Dimethylthioarsenicals as Arsenic Metabolites and Their Chemical

Chiba 263-8555, Japan, Chemical Analysis Center, Chiba University, Chiba ... Nihon University College of Pharmacy, Funabashi, Chiba 274-8555, Japan, ...
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Dimethylthioarsenicals as Arsenic Metabolites and Their Chemical Preparations Kazuo T. Suzuki,*,† Badal K. Mandal,†,‡ Akio Katagiri,† Yoko Sakuma,† Ayumi Kawakami,† Yasumitsu Ogra,† Kentaro Yamaguchi,§ Yoshihisa Sei,§ Kenzo Yamanaka,| Kazunori Anzai,‡ Masayoshi Ohmichi,⊥ Hiromitsu Takayama,# and Noriro Aimi# Department of Toxicology and Environmental Health, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan, National Institute of Radiological Sciences, Chiba 263-8555, Japan, Chemical Analysis Center, Chiba University, Chiba 263-8522, Japan, Nihon University College of Pharmacy, Funabashi, Chiba 274-8555, Japan, Chiba City Institute of Health and Environment, Chiba 261-0001, Japan, and Department of Molecular Structure and Biological Function, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 263-8522, Japan Received January 20, 2004

Two unidentified arsenic metabolites were detected in the liver of rats on a gel filtration column by HPLC inductively coupled argon plasma mass spectrometry after an injection of dimethylarsinic (DMAV), dimethylarsinous (DMAIII), monomethylarsonic (MMAV), or monomethylarsonous (MMAIII) acid. The same arsenicals were also produced in vitro by incubation of DMAIII in the liver supernatant but not by DMAV. The two arsenic metabolites eluted at the same retention times as those of the two arsenicals prepared by reaction of DMAV with either thiosulfate plus disulfite or hydrogen sulfide or sodium sulfide plus sulfuric acid. The faster and slower eluting products on a gel filtration column were assigned as dimethyldithioarsinic acid (dimethylarsinodithioic acid) (DMTAV) and dimethylthioarsinous acid (DMTAIII) from mass spectrometric data at m/z ) 170 and 138 by electrospray ionization mass spectrometry with negative and positive ion modes, respectively. They were prepared selectively by reacting DMAV with hydrogen sulfide or sodium sulfide plus sulfuric acid under different reaction conditions. DMAIII but not DMAV was transformed to DMTAIII and DMTAV in the presence of sodium sulfide in vitro, suggesting that DMAV is reduced to DMAIII with hydrogen sulfide, thiolated to DMTAIII, and then further thiolated oxidatively to DMTAV. Metabolically, it is assumed that DMAIII is transformed to DMTAIII in the presence of sulfide ions, and then, DMTAIII is oxidatively thiolated to DMTAV. As the chemical species produced by reduction with the Reay and Asher method are DMTAIII and DMTAV, and different from DMAIII, the studies carried out with DMAIII with the Reay and Asher method have to be reexamined.

Introduction Several millions of people in the world are now affected by arsenic from drinking naturally occurring arseniccontaminated groundwater (1). The chemical species of arsenic in arsenic-contaminated drinking water are inorganic arsenicals, mostly iAsIII 1 and iAsV, which are known to cause acute and chronic effects including cancers in humans (2-9). Inorganic arsenicals are transformed in the body by consecutive reduction and oxidative methylation reactions leading to dimethylated arsenicals (10-13), as schematically demonstrated as a part of the proposed metabolic pathway in Scheme 1. Arsenic taken up by the * To whom correspondence should be addressed. Tel/Fax: +81-43226-2865. E-mail: [email protected]. † Department of Toxicology and Environmental Health, Chiba University. ‡ National Institute of Radiological Sciences. § Chemical Analysis Center, Chiba University. | Nihon University College of Pharmacy. ⊥ Chiba City Institute of Health and Environment. # Department of Molecular Structure and Biological Function, Chiba University.

body is mostly excreted into the urine in the form of DMAV (12-17). In the liver, it is known that about 50% of the iAsIII taken up by the liver is excreted into the bile after being conjugated with GSH to iAsIII(GS)3 and MMAIII(GS)2 through MRP2/cMOAT (18-20). The GSHconjugated arsenicals get into the hepato-enteric circulation or are excreted into feces (21). On the other hand, DMAIII or its GSH-conjugated form [DMA(SG)] is not excreted into the bile (18, 21, 22). Therefore, it is assumed that DMA is excreted in some form from the liver, mainly into the bloodstream, and then into urine. Recently, Kala et al. (23) reported that while iAsIII and MMAIII were 1 Abbreviations: AsB, arsenobetaine; iAsV, arsenate; iAsIII, arsenite; DMI, dimethylarsinous iodide; DMAIII, dimethylarsinous acid; DMAV, dimethylarsinic acid; DMTAIII, dimethylthioarsinous acid; DMTAV, dimethyldithioarsinic acid or dimethylarsinodithioic acid; MMI2, monomethylarsonous diiodide; MMAIII, monomethylarsonous acid; MMAV, monomethylarsonic acid; GSH, glutathione; iAsIII(GS)3, arsenotriglutathione; MMAIII(GS)2, monomethylarsenodiglutathione; DMAIII(GS), dimethylarsenoglutathione; MRP2/cMOAT, multidrug resistance protein 2/canalicular multispecific organic anion transporter; ROS, reactive oxygen species; RBCs, red blood cells; HPLC, high-performance liquid chromatography; ICP MS, inductively coupled argon plasma mass spectrometry.

