Toxicological Sciences 70, 13-19 (2002)
Copyright © 2002 by the Society of Toxicology
BIOTRANSFORMATION AND TOXICOKINETICS |
Purine Nucleoside Phosphorylase as a Cytosolic Arsenate Reductase
Department of Pharmacology and Pharmacotherapy, University of Pécs, Medical School, Szigeti út 12, H-7624, Pécs, Hungary
Received April 5, 2002; accepted June 3, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The findings of the accompanying paper (Németi and Gregus, Toxicol. Sci. 70, 4–12) indicate that the arsenate (AsV) reductase activity of rat liver cytosol is due to an SH enzyme that uses phosphate (or its analogue, arsenate, AsV) and a purine nucleoside (guanosine or inosine) as substrates. Purine nucleoside phosphorylase (PNP) is such an enzyme. It catalyzes the phosphorolytic cleavage of 6-oxopurine nucleosides according to the following scheme: guanosine (or inosine) + phosphate
guanine (or hypoxanthine) + ribose-1-phosphate. Therefore, we have tested the hypothesis that PNP is responsible for the thiol- and purine nucleoside-dependent reduction of AsV to AsIII by rat liver cytosol. AsIII formed from AsV was quantified by HPLC-hydride generation–atomic fluorescence spectrometry analysis of the deproteinized incubates. The following findings support the conclusion that PNP reduces AsV to AsIII, using AsV instead of phosphate in the reaction above: (1) Specific PNP inhibitors (CI-1000, BCX-1777) at a concentration of 1 µM completely inhibited cytosolic AsV reductase activity. (2) During anion-exchange chromatography of cytosolic proteins, PNP activity perfectly coeluted with the AsV reductase activity, suggesting that both activities belong to the same protein. (3) PNP purified from calf spleen catalyzed reduction of AsV to AsIII in the presence of dithiothreitol (DTT) and a 6-oxopurine nucleoside (guanosine or inosine). (4) AsV reductase activity of purified PNP, like the cytosolic AsV reductase activity, was inhibited by phosphate (a substrate of PNP alternative to AsV), guanine and hypoxanthine (products of PNP favoring the reverse reaction), mercurial thiol reagents (nonspecific inhibitors of PNP), as well as CI-1000 and BCX-1777 (specific PNP inhibitors). Thus, PNP appears to be responsible for the AsV reductase activity of rat liver cytosol in the presence of DTT. Further research should clarify the mechanism and the in vivo significance of PNP-catalyzed reduction of AsV to AsIII. Key Words: arsenate; arsenite; purine nucleoside phosphorylase; reduction; thiols.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Arsenate (AsV), the environmentally prevalent form of arsenic, can be readily reduced to arsenite (AsIII) in living organisms ranging from bacteria (Ji and Silver, 1992
) to mammals (Gregus et al., 2000; Radabaugh and Aposhian, 2000; Thomas et al., 2001; Vahter, 1983). This process is regarded as toxification, because AsIII is more toxic than AsV, owing to facile covalent reaction of the trivalent arsenic with thiols, especially dithiols (Knowles and Benson, 1983). In spite of the toxicological significance of AsV reduction, AsV reductases have been identified only in bacteria and yeast (Bennett et al., 2001; Mukhopadhyay and Rosen, 2001; Zegers et al., 2001), but not in mammals.
The accompanying paper (Németi and Gregus, 2002) has characterized an AsV reductase activity in rat liver cytosol. This activity, which depended on the presence of an appropriate exogenous thiol compound, was enhanced up to 100-fold in the presence of 6-oxopurine nucleosides, i.e., inosine and guanosine, and was inhibited significantly by mercurial thiol reagents, inorganic phosphate, that is an analogue of AsV (Dixon, 1997), as well as by 6-oxopurine nucleobases, i.e., hypoxanthine and guanine. These findings have prompted the tentative conclusions that the cytosolic AsV reductase may contain functionally important thiol group(s), may accept inosine or guanosine as well as inorganic phosphate as substrates and may yield hypoxanthine or guanine as products. The enzyme that fits the deducted characteristics is known as purine nucleoside phosphorylase (PNP).
