Reduction of Dimethylarsinic Acid to the Highly Toxic

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Reduction of Dimethylarsinic Acid to the Highly Toxic Dimethylarsinous Acid by Rats and Rat Liver Cytosol Balázs Németi and Zoltán Gregus* Department of Pharmacology and Pharmacotherapy, Toxicology Section, University of Pécs, Medical School, Pécs, Hungary ABSTRACT: Dimethylarsinic acid (DMAsV), the major urinary metabolite of inorganic arsenic, is weakly cytotoxic, whereas its reduced form, dimethylarsinous acid (DMAsIII), is highly toxic. Although glutathione S-transferase omega 1 (GSTO1) and arsenic methyltransferase have been shown or thought to catalyze DMAsV reduction, their role in DMAsV reduction in vivo, or in cell extracts is uncertain. Therefore, the reduction of DMAsV to DMAsIII in rats and in rat liver cytosol was studied to better understand its mechanism. To assess DMAsV reduction in rats, a novel procedure was devised based on following the accumulation of red blood cell (RBC)-bound dimethylarsenic (DMAs), which represents DMAsIII, in the blood of DMAsVinjected anesthetized rats. These studies indicated that rats reduced DMAsV to DMAsIII to a significant extent, as in 90 min 31% of the injected 50 μmol/kg DMAsV dose was converted to DMAsIII that was sequestered by the circulating erythrocytes. Pretreatment of rats with glutathione (GSH) depletors (phorone or BSO) delayed the elimination of DMAsV and the accumulation of RBC-bound DMAs, whereas the indirect methyltransferase inhibitor periodate-oxidized adenosine was without effect. Assessment of DMAsV-reducing activity of rat liver cytosol revealed that reduction of DMAsV required cytosolic protein and GSH and was inhibited by thiol reagents, GSSG and dehydroascorbate. Although thioredoxin reductase (TRR) inhibitors (aurothioglucose and SbIII) inhibited cytosolic DMAsV reduction, recombinant rat TRR plus NADPH, alone or when added to the cytosol, failed to support DMAsV reduction. On ultrafiltration of the cytosol through a 3 kDa filter, the reducing activity in the retentate was lost but was largely restored by NADPH. Such experiments also suggested that the reducing enzyme was larger than 100 kDa and was not GSTO1. In summary, reduction of DMAsV to the highly toxic DMAsIII in rats and rat liver cytosol is a GSH-dependent enzymatic process, yet its mechanism remains uncertain.

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promoted tumorigenesis in adult mice.15 In cultured cells, DMAs V caused apoptosis, which could be prevented, surprisingly, by the depletion of cellular glutathione (GSH).16 The toxicity of DMAsV is attributed largely to its partial reduction into its thiol-reactive trivalent form, dimethylarsinous acid (DMAsIII, (CH3)2AsOH).7,12,17,18 Because DMAsIII is 100−10,000 times more cytotoxic than DMAsV,19,20 the reduction of DMAsV to DMAsIII is a very effective toxification process. On the basis of the high cytotoxicity of DMAsIII, thiol complexes of DMAsIII (e.g., darinaparsine, the DMAsIIIglutathione complex), which are more stable than the spontaneously oxidizable DMAsIII, are being considered as anticancer drugs.21 The low acute toxicity of DMAsV is partly ascribed to rapid urinary excretion of this arsenical in most species. In rats, however, DMAsV is eliminated slowly, as indicated by the 90day long half-life of arsenic in DMAsV-dosed rats.22 This is due to the fact that rat erythrocytes sequester and retain arsenic22,23 in the form of DMAsIII bound to rat hemoglobin.24,25 In vitro studies indicate that DMAsV does not accumulate in the red blood cells (RBC), whereas DMAsIII is taken up and retained

INTRODUCTION Dimethylarsinic acid (DMAsV, (CH3)2As(O)OH, or cacodylic acid) is an organic pentavalent arsenical. DMAsV is the major urinary metabolite of inorganic arsenic (e.g., arsenite and arsenate), representing the most abundant arsenic species in the urine of humans and most animals,1−3 and is formed from arsenite in two sequential steps of methylation catalyzed by arsenic (+3 oxidation state) methyltransferase (As3MT).4 Therefore, exposure to inorganic arsenic through contaminated food and water, which is of great concern due to the associated chronic toxicity and carcinogenicity,5 is the most common source of DMAsV in the body. In addition, DMAsV is also a metabolite of some organic arsenicals, such as arsenosugars6 that are present in edible sea weed and dimethylthioarsenicals7 that are produced by intestinal bacteria. Furthermore, DMAsV is a nonselective herbicide and has been used for agricultural and military purposes.8 Therefore, DMAsV is an environmental contaminant, which may also occur in some food products such as brown rice syrup.9 The acute toxicity of DMAsV is comparatively low, yet DMAsV in large doses caused renal and bladder injury.10,11 Its prolonged high dose administration induced urinary bladder tumors in rats1,12,13 and lung and as well as skin tumors in mice.14 When initiated by prenatal arsenite exposure, DMAsV © 2013 American Chemical Society

Received: December 15, 2012 Published: February 17, 2013 432

dx.doi.org/10.1021/tx300505v | Chem. Res. Toxicol. 2013, 26, 432−443

Chemical Research in Toxicology

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by the RBC of rats (but not of other species)26 by avidly binding to the highly reactive thiol group of Cys-13α in rat hemoglobin.25 When GSH and DMAsV are incubated at extremely high concentrations, the thiol reduces DMAsV to DMAsIII and subsequently forms a DMAsIII−glutathione conjugate.27,28 At physiological GSH concentrations (1−10 mM), however, reduction of DMAsV is slow. Some enzymes have been shown or postulated to facilitate DMAsV reduction. Glutathione S-transferase omega 1 (GSTO1) was first described as monomethylarsonate reductase;29,30 however, later it was shown to catalyze the reduction of arsenate and DMAsV as well.31,32 DMAs V can also be reduced by a poxviral glutaredoxin.33 In addition, As3MT has been proposed to reduce pentavalent methylated metabolites into trivalent forms because this enzyme can transform arsenite into trimethylarsine oxide in serial reactions that putatively involve production of pentavalent mono- and dimethylated metabolites which are reduced into trivalent forms before the subsequent oxidative methylation takes place.4,17 Nevertheless, the role of these enzymes in the reduction of DMAsV to DMAsIII in the body is uncertain. For example, GSTO1 knockout mice exhibited only marginal alterations in arsenic metabolism,34 and according to a recent concept, As3MT-catalyzed stepwise methylation of arsenic does not involve the formation of pentavalent methylated metabolites and their reduction.35,36 The present work aimed at studying the reduction of DMAsV to the much more toxic DMAsIII both in vivo in rats and in vitro in rat liver cytosol in order to better understand its mechanism. For this purpose, we designed experiments for assessing the involvement in DMAsV reduction of (1) GSH, using rats pretreated with the GSH depletor phorone or BSO; (2) As3MT, using rats pretreated with the indirect methyltransferase inhibitor periodate-oxidized adenosine (PAD); (3) GSTO1, by examining the correlation of DMAsV-reducing and GSTO1 activities in fractions of rat liver cytosol obtained by ultrafiltration; and (4) other cytosolic enzymes, such thiol enzymes in general, some arsenolytic enzymes that facilitate reduction of arsenate to arsenite,37,38 and thioredoxin reductase 1 (TRR) that can reduce several xenobiotics,39 using their chemical inhibitors. For assessment of DMAsV reduction in rats, we devised a novel procedure. This is based on following the time course of the accumulation in the blood of the RBCbound dimethylarsenic (DMAs), which represents DMAsIII. For this purpose, the concentration of RBC-bound DMAs was periodically determined in the blood of anesthetized DMAsVinjected rats whose renal pedicles had been ligated, lest variations in the urinary excretion of DMAsV should confound the assessment of DMAsV reduction. Reduction of DMAsV by rat liver cytosol was assayed by extraction of DMAsIII from the incubations of cytosol with DMAsV and quantification by HPLC-HG-AFS.

