Selenium Compounds Modulate the Activity of Recombinant Rat AsIII

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Chem. Res. Toxicol. 2003, 16, 261-265

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Communications Selenium Compounds Modulate the Activity of Recombinant Rat AsIII-Methyltransferase and the Methylation of Arsenite by Rat and Human Hepatocytes Felecia S. Walton,† Stephen B. Waters,‡ Summer L. Jolley,§ Edward L. LeCluyse,§ David J. Thomas,| and Miroslav Styblo*,†,⊥ Department of Pediatrics, Curriculum in Toxicology, Division of Drug Delivery and Disposition, and Department of Nutrition, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, Pharmacokinetics Branch, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U. S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711 Received October 18, 2002

Formation of methylated metabolites is a critical step in the metabolism of inorganic arsenic or selenium. We have previously shown that under conditions of a concurrent exposure sodium selenite inhibits methylation of arsenite by cultured rat hepatocytes. Here, we compare the effects of sodium selenite and mono-, di-, and trimethylated selenium compounds on the methylation of arsenite by purified recombinant rat AsIII-methyltransferase (Cyt19) and by primary rat and human hepatocytes. Among these compounds, sodium selenite was the most potent inhibitor of the methylation of arsenite by the recombinant enzyme (Ki ) 1.4 µM) and by cultured cells. In both systems, methylseleninic acid was an order of magnitude less potent an inhibitor (Ki ) 19.4 µM) than was sodium selenite. Dimethylselenoxide and trimethylselenonium iodide were weak activators of recombinant AsIII-methyltransferase activity but were weak inhibitors of arsenite methylation in hepatocytes. These data suggest that selenite, rather than its methylated metabolites, is responsible for inhibition of arsenite methylation in cultured hepatocytes and that inhibition may involve direct interactions between selenite and AsIII-methyltransferase.

Introduction In cultured rat hepatocytes concurrently exposed to iAsIII 1 and SeIV, cellular accumulation of iAs is increased, the production of methylated metabolites of iAsIII is suppressed, and the cytotoxicity of iAsIII is potentiated (1). Similar interactions between these metalloids in cultured cells or in laboratory animals have frequently been reported (2, 3). For example, iAs antagonizes the toxic effects of inorganic selenium (3, 4) but potentiates * To whom correspondence should be addressed. Tel: (919)966-5721. E-mail: [email protected]. † Department of Pediatrics, The University of North Carolina at Chapel Hill. ‡ Curriculum in Toxicology, The University of North Carolina at Chapel Hill. § Division of Drug Delivery and Disposition, The University of North Carolina at Chapel Hill. | U. S. Environmental Protection Agency. ⊥ Department of Nutrition, The University of North Carolina at Chapel Hill. 1Abbreviations: arsenite, iAsIII; inorganic arsenic, iAs; selenite, SeIV; dimethylarsinic acid, DMAs; methylarsonic acid, MAs; S-adenosylmethionine, AdoMet; S-adenosyl-L-methionine, arsenic(III) methyltransferase, Cyt19; methylseleninic acid, MSe; dimethyl selenoxide, DMSeO; trimethylselenonium iodide, TMSe; optical density, OD; native binding buffer, NBB; thiazolyl blue, MTT; concentration of inhibitor that decreases enzyme activity by 50%, IC50.

the toxicity of methylated selenium compounds (5). Conversely, metabolism, retention, and toxicity of iAs or methylated arsenicals in organisms or cells are modified by exposure to selenium (6-8). Interactions between arsenic and selenium could originate in common features of their metabolism. Both are reduced to lower oxidation states (9-13), and both are enzymatically methylated to mono-, di-, and trimethylated derivatives by distinct AdoMet-dependent enzymes (14-18). SeIV inhibits the methylation of iAsIII by rat liver cytosol (19) and by an arsenic methyltransferase purified from rabbit liver (17). iAsIII inhibits production of dimethylselenide from SeIV by a cytosolic fraction of mouse liver or kidney (14). In contrast, production of trimethylselenonium ion from dimethyselenide catalyzed by thioether-S-methyltransferase is not inhibited by iAsIII (16). A previous study (1) described kinetics of the inhibition of iAsIII methylation in rat hepatocytes concurrently exposed to SeIV. Here, we have examined effects of SeIV and methylated selenium compounds, homologues of identified methylated metabolites of SeIV, on the methylation of iAsIII by recombinant rat AsIII-methyltransferase (arsenic(III) methyltransferase, Cyt19) and by primary rat and human hepatocytes. These studies found SeIV to

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be the most potent inhibitor of iAsIII methylation by Cyt19 and by cultured hepatocytes. MSe was less potent than SeIV as an inhibitor of iAsIII methylation. DMSeO and TMSe had minimal effect on the methylation patterns of iAsIII. Thus, inhibition of Cyt19 activity by SeIV, and to a lesser extent by its methylselenol metabolites, is likely responsible for the decreased production of methylated arsenicals in hepatocytes concurrently exposed to iAsIII and SeIV.