10.1021/tx049963s CCC: $27.50 © 2004 American Chemical Society Published on Web 06/10/2004

Dimethylthioarsenicals in As Metabolism Scheme 1. Proposed Metabolic Pathway for Arsenic in the Liver and a Possible Role of DMTAsa

a DMTAIII (10), Me AsSH; DMMTAV (11), dimethylthioarsinic 2 acid [Me2As(dS)OH] or dimethylarsinothioic acid [Me2As(dO)SH]; V DMTA (12), Me2As(dS)SH.

excreted in the GSH-conjugated forms into urine in mice deficient in γ-glutamyl transpeptidase, DMAIII was not detected in a form conjugated with GSH. Thus, the precise metabolic pathway by which the major portion of arsenic taken up by the body is excreted into urine in the form of DMAV is not yet known. Namely, it is not clear how arsenic is effluxed from the liver after the first pass and excreted into the urine or redistributed in other organs/tissues before being excreted into urine. While trying to reveal the precise metabolic pathway of arsenic based on chemical speciation of each metabolite, we detected two unidentified metabolites in the liver after injections of DMAV and MMAV (21). In parallel with the identification of these unidentified metabolites, we have tried several methods to prepare DMAIII by a convenient procedure. Although a single arsenic peak was detected by reduction of DMAV according to the Reay and Asher method (24) on an anion exchange column (25, 26), it was separated into two peaks on a gel filtration column. However, neither of the two arsenicals was identical with DMAIII of DMI (27) origin (our unpublished data and the present study). Furthermore, the two synthetic arsenicals were identical with the two unidentified metabolites, which were reported by Suzuki et al. (21). Our present study was focused on the identification of the two unidentified metabolites in relation to the chemical preparation of the two arsenicals. The reduction of DMAV was carried out according to the Reay and Asher method (24) with modification of reaction conditions. Although a single reaction product was detected with differing yields on an anion exchange column, it was not quantitative. Furthermore, as neither of the two peaks separated on a gel filtration column was identical with the authentic DMAIII of DMI (27) origin, we tried to reduce DMAV with more vigorous reduction

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conditions using hydrogen sulfide or sodium sulfide plus sulfuric acid. These conditions gave quantitatively the same two arsenicals as those of the Reay and Asher method with different ratios between the two products. However, DMAIII was not detected in the reaction mixtures. Then, an alternative convenient preparation method for DMAIII was proposed in this communication by reduction with L-cysteine (Cys). Meanwhile, the two reaction products were determined to be identical with the two unidentified metabolites on a gel filtration and an anion exchange column, and the determination of their chemical structures was carried out. The two reaction products were shown to contain sulfur by simultaneous determination of arsenic and sulfur by HPLC-ICP MS, which showed the best fit S/As ratios as 1/1 and 2/1 (data not shown), and they were named as dimethylthioarsenicals (DMTAs). During our study on DMTA, Yoshida et al. (28) reported that one of the two arsenic metabolites transformed in the intestine after long-term oral administration of DMAV was monothioarsenical with a molecular mass of 154, having a higher mutagenicity than the other one, which was confirmed to be a dithioarsenical with a molecular mass of 170 (29). Still now, their structures have not been established or identified. The major problem in the determination of chemical structure of trivalent arsenicals such as MMAIII and DMAIII is the difficulty in obtaining mass spectral data. In the present study, we show the structures of the two unidentified metabolites (DMTA-1 and DMTA-2) as DMTAIII and DMTAV, respectively. The chemical synthesis of DMTA and related reactions are described, including a convenient preparation of DMAIII. The metabolic significance of the two DMTAs was also discussed.