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tris(2-carboxyethyl)phosphine hydrochloride (TCEP), tocopherol succinate, N-ethylmaleimide (NEM), 5,5′-dithio-bis-nitrobenzoic acid (DTNB), and 4-(chloromercuri)benzenesulfonic acid sodium salt (PCMBS) were from Sigma. Polyethylene glycol 20000 (PEG) was from Fluka. Recombinant rat liver thioredoxin reductase 1 (TRR; TR03-B) was from IMCO Corporation Ltd. AB (Stockholm, Sweden). Aurothioglucose was a gift from Schering-Plough (Kenilworth, NJ) and BCX-1777 from BioCryst Pharmaceuticals (Birmingham, AL), whereas koningic acid was from Professor Keiji Hasumi (Tokyo Noko University, Tokyo, Japan). Periodate-oxidized adenosine (PAD) was synthesized according to Hoffman40 and S-(4-nitrophenacyl)glutathione according to Board and co-workers.41 The sources of chemicals used in arsenic speciation have been given elsewhere.42,43 All other chemicals were of the highest purity available. Assessment of the Reduction of DMAsV in Vivo. Male Wistar rats from the SPF breeding house of the University of Pécs (Hungary) weighing 270−330 g were used. The rats were kept at 22−25 °C, at 55−65% relative air humidity, and on a 12-h light/dark cycle, and were provided with rodent lab chow and tap water ad libitum. All procedures were carried out on animals according to the Hungarian Animals Act, and the study was approved by the Ethics Committee on Animal Research of the University of Pécs. To deplete hepatic GSH, some rats were injected ip with phorone (2 mmol/kg in 2 mL/kg corn oil) or BSO (4 mmol/kg in 10 mL/kg saline) at 2 or 5 h before DMAsV administration, respectively. To inhibit methyltransferases, some rats were pretreated with ip injection of PAD (50 μmol/kg in 5−10 mL/kg saline) at 40 min before DMAsV administration. The PAD solution was prepared in saline before use, and its concentration was determined spectrophotometrically as described.44 Control rats were injected with corn oil or saline. At approximately 30 min before the injection of DMAsV, the animals were anesthetized with urethane (1.2 g/kg ip in 5 mL/kg water), and their body temperature was maintained by heating. The right carotid artery of the rats was cannulated, and the renal pedicles were ligated. DMAsV (50 μmol/kg in 2 mL/kg saline) was then injected into the left saphenous vein. Blood samples (170−200 μL) were collected through the carotid cannula into heparinized tubes before DMAsV administration and at 5, 20, 40, 60, and 90 min thereafter. At 90 min after DMAsV injection, the animals were exsanguinated, and the liver was sampled to determine hepatic DMAs and GSH concentrations. For determining GSH concentrations in blood, 150 μL of blood taken from the carotid artery at 90 min was mixed with an equal volume of 0.6 M perchloric acid and then stored at −70 °C. For measuring total DMAs in blood, 75 μL of whole blood was hemolyzed by mixing with 1/10 volume of 5.5% Triton X-100, then the protein-bound DMAs was displaced by adding 1/10 volume of 150 mM HgCl2. After keeping on ice for 1 min, the sample was deproteinized by mixing with 75 μL of 0.67 M perchloric acid (0.3 M final concentration) and centrifuged for 30 s at 10,000g. The supernatant fraction was diluted 5- or 10-fold and its DMAs concentration was determined by HPLC-HG-AFS. For measuring RBC-bound DMAs, rat RBC were isolated and washed with saline, which contained 1% (m/v) PEG-20000 in order to prevent hemolysis.45 First, 75 μL of blood was mixed with 100 μL of saline-PEG solution and centrifuged at 10,000g for 30 s. The resultant supernatant liquid was carefully removed and discarded, and the RBC sediment was washed three more times with 0.5 mL of saline-PEG solution by serially resuspending the RBC and sedimenting them by centrifugation. The final RBC pellet was measured gravimetrically, and its volume was brought to 75 μL by adding 0.9% NaCl solution. As described above, this washed RBC suspension was hemolyzed, mixed with HgCl2, and was deproteinized before analysis of RBC-bound DMAs. For quantifying hepatic DMAs and GSH, the liver samples were homogenized in 2.5 volumes of saline with continuous argon purging to minimize the oxidation of trivalent arsenicals. Because trivalent arsenicals are bound to thiol groups in proteins and GSH, whereas pentavalent ones remain unbound,35 the hepatic concentrations of total, bound, and unbound DMAs were determined. For total DMAs

EXPERIMENTAL PROCEDURES

Caution: The arsenic species included in this study are toxic and are potential human carcinogens; therefore, they should be handled with great care. Chemicals. Reduced and oxidized glutathione (GSH and GSSG, respectively), reduced β-nicotinamide-adenine-dinucleotide phosphate (NADPH), and phorone were from Reanal Ltd. (Budapest, Hungary). HEPES, glutathione reductase (GR) from baker’s yeast, L-buthionineS,R-sulfoximine (BSO), diethyldithio-carbamic acid diethylammonium salt (DDDC), potassium antimony(III) tartrate, cacodylic acid (dimethylarsinic acid) sodium salt, dehydroascorbic acid (DHA), 433