PNP is a soluble enzyme localized in the cytosol and contains critical thiol groups (Parks and Agarwal, 1972; Bzowska et al., 2000; Erion et al., 1997). As shown in Figure 1, this enzyme catalyzes the phosphorolytic cleavage of 6-oxopurine nucleosides, utilizing inorganic phosphate. PNP can cleave inosine to hypoxanthine, or guanosine to guanine, yielding, in either case, ribose-1-phosphate. This reaction is readily reversible; however, the forward reaction is favored in vivo because of rapid elimination of the products. Like many phosphate-utilizing enzymes, PNP also accepts AsV instead of phosphate and produces the purportedly unstable ribose-1-arsenate during arsenolysis of 6-oxopurine nucleosides (Kline and Schramm, 1993).
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The aim of this study was to test the hypothesis that PNP in rat liver cytosol could indeed work as an AsV reductase when catalyzing the cleavage of 6-oxopurine nucleosides in the presence of AsV. For this purpose, we have tested (1) whether specific and highly potent inhibitors of PNP, i.e., CI-1000 (Gilbertsen et al., 1992
) and BCX-1777 (Miles et al., 1998; Bantia et al., 2001) inhibit the cytosolic AsV reductase activity; (2) whether there is an association between the hepatic AsV reductase and PNP activities among various species and during chromatography of cytosolic proteins; (3) whether purified PNP can catalyze reduction of AsV to AsIII, and if it can, (4) whether the AsV reductase activity of purified PNP exhibits similar responsiveness to various compounds as the cytosolic AsV reductase activity did (Németi and Gregus, 2002). |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
BCX-1777 (also called immucillin-H) and CI-1000 (also called PD 141955) were gifts from BioCryst Pharmaceuticals (Birmingham, AL) and Pfizer (Ann Arbor, MI), respectively. Structural formulae of these PNP inhibitors are in Bzowska et al. (2000)
. Acyclovir (Herpesin®) was obtained from Lachema, whereas PNP (nucleoside phosphorylase, EC 2.4.2.1) from calf spleen (N 3003) was purchased from Sigma and xanthine oxidase from bovine milk from ICN Biomedicals. Immediately before use, the sulfate content was removed from the PNP preparation by centrifugal ultrafiltration at 4°C with a Microcon-10 filter (Amicon), after which the retained enzyme was taken up in ice-cold incubation buffer (see below). The sources of chemicals used in the AsV reductase assay (Németi and Gregus, 2002) and arsenic analysis (Gregus et al., 2000) have been given.
Preparation of hepatic cytosol.
Cytosol from the liver of male Wistar rats (270–330 g), CFLP mice (28–35 g), English shorthair guinea pigs (450–550 g), golden Syrian hamsters (90–115 g), and New Zealand white rabbits (1.6–1.8 kg) was prepared by differential centrifugation of the 33% homogenate made in 150 mM KCl–50 mM TRIS (pH 7.0), as described (Németi and Gregus, 2002). Cytosolic protein concentration was determined by the bicinchoninic acid method (Brown et al., 1989). The cytosol was stored in 1-ml aliquots at –80°C until use. For anion exchange chromatography (see below), 500 µl cytosol (30 mg protein/ml) was desalted by centrifugal ultrafiltration at 4°C using a Microcon-30 filter. The retentate was washed twice with 20 mM TRIS (pH 7.6), after which the final retentate was taken up in 300 µl of 20 mM TRIS, pH 7.6.
Enzymatic assays.