Experimental Procedures Caution: iAs has been classified as a human carcinogen (20) and should be handled accordingly. Arsenic and Selenium Compounds. iAsIII and SeIV (sodium salts) were purchased from Sigma (St. Louis, MO). TMSe was obtained from Organometallics, Inc. (East Hampstead, NH). Custom-synthesized MSe (potassium salt) and DMSeO were kindly provided by Professor Howard Ganther (University of Wisconsin-Madison). Radiolabeled [73As]arsenate was purchased from Los Alamos Meson Production Facility (Los Alamos, NM). [73As]iAsIII was prepared from [73As]arsenate by reduction with metabisulfite/thiosulfate reagent (21). The yield of [73As]iAsIII in this reaction as determined by TLC (22) typically exceeded 95%. Recombinant Rat Cyt19. Rat liver cDNA (Clontech, BD Biosciences, Palo Alto, CA) was polymerase chain reaction (PCR)-amplified using primers derived from the published Cyt19 mRNA sequence (18): 5′GATCGACTCGAGTTCCTGAGACCCTCTGCCAAC-3′ and 5′-GATCGAGAATTCCTAGCAGCTTTTCCTTTTGCC-3′. The PCR product and the vector pRSETa (Invitrogen, Carlsbad, CA) were digested with XhoI and EcoRI restriction enzymes (Promega, Madison, WI). Digested products were ligated together and used to transform Escherichia coli strain DH5R. Ampicillin resistant colonies were screened, and a clone containing the complete rat Cyt19 sequence was identified and designated pRSET-rcyt19. E. coli strain BL21(DE3)pLysS was transformed with pRSET-rcyt19. Luria-Bertani broth (250 mL) was inoculated with 6 mL of an overnight culture and grown to an OD600 of 0.4-0.5. Expression of recombinant Cyt19 was induced by 1 mM isopropyl-1-thio-β-D-galactoside. Bacteria were pelleted after 3 h of incubation. Pellets were resuspended in 25 mL of NBB, 50mM NaPO4, 50 mM NaCl, pH 7.4, and lysed by French press. Lysates were clarified by centrifugation at 20 000g for 20 min. The His-tagged cyt19 protein was purified by passage over a probond Ni-NTA resin column (Invitrogen). The column was washed with 3 vol (8 mL each) of NBB containing 20 mM imidazole. Cyt19 protein was eluted from the column with NBB containing 300 mM imidazole. The purified Cyt19 separated by SDS-PAGE yielded a single Coomassie blue reactive band. Bovine serum albumin was used for protein calibration during the gel electrophoresis. In Vitro Methylation Assay. The in vitro reaction mixture (50 µL) contained 100 mM phosphate buffer (pH 7.4), 1 mM dithiothreitol (Sigma), 1 mM AdoMet (Sigma), 0.1 µM [73As]iAsIII (0.25 µCi), and 4 µg of recombinant Cyt19. Phosphate buffer or selenium compounds were added to reaction mixtures to reach a final volume of 50 µL. Reaction mixtures were incubated in capped tubes at 37 °C for up to 2 h, and the methylation reaction was stopped by addition of 25 µL of 30% hydrogen peroxide (Sigma). Cell Cultures and Treatments. Rat hepatocytes were prepared (23) at the Advanced Cell Technologies Core, UNC, Chapel Hill, from adult male Fischer 344 rats (Charles River Laboratory, Raleigh, NC). Human hepatocytes were isolated (24, 25) from normal hepatic tissue obtained from a 57 year old female patient during a resection carried out in the University of North Carolina Memorial Hospital. The protocol for use of human hepatocytes in this study has been approved by the UNC Institutional Review Board. Both rat and human hepatocytes were plated (250 000 cells/well) in 24 well plates coated with

