Human Monomethylarsonic Acid (MMAV) Reductase Is a Member of

MMAIII is more toxic than arsenite in Chang human hepatocytes (14). .... 1 mM EDTA, 4 μg glutathione reductase, 0.7 mM HED, and 7.6 μg of fraction V...
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Chem. Res. Toxicol. 2001, 14, 1051-1057

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Human Monomethylarsonic Acid (MMAV) Reductase Is a Member of the Glutathione-S-transferase Superfamily Robert A. Zakharyan,† Adriana Sampayo-Reyes,† Sheila M. Healy,† George Tsaprailis,‡,§ Philip G. Board,| Daniel C. Liebler,‡,§ and H. Vasken Aposhian*,†,§ Department of Molecular and Cellular Biology, Department of Pharmacology and Toxicology, Center of Toxicology, The University of Arizona, Tucson, Arizona 85721, and Molecular Genetics Group, John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory 2601, Australia Received March 2, 2001

The drinking of water containing large amounts of inorganic arsenic is a worldwide major public health problem because of arsenic carcinogenicity. Yet an understanding of the specific mechanism(s) of inorganic arsenic toxicity has been elusive. We have now partially purified the rate-limiting enzyme of inorganic arsenic metabolism, human liver MMAV reductase, using ion exchange, molecular exclusion, and hydroxyapatite chromatography. When SDS-βmercaptoethanol-PAGE was performed on the most purified fraction, seven protein bands were obtained. Each band was excised from the gel, sequenced by LC-MS/MS and identified according to the SWISS-PROT and TrEMBL Protein Sequence databases. Human liver MMAV reductase is 100% identical, over 92% of sequence that we analyzed, with the recently discovered human glutathione-S-transferase Omega class hGSTO 1-1. Recombinant human GSTO1-1 had MMAV reductase activity with Km and Vmax values comparable to those of human liver MMAV reductase. The partially purified human liver MMAV reductase had glutathione S-transferase (GST) activity. MMAV reductase activity was competitively inhibited by the GST substrate, 1-chloro 2,4-dinitrobenzene and also by the GST inhibitor, deoxycholate. Western blot analysis of the most purified human liver MMAV reductase showed one band when probed with hGSTO1-1 antiserum. We propose that MMAV reductase and hGSTO 1-1 are identical proteins.

Introduction The World Health Organization has estimated that at least 70 million people in Bangladesh are at risk of getting cancer because they drink water containing large amounts of inorganic arsenic. Other areas of the world such as Romania, Taiwan, China, Chile, Mexico, Inner Mongolia, and Hungary have similar arsenic drinking water problems (1, 2). To help understand the mechanism of arsenic carcinogenicity and toxicity, the enzymes that catalyze the biotransformation of inorganic arsenic to DMAV have been partially purified (3). This multistep biotransformation (Figure 1) is catalyzed by arsenate reductase (4), arsenite methyltransferase (5, 6), monomethylarsonate (MMAV)1 reductase (7) and monomethylarsenous acid (MMAIII) methyltransferase (8). In this pathway, MMAV reductase catalyzes the reduction of MMAV to MMAIII in a reaction that has an absolute requirement for GSH and is thought to be the ratelimiting step in inorganic arsenic biotransformation (7). * To whom correspondence should be addressed. (520) 621-7565. Fax: (520) 621-3709. E-mail: [email protected]. † Department of Molecular and Cellular Biology. ‡ Department of Pharmacology and Toxicology. § Center of Toxicology. | Molecular Genetics Group. 1 Abbreviations: MMA, a generic term including MMAIII and MMAV; MMAIII, monomethylarsonous acid; MMAV, monomethylarsonic acid, hGSTO 1-1, recombinant human glutathione-S-transferase class Omega; GST, glutathione-S-transferase; SAM, S-adenosyl-L-methionine; SAHC, S-adenosyl-L-homocysteine.

Figure 1. Inorganic arsenic biotransformation.