For assaying AsV reductase activity, hepatic cytosol (5 mg protein/ml) or calf spleen PNP (50 mU/ml) was preincubated at 37°C for 5 min in 150 mM KCl–50 mM TRIS (pH 7.6) containing a nucleoside (typically inosine) at a concentration indicated in the legends of figures and footnotes of tables. When the effect of inhibitor compounds was tested on AsV reductase activity, these compounds (except phosphate) were also present during preincubation, at concentrations specified (see Fig. 2 and Table 1). Subsequently, the incubation was started by addition of a thiol compound (typically dithiothreitol, DTT), at a concentration specified in the legends, and AsV (25 µM). At 10 min after AsV addition, the reaction was stopped by addition of mersalyl (20 mM) to displace thiol-bound AsIII and, 15 seconds later, 3 volumes of ice-cold deoxygenated methanol to precipitate proteins. The methanolic incubates were then stored at –80°C until arsenic analysis. AsIII in the deproteinized methanolic incubates was separated from AsV and quantified by HPLC-HG-AFS, essentially as described for speciation of arsenic metabolites (Gregus et al., 2000). In the present work, however, an isocratic elution, rather than gradient elution, was performed with 60 mM potassium phosphate (pH 5.75) at 1 ml/min flow rate.
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PNP activity was assayed, based on the xanthine oxidase-coupled spectrophotometric method of Kalckar et al. (1947)
. Briefly, cytosol (0.5 mg protein/ml) was incubated at 25°C in a 0.5-ml cell of a recording spectrophotometer (DU-64, Beckman) containing 100 mM potassium phosphate buffer (pH 7.4), inosine (0.5 mM) and xanthine oxidase (40 mU/ml), and the change in absorbance at 293 nm was recorded for 5 min. PNP activity was calculated on the basis of millimolar absorption coefficient of uric acid (12.0). In both assays, product formation was linear with respect to time and protein or enzyme concentrations.
Anion exchange chromatography of cytosol.
Rat liver cytosol was fractionated using a Superformance 150–10 glass column (Merck) filled with Fractogel EMD TMAE (S) anion-exchange resin to obtain a 100 x 10-mm gel column, 20 mM TRIS, pH 7.6 (eluent A) and 20 mM TRIS (pH 7.6)–500 mM KCl (eluent B). The ice-cold eluents were pumped at a combined flow rate of 1 ml/min with 2 Waters-501 HPLC pumps operated under control of Millennium Chromatography Manager (Waters), through an injector (Rheodyne 7125) equipped with a 100-µl sample loop and then through the column, maintained at 4°C. After equilibrating the column by pumping eluent A for 30 min, 100 µl desalted cytosol (see above) was injected onto the column and eluted with 100% eluent A for 5 min. By 35 min, 100% eluent A was changed linearly to 100% eluent B, after which 100% eluent B was maintained for 10 min. During the 45-min elution, the eluate was collected in 1-ml fractions. The fractions stored on ice were assayed for PNP activity and AsV reductase activity within 3 h.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effect of PNP inhibitors on cytosolic AsV reductase activity.
The concentration-dependent effects of CI-1000 and BCX-1777, preincubated with rat liver cytosol for 5 min, on formation of AsIII from AsV in the presence of DTT, is depicted in Figure 2
. Both PNP inhibitors decreased AsIII formation in a concentration-dependent manner, causing almost complete cessation of AsV reduction at 0.5 µM. At lower concentrations; however, CI-1000 appeared slightly more potent than BCX-1777 (approximate IC50 values were 125 nM and 175 nM, respectively).
The PNP inhibitors also markedly inhibited the DTT-supported AsV reductase activity of hepatic cytosol prepared from other species (Table 1). BCX-1777, at 1 µM concentration, practically abolished AsV reductase activity of rat, mouse, and hamster cytosols, whereas it decreased the activity to 2% and 13% of control, respectively, in the cytosols of guinea pigs and rabbits. At this concentration CI-1000 proved to be slightly less effective, lowering AsV reductase activities to 1, 4, 8, 15, and 14% of control, respectively, in the cytosols of rats, mice, guinea pigs, hamsters and rabbits (Table 1).
Association between cytosolic AsV reductase and PNP activities.