Materials and Methods Reagents. All reagents were of analytical grade. Milli-Q water (Millipore) was used throughout. Cys, sodium sulfide (Na2S), sodium thiosulfate, sodium disulfite, sodium iAsIII (NaAsO2) (iAsIII), and DMAV [(CH3)2AsO(OH)] were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), while MMAV [(CH3)AsO(OH)2] and AsB were purchased from Tri Chemical Laboratories, Inc. (Yamanashi, Japan). The arsenic standard solution (1000 ppm) for ICP MS was purchased from SPEX CentiPrep (Metuchen, NJ). Stock solutions of all arsenic compounds (10 mmol/L) were prepared from the respective standard compounds. All stock solutions were stored in the dark at 0 °C. Dilute standard solutions for analysis were prepared daily prior to use. Apparatus. ICP MS (HP4500; Yokogawa Analytical Systems, Hachiouji, Japan), HPLC (PU 610; GL Sciences Co., Tokyo, Japan), 1H NMR (600 MHz; JEOL JNM-ECP 600, Tokyo), and an electrospray ionization mass spectrometer (ESI-MS) (JMS700, JEOL, Tokyo) with negative and positive ion modes were operated under the respective standard conditions. Analytical Procedures. For HPLC-ICP MS analysis, a 20 µL aliquot of a sample solution was applied to a polymer-based gel filtration column (Shodex Asahipak GS-220 HQ, 300 mm × 7.6 mm i.d., exclusion limit >3000, Showa Denko, Tokyo) or an anion exchange column (Shodex Asahipak ES-502N 7C, 100 mm × 7.6 mm i.d.). The column was eluted with 50 mM ammonium acetate buffer (pH 6.5 at 25 °C) or 15 mM citric acid buffer (pH 2.0 at 19 °C) for the gel filtration or anion exchange column, at a flow rate of 0.6 or 1.0 mL/min. The outlet of the HPLC system was coupled directly (with 300 mm × 0.25 mm i.d. long PEEK tubing) to the inlet of the ICP nebulizer. The signal at m/z 75 was monitored for arsenic. The signal at m/z 77 was also

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monitored to compensate the molecular interference of ArCl+. On-line ICP MS data were processed with software developed in-house. ESI MS Analyses. ESI MS analyses were performed in both negative and positive modes using a four-sector (BE/BE) tandem mass spectrometer (JMS-700, JEOL) equipped with a cold-spray ionization (CSI) source (30). Nitrogen was used as a nebulizing gas at a pressure of 50 psi and a temperature of 200 °C. Typical ESI measurement conditions were as follows: acceleration voltage, 5.0 kV; needle voltage, 1.6 kV; needle current, -100-0 nA; orifice voltage, 40-73 V; resolution (10% valley definition), 1000; sample flow rate, 0.5 mL/h; solvent, methanol:ethanol (1:9); concentration, 0.25 mmol; spray temperature, 20 °C; ion source temperature, 200 °C for negative mode (ESI-); and acceleration voltage, 5.0 kV; needle voltage, 3.6 kV; needle current, 6000-7000 nA; orifice voltage, 70-94 V; resolution (10% valley definition), 1000; sample flow rate, 0.5 mL/h; solvent, chloroform; concentration, 0.20 mmol; spray temperature, 20 °C; ion source temperature, 200 °C for positive mode (ESI+). Simultaneously, we checked our compounds using a CSI source. Animal Experiments. Male Wistar rats were purchased at 5 weeks of age from a breeder (Clea Japan Co., Tokyo). They were housed in a humidity-controlled room, maintained at 2225 °C with a 12 h light-dark cycle. The animals were fed a commercial diet (CE-2; Clea Japan Co.) and tap water ad libitum. Following a 1 week acclimation period, the animals, at 6 weeks of age and weighing 160-180 g, were used for experiments. The rats were injected with arsenic at a single dose of 0.5 mg As/kg body weight through the portal vein under pentobarbital anesthesia and then sacrificed 5 min after the injection by heart puncture. The liver was excised after the whole body perfusion. Immediately before the injection, DMAV and MMAV were dissolved in saline (Otsuka Pharmaceutical Co., Ltd., Naruto, Japan). DMAIII and MMAIII were prepared by reducing DMAV and MMAV, respectively, with 5 molar equiv of Cys in distilled water at 70 °C for 1.5 h. They were confirmed by comparing with those prepared from their iodide forms (27) on a gel filtration column by HPLC-ICP MS. The arsenic solutions were adjusted to 1.0 mL/kg body weight. The rats were sacrificed 5 min after the injection. Whole Body Perfusion. As arsenic accumulates preferentially in RBCs in rats, the concentrations of arsenic in organs and tissues were determined after completely removing blood (RBCs) by whole body perfusion. Whole body perfusion was performed as already reported elsewhere (26) except that the duration of the perfusion was shortened to 20 min. Preparation of Liver Supernatant. Livers were homogenized in 4 vol of the extraction buffer (50 mM ammonium acetate buffer, pH 7.4) with a glass-Teflon homogenizer in an atmosphere of nitrogen. The homogenates were centrifuged at 105000g for 60 min at 4 °C to obtain a supernatant fraction. Preparation of DMTAs by Hydrogen Sulfide. DMTAIII (DMTA-1) was prepared by stepwise addition of H2SO4 to a purified water solution containing DMAV and Na2S at the final molar ratio of DMAV:Na2S:H2SO4 ) 1:1.6:1.6 in a nitrogen atmosphere and was allowed to stand for 1 h. DMTAIII was extractable in organic solvents such as ether and chloroform, while DMAV was not. DMTAV (DMTA-2) was prepared similarly to DMTAIII except for the molar ratio of DMAV:Na2S:H2SO4 ) 1:7.5:7.5 and for a prolonged reaction time (1 day) in an air atmosphere or simply by bubbling H2S gas into an ethanol solution of DMAV for 1 day. The reduction of DMAV by sodium thiosulfate and sodium disulfite (the Reay and Asher method) was carried out according to Reay and Asher (24). Typically, an equivolume of a 10 mM DMAV solution and a reducing solution were prepared by adding 2 mL of 1% sodium thiosulfate and 0.1 mL of concentrated H2SO4 to a 280 mg of sodium metabisulfite solution in 15 mL of water.