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analysis, the homogenate was first mixed with HgCl2 (10 mM) in order to displace SH-group-bound DMAsIII and 60 s later with perchloric acid (0.3 M) to precipitate proteins. For quantitation of DMAs unbound to thiol groups, the homogenate was mixed with water instead of HgCl2 and then was deproteinized with perchloric acid. The deproteinized liver homogenate was centrifuged, and the supernatant fraction was used for arsenic analysis. Aliquots of the homogenates treated with perchloric acid (0.3 M) were stored at −70 °C until GSH analysis. Preparation of Rat RBC and Rat Liver Cytosol for Assessing DMAsV Reduction in Vitro. For the preparation of washed RBC, arterial blood was collected from anesthetized rats into heparinized tubes, centrifuged at 1,000g at 4 °C for 10 min, and the plasma and buffy coat were discarded. The RBC pellet was resuspended in 3 volumes of a buffer containing 250 mM sucrose, 25 mM HEPES, 5 mM MgCl2, 5 mM glucose, 2 mM EGTA, and 1% (m/v) PEG-20000 at pH 7.4, and centrifuged. This washing procedure was repeated two more times. The final RBC pellet was resuspended in equal volumes of the above buffer and kept on ice until use within 3 h. In order to prepare hepatic cytosol, rats were euthanized and exsanguinated, and their liver was removed and weighed. Great care was taken to remove blood from the liver. Therefore, the liver was chopped up in ice-cold homogenization buffer into small pieces, which were then washed three times by decanting and refilling. Thereafter, the liver was homogenized in 3 volumes of a buffer containing 250 mM sucrose, 25 mM HEPES, 5 mM MgCl2, and 2 mM EGTA, pH 7.4 (sucrose buffer), using a glass homogenization tube first with a looser then with a tighter motor-driven Teflon pestle. The homogenate was centrifuged at 10,000g at 4 °C for 20 min to obtain the postmitochondrial supernatant fraction. After removing the fatty top layer by suction, the postmitochondrial supernatant fraction was subjected to ultracentrifugation (at 100,000g at 4 °C for 75 min). The supernate corresponding to the cytosolic fraction was stored in aliquots at −70 °C until use. The protein concentration of the cytosol was determined by the bicinchoninic acid method.46 Assessment of the Reduction of DMAsV by Rat Liver Cytosol. Rat liver cytosol (typically 5 mg protein/mL) was incubated with DMAsV (typically 50 μM) with GSH (typically 10 mM) in sucrose buffer at 37 °C for 20 min. The incubations were started by adding DMAsV. Deviations from these conditions are given in the figure legends. For quantifying DMAsIII formed in the incubations, two procedures were used. In some incubations, freshly prepared washed rat RBC were added (50 μL packed cell/300 μL incubation volume) in order to sequester DMAsIII. In these assays, the incubation buffer was supplemented with 5 mM glucose and 1% PEG-20000 for providing RBC with fuel and to prevent hemolysis, respectively. After the 20-min incubation, the RBC were quickly sedimented from the incubation mixtures by centrifugation, the supernatant liquid was discarded, and the RBC were washed 3 times with 500 μL of incubation buffer containing glucose and PEG. After the last washing, the RBC pellet was hemolyzed by adding 50 μL of water and 10 μL of 5.5% Triton X100. DMAsIII bound to rat hemoglobin was displaced by HgCl2 (10 mM), followed by protein precipitation with perchloric acid (0.3 M) and centrifugation. During this procedure, DMAsIII became oxidized to DMAsV, which was then quantified in the deproteinized samples by HPLC-HG-AFS. In most incubations, the DMAsIII formed was extracted as a DDDC complex. Because DMAsIII is highly oxidizable,47 the incubation mix dispensed into 1.5-mL Eppendorf tubes was purged with argon. The tubes were kept closed and were refilled with argon and closed whenever opened for adding cytosol, DMAsV, or reagents. At termination of the incubations, DMAsIII was extracted by a method described previously48,49 with slight modifications. Briefly, 4 incubation volumes of ice-cold 10 mM DDDC dissolved in carbon tetrachloride was added to the incubation mixtures, then the tubes were shaken at 1400/min for 5 min at 20 °C in a Thermomixer (Eppendorf). DDDC forms a stable complex with trivalent arsenic compounds at near-neutral pH that is readily soluble in CCl4. Immediately after shaking, the samples were centrifuged for 15 s. The

upper aqueous phase was carefully removed by aspiration, and the lower CCl4 phase containing DMAsIII complexed with DDDC was transferred into a clean tube. To the CCl4 extract, half of its volume of 0.1 M NaOH solution was added and was then shaken again for 5 min. At the highly basic pH, the DDDC-DMAsIII complex dissociates, and the free arsenical (largely in oxidized form) is back-extracted into the aqueous phase. After shaking, the two phases were separated by a short centrifugation, and the aqueous upper phase was transferred into a clean tube. This back-extraction step was repeated. To the combined aqueous extracts, H2O2 was added at a final concentration of 0.25% to convert any remaining DMAsIII to DMAsV, which was then measured by HPLC-HG-AFS. Ultrafiltration of Cytosol. Rat liver cytosol was subjected to ultrafiltration using Amicon Ultra 3K and 100K centrifugal filtration devices with 3 kDa and 100 kDa molecular weight cutoff, respectively. The filtrate after the first filtration step was kept on ice until use, whereas the retentate was washed 3 times with sucrose buffer at volumes equal to that of the cytosol processed. The final retentate was measured gravimetrically, and buffer was added to it to match the volume of the original cytosol. The filtrate and the final retentate were assayed for DMAsV-reducing and GSTO1 activities. Analyses. GSH in blood and liver was quantified spectrophotometrically according to Tietze50 by measuring the GSH-limited reduction of DTNB in the presence of glutathione reductase and NADPH. The GSTO1 activity in liver cytosols as well as the retentates and filtrates prepared by ultrafiltration of cytosol was assayed as described.41 This spectrophotometric assay follows the GSTO1limited conversion of S-(4-nitrophenacyl)glutathione to 4-nitroacetophenone in the presence of 2-mercaptoethanol at 305 nm. The activity of purified recombinant TRR was determined according to Holmgren and Björnstedt.51 The spectrophotometric assay measures the TRR-limited reduction of DTNB (5 mM) in the presence of 0.2 mM NADPH. The measurements were run in a buffer containing 100 mM potassium phosphate and 10 mM EDTA, pH 7.0, in a final volume of 0.1 mL. The assay mixture contained 0.02% bovine serum albumin in order to prevent adsorption of TRR to the spectrophotometer cells. When the inhibitory effects of ATG and SbIII on TRR were compared, these compounds were preincubated with TRR for 4 min before starting the assay with the addition of NADPH. The TRR activity in rat liver cytosol was measured spectrophotometrically, according to Hill and co-workers 52 with minor modifications. GSH and small proteins with reactive SH-groups (e.g., thioredoxin) were removed from the cytosols through a 30 kDa MWCO centrifugal filter, as they would interfere with the TRR assay. The three times washed retentate was assayed for TRR activity with DTNB and NADPH as described above. These assays were run in the absence and presence of the specific TRR inhibitor ATG (20 μM), and the TRR activity was calculated from the difference in the rates of absorbance increase at 412 nm measured under these conditions. One unit of TRR is defined as the quantity that reduces 1 μmol of DTNB in 1 min. DMAs in the blood and liver samples of rats prepared as described above as well as in incubation extracts was measured by HPLC-HGAFS largely as reported.43,53 Such analyses indicated that the samples contained only DMAsV. Therefore, in subsequent analyses the analytical system was used without HPLC column. Highly purified water was pumped with an HPLC pump at a flow rate of 2.2 mL/min through an injector equipped with a 20-μL sample loop. To the effluent exiting from the injector, first 1.5 M HCl then 1.5% sodium borohydride in 0.1 M NaOH were mixed (both at 1 mL/min flow rate) to convert DMAs V into dimethylarsine. The gaseous dimethylarsine was separated from the aqueous phase by a gas−liquid separator and transferred to the atomic fluorescence detector (PS Analytical Excalibur, PSA, Kent, UK) by a mixed stream of argon (300 mL/min) and hydrogen (60 mL/min) through a hygroscopic membrane dryer tube. The fluorescence signal was recorded by a computer using Millennium Chromatography Manager (Waters). Quantification of DMAsV was based on the peak areas in the samples and in authentic standards. 434



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Statistics. Data were analyzed using one-way ANOVA followed by Dunnett’s t test with p < 0.05, as the level of significance.