We sought for association between cytosolic AsV reductase and PNP activities in 2 ways; first, AsV reductase activities in the hepatic cytosol of various species that were related to the PNP activities assayed in the same cytosols (Fig. 3). When AsV reductase activity was assayed without added inosine, a correlation appeared between these 2 activities in the hepatic cytosol of rats, guinea pigs, mice and rabbits, with decreasing PNP and AsV reductase activities in this order (Fig. 3, left). However, the hamster, with its extremely low AsV reductase activity relative to its PNP activity, did not fit into this interspecies correlation. Nevertheless, when AsV reductase activity was assayed with added inosine, which necessitated the use of 50-fold diluted cytosols (Fig. 3, right), the AsV reductase activity of the hamster rose much more than that of the other species. In this case also, only a rough interspecies correlation was observed, with the rabbit exhibiting the lowest PNP and lowest AsV reductase activities, whereas the other species with high PNP activities ranging from 115 to 225 nmol/min • mg protein exhibited high, but comparable AsV reductase activities.
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Second, association between the PNP and AsV reductase activities was examined by applying rat liver cytosol to a chromatographic column in order to determine whether or not the two activities coelute. Figure 4
demonstrates that upon fractionation of rat liver cytosol from an anion exchange column with a potassium chloride gradient, AsV reductase activity and PNP activity appeared in the very same eluate fractions. Perfect coelution of these two enzymatic activities was observed, not only in this experiment but also when different eluents (A = 10 mM potassium citrate, pH 6.0 and B = 10 mM potassium citrate–0.5 M KCl, pH 6.0 or these eluents at pH 7.0) were used (not shown).
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AsV reduction by purified PNP.
In order to test directly whether PNP could reduce AsV to AsIII, PNP purified from calf spleen was incubated with AsV. PNP readily formed AsIII from AsV when both its nucleoside substrate (e.g., inosine) and an appropriate thiol (e.g., DTT) were present (Fig. 5
, bottom). However, the purified PNP failed to reduce AsV in the presence of DTT without added inosine (Fig. 5, top), or in the presence of inosine without added DTT (Table 2).
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The effect of several compounds known to influence the rat liver cytosolic AsV reductase activity (Németi and Gregus, 2002
) was tested on the AsV reductase activity of the purified PNP. Of the thiols investigated, it was only DTT, dimercaptopropanol, dimercaptopropane sulfonate, and mercaptoethanol that supported reduction of AsV by calf spleen PNP in the presence of inosine, whereas glutathione and dimercaptosuccinate were practically inactive (Table 2). Of the nucleosides, the pyrimidine nucleosides were inactive and adenosine was slightly active, whereas the 6-oxopurine nucleoside inosine and guanosine were highly active in facilitating the AsV reductase activity of purified PNP in the presence of DTT (Table 3). In the presence of inosine and DTT, inorganic phosphate (a PNP substrate alternative to AsV) as well as the 6-oxopurine bases, hypoxanthine and guanine (PNP products), markedly diminished AsIII formation from AsV by calf spleen PNP (Table 4). AsV reduction by the purified PNP was also dramatically decreased by the nonspecific PNP inhibitor mercurials, as well as by the specific PNP inhibitors, most notably CI-1000 and BCX-1777 (Table 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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On the basis of considerations outlined above, we tested the hypothesis that PNP in rat liver cytosol was responsible for the thiol- and purine nucleoside-dependent reduction of AsV to AsIII characterized in the accompanying paper (Németi and Gregus, 2002
). The following pieces of evidence support this hypothesis:
- Specific and highly potent inhibitors of PNP, i.e., BCX-1777 and CI-1000, decreased AsV reductase activity of rat liver cytosol in a concentration-dependent fashion, causing, at 1 µM concentrations, complete or near complete inhibition of AsV reduction in the hepatic cytosols of the rat, mouse, hamster, guinea pig, and rabbit.