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Results Detection of Two Unidentified Arsenic Metabolites in Vivo in the Liver after a Single Intravenous Injection of Methylated Arsenicals. The trivalent arsenicals (DMAIII and MMAIII) prepared by dissolving and hydrolyzing the corresponding iodide forms (DMI and MMI2) (27) in water were partly but readily oxidized to the corresponding pentavalent forms during the preparation procedure for the administration, and it was hard to avoid the pentavalent form. Therefore, DMAIII and MMAIII were prepared by reducing their pentavalent forms with 5 molar equiv of Cys at 70 °C for 1.5 h and were administered to rats in the presence of the unreacted Cys and oxidized cystine. DMAIII and MMAIII in these solutions were stable during the administration procedure, as analyzed by the HPLC-ICP MS method. A single injection (via the portal vein) of four kinds of methylated arsenicals (DMAV, DMAIII, MMAV, and MMAIII) was administered to rats at a dose of 0.5 mg As/ kg body weight, and the rats were sacrificed 5 min after the injection. Arsenic was taken up by the liver at less than 10% of the dose in all arsenicals due to preferential distributions to the urine in the case of the pentavalent arsenicals and to RBCs in the case of the trivalent ones. The following sample preparation and analytical procedures were carried out in an atmosphere of nitrogen after removing dissolved air in the extraction and elution buffer solutions by bubbling nitrogen gas or degassing dissolved air to minimize the possible oxidation of the metabolites. The supernatants prepared from the 20% liver homogenates were subjected to the HPLC-ICP MS analysis on a gel filtration column of GS 220 to reveal the distribution of arsenic after the administration of the four arsenicals. The present column is basically a size exclusion column for the separation of low molecular weight compounds (exclusion limit >3000). Low molecular weight arsenicals were well-separated under the present conditions, as seen in Figure 1: DMAV (14.7), DMAIII (29.8), MMAV (13.8), and MMAIII (21.5 min), the void volume of the column and AsB of diet origin being 9.8 and 15.4 min, respectively. The distributions of arsenic in the corresponding liver supernatants are presented in Figure 1 for the administration of MMAV (A), MMAIII (B), DMAV (C), and DMAIII (D). The arsenic peaks were detected commonly at the void volume of the column (9.8), AsB (15.4), DMAV (14.7), and MMAV (13.8 min). In addition, an unidentified peak named DMTA-2 was detected at 19.5 min, and one tiny peak named DMTA-1 at 23.8 min was in the three profiles except for the MMAV. The retention times of the two unidentified arsenic peaks (19.5 and 23.8 min) are different from the four methylated arsenicals administered in the present experiment and also from iAsIII and iAsV. As the two unidentified peaks were detected after the administration of mono- and dimethylated arsenicals, these two metabolites were assumed to be dimethylated arsenicals, and it was confirmed by the following in vitro experiment. Transformation of DMAIII to the Two Unidentified Arsenic Metabolites by Incubation in Vitro with Liver Supernatant. DMAIII prepared by reducing DMAV with 5 molar equiv of Cys was incubated in 20% control liver supernatant at the final concentration of 30, 45, and 90 µM at 37 °C for 30 min. The arsenic peaks