MMAIII has been recently isolated and identified in tissues of hamsters exposed to inorganic arsenate (9) and in human urine (10-13). MMAIII is more toxic than arsenite in Chang human hepatocytes (14). Because of

10.1021/tx010052h CCC: $20.00 © 2001 American Chemical Society Published on Web 07/13/2001

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Table 1. Partial Purification of MMAV Reductase from Human Liver Cytosola

fraction

volume (mL)

protein (mg/mL)

total protein (mg)

activity (nmol/mL)

specific activity (nmol/mg)

total activity (nmol)

fold purification

recovery (%)

(I) cytosol (II) DEAE-Sepharose (III) Sephadex G-200 (IV) CM-Sepharose (V) hydroxyapatite

100 690 285 260 160

32.4 1.18 1.24 0.268 0.048

3240 821 354 69.8 7.96

0.9 0.163 0.25 0.27 0.35

0.027 0.137 0.2 1.03 7.29

90.8 113 72.2 72.1 56

5 7.4 38 270

125 80 80 62

a Activity is the enzyme activity expressed as nanomoles per milliliter. Total activity is the volume of the fraction multiplied by the activity per milliliter. MMAV reductase was assayed according to the procedure in Experimental Procedures.

this, the methylation of inorganic arsenic can no longer be assumed to be a detoxication step. A number of glutathione-S-transferase gene families such as Alpha, Mu, Theta, Pi, Zeta, and Omega have been identified (15). Board et al., (16) have pointed out that hGSTO 1-1, one of the most recently identified enzymes of this family has properties unlike those of the other GSTs. We have characterized and determined the amino acid sequence of human liver MMAV reductase, the ratelimiting enzyme of inorganic arsenic biotransformation (7) and show here that it is identical to the recently reported human Omega class glutathione transferase (16).

Experimental Procedures Caution: Arsenic has been classified as a human carcinogen by the International Agency for Research on Cancer (17). Reagents. [14C]MMAV (0.183 µCi/nmol) was synthesized according to the method of Klinger and Kreutz (18). Disodium methylarsenate was obtained from ChemService Inc. (West Chester, PA); DEAE Sepharose and CM Sepharose from Amersham Pharmacia Biotech (Piscataway, NJ); hydroxyapatite from Bio-Rad (Hercules, CA) and YM-10 ultrafiltration membranes from Amicon (Bedford, MA). Monoflow-3 scintillation cocktail was from National Diagnostics (Atlanta, GA); carbon tetrachloride and diethylammonium diethyldithiocarbamate from Aldrich (Milwaukee, WI); 1-chloro-2,4 dinitrobenzene, ethacrynic acid, sodium deoxycholate, dehydro-L-(+)-ascorbic acid, dichloromethane, human erythrocyte superoxide dismutase (EC 1.15.11), rabbit muscle triosephosphate isomerase (EC 5.3.11), Thermoanaerobium brockii alcohol dehydrogenase NADP+ (EC. 1.1.1.2), pig heart isocitrate dehydrogenase (NADP cytoplasmic, EC 1.1.1.4.2.), and goat anti-rabbit IgG alkaline phosphatase-conjugate were purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant human GST omega (hGSTO 1-1) and rabbit antiserum raised against this protein were prepared as described previously (16). Human liver was purchased from The Association of Human Tissue Users (Tucson, AZ). The donors did not die of a liverrelated disease and the cold ischemia time was e16 h. MMAV Reductase Assay. Unless otherwise noted, MMAV reductase was assayed as previously described by Zakharyan and Aposhian (7). MMAV Reductase Purification. Human liver cytosol was prepared at 4 °C as described by Radabaugh and Aposhian (4) and stored at -70 °C. Cytosol (100 mL, 3.24 g of protein) was applied to a DEAE-Sepharose column (2.5 × 27 cm) which had been preequilibrated with 10 mM Tris-HCl, pH 7.5 at 4 °C. The column was washed with equilibration buffer until no protein could be detected by UV absorbance. A 600 mL linear gradient consisting of 300 mL of 0.01 M Tris-HCl (pH 7.5, 4 °C) and 300 mL of 0.01 M Tris-HCl (pH 7.5, 4 °C)-0.5 M NaCl was then applied to the column. Fractions with MMAV reductase activity were pooled and precipitated by the addition of ammonium sulfate to 100% saturation. The precipitate was dissolved in