blood. As shown in Figure 2A, the RBC-unbound DMAs concentrations progressively declined with time in control rats. This decline was significantly slower in rats pretreated with phorone than in the controls; therefore, the unbound DMAs concentrations became significantly higher in the phoronetreated rats at 40−90 min. In the BSO-treated animals, the decline in the RBC-unbound DMAs concentration tended to be slower. In contrast, the RBC-bound DMAs concentrations increased at a considerably slower rate in the GSH-depleted rats as compared to the controls and were significantly lower from 5 min on in rats dosed with phorone and from 20 min on in the BSO-treated animals (Figure 2B). Phorone was more effective than BSO in delaying both the elimination of RBC-unbound DMAs and the accumulation of RBC-bound DMAs in the blood of DMAsV-injected rats. At 90 min after DMAsV injection, the hepatic GSH levels in control, phorone-, and BSO-treated rats were 4.99 ± 0.23 (n = 9), 0.14 ± 0.04 (n = 4), and 0.94 ± 0.08 μmol/g (n = 5), respectively, indicating that the treatments markedly lowered the GSH content of the liver. However, the blood GSH concentrations were decreased insignificantly by these chemicals, as GSH in the blood of control, phorone-, and BSO-treated rats was 1.27 ± 0.09, 1.08 ± 0.15, and 1.01 ± 0.05 μmol/mL whole blood, respectively. The role of As3MT in DMAsV reduction was assessed by examining the effect of PAD pretreatment on the fate of DMAsV in blood. Pretreatment of rats with PAD at a dose that abolished the formation of methylated arsenite metabolites from arsenite or arsenate53,54 resulted in no significant change in the concentrations of either the RBC-unbound (Figure 2C) or the RBC-bound DMAs (Figure 2D). PAD pretreatment did not influence the GSH concentration in the liver and the blood at 90 min after DMAsV injection (not shown). At the end of the 90-min experiment, the unbound and the SH-group-bound DMAs concentrations were also determined in the liver of rats treated with phorone, BSO, and PAD. The unbound DMAs level markedly increased, whereas the thiolbound DMAs concentration (putatively representing trivalent arsenical) significantly decreased as a result of phorone and BSO pretreatment (Figure 3), albeit the latter appeared less effective. PAD slightly diminished the concentration of the unbound DMAs without influencing that of the bound one. Reduction of DMAsV by Rat Liver Cytosol: Dependence on GSH and Protein Thiols. In order to further substantiate our postulation that reduction of DMAsV to DMAsIII by rat tissues may be assessed from the increase in erythrocyte-bound DMAs in rat blood, we incubated DMAsV with rat liver cytosol (that may contain DMAsV-reducing enzymes, such as GSTO1) in the presence of washed rat RBC and measured the DMAs sequestered by the RBC. Rat erythrocytes always contain DMAs due to some minimal arsenic exposure of the animals with food and drinking water. Therefore, the RBC isolated from incubations with buffer without any DMAsV added contained a basal concentration of DMAs that is indicated by the horizontal stripe in Figure 4. The RBC-bound DMAs concentration increased only insignificantly above this basal value when rat RBC were incubated with DMAsV alone, or DMAsV together with GSH, or DMAsV together with hepatic cytosol. In contrast, incubation of DMAsV with RBC in the presence of both cytosol and GSH increased the RBC-bound DMAs concentration significantly (Figure 4). The net increase in RBC-bound DMAs concentration brought about by the combined presence of cytosol, GSH, and DMAsV indicated that the GSH-fortified

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RESULTS Reduction of DMAs V by Rats: Assessment by Quantitation of RBC-Bound DMAs in the Blood. Figure 1 demonstrates the time courses of total, RBC-bound, and

Figure 1. Time courses of total, RBC-bound, and RBC-unbound DMAs in the blood of rats injected with DMAsV. Urethaneanesthetized rats with cannulated right carotid artery and ligated renal pedicles were injected with DMAsV (50 μmol/kg, iv) at time zero, and blood samples were collected through the carotid cannula at the times indicated. The concentration of DMAs in whole blood and in washed RBC was measured as total and RBC-bound DMAs, respectively. The concentration of the RBC-unbound DMAs was calculated as the difference between the total and the RBC-bound concentration. Symbols represent DMAs concentrations (mean ± SE) in the blood of nine rats.

RBC-unbound concentrations of DMAs in the blood of renal pedicle-ligated rats injected with DMAsV. As shown in the figure, the blood of rats contained 13.7 ± 1.29 nmol/mL DMAs bound to RBC even before administering DMAsV. After DMAsV injection, the concentration of RBC-bound DMAs (which putatively represents DMAsIII) increased linearly for 90 min. No RBC-unbound arsenic was detected in rat blood before arsenic administration. The concentration of RBCunbound DMAs was high early after the injection of DMAsV and kept decreasing thereafter. The DMAs concentration of the whole blood exhibited a biphasic time course, as it remained unchanged or declined slightly between 5 and 20 min but increased steadily thereafter, in parallel with the increase in RBC-bound DMAs. Reduction of DMAs V by Rats: Effects of GSH Depletion and Methyltransferase Inhibition. In order to test whether hepatic GSH depletion influences the reduction of DMAsV, control rats and animals pretreated with phorone or BSO were injected with DMAsV, and the RBC-bound and RBC-unbound DMAs concentrations in blood were measured from 0 to 90 min. At 90 min, the effectiveness of GSH depletors was evaluated by quantifying GSH in the liver and 435



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Figure 2. Effects of GSH depletion by phorone or BSO and of methyltransferase inhibition by PAD on the time courses of the RBC-unbound and RBC-bound DMAs in rats injected with DMAsV. Rats were pretreated with phorone (2 mmol/kg, ip), BSO (4 mmol/kg, ip), or PAD (50 μmol/kg, ip) 120, 300, and 40 min before DMAsV administration (50 μmol/kg, iv), respectively. Shortly before DMAsV injection, rats were subjected to urethane anesthesia, carotid artery cannulation, and renal pedicle ligation. After DMAsV injection at time zero, blood samples were collected at the times indicated for the determination of RBC-unbound DMAs (panels A and C) and RBC-bound DMAs (panels B and D), as described in the legend to Figure 1. Symbols represent DMAs concentrations (mean ± SE) in chemical-treated rats (five in each group) and nine vehicle-treated controls. Asterisks indicate DMAs concentrations significantly different (p < 0.05) from those observed in control rats.