- Although interspecies correlation between hepatic cytosolic PNP and AsV reductase activities was imperfect, possibly due to species variations in the composition of these cytosols, the AsV reductase activity perfectly and consistently coeluted with the PNP activity during fractionation of rat liver cytosol on an anion exchange column. This latter finding represents strong circumstantial evidence that both activities belong to the same protein.
- Purified PNP readily catalyzed AsV reduction, provided that its nucleoside substrate and an appropriate thiol were present simultaneously, proving directly that PNP can function as an AsV reductase.
- Various compounds similarly influenced the reduction of AsV by rat liver cytosol (Németi and Gregus, 2002) and the reduction of AsV by purified PNP. Both were activated by the simultaneous presence of a purine nucleoside (especially a 6-oxopurine nucleoside, but not pyrimidine nucleoside) and an appropriate thiol (especially DTT and dimercaptopropanol, but barely by glutathione). Both were inhibited by inorganic phosphate, 6-oxopurine bases, mercurial thiol reagents, and PNP inhibitors. The strong PNP inhibitors BCX-1777 (Bantia et al., 2001; Miles et al., 1998) and CI-1000 (Gilbertsen et al., 1992) were far more potent in decreasing PNP-catalyzed AsIII formation from AsV than was the weak PNP inhibitor acyclovir (Bzowska et al., 2000; Tuttle and Krenitsky, 1984).
Collectively, these observations constitute compelling evidence that the DTT-supported AsV reductase activity in the hepatic cytosol of rats, and other species tested here, is ascribable, depending on the species, completely or almost completely to PNP, because PNP can work as an AsV reductase and because specific PNP inhibitors completely or almost completely abolished the cytosolic AsV reductase activities in the species investigated. It appears most likely that the AsV reductase activity found recently in the liver of humans (Radabaugh and Aposhian, 2000) and nonhuman primates (Wildfang et al., 2001) is also ascribable, at least in part, to PNP, because that AsV reductase was also supported by DTT (but barely by GSH) and required a heat-stable endogenous compound or compounds less than 3 kDa in size, which is likely to be inosine and/or guanosine, essential for the PNP-catalyzed AsV reduction.
PNP is a ubiquitous enzyme that cleaves purine nucleosides, thereby contributing to salvage of the released purine bases for reutilization in the synthesis of purine nucleotides (Bzowska et al., 2000; Parks and Agarwal, 1972). This soluble enzyme consists of 3 identical subunits of approximately 32 kDa, contains 12 cysteines, and is sensitive to inactivation by p-chloromercuribenzoate (Parks and Agarwal, 1972). Mammalian PNPs exhibit a very high degree of sequence homology and identical amino acids constitute the substrate binding sites in the human, bovine, rat, and mouse enzymes (Bzowska et al., 2000; Erion et al., 1997). PNP exhibits high substrate specificity for inosine and guanosine (6-oxopurines), whereas the 6-aminopurine adenosine is a very weak substrate (Bzowska et al., 2000). For the second substrates phosphate and AsV, the human erythrocytic enzyme has KM values of 0.74 and 1.8 mM, respectively (De Verdier and Gould, 1963).
The observations presented here indicate that PNP-catalyzed AsV reduction takes place during or as a consequence of the arsenolytic reaction, i.e., the forward reaction depicted in Figure 1. This conclusion is supported in part by the findings that AsV reduction is inhibited by phosphate that competes with AsV in the arsenolytic reaction (based on structural similarities of these oxyanions) and also by the observation that 6-oxopurine nucleoside substrates of PNP, which support the forward reaction, were required for PNP-catalyzed AsV reduction. Interestingly, adenosine, which is an extremely poor substrate for PNP, also supported to some extent the reduction of AsV by calf spleen PNP. This seemingly controversial finding is attributable to the fact that, according to the manufacturer, this enzyme preparation may contain up to 0.5% adenosine deaminase, which can convert the poor PNP substrate adenosine into the good substrate inosine. Furthermore, the observation that hypoxanthine and guanine, strong inhibitors of the phosphorolytic cleavage of purine nucleosides by PNP (Glantz and Lewis, 1978; Parks and Agarwal, 1972), strongly inhibited the PNP-catalyzed AsV reduction, also indicates that the forward reaction is essential for reduction of AsV to AsIII by this enzyme. It is unknown, however, whether the arsenolytic cleavage of 6-oxopurine nucleosides is a prerequisite for the PNP-catalyzed reduction of AsV, because 6-oxopurine nucleoside binding induces a favorable conformational change in the enzyme (Bzowska et al., 2000; Mao et al., 1998), and/or because, in this reaction, the AsV oxyanion becomes susceptible to reduction and/or it becomes accessible to the reducing moieties of the enzyme protein.
