaka, Japan). Reduction of DMAV. Reduction of DMAV was carried out with metabisulfite-thiosulfate reagent essentially according to procedures previously described by Reay and Asher (25). Briefly, the reducing solution (0.28 g of sodium metabisulfite, 15 mL of water, 2 mL of 1% sodium thiosulfate, and 0.1 mL of 36 N H2SO4) was added in a ratio of 2:1 (v/v) to 10 mM DMAV. The reduced mixture was allowed to stand at room temperature for 3 h in a tightly capped tube. This reaction mixture was analyzed by LC-ICP-MS and LC-MS. Microorganism Culture. E. coli A3-6 (24) suspension was prepared as follows. E. coli A3-6 was precultured overnight in bouillon medium with Cys added. E. coli A3-6 was suspended in phosphate buffer (pH 7.4) of equal volume to the bouillon medium after washing twice by centrifugation. To examine the effects of Cys concentration on conversion of DMAV or TMAO, 1 mM DMAV or TMAO and various concentrations of Cys were added to E. coli A3-6 suspension at 37 °C. At 1 or 6 h after incubation, the culture media were centrifuged. The supernatants were ultrafiltered using an Ultrafree-MC (Millipore, MA) with a cutoff value of 10 kDa, and the filtrate was stored at -20 °C until analysis. To study the temporal pattern of conversion of DMAV to M-2 or M-3 by E. coli A3-6, 1 mM DMAV and 2 or 3 mM Cys were added to the E. coli A3-6 suspension at 37 °C. At 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, and 6 h after incubation, a portion of culture media was withdrawn and centrifuged. The supernatants were ultrafiltered using an Ultrafree-MC, and the filtrate was stored at -20 °C until analysis. Separation of Arsenic Compounds by LC-ICP-MS. Analysis of arsenic compounds using LC-ICP-MS was carried out as reported previously (21). A model HP4500 ICP-MS (HewlettPackard, DE) was used for arsenic specific detection. A model IC (Yokogawa Analytical Systems, Tokyo, Japan) was used for separating arsenic species. For separation of arsenic compounds, two separation modes, cation and anion exchange, were used. The cation mode experiment, using a Shodex RSpak NN-614 column (150 mm × 4.6 mm i.d.) packed with cation exchange resin (Showadenko, Tokyo, Japan), was performed under the following conditions: mobile phase, 5 mM HNO3-6 mM NH4NO3; flow rate, 0.8 mL/min; and injection volume, 50 µL. The anion mode experiment, using an Excelpak ICS-A13 column (75 mm × 4.6 mm i.d.) packed with anion exchange resin (Yokogawa Analytical Systems), was performed under the following conditions: mobile phase, 3 mM NaH2PO4 at pH 6 with NaOH; flow rate, 0.8 mL/min; and injection volume, 50 µL. The ICP-MS detection mass was set to m/z 75 (75As+), m/z 72 (72Ge+), and m/z 77 (40Ar37Cl+). The ion intensity at m/z 72 was of an internal standard value to obtain precise measurements. The ion intensity at m/z 77 was of diagnostic value only in the examination for the possible occurrence of 40Ar35Cl+ interference at m/z 75. A standard solution of DMAIII was prepared by dissolving iododimethylarsine in pure water and stirring for 30 min under a nitrogen atmosphere just before use. Solutions of other standard arsenic compounds, sodium AsIII, sodium AsV, MMAV, DMAV, TMAO, and TeMA iodide, were prepared as described previouly (21). The final diluted aqueous standard mixtures were prepared from each stock standard just before use. Analysis of Reactants of DMAV with MetabisulfiteThiosulfate Reagent by LC-MS. The LC-MS system consisted of an L7000 series HPLC instrument (Hitachi, Tokyo, Japan) and LCQ ion trap mass spectrometer (ThermoFinnigan, Waltham, MT) equipped with an electrospray ionization LC-MS interface. The ion source was operated in the positive ion mode. The chromatography was performed using an RSpak NN-614 cation exchange column (Shodex; 150 mm × 4.6 mm i.d.) at 40 °C equilibrated with a mobile phase consisting of 36 mM formic acid-2 mM ammonium formate buffer, a flow rate of 0.8 mL/ min, and an injection volume of 5 µL. Full scan LC-MS spectra were recorded, and the protonated molecules were identified.