cytosol formed DMAsIII at a rate of 32.4 ± 2.5 pmol/min/mg cytosolic protein (n = 5). Thus, the cytosolic DMAsV-reducing activity could be assessed based on the sequestration of DMAsIII by washed rat erythrocytes. However, the sensitivity and accuracy of such assays are compromised by the basal DMAs content of rat RBC. Therefore, in subsequent experiments DMAsV reduction by rat liver cytosol was assayed by a method based on complexation of the formed DMAsIII by DDDC.48,49 In order to establish the optimal incubation conditions for cytosolic DMAsV-reducing activity, we examined the time course of the formation of DMAsIII from DMAsV and its dependence on GSH and cytosolic protein concentrations. GSH supported the cytosolic DMAsV reduction in an asymptotic fashion: addition of 2.5 mM GSH greatly enhanced the formation of DMAsIII, whereas further GSH concentration increases resulted in comparatively smaller elevations of the DMAsV reduction rate (Figure 5A). In contrast, DMAsIII production in the presence of 10 mM GSH increased linearly as a function of cytosolic protein concentration (Figure 5B) and time (Figure 5C). Because the plot of the cytosolic DMAsIII formation rate against the GSH concentration exhibited an apparent saturation curve (Figure 5A), it was of interest to test whether oxidized glutathione (GSSG; the product of GSH-dependent DMAsV reduction) and dehydroascorbic acid (whose reduction to ascorbate generates GSSG) affect the cytosolic DMAsVreducing activity. In the presence of 10 mM GSH, both GSSG and DHA inhibited DMAsV reduction by rat liver cytosol in a concentration-dependent manner (Figure 6A), and their IC50 values were approximately 0.25 mM and 0.5 mM, respectively. To test whether or not GSSG production was responsible for the observed inhibition of DMAsV reduction by DHA, incubations containing 1 mM DHA were also carried out

in the presence of GR, NADPH, or both. As shown in Figure 6B, 1 mM DHA inhibited the formation of DMAsIII by more than 90%. GR alone tended to diminish the inhibitory effect of DHA, and NADPH markedly attenuated it, whereas in the combined presence of GR plus NADPH, DHA decreased DMAsV reduction by rat liver cytosol only by 25%. Importantly, GR, NADPH, or their combination did not influence the rate of DMAsIII formation when the cytosolic DMAsV reduction was measured in the absence of DHA (Figure 6B). To assess the role of protein SH-groups in the cytosolic reduction of DMAsV, incubations were performed with cytosol that was preincubated with NEM or PCMBS. These experiments revealed that both SH-reagents inhibited DMAsIII formation, decreasing it by more than 95% at a concentration of 0.5 mM (Table 1). Reduction of DMAsV by Rat Liver Cytosol: Role of PNP, GAPDH, TRR, and GSTO1. Using chemical inhibitors of enzymes, we tested whether or not PNP, GAPDH, TRR, or GSTO1 contributed to the cytosolic DMAsV reduction. The PNP inhibitor BCX-1777, the GAPDH inhibitor koningic acid, as well as the GSTO1 inhibitor tocopherol succinate at concentrations well exceeding those causing complete inactivation of these enzymes failed to affect the formation of DMAsIII from DMAsV by rat liver cytosol (Table 1). The known TRR inhibitor ATG and SbIII at 1 μM concentration inhibited the activity of the purified recombinant rat liver TRR by 93% and 89%, respectively (observations not shown). These two chemicals also inhibited the cytosolic DMAsV reduction in a concentration-dependent manner at concentrations 5 μM and above (Figure 7). However, when incubated in the presence of GSH (10 mM) and NADPH (2 mM), rat recombinant TRR at 400 mU/mL final concentration did not catalyze the formation of DMAsIII from DMAsV. For comparison, the TRR activity in the cytosol incubations 436



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Figure 4. Glutathione-dependent reduction of DMAsV by rat liver cytosol as assessed by the increase in the amount of DMAs bound to rat RBC. In 300 μL incubation volume, washed rat RBC (50 μL packed cell) were incubated with DMAsV (50 μM) alone or together with GSH (10 mM), rat liver cytosol (5 mg protein/mL), or GSH plus cytosol at 37 °C in a buffer containing sucrose (250 mM), HEPES (25 mM), MgCl2 (5 mM), glucose (5 mM), EGTA (2 mM), and PEG20000 (1%) at pH 7.4. The incubations were started by adding DMAsV. Twenty minutes later, the RBC were quickly removed from the incubation mixture by centrifugation and washed three times, and their DMAs content was measured. Bars represent the concentration of RBC-bound DMAs (mean ± SE) in five incubations with RBC and liver cytosol prepared from different rats. The horizontal stripe indicates DMAs concentration in rat RBC (mean ± SE; n = 5) not incubated with DMAsV. The asterisk indicates significantly higher DMAs concentrations in RBC incubated with DMAsV, GSH, and cytosol (p < 0.05) than in RBC removed from incubations lacking GSH, cytosol, or both.

Figure 3. Effects of GSH depletion by phorone or BSO and of methyltransferase inhibition by PAD on the DMAs concentrations in the liver of rats injected with DMAsV. Rats were pretreated with phorone (2 mmol/kg, ip), BSO (4 mmol/kg, ip), or PAD (50 μmol/ kg, ip) 120, 300, and 40 min before DMAsV injection (50 μmol/kg, iv), respectively. Shortly before DMAsV injection, rats were subjected to urethane anesthesia, carotid artery cannulation, and renal pedicle ligation. At 90 min after DMAsV injection, liver samples were taken. Unbound DMAs was measured from the supernatant fraction of the liver homogenates deproteinized with perchloric acid. Total DMAs was determined from the same homogenates treated with HgCl2 prior to deproteinization. Bound DMAs was calculated as the difference between the total and unbound DMAs. Bars represent the unbound and the bound DMAs concentration (mean ± SE) in the liver of chemical-treated rats (five in each group) and nine vehicle-treated controls. Asterisks indicate DMAs concentrations significantly different (p < 0.05) from those observed in control rats.

containing 5 mg of cytosolic protein/mL was only 21.2 ± 1.1 mU/mg cytosolic protein (n = 6). Furthermore, when rat recombinant TRR (400 mU/mL) was added together with 10 mM GSH and 2 mM NADPH into cytosolic incubations, the DMAsV-reducing activity (236 ± 4 pmol/min/mL incubation mixture; n = 3) was comparable to that observed in similar incubations without recombinant TRR supplementation (247 ± 10 pmol/min/mL incubation mixture; n = 4). Because GSTO1 has been shown to reduce DMAsV, we further assessed its contribution to the cytosolic DMAsV reduction by searching for the correlation between the GSTO1- and DMAsV-reducing activities in rat liver cytosols. Unexpectedly, GSTO1 activity was undetectable in rat liver cytosol (Figure 8); however, it was present in the retentate of the cytosol obtained after its ultrafiltration. Interestingly, GSTO1 activity was present not only in the 3 kDa retentate but also in the retentate prepared with a 100 kDa molecular weight cutoff filter, albeit this activity was lower than in the 3 kDa retentate. The filtrate fractions originating from either the 3 kDa or the 100 kDa ultrafiltration did not exhibit any GSTO1 activity; however, they diminished the GSTO1 activity of the retentate after recombining the filtrate with the retentate at amounts corresponding to the unfiltered cytosol (Figure 8). Prompted by these unexpected results, we examined how the ultrafiltration of cytosol influenced its DMAsV-reducing activity.

As shown in Table 2, ultrafiltration of the cytosol through either a 3 kDa or a 100 kDa filter yielded retentates and filtrates with minimal DMAsV-reducing activities. On recombining the retentate with the filtrate, the DMAsV-reducing activity could be restored to approximately 80% of the unfiltered cytosol. Interestingly, the activity of the 100 kDa retentate could also be restored partially when it was incubated in the presence of a 3 kDa filtrate prepared from the same cytosol. In addition, NADH, NADPH, or TCEP could also partially restore the DMAsV-reducing activity of the retentate. Of these, NADH was the weakest, causing recovery of approximately 50% and 35% activity of the unfiltered cytosol when added to the retentates of the 3 kDa and the 100 kDa filters, respectively. Addition of NADPH to the retentate supported DMAsV reduction more effectively, resulting in approximately 75% reducing activity, whereas the DMAsIII formation by the TCEP-supplemented retentate almost reached that of the unfiltered cytosol (Table 2).