Dimethylthioarsenicals in As Metabolism

Figure 1. Distributions of arsenic in the liver supernatants of rats 5 min after an intravenous injection of four kinds of methylated arsenicals on a gel filtration column by HPLC-ICP MS. MMAV (A), MMAIII (B), DMAV (C), and DMAIII (D) were injected intravenously to rats at a dose of 0.5 mg As/kg body weight. The animals were sacrificed 5 min after the injection by removing the blood, and the livers were obtained after whole body perfusion with buffer. The livers were homogenized in 4 vol of the extraction buffer in an atmosphere of nitrogen with a glass-Teflon homogenizer, and the homogenates were centrifuged at 105000g for 60 min at 4 °C to obtain the supernatant fractions. A 20 µL portion of each supernatant was applied to a gel filtration GS 220 column, and the column was eluted with 50 mM ammonium acetate buffer (pH 6.5 at 25 °C) at a flow rate of 0.6 mL/min. Distributions of arsenic were determined with on-line detection by ICP MS. The authentic samples of DMAV, MMAV, and AsB are commercially available ones. DMAIII and MMAIII were prepared by reducing DMAV and MMAV with 5 molar equiv of Cys at 70 °C for 1.5 h and also by hydrolyzing DMI and MMI2.

Figure 2. Distributions of arsenic in the supernatant after incubation in vitro with DMAIII on a gel filtration column by HPLC-ICP MS. DMAIII was freshly prepared by reducing DMAV with Cys and then incubated with 20% liver supernatant prepared from control rats at the final concentrations of 30 (B), 45 (C), and 90 µM (D) at 37 °C for 30 min. (A) Distribution of arsenic in the control liver supernatant without adding arsenicals. (E) Distribution of arsenic in the DMAIII solution. The distributions of arsenic were determined on a gel filtration GS 220 column by HPLC-ICP MS.

were detected at the retention times of the void volume (9.8), DMAV (14.7), and the two unidentified peaks (DMTA-1 at 23.8 and DMTA-2 at 19.8 min) together with DMAIII (29.8 min), as shown in Figure 2. DMAIII was transformed almost quantitatively to DMTA-2; arsenic

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Figure 3. Reaction products of DMAV under various reducing conditions. DMAV was reduced under various conditions, and the products were determined on a gel filtration GS 220 column by HPLC-ICP MS under the same analytical conditions, as described in the legend to Figure 1. A; DMAV was reduced by sodium thiosulfate and sodium bisulfite according to the Reay and Asher method (24). B; DMAV was reduced by dropwise addition of H2SO4 to a purified water solution containing DMAV and Na2S at the final molar ratio of DMAV:Na2S:H2SO4 ) 1:1.6: 1.6 for 1 h in a nitrogen atmosphere. C; DMAV was reduced by dropwise addition of H2SO4 to a purified water solution containing DMAV and Na2S at the final molar ratio of DMAV:Na2S: H2SO4 ) 1:7.5:7.5 for 1 day in an air atmosphere or by bubbling H2S gas into an ethanol solution of DMAV.

peaks at the void volume, DMTA-1, and DMAIII were increased depending on the dose (Figure 2B-D). DMTA-1 and DMTA-2 were stable to heat treatment at 95 °C for 5 min. DMAV incubated with a supernatant in a separate experiment was recovered in its intact form (data not shown), indicating that the unidentified peaks were transformed directly from trivalent but not from pentavalent dimethylated arsenical. The results suggest that DMAIII but not DMAV is transformed to DMTA-1 and DMTA-2 in a liver supernatant. DMTA-1 can be present when the amount of DMAIII relative to a liver supernatant is high. Reaction Products of DMAV under Various Reducing Conditions. Reduction of DMAV with sodium thiosulfate and sodium disulfite according to the Reay and Asher method (24) produced a different arsenic peak from the original DMAV on an anion exchange column (25). The reduced product gave a different metabolic distribution from that of DMAV (not excreted into urine but taken up preferentially by RBCs) when it was administered to rats (data not shown) and also a different behavior from that of DMAV in the uptake by RBCs (26). Therefore, the reaction product was assumed to be DMAIII according to the reported assignment. However, later experiments and finally by comparison with the authentic sample prepared by hydrolysis of DMI (21, 27) posed a doubt on its identification as DMAIII. In addition, simultaneous detection of arsenic and sulfur by HPLC-ICP MS suggested the presence of sulfur in the reduction product. Furthermore, the reaction product detected as a single peak on an anion exchange column was separated into two peaks on the present gel filtration column of GS 220, as shown in Figure 3A. DMAIII prepared by dissolving DMI in distilled water under nitrogen atmosphere was eluted at 29.8 min under the present conditions.