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Figure 1. Conversion of DMAV to M-2 and M-3 by E. coli A36. One millimolar DMAV and (A) 2 or (B) 3 mM Cys were added to E. coli A3-6 suspension in phosphate buffer at 37 °C. At 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, and 6 h after incubation, a portion of the culture media was withdrawn and centrifuged. Cell Viability Assay. HL-60 cells (Institute for Fermentation, Osaka, Japan) were seeded at 20 × 104 cells/mL in 96 well microplates (Coster) and were preincubated for 22 h in PRMI1640 medium (Sigma-Aldrich, Japan). The cells were treated with the culture mixtures of DMAV, Cys, and E. coli A3-6 obtained by the foregoing procedure and were incubated for 24 h in the presence or absence of antioxidants, SOD, catalase, or a scavenger of free radicals, PBN. Cell viability was assayed by ATP bioluminescense assay (VIALIGHT HS, Lumitech Ltd., U.K.) using a Wallac 1420 ARVOsx (Amersham Pharmacia Biotech, Japan).

Results Conversion of DMAV or TMAO by E. coli. The effects of Cys concentrations on conversion of DMAV to M-2 and M-3 by E. coli A3-6 after 6 h of incubation were investigated. When the Cys/DMAV molar ratio was increased from 0.5 to 3, the concentration of DMAV in culture media decreased rapidly. The maximum concentration of M-2 was observed at a Cys/DMAV ratio of 2, and the maximum of M-3 was observed when the ratio exceeded 3 (data not shown). The time course of change in metabolites of DMAV by E. coli A3-6 in the presence of Cys was investigated. As shown in Figure 1A, DMAV was rapidly converted to M-2 and was converted to M-3 more gradually at a Cys/DMAV ratio of 2. At a Cys/DMAV ratio of 3 (Figure 1B), the maximum concentration of M-2 was observed between

Yoshida et al.

45 and 90 min, while that of M-3 was after 120 min. DMAIII appeared within 45 min. These results indicated that E. coli A3-6 reduced DMAV to DMAIII to form M-2 in the presence of Cys and that M-2 was further converted to M-3. The effects of Cys concentrations on conversion of TMAO to M-1 by E. coli A3-6 were investigated after 1 h of incubation and after 6 h of incubation. After 1 h of incubation, approximately 100% of TMAO was converted to M-1 at Cys/TMAO ratios above 2, whereas after 6 h of incubation approximately 100% of TMAO was converted to M-1 at ratios above 1. The rate of conversion of TMAO to M-1 by E. coli A3-6 was faster than the rate of conversion of DMAV to M-2 and M-3 (data not shown). Cytotoxicity of Unidentified Metabolites. In preliminary experiments, we examined the relative toxicity of metabolites to which E. coli A3-6 converted DMAV and TMAO in the presence of Cys, using hemocytometry in V79 cells. The samples were tested without purification because arsenic metabolites produced by E. coli A3-6 were rather unstable. The experiments showed that samples rich in M-2 were more toxic than DMAV. The samples rich in M-1 or M-3 did not exhibit cytotoxicity. These results indicated that M-2 was the most toxic of all of the metabolites produced by E. coli A3-6. To further investigate the cytotoxicity of M-2, HL-60 cells were incubated with the metabolites produced by E. coli A3-6 at a Cys/DMAV ratio of 1 or 3 and with DMAV at various concentrations for 24 h, and cell viability was determined at the end of incubation by ATP bioluminescense assay. The ID50 values, that is, the arsenic concentrations of samples that inhibited growth of the cells by 50% as compared to growth of untreated control cells, for metabolites produced by E. coli A3-6 at a Cys/DMAV ratio of 1 and 3 were 10.7 and 10.6 µM, respectively, whereas that of DMAV was 610 µM. We examined the effects of antioxidants and a scavenger of free radicals on the cytotoxicity of DMAV and the metabolites produced by E. coli A3-6 at a Cys/DMAV ratio of 1 or 3. As shown in Figure 2, suppression of cytotoxicity by SOD was observed. The other antioxidant and PBN, a scavenger of hydroxyl radicals, had no effects on cytotoxicity. Chemical Identification of Unidentified Metabolites by LC-ICP-MS. Several approaches were used to investigate the chemical properties of M-1, M-2, and M-3 in the cultures. To determine whether the metabolites are tri- or pentavalent arsenicals, the culture mixtures prepared for studies of conversion of DMAV or TMAO by E. coli A3-6 were oxidized by hydrogen peroxide and reanalyzed by LC-ICP-MS. After exposure to hydrogen peroxide, the peaks of M-2 and M-3 disappeared on the chromatograms and only the DMAV peak appeared in the sample obtained from the culture mixtures of DMAV, Cys, and E. coli A3-6, suggesting that hydrogen peroxide oxidized M-2 and M-3 to DMAV. Hydrogen peroxide also eliminated the M-1 peak from the culture mixture of TMAO, Cys, and E. coli A3-6 and resulted in the appearance of the TMAO peak alone. Because this observation indicated that M-2 and M-3 may be a reduced form of DMAV, the chromatographic properties of M-2 and M-3 were compared with those of DMAIII or the reactants of DMAV reduced by metabisulfite-thiosulfate reagent by LC-ICP-MS using two separation modes. Metabisulfite-thiosulfate reagent is used by some researchers to prepare trivalent methylated