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DISCUSSION The observation that the erythrocytes apparently accumulate DMAsV in both untreated rats (derived from arsenic present in chow and drinking water at acceptable levels) and in rats injected with arsenite or arsenate54 made us suggest that rats 437



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Figure 5. Reduction of DMAsV by rat liver cytosol: dependence on GSH and cytosolic protein concentrations and on incubation time. Rat liver cytosol (5 mg protein/mL or as indicated in panel B) was preincubated with GSH (10 mM or as indicated in panel A) at 37 °C for 5 min in a buffer containing sucrose (250 mM), HEPES (25 mM), MgCl2 (5 mM), EGTA (2 mM) at pH 7.4 (sucrose buffer). Thereafter, DMAsV (50 μM) was added to start the incubation lasting 20 min or as shown in panel C. At the end, the DMAsIII produced was extracted from the incubation mixtures with DDDC and was measured as described in Experimental Procedures. Symbols represent DMAsIII formation rates (mean ± SE) in four incubations with liver cytosol prepared from different rats. The equations and the r2 values of the linear regression lines fitted to the data points in panels B and C are y = 45.8x − 36.4 (r2 = 0.988) and y = 36.1x − 14.4 (r2 = 0.998), respectively.

are useful to study the in vivo formation of DMAsV by quantifying the buildup of this metabolite in the blood. Later it turned out that the hemoglobin-bound dimethylated arsenic is in the trivalent form,25 which is rapidly oxidized to DMAsV upon its displacement for analysis. On recognizing this, we now propose that rats are uniquely useful to study the in vivo reduction of DMAsV by quantifying the time-dependent accumulation of DMAsIII in the RBC after injection of DMAsV. Even if most or all species may have the capacity to reduce DMAsV, no other species is known in which RBCsequestered DMAsIII could serve as a quantitative biomarker of DMAsV reduction. Thus, while the rat is not regarded as a good model to study arsenic disposition in general because arsenic is bound to rat RBC in the form of DMAsIII, it is precisely this peculiar feature that makes the rat suitable for studying the conversion of DMAsV to DMAsIII in vivo. Binding of DMAsIII to rat hemoglobin is virtually permanent as suggested by the 90day-long half-life of arsenic in DMAsV-dosed rats,22 indicating that the rat RBC retains DMAsIII during the cell’s lifetime. On the basis of the information cited in the Introduction, the steps leading to the sequestration of arsenic in the erythrocytes of rats exposed to DMAsV are envisaged as follows: (1) uptake of DMAsV from the blood plasma into tissues, (2) reduction of DMAsV to DMAsIII (perhaps preferentially by the liver), (3) covalent reaction of DMAsIII with intracellular GSH and protein thiols, (4) export of the DMAsIII−glutathione conjugate into the blood, (5) hydrolysis of this conjugate in the GSHpoor plasma, (6) uptake of the membrane permeable DMAsIII

by the erythrocytes, and (7) covalent reaction of DMAsIII with rat hemoglobin. This scenario is compatible with our observations on DMAsV-injected rats, including the timeproportional increase in RBC-bound DMAs in the blood (Figure 1) and the presence of over 50% of DMAs in the liver in bound form (bound in trivalent form to protein-SH and GSH; Figure 3), as well as with the findings of the in vitro studies, demonstrating that upon incubation of DMAsV with rat liver cytosol and GSH (ingredients required for DMAsV reduction) a substantial amount of DMAs accumulates in the coincubated rat RBC (Figure 4). This work demonstrates that reduction of DMAsV to DMAsIII in renal-pedicle-ligated, DMAsV-injected rats is a rapid process. In DMAsV-injected rats, the concentration of the RBC-bound DMAs (representing DMAsIII) increased linearly from 14 nmol/mL at the time of injection to 274 nmol/mL at 90 min after injection (Figure 1). Thus, the net increase in RBC-bound DMAs concentration over 90 min was 260 nmol/ mL blood. Considering that the blood volume of rats is approximately 60 mL/kg body wt, the rats formed 15.6 μmol/ kg RBC-bound DMAs in 90 min, thus they converted 31% of the injected 50 μmol/kg DMAsV dose into DMAsIII that was eventually sequestered by the circulating erythrocytes. This figure is a minimal estimate for the extent of DMAsV reduction in rats because not all DMAsIII formed may be captured by the erythrocytes; some are bound to protein thiols in tissues,35 and some may be reoxidized to DMAsV in the plasma after being hydrolyzed from DMAsIII−glutathione and before being 438



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Figure 6. Reduction of DMAsV by rat liver cytosol: inhibition by GSSG and DHA (A) and attenuation of the inhibitory effect of DHA in the presence of glutathione reductase (GR), NADPH, or both (B). Rat liver cytosol (5 mg protein/mL) was preincubated at 37 °C for 5 min with GSSG or DHA (at concentrations indicated) in sucrose buffer. Thereafter, GSH (10 mM) and DMAsV (50 μM) were added in rapid succession to start the incubation lasting for 20 min. GR (0.5 U/0.3 mL), NADPH (2 mM) or both were added to the incubations immediately before DMAsV. The DMAsIII produced was extracted from the incubation mixtures with DDDC and was measured as described in Experimental Procedures. Symbols and bars represent DMAsIII formation rates (mean ± SE) in three incubations with rat liver cytosol prepared from different animals. Asterisks indicate DMAsIII formation significantly lower (p < 0.05) than that observed in the absence of GSSG and DHA. # in the right panel indicates DMAsIII formation significantly higher (p < 0.05) than that observed in the presence of 1 mM DHA alone.

Table 1. Reduction of DMAsV by Rat Liver Cytosol: Effects of Thiol Reagents and Inhibitors of PNP, GAPDH, and GSTO1a DMAsIII formation % control control NEM, 0.5 mM NEM, 2 mM PCMBS, 0.1 mM PCMBS, 0.5 mM BCX-1777, 0.02 mM koningic acid, 0.1 mM tocopherol succinate, 0.025 mM tocopherol succinate, 0.05 mM

100 ± 4.05 5.00 ± 0.38 1.30 ± 0.51 24.9 ± 2.21 3.10 ± 0.12 106 ± 9.78 103 ± 4.78 104 ± 2.96 96.1 ± 3.77

* * * *

a

Rat liver cytosol (5 mg protein/mL) was preincubated in sucrose buffer at 37 °C for 5 min in the presence of one of the inhibitors. Thereafter, GSH (10 mM) and DMAsV were added in rapid succession to start the incubations lasting for 20 min. The DMAsIII produced was extracted from the incubation mixtures with DDDC and was measured as detailed in Experimental Procedures. DMAsIII formation in the control incubations was 37.53 ± 1.52 pmol/min/ mg of protein (n = 3). Values are the percentages of the control DMAsIII formation rates (mean ± SE) measured in three incubations with liver cytosol prepared from different rats. Asterisks indicate DMAsIII formation significantly different (p < 0.05) from that observed in the absence of inhibitors.