buffer A which was 100 mM potassium phosphate (pH 6.85), desalted by ultrafiltration using an ultrafiltration membrane (YM-10) and, in a final volume of 40 mL buffer A, loaded onto a Sephadex G-200 (5 × 60 cm) column. Active fractions eluting with 50 mM potassium phosphate (pH 6.85) were pooled and passed through a carboxymethyl (CM)-Sepharose column (2.5 × 20 cm) that had been preequilibrated with 50 mM potassium phosphate (pH 6.85). Less than 1% of MMAV reductase activity was bound to the CM-Sepharose column after washing with 260 mL of equilibration buffer. Pooled MMAV reductase activity was concentrated by ultrafiltration using an Amicon membrane YM10, resuspended in 100 mL of 10 mM potassium phosphate (pH 6.75) and applied to a hydroxyapatite column (1.8 × 15 cm) which had been preequilibrated with 10 mM potassium phosphate (pH 6.75). The column was washed with 10 mM potassium phosphate buffer (pH 6.75) until no protein could be detected in the effluent. More than 75% of MMAV reductase activity was collected in the pass through plus wash and designated as Fraction V (Table 1). Mass Spectrometry Analysis of MMAV Reductase. Mass spectrometric analysis of MMAV reductase was carried out in the Analytical Core Facility of the Southwest Environmental Health Sciences Center at The University of Arizona. MMAV reductase, fraction V, was concentrated and analyzed by reducing- SDS-PAGE (19). Three major bands and four minor bands were detected with coomassie blue staining. Each protein band was excised from the gel and digested with trypsin or chymotrypsin (20). Extracted peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a quadrupole ion trap Finnigan LCQ Classic mass spectrometer equipped with a Finnigan MAT Spectra System quaternary pump P4000 HPLC and a Finnigan electrospray ion source (Thermoquest, San Jose, CA). Peptides were eluted from a reversed-phase C18 micro-column (Vydac 250 mm × 1 mm, Hesperia, CA) with a gradient of 3 to 95% acetonitrile in 0.5% formic acid, 0.01% trifluoroacetic acid over 150 min at a flow rate of 15 µL/min. Tandem MS spectra of peptides were analyzed with the Sequest program (v. C1) to assign peptide sequences to the spectra (21). Sequest analyses were performed against the OWL database SWISS-PROT with PIR. Assignments made by Sequest were verified by manual inspection of the spectra and according to criteria described previously (22). Other Enzyme Assays. The GSH-dependent dehydroascorbate (DHA) reductase activity was measured by the spectrophotometric assay of Stahl et al. (23), which monitors the change in A265 associated with the formation of ascorbate. A molar absorption coefficient of 14 800 was used. The assay was performed by incubating 0.25 mM DHA and 1 mM GSH in 100 mM potassium phosphate buffer pH 6.7 with 7.6 µg of enzyme (fraction V) at 30 °C. Thiol-transferase activity was evaluated by measuring the reduction of hydroxyethyl disulfide (HED) in 0.1 M Tris buffer (pH 8.0). The 1 mL reaction mixture contained 1 mM GSH, 0.2 mM NADPH, 1 mM EDTA, 4 µg glutathione reductase, 0.7 mM HED, and 7.6 µg of fraction V and was incubated at 30 °C. Activity was measured as the decrease in NADPH absorbance at 340 nm (24). 1-Chloro-2,4 dinitrobenzene (CDNB) GSH thioether formation was determined by monitoring the change in A340 of a reaction