Regarding the mechanism of AsV reduction by PNP, views on the mechanism of AsV reduction by certain microbial AsV reductases may be enlightening. It has been proposed recently that the identical AsV reductases of Bacillus subtilis and Staphylococcus aureus work as a "triple cysteine redox relay" or a "disulfide cascade," using Cys-10, Cys-82, Cys-89, Arg-16, and Asp-105, as essential residues in the catalytic cycle, and the small dithiol protein, thioredoxin (Bennet et al., 2001; Zegers et al., 2001). Briefly, the reduction process is thought to be initiated by the nucleophilic attack of Cys-10 thiolate anion on AsV, after which AsV is reduced with the contribution of Cys-82 thiolate, resulting in formation of AsIII and a disulfide bond between these cysteines. This disulfide bond will then be broken by Cys-89, with recovery of Cys-10 thiolate group and formation of a new disulfide bond between Cys-82 and Cys-89, which in turn are reduced by thioredoxin, thus reactivating the enzyme. Importantly, Cys-10, which initiates AsV reduction by this bacterial AsV reductase, is part of a CX5R signature motif (in which C and R represent Cys-10 and Arg-16), which is also found in the phosphate-binding loop of low molecular weight protein tyrosine phosphatases (Bennett et al., 2001; Zegers et al., 2001). The CX5R motif is also present in the AsV reductase of Saccharomyces cerevisiae and is essential for its catalytic function (Mukhopadhyay and Rosen, 2001). It is important to point out that the human, bovine, rat, and mouse PNPs also contain one CX5R motif in each subunit, with Cys-78 and Arg-84 representing C and R in this motif (Bzowska et al., 2000; Erion et al., 1997). Moreover, Arg-84 is involved in the phosphate (and AsV) binding site of PNP (Mao et al., 1998; Bzowska et al., 2000). Therefore, it is tempting to hypothesize that the CX5R motif represents the catalytic center of PNP when functioning as an AsV reductase, just as it does in the above-mentioned microbial AsV reductases. It remains to be analyzed whether this hypothesis is verifiable, whether other cysteines in PNP can assume the same role as Cys-82 and Cys-89 can in bacterial AsV reductase (see above), and whether DTT and some other thiols assume the function of thioredoxin (or some other endogenous thiols) in returning the protein into an active form capable of reducing AsV.
In summary, this paper demonstrates that PNP can fortuitously function as an AsV reductase when catalyzing the arsenolytic cleavage of inosine or guanosine in the presence of an appropriate thiol. Furthermore, the findings of this and the accompanying work (Németi and Gregus, 2002) indicate that it is the PNP that is largely or exclusively responsible for the thiol-dependent reduction of AsV to AsIII in the hepatic cytosol of rats, mice, hamsters, guinea pigs, and rabbits. Further research is warranted to clarify the mechanism of PNP-catalyzed AsV reduction and the in vivo role of PNP in forming the toxic AsIII from the less toxic and environmentally prevalent AsV.
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ACKNOWLEDGMENTS |
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This publication is based on work supported by the Hungarian National Scientific Research Fund (OTKA) and the Hungarian Ministry of Health. The authors thank István Schweibert for excellent assistance in the analytical work.