Figure 7. Reduction of DMAsV by rat liver cytosol: effects of aurothioglucose (ATG) and potassium antimony tartrate (SbIII). Rat liver cytosol (5 mg protein/mL) was preincubated at 37 °C for 5 min with ATG or potassium antimony tartrate (SbIII) at concentrations indicated in sucrose buffer in the presence of GSH (10 mM). Thereafter, DMAsV (50 μM) was added to start the incubation lasting for 20 min. The DMAsIII produced was extracted from the incubation mixtures with DDDC and was measured as described in Experimental Procedures. Symbols represent DMAsIII formation rates (mean ± SE) in three incubations with rat liver cytosol prepared from different animals. Asterisks indicate DMAsIII formation significantly lower (p < 0.05) than that observed in the absence of TRR inhibitors.

sequestered by hemoglobin in the RBC. If reduction of DMAsV took place in the liver (which is a tentative assumption), then the reduction rate estimated from the quantity of RBC-bound DMAs is approximately 5 nmol/min/g liver (taking 35 g/kg body wt as the specific liver weight in rats). Expressed on a hepatic cytosolic protein amount basis, this rate is 50 pmol/ 439



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Table 2. Reduction of DMAsV by Rat Liver Cytosol: Effects of Ultrafiltrationa DMAsIII formation % control unfiltered cytosol 3 kDa retentate 3 kDa filtrate 3 kDa retentate +3 kDa filtrate 3 kDa retentate + NADH 3 kDa retentate + NADPH 3 kDa retentate + TCEP 100 kDa retentate 100 kDa filtrate 100 kDa retentate +100 kDa filtrate 100 kDa retentate +3 kDa filtrate 100 kDa retentate + NADH 100 kDa retentate + NADPH 100 kDa retentate + TCEP

100 ± 6.27 8.18 ± 1.18 0.35 ± 0.35 83.7 ± 7.77 49.0 ± 5.23 78.9 ± 7.42 98.3 ± 3.05 5.99 ± 1.20 3.61 ± 2.08 82.6 ± 5.05 68.8 ± 8.80 34.0 ± 2.69 59.7 ± 4.23 91.4 ± 5.54

Rat liver cytosol was filtered in either a 3 kDa or a 100 kDa centrifugal filtration device as described in Experimental Procedures. After washing the retentate with sucrose buffer 3 times, it was reconstituted with sucrose buffer that matched the volume of the original cytosol subjected to ultrafiltration. Rat liver cytosol (5 mg protein/mL), the retentate, the filtrate, or both (each at amounts corresponding to 5 mg protein/mL of the unfiltered cytosol) were preincubated at 37 °C for 5 min in sucrose buffer in the presence of GSH (10 mM). Thereafter, NADH (2 mM), NADPH (2 mM), or TCEP (1 mM) plus DMAsV (50 μM) was added in rapid succession to start the incubations lasting for 20 min. The DMAsIII produced was extracted from the incubation mixtures with DDDC and was measured as detailed in Experimental Procedures. Values are the percentages of the DMAsIII formation rates (mean ± SE) measured in the unfiltered cytosol (33.47 ± 1.65 pmol/min/mg protein). The filtration experiments and incubations were performed with four rat liver cytosols prepared from different animals. a

Figure 8. Effect of ultrafiltration on the GSTO1 activity of rat liver cytosol. Rat liver cytosol was filtered in either a 3 kDa or a 100 kDa centrifugal filtration device as described in Experimental Procedures. After washing the retentate with sucrose buffer 3 times, it was reconstituted with sucrose buffer in a volume that matched the volume of the original cytosol subjected to ultrafiltration. In addition to the unfiltered cytosol, the filtrate obtained after the first centrifugation and the reconstituted retentate were assayed for GSTO1 activity. Bars represent GSTO1 activity (mean ± SE; n = 3) related to the protein concentration of the unfiltered cytosol.

min/mg cytosolic protein (as the protein concentration in the liver cytosols we prepared was approximately 100 mg/g wet liver). This rate compares well with the in vitro rate of DMAsV reduction in the incubations containing rat liver cytosol, 10 mM GSH, and 50 μM DMAsV, which is approximately 35 pmol/ min/mg protein (Figure 5A). Our investigations also indicate that reduction of DMAsV to DMAsIII requires GSH. The following findings support this conclusion. (1) Pretreatment of rats with GSH depletors delayed both the accumulation of RBC-bound DMAs (representing DMAsIII) in blood and the elimination of RBCunbound DMAs (putatively largely DMAsV) from blood (Figures 2A and B). (2) GSH depletors also decreased the hepatic concentration of bound (trivalent) DMAs while increasing that of the unbound (largely pentavalent) DMAs (Figure 3). (3) Both the aforementioned changes in DMAsV disposition were more pronounced in phorone-treated rats than in BSO-dosed animals (Figures 2A,B and 3), in accordance with the observation that phorone depleted the liver of GSH more than BSO. (4) Reduction of DMAsV by rat liver cytosol was negligible in the absence of GSH and increased in a GSH concentration-dependent fashion (Figures 4 and 5A). The diminished accumulation of RBC-bound DMAs in the blood of rats pretreated with GSH depletors might have resulted from both impaired hepatic reduction of DMAsV into DMAsIII and impaired hepatovascular transport of DMAsIII as its GSH conjugate. Yet, the observation that the GSH depletors markedly diminished (rather than increased) the concentration of bound DMAs in the liver indicates that the former rather than the latter effect prevails. Dependence of DMAsV reduction on GSH readily explains why depletion of cellular GSH

ameliorated the apoptotic effect of DMAsV in cultured cells,16 as GSH shortage should have decreased the formation of the highly cytotoxic DMAsIII. The present study on rats pretreated with PAD, an indirect inhibitor SAM-dependent methyltransferases,40 does not support the hypothesis that As3MT plays a role in the disposition of DMAsV. Under similar conditions, PAD virtually abolished the formation of all methylated metabolites in rats injected with arsenite or arsenate,53,54 indicating that it strongly inhibits As3MT. However, PAD pretreatment neither diminished the accumulation of RBC-bound DMAs in the blood and the concentration of thiol-bound DMAs in the liver nor increased the retention of unbound DMAs in the blood and the liver (Figures 2C,D and 3). This may suggest that during the time frame of our study As3MT contributed significantly neither to the reduction of DMAsV into the trivalent form nor to its further methylation into trimethylarsine oxide. Dependence of DMAsV reduction rate on the cytosolic protein concentration (Figure 5B) indicates the involvement of at least one cytosolic enzyme in this biotransformation. This enzyme apparently possesses a catalytically important thiol group because preincubation of rat liver cytosol with the thiolreagent NEM and PCMBS nearly abolished the DMAsV reducing activity (Table 1). This enzyme is also sensitive to GSSG, which may form a mixed disulfide with reactive protein thiols because GSSG as well as the GSSG-forming DHA inhibited cytosolic DMAsV reduction considerably, and the 440