Communications collagen-I (Becton Dickinson, Bedford, MA) and cultured in a supplemented William’s medium E (26). For metabolic studies, cells were incubated for up to 24 h with 0.1 µM [73As]iAsIII alone or in the presence of SeIV, MSe, DMSeO, or TMSe. Selenium compounds were added concurrently with [73As]iAsIII. Cell viability was examined by a MTT assay (27) in parallel cultures that were treated with selenium compounds and with 0.1 µM iAsIII. Analysis of Radioactive Metabolites. For speciation analysis, in vitro reaction mixtures were oxidized by 10% H2O2 to convert all arsenic metabolites to pentavalency. Culture media and cells were treated with 0.2 M CuCl (pH 1) at 100 °C to release protein-bound metabolites (28). The metabolites in CuCl extracts were oxidized by 10% H2O2. Pentavalent metabolites, arsenate, MAs, and DMAs were analyzed in oxidized in vitro reaction mixtures and CuCl extracts by TLC (22, 28). Distribution of radioactivity on TLC plates was analyzed with a FLA 2000 imaging system (FujiFilm, Stamford, CT). Statistical Analysis. Differences in metabolic yields in the in vitro and cell culture methylation systems and in cell viability were analyzed with Instat software package (GraphPad Software, San Diego, CA). Data were evaluated by one way ANOVA with Dunnett’s multiple comparisons posttest. Differences with p values smaller than 0.05 were considered statistically significant.

Results and Discussion Effects of Selenium Compounds on Methylation of iAsIII by Cyt19. The Cyt19 gene encodes an AdoMetdependent methyltransferase found in mouse, rat, and human genomes. This enzyme catalyzes the methylation of iAs to MAs and DMAs (18). Cyt19 is expressed in a variety of rat tissues and in human hepatocytes (18). The kinetics of in vitro methylation catalyzed by Cyt19 are consistent with the qualitative and quantitative characteristics of the methylation of iAsIII by liver cytosol (19, 29) and by cultured hepatocytes (26). Thus, Cyt19 may be the rate-limiting enzyme in this pathway. Like Cyt19 purified from rat liver (18), the recombinant Cyt19 requires AdoMet and a dithiol for full activity. The recombinant enzyme efficiently methylates iAsIII at concentrations up to 10 µM. At higher iAsIII concentrations, the activity of Cyt19 is inhibited (unpublished results). In the present study, the methylation activity of Cyt19 was assayed using 0.1 µM iAsIII. Because higher concentrations of iAsIII may be cytotoxic (30), 0.1 µM iAsIII was also used in the methylation experiments in cultured rat and human hepatocytes. To determine whether Cyt19 is a target for inhibitory effects of SeIV or its methylated metabolites, we examined the methylation of 0.1 µM [73As]iAsIII by recombinant rat Cyt19 in the absence or presence of up to 1000 µM SeIV, MSe, DMSeO, or TMSe. Methylation rates after a 90 min incubation at 37 °C were calculated as mole equivalents of methyl groups transferred from AdoMet to iAsIII (i.e., 1 pmol CH3 per 1 pmol MAs or 2 pmol CH3 per 1 pmol DMAs). The average methylation rate in the absence of selenium compounds was 285 pmol CH3 per mg of protein (Cyt19) per hour. Addition of SeIV or MSe decreased methylation rates in a concentration-dependent manner (Figure 1). The IC50 values for SeIV and MSe were 2 and 50 µM, respectively. In contrast, addition of DMSeO or TMSe to reaction mixtures slightly, but significantly, increased methylation rates as compared to controls. To determine whether either DMSeO or TMSe donated methyl groups to methylate iAsIII, reaction mixtures with all components except AdoMet were incubated with up to 1000 µM DMSeO or

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Figure 1. Modulation of the AsIII-methyltransferase activity of the recombinant rat Cyt19 by SeIV (O), MSe (b), DMSeO (0), and TMSe (9). The assay mixtures were incubated for 90 min. *Methylation rate is significantly different from that in the control assay mixture incubated without selenium compounds.