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NOTES |
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This study was presented at the 41st annual meeting of the Society of Toxicology, March 2002, Nashville, Tennessee. The Abstract was published in Toxicol. Sci. 66(Supp.), 2002, p. 84.
1 To whom correspondence should be addressed. Fax: 36-72-536-218. E-mail: zoltan.gregus{at}aok.pte.hu. 
Note added in proof: After acceptance of this work for publication, it was reported (Radabaugh, T. R., Sampayo-Reyes, A., Zakharyan, R. A., and Aposhian, H. V. [2002]. Arsenate reductase II. Purine nucleoside phosphorylase in the presence of dihydrolipoic acid is a route for reduction of arsenate to arsenite in mammalian systems. Chem. Res. Toxicol. 15, 692–698) that the purified human liver arsenate reductase characterized earlier by Radabaugh et al. (2000) has an amino acid sequence identical with that of human purine nucleoside phosphorylase.
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B. Nemeti, I. Csanaky, and Z. Gregus Effect of an Inactivator of Glyceraldehyde-3-Phosphate Dehydrogenase, a Fortuitous Arsenate Reductase, on Disposition of Arsenate in Rats Toxicol. Sci., March 1, 2006; 90(1): 49 - 60. [Abstract] [Full Text] [PDF]
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Z. Gregus and B. Nemeti The Glycolytic Enzyme Glyceraldehyde-3-Phosphate Dehydrogenase Works as an Arsenate Reductase in Human Red Blood Cells and Rat Liver Cytosol Toxicol. Sci., June 1, 2005; 85(2): 859 - 869. [Abstract] [Full Text] [PDF]
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B. Nemeti and Z. Gregus Reduction of Arsenate to Arsenite by Human Erythrocyte Lysate and Rat Liver Cytosol - Characterization of a Glutathione- and NAD-Dependent Arsenate Reduction Linked to Glycolysis Toxicol. Sci., June 1, 2005; 85(2): 847 - 858. [Abstract] [Full Text] [PDF]
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E. M. Kenyon, L. M. Del Razo, and M. F. Hughes Tissue Distribution and Urinary Excretion of Inorganic Arsenic and Its Methylated Metabolites in Mice Following Acute Oral Administration of Arsenate Toxicol. Sci., May 1, 2005; 85(1): 468 - 475. [Abstract] [Full Text] [PDF]
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B. Nemeti and Z. Gregus Glutathione-Dependent Reduction of Arsenate in Human Erythrocytes--a Process Independent of Purine Nucleoside Phosphorylase Toxicol. Sci., December 1, 2004; 82(2): 419 - 428. [Abstract] [Full Text] [PDF]
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 J.-W. Ting, M.-F. Wu, C.-T. Tsai, C.-C. Lin, I.-C. Guo, and C.-Y. Chang Identification and characterization of a novel gene of grouper iridovirus encoding a purine nucleoside phosphorylase J. Gen. Virol., October 1, 2004; 85(10): 2883 - 2892. [Abstract] [Full Text] [PDF]
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 E. M. Leslie, A. Haimeur, and M. P. Waalkes Arsenic Transport by the Human Multidrug Resistance Protein 1 (MRP1/ABCC1): EVIDENCE THAT A TRI-GLUTATHIONE CONJUGATE IS REQUIRED J. Biol. Chem., July 30, 2004; 279(31): 32700 - 32708. [Abstract] [Full Text] [PDF]
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B. Nemeti, I. Csanaky, and Z. Gregus Arsenate Reduction in Human Erythrocytes and Rats--Testing the Role of Purine Nucleoside Phosphorylase Toxicol. Sci., July 1, 2003; 74(1): 22 - 31. [Abstract] [Full Text] [PDF]
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B. Nemeti and Z. Gregus Reduction of Arsenate to Arsenite in Hepatic Cytosol Toxicol. Sci., November 1, 2002; 70(1): 4 - 12. [Abstract] [Full Text] [PDF]
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