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Nevertheless, because the retentate could be reactivated by the disulfide reducing reagent TCEP completely and by the addition of 2 mM NADPH or NADH incompletely (Table 2), it may be speculated that the cofactor molecule is required for the reduction of an enzyme protein. We may also suggest that the cytosolic reduction of DMAsV is not mediated by proteins significantly smaller than 100 kDa because the DMAsV reducing activity was largely recovered when the 100 kDa retentate was supplemented with the 3 kDa filtrate (Table 2). Therefore, this finding calls into question not only the role of GSTO1 and As3MT but also the importance of several smaller cytosolic reducing proteins, such as thioredoxin, glutaredoxin, peroxiredoxins, sulfiredoxin, in the reduction of DMAsV, unless such a small protein interacts with another to form a large complex. While the ultrafiltration experiments appear to discount the role of the above-mentioned enzymes in the reduction of DMAsV by rat liver cytosol, they may point to the importance of TRR, as the dimeric molecular mass of TRR exceeds 100 kDa. Other observations that are compatible with the involvement of TRR, a NADPH-dependent enzyme,39 include the sensitivity of the cytosolic reduction of DMAsV to thiol reagents (Table 1), to the NADPH utilizing compounds GSSG and DHA (Figure 6), as well as to the TRR inhibitor ATG.39,52 Potassium antimony tartrate, which contains trivalent antimony (SbIII), was almost equipotent with ATG in inhibiting cytosolic DMAsV reduction (Figure 7). This work demonstrated that SbIII is also a TRR inhibitor, similarly to the chemically related trivalent arsenicals,59,60 the testing of which was precluded by their interference with the DMAs analysis. TRR can in fact directly reduce a number of compounds, including selenite and DHA.39 Yet, we found that in four times larger quantity than that present in the hepatic cytosol of rats, recombinant rat TRR supplemented with NADPH and GSH neither mediated DMAsIII formation from DMAsV nor enhanced the cytosolic DMAsV reduction when added to rat liver cytosol. Therefore, TRR may only play an indirect role in the cytosolic reduction of DMAsV, and it is not rate limiting at the quantity present in the hepatic cytosol of rats. Alternatively, another ATG- and SbIIIsensitive selenoenzyme might be involved in the reduction. In summary, this work demonstrates that the reduction of DMAsV to the much more toxic DMAsIII in rats (as assessed from the accumulation of RBC-bound DMAs) and in rat liver cytosol is a GSH-dependent and thiol-reagent sensitive enzymatic process. DMAsV reduction in rat liver cytosol does not appear to be mediated by GSTO1. Rather, it seems to require a small cofactor, an enzyme larger than 100 kDa and, possibly, the indirect involvement of TRR. The precise mechanism of DMAsV reduction remains to be defined. Therefore, further studies are warranted to elucidate the enzymatic mechanism of this powerful toxification reaction in arsenic metabolism.

inhibitory effect of DHA was largely reversed by NADPH plus glutathione reductase (Figure 6). Another positive finding on cytosolic DMAsV reduction is that both aurothioglucose, a known inhibitor of TRR, and antimony potassium tartrate, the TRR inhibitory potency of which we demonstrated (see Results), potently inhibited the reduction of DMAsV in vitro (Figure 7), suggesting that TRR plays a role in this process (to be further discussed below). Our study does not support the involvement in DMAsV reduction of some enzymes whose potential role might have been surmised. Cytosolic enzymes catalyzing phosphorolytic and arsenolytic reactions, such as purine nucleoside phosphorylase (PNP) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), markedly facilitate the reduction of arsenate in the presence of a thiol, such as GSH, because they catalyze the formation of arsenate esters or anhydrides, which are highly susceptible to reduction by the thiol into arsenite.37,38 While the PNP inhibitor BCX-1777 and the GAPDH inhibitor koningic acid markedly inhibited the reduction of arsenate by rat liver cytosol,55,56 these inhibitors did not influence the cytosolic reduction of DMAsV, suggesting that PNP and GAPDH do not assist DMAsV reduction. Human GSTO1 has been reported to catalyze the reduction of DMAsV when incubated with 5 mM GSH and 10 mM DMAsV.31,32 This enzyme uses GSH as a cosubstrate, contains a catalytic cysteine, and has DHA-reducing activity;29,57 therefore, certain features of cytosolic DMAsV reduction, such as dependence on GSH and its inhibition by NEM, PCMBS, GSSG, as well as DHA (Figure 6 and Table 1), are compatible with the involvement of GSTO1. However, other observations do not support the role of GSTO1 in DMAsV reduction by rat liver cytosol. For example, tocopherol succinate that inhibited DMAsV reduction by human GSTO1 with an IC50 of 3 μM58 failed to inhibit DMAsV reduction by rat liver cytosol even at 50 μM (Table 1). Evidence against the role of GSTO1 has also been provided by the experiments in which we sought for correlation between the GSTO1 activity and the DMAsV reducing activity in the fractions of rat liver cytosol obtained by ultrafiltration. Interestingly, while rat liver cytosol catalyzed DMAsV reduction, it failed to exhibit any GSTO1 activity as assayed through reduction of S-(4-nitrophenacyl)glutathione according to Board and collegues.41 Conversely, when the cytosol was subjected to ultrafiltration the retentate had GSTO1 activity (Figure 8), but almost no DMAsV reducing activity (Table 2). Further, readdition of the filtrate (which was devoid of both activities) to the retentate inhibited the GSTO1 activity of the retentate (Figure 8) but largely restored the DMAsV reducing activity of the retentate (Table 2). Some unknown differences in structure and function between human and rat GSTO1 might account for the contradictory findings. It also remains to be deciphered what small molecule inactivates GSTO1 in rat liver cytosol and why the 100 kDa retentate of the cytosol has GSTO1 activity (Figure 8), which is unexpected given the 55 kDa dimeric molecular mass of GSTO1.29 The ultrafiltration experiments also indicate that the cytosolic DMAsV reducing activity in the presence of GSH requires a 100 kDa protein because the 3 kDa retentate of the cytosol has negligible DMAsV reducing activity and because recombination of the 3 kDa filtrate with 100 kDa retentate (both of which are practically inactive alone) restored the activity of the retentate up to 70% of the DMAsV reducing activity of the cytosol (Table 2). Both of the 100 kDa protein remain to be identified.

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AUTHOR INFORMATION

Corresponding Author

*Department of Pharmacology and Pharmacotherapy, Toxicology Section, University of Pécs, Medical School, Szigeti út 12, H-7624 Pécs, Hungary. Tel: +36-72-536-001/31645. Fax: +3672-536-218. E-mail: [email protected]. Funding

This work was supported by the Hungarian National Research Fund (OTKA No. K73228), the Hungarian Ministry of Health 441



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(ETT No. 070/2009), and TÁ MOP/SROP-4.2.2/B-10/12010-0029.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank István Schweibert for his excellent assistance in the experimental work. ABBREVIATIONS As3MT, arsenic (+3 oxidation state) methyltransferase; ATG, aurothioglucose; BSO, L-buthionine-S,R-sulfoximine; DHA, dehydroascorbic acid; DMAs, dimethylated arsenic; DMAsIII, dimethylarsinous acid; DMAsV, dimethylarsinic acid; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; GSTO1, glutathione S-transferase omega 1; PAD, periodate-oxidized adenosine; RBC, red blood cells; TCEP, tris(2-carboxyethyl)phosphine; TRR, thioredoxin reductase

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Reduction of Dimethylarsinic Acid to the Highly Toxic

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