MMAV Reductase is GSTΩ mixture containing in 0.1 M sodium phosphate (pH 6.5), 1 mM GSH, 1 mM CDNB, and 3.8 µg of fraction V and incubating at 25 °C (25). The activity of fraction V on dichloromethane (DCM) was determined by the formation of formaldehyde. The reaction mix contained 10 mM GSH, 40 mM DCM, 0.1 M potassium phosphate (pH 7.4), and 3.8 µg of fraction V in a final volume of 1 mL and was incubated for 1 h at 37 °C. The reaction was stopped by adding 200 µL of 10% TCA (26). The formaldehyde product was determined using known concentrations of formaldehyde as standard (27). Conjugation of ethacrynic acid with GSH was assayed at 270 nm in 0.1 M phosphate buffer (pH 6.5), 0.25 mM GSH, 0.2 mM ethacrynic acid, and 3.8 µg of fraction V at 25 °C (28). GSH peroxidase activity of fraction V, using cumene hydroperoxide as the substrate, was assayed in a 1 mL reaction mixture containing 4 mM GSH, 0.4 mM NADPH, 4 µg of GSH reductase, 0.12 mM cumene hydroperoxide, 160 mM Tris-HCl (pH 7.0), 0.82 mM EDTA, and 3.8 µg of fraction V at 37 °C for 5 min (29). Nonenzymatic reaction rates in the absence of protein were subtracted for all MMAV and GST activities. Superoxide dismutase, triosephosphate isomerase, alcohol dehydrogenase. or isocitrate dehydrogenase was tested for MMAV reductase activity using the standard MMAV reductase assay (7). Protein Assay. Protein concentrations were determined according to the Bradford method (30) using bovine serum albumin as the standard. SDS-β-Mercaptoethanol-PAGE and Western Blot Analysis. The proteins of fraction V were separated using reducing SDS-PAGE (10%) and electrophoretically transferred to unmodified nitrocellulose (0.45 µm) according to Burnette (31). Western blots were developed using antiserum (1:400) raised against recombinant hGSTO1-1 in rabbit (16), and goat antirabbit IgG (1:15000) alkaline phosphatase conjugate (32).

Results Purification and Amino Acid Sequence of Human Liver MMAV Reductase. MMAV reductase was purified 270-fold from human liver using ion exchange, molecular exclusion and hydroxyapatite chromatography (Table 1). When fraction V was subjected to SDS-β-mercaptoethanol-PAGE, three major and four minor bands were seen after coomassie staining (Figure 2). Each band was excised, digested with trypsin and protein identification was performed by Sequest using the MS/MS data for the tryptic peptides. The seven proteins were identified using the SWISS-PROT, TrEMBL Protein Sequence Database or the National Center for Biotechnology Information Database, Washington. Band A was superoxide dismutase (P00441); band B, triosephosphate isomerase (P00938); band C, glutathione-S-transferase omega (P78417 and AAF73376); band D, carbonyl reductase (P16152); band E, esterase-D (P10768); band F, alcohol dehydrogenase (P14550); and band G, isocitrate dehydrogenase (O075874) (Figure 2). Human MMAV Reductase and Human Omega Class Glutathione-S-Transferase (hGSTO 1-1) Are the Same Protein. Band C based on the PAGE markers had an approximate molecular weight of 28 000 Da and was digested also with chymotrypsin for more comprehensive mass spectrometric data. On the basis of a combined 92% coverage using trypsin and chymotrypsin, band C had 100% sequence identity with human glutathione S-transferase class omega (Figure 3). Board et al. (16) have recently identified hGSTO 1-1 and shown by sequence analysis, size exclusion chromatography and

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Figure 2. SDS-β-mercaptoethanol polyacrylamide gel analysis of MMAV reductase (fraction V). Lane 1, purified recombinant 6× His-tag hGSTO 1-1 (2 µg), lane 2, molecular mass marker (10 µg), and lane 3, partially purified human liver MMAV reductase (fraction V) 6 µg. Band A: Superoxide dismutase (EC 1.15.11), 15 804 Da. Band B: Triose phosphate isomerase (EC 5.3.11), 26 538 Da. Band C: Glutathione-S-transferase-Omega, 27 566 Da. Band D: Carbonyl reductase (NADPH, EC 1.1.184), 30 244 Da. Band E: Esterase-D (EC 3.1.1.1), 31 463 Da. Band F: Alcohol dehydrogenase (NADP+, EC 1.1.1.2), 36 442 Da. Band G: Isocitrate dehydrogenase (NADPH cytoplasmic, EC 1.1.1.4.2.), 46 688 Da.