TMSe and with or without Cyt19. Neither chemical nor enzymatically catalyzed methylation of iAsIII was detected under these conditions (data not shown). Kinetic analyses characterized the inhibition of Cyt19 by SeIV and by MSe over a range of [73As]iAsIII concentrations from 0.167 to 4 µM. Double reciprocal (1/V vs 1/[iAsIII]) plots (Figure 2) show both SeIV and MSe to be competitive inhibitors of the methylation of iAsIII by Cyt19. The Ki values of 1.44 µM for SeIV and 19.4 µM for MSe were calculated from the replots of the slopes of the corresponding double reciprocal plots vs the concentration of the inhibitor (Figure 2, insets). A 24 h dialysis of the Cyt19-SeIV enzyme-inhibitor complex against 100 mM phosphate buffer (pH 7.4) with 1 mM DTT at 4 °C partially reversed the inhibition of Cyt19 activity. The presence of DTT in the dialysis buffer was essential for the recovery Cyt19 activity. In contrast, dialysis did not reverse the inhibitory effect of MSe (data not shown). Thus, SeIV is a potent, but reversible, inhibitor of the methylation of iAsIII by Cyt19 and MSe, although less potent than SeIV, inhibits Cyt19 in an irreversible manner. Mechanisms by which SeIV and MSe inhibit Cyt19 activity are unclear. In cells, inhibition of the methylation of iAsIII by selenium compounds may involve competition for AdoMet as a methyl group donor or for methyltransferases. Although the present study did not examine the methylation of SeIV or MSe by Cyt19, the distinct garlic odor of volatile methylated selenium metabolites was not detected. Hence, under these experimental conditions, Cyt19 does not methylate these selenium compounds. Given the high affinity of SeIV for thiols (31), the inhibition of Cyt19 by this compound probably involves interactions with one or several of the 13 Cys residues in the enzyme. The competitive character of the inhibition suggests catalytically active Cys(s) in Cyt19 to be primary targets for SeIV and MSe. However, the mechanistic basis for these interactions in the presence of DTT, a potent reductant, must be determined. Effects of Selenium Compounds on Methylation of iAsIII by Rat and Human Hepatocytes. Here, rat hepatocytes were concurrently exposed to 0.1 µM [73As]iAsIII and up to 50 µM SeIV, MSe, DMSeO, or TMSe. Cells exposed to 0.1 µM [73As]iAsIII in the absence of

Figure 2. Double reciprocal plots for the inhibition of the recombinant rat Cyt19 by SeIV and MSe. The assay mixtures were incubated for 120 min. Upper panel: plots for 0 (b), 1 (0), 3 (O), and 10 µM (9) SeIV. Lower panel: plots for 0 (b), 30 (0), 90 (O), and 300 µM (9) MSe. Corresponding replots of slopes vs concentration of inhibitor are shown in the insets.

selenium compounds were the controls. The radiolabeled metabolites were analyzed in cells and in culture medium after a 3 or 6 h incubation. Rat hepatocytes incubated for 3 h with [73As]iAsIII in the presence of SeIV retained 6-10-fold greater amounts of iAs than did control cells (Table 1). Similarly, incubation with MSe increased cellular retention of iAs 3-9-fold. Exposures to DMSeO or TMSe did not significantly affect iAs retention in hepatocytes. Methylation rates were significantly lower in hepatocytes exposed to SeIV or MSe (Table 1). At equimolar concentrations, SeIV had a greater effect on iAsIII methylation than did MSe. A small, but statistically significant, decrease in the methylation rate was also detected in cultures exposed to 10 or 50 µM DMSeO and to 5 or 10 µM TMSe. After a 6 h incubation, the effects of SeIV or MSe on cellular retention of iAs and on methylation yields were statistically significant but less pronounced (data not shown). Neither cellular retention nor methylation rates was affected in cultures incubated for 6 h with DMSeO or TMSe (data not shown). A 30% decrease in cell viability was found in cultures treated for 3-6 h with 50 µM SeIV or MSe; cell viability was not significantly changed in the other treatment groups. Because few human hepatocytes were available, only effects of the two most potent inhibitors, SeIV and MSe, were examined. Human hepatocytes are much less effective methylators of iAs than rat hepatocytes (26). Hence, the incubation period was extended to 24 h to increase production of methylated metabolites. Human cells exposed to either 2 or 10 µM SeIV retained signifi-

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Table 1. Methylation of 0.1 µM iAsIII by Primary Rat Hepatocytes in the Presence or Absence of Se Compounds: Cellular Retention of iAs and Methylation Ratesa Se compd added concn compd (µM) none (control) SeIV

MSe

DMSeO

TMSe

0 1 5 10 50 1 5 10 50 1 5 10 50 1 5 10 50

cellular retention of iAs (% of total As)

methylation rate (pmol CH3/h/106 cells)

3.5 ( 1.05 25.0 ( 1.77b 39.5 ( 4.90b 40.9 ( 4.59b 27.7 ( 5.57b 12.6 ( 0.60b 10.9 ( 1.75b 12.9 ( 1.93b 35.6 ( 0.86b 5.9 ( 1.04 4.6 ( 1.52 7.8 ( 1.90 6.0 ( 0.7 2.7 ( 1.82 6.5 ( 1.53 7.7 ( 1.52 2.1 ( 1.24

99.3 ( 1.87 56.8 ( 6.70b 17.3 ( 1.62b 20.0 ( 1.76b 0.4 ( 0.49b 73.8 ( 2.95b 78.2 ( 3.04b 76.3 ( 6.50b 12.4 ( 0.35b 86.3 ( 2.65 93.2 ( 1.44 86.5 ( 1.41b 86.5 ( 1.87b 103.6 ( 4.98 85.8 ( 1.20b 84.3 ( 1.64b 101.6 ( 1.94

a Cellular retention and methylation rates after 3 h incubation (mean ( SD, n ) 4). b Value significantly (p < 0.05) different from the corresponding control value.