Sulfur-Containing Metabolite in DMAV-Treated Rats

Figure 2. Effects of SOD, catalase, and PBN on cytotoxicity of metabolites of DMAV by E. coli A3-6. E. coli A3-6 and 1 mM DMAV were incubated with 1 or 3 mM Cys in phosphate buffer at 37 °C for 6 h. E. coli A3-6 was removed by centrifugaion and ultrafiltration. The solution was diluted with water and administered to HL-60 cells with SOD (0.1 mg/mL), catalase (0.3 mg/ mL), or PBN (100 µM). Afer 24 h, cell viability was assayed by ATP asssay. Values represent the mean ( SD (n ) 4). (**) Cell viability is statistically different (p < 0.01) from control (antioxidant- or PBN-untreated) cells by a Student’s t-test. (*) Cell viability is statistically different (p < 0.05) from control (antioxidant- or PBN-untreated) cells by a Student’s t-test.

arsenicals (MMAIII and DMAIII) by reducing the corresponding pentavalent arsenic species (15-17, 26). Reduction of DMAV by metabisulfite-thiosulfate reagent produced a single product peak, P-X, and a trace of unchanged DMAV. The retention time of M-2 was consistent with the peak P-X but not that of DMAIII on anion exchange LC-ICP-MS chromatograms (Figure 3). These findings were also obtained in cation exchange LC-ICP-MS analysis (data not shown). These results indicated that M-2, which was the most toxic of all the unknown metabolites, was the same compound as P-X. Chemical Characteristic of Unidentified Metabolite M-2. Figure 4 shows the LC-MS spectra for P-X of reactants of DMAV reduced by the metabisulfite-thiosulfate reagent. The protonated molecule [M + H]+ signal of the unknown metabolite was detected at m/z 154.9. This result indicated that the molecular weight of M-2 is 154. We also examined M-2 in the sample obtained from the culture mixtures of DMAV, Cys, and E. coli A3-6 by LC-MS. In the negative spectrum of M-2, a strong fragment was detected at m/z 153 (Figure 5). The result

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Figure 3. Comparison of M-2 with the reactant P-X of DMAV reduced by metabisulfite-thiosulfate reagent and DAMIII. Anion exchange LC-ICP-MS chromatograms of (A) the culture mixture of DMAV by E. coli A3-6 in the presence of Cys, (B) the reactants of DMAV reduced by metabisulfite-thiosulfate reagent, and (C) DMAIII standard solution. Column, Excelpak ICS-A13 column (75 mm × 4.6 mm i.d.), was performed under the following conditions: mobile phase, 3 mM NaH2PO4 at pH 6 with NaOH; flow rate, 0.8 mL/min.

Figure 4. LC-MS spectra of P-X in the positive ion mode. Chromatography was performed using an RSpak NN-614 cation exchange column (150 mm × 4.6 mm i.d.) and a mobile phase of 36 mM formic acid-2 mM ammonium formate at 40 °C and a flow rate of 0.8 mL/min.

indicated that the molecular weight of M-2 is 154. These results clearly indicated that M-2 was the same compound as P-X. To examine whether M-2 contained sulfur or not, the detection mass was set to m/z 75 (75As+) and m/z 34 (34S+), and M-2 was determined by LC-collision cell ICP-MS.

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Figure 5. LC-MS spectra of M-2 in the negative ion mode. Chromatography was performed using an Excelpak ICS-A23 anion exchange column (75 mm × 4.6 mm i.d.) and a mobile phase of 6 mM formate buffer (pH 5.5) at 40 °C and a flow rate of 1.0 mL/min.