X-ray crystallography that it is a 56 kDa homodimer composed of 27.5 kDa subunits. Proteins representing four of the other gel bands were obtained commercially and when assayed were found to be devoid of MMAV reductase activity. These proteins were human erythrocyte superoxide dismutase, rabbit muscle triosephosphate isomerase, alcohol dehydrogenase from thermoanaerobium brockii, and pig heart isocitrate dehydrogenase. Esterase-D and carbonyl reductase could not be obtained. Human MMAV Reductase and Omega Class Glutathione-S-Transferase Have Similar Properties. Although they may be distinguished from each other by their characteristic substrate activity spectrums, many GSTs are active with CDNB. Fraction V (Table 1) had GST activity. It catalyzed CDNB-GSH conjugation, GSHdependent thiol transfer and dehydroascorbate reduction (Table 2). Fraction V did not catalyze GSH conjugation with ethacrynic acid. Some glutathione-S-transferases have GSH peroxidase activity but cumene hydroperoxide was not active as a substrate for fraction V. Dichloromethane is a substrate for some specific isoforms of theta class GST (33). Fraction V was not active toward DCM (Table 2). The pH optimum of MMAV reductase or hGSTO 1-1 was 8.0-8.2 using MMAV as the substrate. The rate of reaction of each is linear over a 60 min period (Figure 4). MMAV reductase activity was competitively inhibited by CDNB suggesting that the GST substrate binds at the same site as MMAV (Figures 5 and 6). MMAV reductase activity was inhibited by sodium deoxycholate, a GST inhibitor, in a dose-dependent manner (Figure 5).

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Table 2. Activity of Human Liver MMAV Reductase with Various hGSTO 1-1 Substrates

1-chloro-2,4-dinitrobenzene dehydroascorbate ethacrynic acid 2-hydroxyethyl disulfide MMAV c dichloromethane cumene hydroperoxide

product formeda by MMAV reductase (µmol/min/mg of protein)

pH

1.46 ( 0.032 0.540 ( 0.007 ND ND ND ND ND

6.5 6.7 6.5 6.5 6.5 7.4 7.0

product formed by MMAV reductase (µmol/min/mg of protein) 4.6 ( 0.13 ND 1.36 ( 0.001 0.317 ( 0.010

pH 8.0 8.0 8.0 8.0 8.0

product formed by hGSTO 1-1b (µmol/min/mg of protein)

pH

0.18 0.16 ND 2.9 0.501 ( 0.023 ND ND

6.5 6.7 6.5 8.0 8.0 7.4 7.0

a All values are the mean ( SE of three determinations; ND ) not detectable. b These values except the one for MMAV are from Board et al. (16). All assays in this column used the recombinant enzyme, hGSTO 1-1. c MMAV at a concentration of 96 mM was used as the substrate.

Figure 4. Time course of human liver MMAV reductase or hGSTO 1-1 recombinant enzyme. MMAV reductase activity was assayed with 0.6 µCi [14C]MMAV, 20 mM MMAV, 5 mM GSH, and 5.7 µg of fraction V or 4 µg of hGST0 1-1 at 37 °C.

Figure 3. Sequence identity between human MMAV reductase and hGSTO 1-1. SDS-β-mercaptoethanol polyacrylamide gel band C was digested with trypsin and chymotrypsin separately and the respective peptide fragments analyzed by LC-MS/MS. The amino acid sequence of hGSTO 1-1(1) is aligned with MMAV reductase peptides (2).

The purified 6× His-tag hGSTO 1-1 and MMAV reductase (fraction V) catalyzed MMAIII formation at a similiar rate (Figure 7). The 6× His-tag hGSTO 1-1 had a Vmax of 35.7 µmol/mg/h and Km of 23.5 × 10 -3 M. These values are comparable with those of fraction V human MMAV reductase: Vmax of 22.7 µmol/mg/h and Km of 23.6 × 10-3 M. Western blot analysis of fraction V developed with rabbit antiserum raised against hGSTO 1-1 revealed one band (Figure 8). Bovine liver GST and rabbit liver GST did not react with the antiserum. Neither did bovine liver GST nor rabbit liver GST reduce MMAV.