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The reduction of DMAV was further examined with an assumed reductant in the Reay and Asher method (24), namely, with hydrogen sulfide, and also with a hydrogen sulfide producing mixture (Na2S and H2SO4). The reduction with hydrogen sulfide by bubbling it into a DMAV solution gave the same single arsenic peak as that produced by the Reay and Asher method on an anion exchange column (data not shown). Although the Reay and Asher method gave a differing yield of the two reaction products depending on the ratio of thiosulfate and disulfite, the starting DMAV remained to some extent. As a result, the Reay and Asher method always gave the two reaction products and the starting material DMAV in the reaction mixture. Furthermore, it was hard to scale-up the reaction or to give a concentrated arsenic solution that can be required for use for in vivo experiments. On the other hand, reduction of DMAV with hydrogen sulfide gave a quantitative reaction product with different relative ratios between the two products without the remaining starting material. The relative ratio can be controlled by the reaction time and also by the relative amount of DMAV to the reducing agent. The slower eluting peak at 23.8 min (DMTA-1) on a gel filtration column was the major product with a shorter reaction time and with a lower sulfide concentration. The stoichiometric relation can be controlled more easily by supplying sulfide with Na2S plus H2SO4 rather than bubbling hydrogen sulfide into a DMAV solution. When the reducing hydrogen sulfide was stoichiometrically controlled by Na2S and H2SO4, stepwise changes were observed in the relative ratio of the two arsenic products. Namely, when the ratio (Na2S + H2SO4)/DMAV was less than one, the starting arsenical (DMAV) remained, and the slower eluting peak (DMTA-1) appeared without accompanying the faster peak (DMTA-2). When the ratio (Na2S + H2SO4)/DMAV was raised to 1.6, the starting arsenical DMAV disappeared, and the slower eluting peak (DMTA-1) became almost a single product as shown in Figure 3B. Increasing the ratio (Na2S + H2SO4)/DMAV to more than 1.6, the faster eluting arsenic peak (DMTA2) started to increase while decreasing the slower eluting peak (DMTA-1). The molar ratio of DMAV:Na2S:H2SO4 ) 1:7.5:7.5 in an air atmosphere or by bubbling H2S gas into an ethanol solution of DMAV for a longer reaction time of 1 day gave an almost single product of the faster eluting arsenical (DMTA-2) as shown in Figure 3C. Correlation of the Unidentified Arsenic Metabolites with the Reduction Products of DMAV. HPLCICP MS profiles under the same column and elution conditions for the detection of arsenic metabolites and the reduction products suggested that the two unidentified arsenic metabolites and the two reduction products of DMAV might be the same arsenicals. The two arsenic peaks of the in vivo metabolites and of the reduction products were eluted at the same retention times on a gel filtration GS 220 column as shown in Figure 4. Identification of the Reduction Products of DMAV as DMTAIII and DMTAV. As the two reduction products (two arsenic metabolites) are supposed to be dimethylated arsenicals containing one or two molar sulfur atoms, their chemical structures are assumed to be DMTA (CH3)2AsSmOn [m ) 1 or 2 depending on the number of oxygens (n ) 1 or 0)], which can be confirmed by elemental analysis. However, the slower eluting arsenic reaction product (DMTA-1) was hygroscopic

Suzuki et al.

Figure 4. Cochromatogram of the unidentified metabolites and synthetic DMTAs on a gel filtration column by HPLC-ICP MS. A liver supernatant was prepared from rats 5 min after injecting DMAV at a dose of 0.5 mg As/kg body weight and then subjected to HPLC-ICP MS analysis as described in the legend to Figure 1. (A). DMTAs were prepared by reacting DMAV (30 mM) with Na2S plus H2SO4 at the molar ratio of 1:7.5:7.5 for 1 day (B). The same volumes of the arsenic solutions A and B were mixed and coeluted on a gel filtration column by HPLC-ICP MS (C).

(when extracted into ether and dried), and elemental analysis was not appropriate, especially to know the presence or absence of oxygen atoms. Therefore, elemental analysis was not carried out. Instead, mass spectrometric data were obtained for DMTA-1 and DMTA-2 by ESI MS with positive and negative ion modes, respectively. Molecular masses for DMTA-1 and DMTA-2 were m/z ) 138 (in positive ion mode, 139) (Figure 5A) and m/z ) 170 (in negative ion mode, 169) (Figure 5B). Thus, the faster eluting arsenical (19.8 min) (DMTA-2) was deduced to be DMTAV [(CH3)2As(dS)SH], while the slower eluting arsenical (23.8 min) (DMTA-1) was deduced to be DMTAIII [(CH3)2AsSH]. Preliminary in vitro experiments with the uptake of DMTAIII and DMTAV by RBCs indicated that DMTAIII was preferentially taken up by RBCs, while DMTAV was not, as in the cases of DMAIII and DMAV, respectively (data not shown), supporting that they may be tri- and pentavalent dimethylarsenicals (DMAs), respectively. Furthermore, although the starting arsenical DMAV was not extractable in ether or chloroform, the slower eluting reduction product (DMTAIII) was extractable into these organic solvents. Metabolic Transformation of DMTAIII to DMTAV in Vivo. DMTAIII was prepared as described in the Materials and Methods section and injected intravenously to rats in the presence of a reaction mixture (Na2S plus resulting Na2SO4 and oxidized products of sulfide). The arsenic distribution in the liver supernatant was obtained according to the sampling and analytical procedures used for those in Figure 1. The distribution of arsenic is similar to those of the two DMAs (DMAV and DMAIII) in Figure 1, as shown in Figure 6. Namely, DMTAV was the major metabolite and DMTAIII was detected marginally, indicating efficient uptake and transformation from DMTAIII to DMTAV in the liver. Chemical Correlation between DMTA and DMA. The two DMTAs identified in the present study were chemically correlated between them and also with DMA. Although DMAIII was readily converted to DMTAIII and DMTAV in the presence of Na2S (without H2SO4) (Figure 7C-E), DMAV remained unchanged under the same condition, as shown in Figure 7A. The transformation of DMAIII to DMTA by sulfide ions was dependent on the ratio of sulfide ions to DMAIII, as shown in Figure 7C-E; DMAIII disappeared from the