Table 2. Methylation of 0.1 µM iAsIII by Primary Human Hepatocytes in the Presence or Absence of Se Compounds: Cellular Retention of iAs and Methylation Ratesa Se compd added concn compd (µM) none (control) SeIV MSe

0 2 10 2 10 50

cellular retention of iAs (% of total As)

methylation rate (pmol CH3/h/106 cells)

42.3 ( 3.99 78.4 ( 3.67b 78.5 ( 3.35b 43.2 ( 2.59 61.9 ( 2.33b 60.9 ( 7.14b

1.67 ( 0.24 0.04 ( 0.05b 0.02 ( 0.04b 1.38 ( 0.14 0.64 ( 0.13b 0.47 ( 0.12b

a

Cellular retention and methylation rates after 24 h incubation (mean ( SD, n ) 4). b Value significantly (p < 0.05) different from the corresponding control value.

cantly more (∼2-fold) iAs than did control cells (Table 2). Cellular retention of iAs also increased in cells treated with 10 and 50 µM MSe. Exposure to 2 or 10 µM SeIV strongly inhibited the methylation of [73As]iAsIII. MSe decreased the methylation rate in a concentrationdependent manner; however, it was less potent than SeIV. Exposure to 10 µM SeIV for 24 h significantly decreased (∼35%) cell viability. Cell viability was unchanged in other treatment groups. Conclusions and Significance. The liver, the largest depot for AdoMet and a major site for methylation reactions (32), is also the major site for methylation of iAs. In vitro, human hepatocytes exposed to iAsIII produce MAs and DMAs in proportions that approximate those found in the urine of individuals exposed to iAs (26). Methylated metabolites produced by human hepatocytes include the methylated trivalent arsenicals, methylarsonous and dimethylarsinous acids (33), which are more potent enzyme inhibitors, cytotoxins, and genotoxins than iAsIII (34). Methylated trivalent arsenicals are also more potent than iAsIII as activators of signal transduction pathways that regulate cell proliferation and apoptosis (35). Thus, methylation of iAs in the liver is a critical step in the activation of iAs as a toxin and carcinogen. Modifying the dynamics of iAs methylation

Communications

may materially affect the toxicity and carcinogenicity of this metalloid. Modifications of these metabolic processes by various environmental, nutritional, and genetic factors may be responsible for variations in the prevalence and severity of adverse health effects within and among human populations chronically exposed to iAs. Notably, selenium has been identified as a factor that might alter the metabolism and toxicity of iAs and possibly modify the risk associated with chronic exposure to iAs (36). The congruence of findings that SeIV and MSe competitively inhibit the methylation of iAsIII by Cyt19 and that exposure to SeIV and MSe compromises the capacity of hepatocytes to produce methylated arsenicals suggests that inhibition of Cyt19 by selenium compounds is a locus for the interaction of these two metalloids. Inhibition of Cyt19 by selenium compounds increases the cellular burden of iAs. Increased accumulation of iAs in hepatocytes exposed to SeIV has been associated with an exacerbation of the cytotoxicity of iAsIII (1). Because SeIV is considerably more potent than MSe, DMSeO, or TMSe as an inhibitor of Cyt19, it can be surmised that methylation of SeIV reduces its efficiency as an inhibitor of iAs metabolism. The relatively low Ki and IC50 values for inhibition of Cyt19 by SeIV indicate that attainable and nontoxic levels of exposure to this metalloid can suppress iAs methylation in hepatocytes. It has been speculated that selenium intake is an important modifier of the toxicity and carcinogenicity of iAs (37). Increasing selenium intake by dietary supplementation has been discussed as prophylaxis and/or therapy for adverse effects of iAs exposure. However, uncertainties about the mechanisms and concentration dependency of interactions between iAs and selenium must be resolved to provide a rational basis for the use of selenium supplements in interventional trials.

Acknowledgment. This study was supported by NIH Grant ES09941 to M.S. and NIH Clinical Nutrition Research Center Grant DK 56350. This paper has been reviewed in accordance with the policy of the U. S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

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