Discussion The purification, identification, and characterization of the enzymes involved in the biotransformation of inorganic arsenic are of critical importance in under-

Figure 5. Inhibition of MMAV reductase activity by CDNB (a glutathione-S-transferase substrate) or sodium deoxycholate (a glutathione-S-transferase inhibitor). MMAV reductase activity was assayed with 0.6 µCi [14C]MMAV, 20 mM MMAV, 5 mM GSH, 7.6 µg of fraction V and increasing concentrations of CDNB (O) or deoxycholate (9) at 37 °C for 1 h.

standing the mechanisms of arsenic toxicity, especially its carcinogenicity. This biotransformation (Figure 1) utilizes S-adenosylmethionine as the cofactor for enzymatic methylation as clearly shown using partially purified methytransferase (3, 5) or methylcobalamin as a nonenzymatic methyl donor (34). When studying this biotransformation, it is advisable to work with human

MMAV Reductase is GSTΩ

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Figure 6. CDNB is a competitive inhibitor of human liver MMAV reductase. The curve representing MMAV reductase activity (9) yielded a Km of 23.6 × 10-3 M and Vmax of 22.7 µmol/ mg/h. MMAV reductase in the presence of 0.8 mM CDNB (O) yielded an apparent Km of 69.8 × 10-3 M and Vmax of 22.2 µmol/ mg/h. Fraction V (5.7 µg of protein) was used for each assay. The assay is described in Experimental Procedure. Figure 8. Western blot of MMAV reductase using anti-hGSTO 1-1. The immunoblot shows the localization of MMAV reductase of fraction V and the 6× His-tag hGSTO 1-1 at an apparent molecular mass of 28 kDa. Lane 1, 8 µg molecular weight marker; lane 2, 6 µg of 6× His-tag hGSTO 1-1; lane 3, 7.6 µg of MMAV reductase (fraction V); lane 4, 5 µg of bovine liver GST; lane 5, 5 µg of rabbit liver GST.

Figure 7. Substrate saturation for MMAV reductase and 6× His-tag hGSTO 1-1. Fraction V (5.7 µg) or 6× His-tag hGSTO 1-1 (4µg) was incubated in the presence of 5 mM GSH and increasing concentrations of MMAV for 60 min at 37 °C. Each point represents the average of duplicate experiments.

tissues and deal with human responses as much as possible. Some of the reasons for this are that humans are much more sensitive to the toxic effects of arsenic; some animals do not methylate inorganic arsenic (3, 3538); and DMA (probably DMAIII) appears to bind only to the red blood cells of the rat and not to those of other species (39). Human Liver MMAV Reductase and Human hGSTO 1-1 Are the Identical Proteins. We have partially purified and characterized rabbit (7) and human liver MMAV reductase. This enzyme is rate limiting for inorganic arsenic biotranformation (7) because its Km is in the millimolar range, while those of the arsenic methyltransferases are in the micromolar range. We have determined its amino acid sequence (Figure 3) and have found that human liver MMAV reductase is 100% identical, over 92% of sequence that we analyzed, with the recently discovered hGSTO 1-1 (16). CDNB was a substrate for MMAV reductase and has been previously reported to be one for hGSTO 1-1 (16). In addition, we found that MMAV reductase was inhibited 50% by 0.8 mM CDNB in the presence of 20 mM MMAV and that CDNB was a competitive inhibitor of MMAV