The peaks at m/z 75 and m/z 34 appeared on the retention time of M-2. The molar ratio of As to S in M-2 was confirmed to be 1:1 (data not shown). The results indicated that M-2 contained a sulfur.

Discussion Our previous studies indicated that long-term exposure to AsIII, MMAV, and DMAV increases excretion of the unidentified metabolites, M-1 and M-2, in urine (20). Thus, studies of the unidentified metabolites may provide clues to the mechanism of carcinogenesis of arsenic in humans. Methylarsenicals are easily reduced to their trivalent derivatives with sulfhydryls such as GSH, Cys, and lipoic acid (27, 28). Tsao and Maki (29) indicated that the reactive derivative (CH3)2AsSR was formed by the reduction of DMAV by a thiol and the derivative bound to EcoRI methyl transferase by mercaptide exchange with a Cys residue located close to a tryptophan site. In the present study, DMAV was reduced to DMAIII to form M-2 by Cys and E. coli A3-6 (Figure 1), suggesting that M-2 might be a trivalent DMA derivative. This hypothesis is supported by our findings that M-2 was oxidized to DMAV by hydrogen peroxide and that the retention time of M-2 on LC-ICP-MS chromatograms was consistent with that of P-X of reactants of DMAV obtained by the metabisulfite-thiosulfate reduction (Figure 3). Recent studies have suggested that trivalent methylated arsenicals are highly cytotoxic (11, 12) and able to interact directly with DNA to produce genotoxic effects (13). DMAIII and MMAIII were more potent inhibitors of sulfhydryl enzyme than their pentavalent analogues or inorganic trivalent arsenic (30). We examined the chemical properties of M-2 by LC-MS and LC-ICP-MS. The molecular weight of the M-2 metabolite of DMA produced by E. coli in the presence of Cys was consistent with that of P-X of the reactants of DMAV obtained by metabisulfite-thiosulfate reduction; the molecular weight of both was 154 (Figures 4 and 5). These results clearly indicate that P-X is the same compound as M-2. P-X was prepared according to the procedures previously described by Reay and Asher (25). The reaction of DMAV with metabisulfite-thiosulfate reagent has been used to prepare the standard of DMAIII by some research groups, although the reduced arsenic species have not been well-characterized (26). In addition, we found that M-2 was the arsenic compound containing a sulfur. It is of interest that a sulfur-containing metabolite is detected in urine following oral administration of DMA to rats. Wanibuchi et al., our co-workers, indicated that DMAV had promoting effects on urinary bladder in rats and that

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a large amount of unidentified metabolite M-2 was excreted in the urine of rats (31). On the basis of these results, they suggested that unidentified metabolite M-2 might play a particular role in the urinary bladder carcinogenesis induced by arsenic in rats (31). The hypothesis was supported by our findings that M-2 was more cytotoxic than DMAV. M-2-induced cytotoxicity was decreased by the addition of SOD (Figure 2). This finding suggests that the production of ROS may play an important role in the cytotoxicity of M-2. Recent studies indicated that oral administration of DMAV to rats increased formation of 8-OHdG, one of the major ROSinduced DNA base-modified products, in liver (32), kidney (33), and urinary bladder (9). 8-OHdG is widely accepted as a sensitive marker of oxidative DNA damage and oxidative stress. The same authors showed that an increase in 8-OHdG level was detected in liver, kidney, and urinary bladder, which are target organs for rat arsenic carcinogenesis. Ahmad et al. (34) reported that DMAV and DMAIII significantly released iron from ferritin with or without ascorbic acid and that DMAIII caused iron-dependent formation of ROS. ROS directly or indirectly caused a broad range of DNA damage, and the DNA damage by DMAIII was increased in the presence of ascorbic acid (35). The authors proposed that this ROS pathway could be a mechanism of action of arsenic carcinogenesis. Oxidative stress may be involved in either initiation, promotion, or progression (36). The results of the present study suggest that ROS produced by M-2 might play a role in DMAV carcinogenesis.

Acknowledgment. We thank Mieko Yoshimura, Department of Preventive Medicine and Environmental Health, Osaka City University Medical School, for her excellent technical assistance. We thank Osaka Laboratory, Sumika Chemical Analysis Service, Ltd., for the excellent LC-MS analyses of reactants of DMAV obtained by metabisulfite-thiosulfate reduction.

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