Dimethylthioarsenicals in As Metabolism

Figure 5. Mass spectra of DMTA-1 (A) and DMTA-2 (B) on an electrospray mass spectrometer. DMTA-1 was prepared by the standard reaction conditions (DMAV:Na2S:H2SO4 ) 1:1.6: 1.6) for 1 h followed by extraction with ether as well as chloroform, while DMTA-2 was prepared by bubbling H2S gas in an ethanol solution of DMAV. DMTA-1 (A) and DMTA-2 (B) were subjected to electrospray mass spectrometry with positive and negative ion modes, respectively.

Figure 6. Distribution of arsenic in the liver supernatant after an intravenous injection of DMTAIII. DMTAIII prepared by the standard reaction condition (DMAV:Na2S:H2SO4 ) 1:1.6:1.6) for 1 h and was injected intravenously to rats, and the rats were sacrificed 5 min after the injection. The 20% liver supernatant was subjected to HPLC-ICP MS analysis on a gel filtration GS 220 column.

reaction mixture at the equimolar ratio to Na2S, and DMTAV seems to be the final transformation product. Reduction with thiosulfate and bisulfite gave preferentially DMTAIII. However, the starting material DMAV remained to some extent in the reaction mixture under various reagents to DMAV ratios and with different thiosulfate-to-bisulfite ratios, DMTAIII being the major

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Figure 7. Distribution of three possible DMTAs by reduction of DMAV with cysteine and then with Na2S. A 20 µL portion of each reaction solution was applied to a gel filtration GS 220 column, and the column was eluted with 50 mM ammonium acetate buffer (pH 6.35 at 25 °C) at a flow rate of 0.6 mL/min. (A) A 1 mmol amount of DMAV was incubated with 2 mmol of Na2S at room temperature for 30 min, and then, the reaction solution was subjected to HPLC-ICP MS analysis. (B) A 1 mmol amount of DMAV was incubated with 5 mmol of cysteine at 72 °C for 1 h to prepare DMAIII. (C-E) A 1 mmol amount of DMAIII was incubated with 0.5 mmol of Na2S (C), 1.0 mmol of Na2S (D), and 2.0 mmol of Na2S (E) for 30 min at room temperature, and then, the reaction solution was subjected to HPLC-ICP MS analysis.

product. Reduction with hydrogen sulfide (including Na2S plus H2SO4) produced DMTAIII and DMTAV at different ratios, DMTAIII being the major product at a shorter reaction time and at a lower reductant concentration. Thus, DMTAIII was assumed to be transformed chemically to DMTAV under more vigorous conditions, thereby accompanying oxidation and assuming the intermediate dimethylmonothioarsinic acid [DMMTAV (11) in Scheme 1; dimethylarsinothioic acid Me2As(dO)SH or dimethylthioarsinic acid Me2As(dS)OH]. This intermediate 11 seems to be eluted between DMAV and DMTAV on the present gel filtration column from their chemical structures. The arsenic distributions in the reaction mixture in Figure 7C-E showed a possible intermediate DMMTAV (11 in Scheme 1) at 16.6 min between DMAV and DMTAV peaks. However, further examinations are required for the assignment.