reductase implying that MMAV and CDNB compete for the same site on the enzyme. Deoxycholate a nonsubstrate inhibitor of GST also inhibited MMAV reductase (Figure 5). GST Omega Class and Other GSTs. The GST Omega class has been identified and characterized by Board et al. (16). The monomer has a mass of 27.5 kDa as deduced from the amino acid sequence. The dimer was 56 kDa based on size exclusion chromatography. The GSTs have an absolute requirement for GSH. MMAV reductase also has an absolute requirement for GSH. No other thiol has been found to replace it. Although arsenate reductase and the arsenic methyltransferases need a thiol present for activity (4, 5), either cysteine, GSH or DTT can satisfy this requirement. Glutathione-S-transferases comprise a large family of enzymes. A number of GST gene families such as Alpha, Mu, Theta, Pi, Zeta, and Omega have been identified (15). Board et al. (16) have pointed out that hGSTO 1-1 has properties unlike those of the other GSTs such as having (a) an active site cysteine that is able to form a disulfide bond with GSH; (b) a unique 19-20 residue N-terminal extension as compared to other GSTs; and (c) a novel structural unit formed by the N-terminal extension and the C-terminus (16). In glutathione-S-transferase, the sulfur atom of GSH makes a nucleophilic attack on the electrophilic groups of endobiotics and xenobiotics. Reduction of MMAV may be achieved via a one electron donation by the thiolate of Cys-32 of MMAV reductase/hGSTO 1-1 and the other electron from the thiolate of GSH. Members of GST super family are considered to be detoxifying enzymes and deficiencies of them can cause an increased risk of some diseases including cancer (40). The GST super family has an important function in protecting cells and there is a large interindividual variability in their expression (41). In humans, there is a high variability of biomarker responses to inorganic

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arsenic (1, 42) which has been speculatively attributed to polymorphism of arsenic methytransferases. Also there are still unanswered questions concerning the mechanism of arsenic tolerance among the arsenic eaters in Styria (43). It is possible that this variability and tolerance may be related to MMAV reductase/hGSTO 1-1 rather than the arsenic methyltransferases. Further investigation will clarify this. Oltipraz is an inducer of the Alpha, Mu, and Pi members of the GST family (44). Since many GSTs are inducible, it is possible that MMAV reductase is inducible by some form of arsenic. The MMAV reductase activity is found in many tissues (9) as is the mRNA for human GST-Omega (16). While many of the GSTs protect cells against toxic compounds by deactivating genotoxic and cytotoxic compounds (44), MMAV reductase catalyzes the formation of MMAIII (7) which is the most toxic compound of the inorganic arsenite biotransformation pathway as shown by Petrick et al. (14) and others (45, 46). It has been suggested that GST π class can facilitate the excretion of arsenic since Chinese hamster ovary cells resistant to arsenic (SA7 cells) overexpress GST π class and accumulate less arsenic (47). In addition, arsenic efflux by these cells was inhibited by ethacrynic acid or Cibacron blue, inhibitors of GST. Cancer, GST, and Arsenic. Some GSTs are known to influence the chemotherapeutic response to cancer chemotherapy agents. Although inorganic arsenic is carcinogenic for humans, it has recently become a most useful chemotherapeutic agent against many cancers (48). Whether MMAV reductase/hGSTO 1-1 is involved in this therapeutic activity of inorganic arsenic is worthy of investigation. In summary, the evidence now supports human MMAV reductase as being identical to hGSTO 1-1. The importance of this critical rate-limiting enzyme of arsenic biotransformation being a member of the glutathione-Stransferase family cannot be minimized. It opens a number of new and unique directions for the investigation of the variability in response, chemotherapy and tolerance of inorganic arsenic compounds.

Acknowledgment. The authors are grateful to Prof. Eugene A. Mash, Jr. and Dr. Reddy B. Aavula in the Department of Chemistry for the Synthesis of [14C]MMA. This work was supported in part by the Superfund Basic Research Program NIEHS Grant Number ES-04940 from the National Institute of Environmental Health Sciences and the Southwest Environmental Health Sciences Center P30-ES-06694.

References (1) National Research Council Report (1999) Arsenic in Drinking Water, National Academy Press, Washington, DC. (2) Arsenic Exposure and Health Effects (1998) In Proceedings of the Third International Conference on Arsenic Exposure and Health Effects (Chappell, W. R., Abernathy, C. O., and Calderon, R. L., Eds.) Elsevier Science Ltd., Oxford. (3) Aposhian, H. V. (1997) Enzymatic methylation of arsenic species and other new approaches to arsenic toxicity. Annu. Rev. Pharmacol. Toxicol. 37, 397-419. (4) Radabaugh, T. R., and Aposhian, H. V. (2000) Enzymatic reduction of arsenic compounds in mammalian systems: Reduction of arsenate to arsenite by human liver arsenate reductase. Chem. Res. Toxicol. 13, 26-30. (5) 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

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