Discussion One of the two DMTAs identified in the present study, DMTAV was found as the most abundant or at least one of the major arsenic metabolites in the liver after an intravenous injection of dimethylated arsenicals. As DMTAV was not the major dimethylated arsenic metabolite in the urine, its presence as the major metabolite in the liver suggests that DMTAV is metabolized more slowly than the normal major metabolites (DMAV and

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DMAIII) in the liver. Then, how can DMTAs be produced metabolically? Inorganic arsenicals are metabolized to the major urinary metabolite, DMAV, by the consecutive reduction and oxidative methylation reactions. DMTAIII and DMTAV seem to be related closely to DMAIII and DMAV, respectively, not only in the chemical structures but also in the metabolic pathway of arsenic. Although DMAV was transformed to DMTAIII and DMTAV in vivo in the liver, it was not in vitro by incubation with liver supernatant, suggesting that DMTAs have to be activated metabolically before being transformed from DMAV. This possibility can be accepted from the observation that DMAIII was easily transformed to DMTAs by incubation in vitro with liver supernatant. Therefore, it is assumed that DMTAs are transformed through DMAIII. This transformation is chemically reasonable, too. Namely, although DMAV was not transformed to DMTAs by incubation with sulfide ions (without H2SO4), DMAIII was transformed easily to DMTAIII and DMTAV under the same condition. These observations suggest the following metabolic pathway for DMTAs; DMAV (5) f DMAIII (6) f DMTAIII (10) f DMTAV (12) in Scheme 1. The transformation of DMTAIII to DMTAV requires an oxidation followed by a substitution with sulfide ions (thiolation), dimethylmonothioarsinic acid [DMMTAV, (11) in Scheme 1; dimethylarsinothioic acid Me2As(dO)SH or dimethylthioarsinic acid Me2As(dS)OH] being assumed as the oxidation product. This oxidation process is assumed to produce ROS as in the case of DMAIII to DMAV (31). DMTAIII was more stable than DMAIII under the present chemical procedures. However, DMTAIII was transformed easily to DMTAV by incubation in vitro in the liver supernatant, suggesting that the production of ROS may occur much more easily in the biological system. Although the metabolic pathway and chemical properties of DMTAIII are considered to be similar to DMAIII, they are different from those of DMAIII, and so, the toxicological significance is urgently required in relation to its toxicity. Sulfide ions are considered to be present normally in the liver to some extent as can be estimated from the present in vitro experiment, namely, thiolation of DMAIII to DMTAV by incubation in liver supernatant. The source of sulfide ions is assumed to be the product of intestinal flora and/or the metabolic product of sulfur-containing amino acids in the liver and other organs. Therefore, DMTAs can be produced when DMAIII is produced in the environment containing sulfide ions. The reduction of DMAV by the Reay and Asher method (24) produced not DMAIII but mostly DMTAIII, while DMTAV was present as a minor product. On the other hand, the reduction of DMAV with hydrogen sulfide (including Na2S + H2SO4) produced the mixture of DMTAIII and DMTAV at a different relative ratio. In both reduction reactions, it is assumed that DMAV is first reduced to DMAIII, and then, DMAIII is transformed to DMTAIII followed by the oxidation to DMMTAV. DMMTAV seems to be readily converted to DMTAV. However, a more precise reaction mechanism is required. Although we identified DMTAs as metabolites in the liver, thioarsenicals have been detected in the urine or feces of rats after long-term oral administration of various arsenic compounds by Endo’s group (28, 29, 32-35), and they are proposed as the microbial products in the enteric flora, Escherichia coli (35). Although the molecular

Suzuki et al.

structure of monothioarsenical M-2 (28, 29) has not been assigned and its molecular size was estimated to be m/z ) 154, it seems to be identical with our DMTAIII because M-2 and DMTAIII are one of the two reaction products by the Reay and Asher method. DMTAIII is much more stable chemically than DMAIII. However, even mass data for DMTAIII are hard to obtain as in the case of other trivalent arsenicals. Also, the molecular size of another unidentified dithioarsenical M-3 in rat feces and/ or in GUM medium in the presence of E. coli A3-6 and Cys (28) was estimated to be 170, but its molecular structure has not been assigned yet. M-3 seems to be DMTAV as identified in the present study. DMTAs, thus, can be produced readily from DMAIII in the presence of sulfide ions. Therefore, although the metabolic form for the excretion of dimethylated arsenicals from liver and other organs to the bloodstream is not known yet, if DMAIII or its equivalent, such as DMA(SG), is produced, it can be transformed to DMTAs in the cells and in the body fluids in the presence of sulfide ions. As DMTAIII is apparently more stable to oxidation than DMAIII, it can be transported and distributed easily through the body fluids to organs/tissues. Thus, DMTAIII seems to be as toxic as that of DMAIII (28) but in a different metabolic and toxic feature. Finally, as the chemical species produced by reduction with the Reay and Asher method are DMTAIII and DMTAV, and different from DMAIII, the studies carried out with DMAIII and MMAIII with the Reay and Asher method have to be reexamined.

Acknowledgment. This study was supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture for K.T.S. and Y.O. (Grants 12000236 and 14657587) and JSPS and JISTEC for B.K.M., K.A., and K.T.S..

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