Arsenate Reduction in Human Erythrocytes and Rats—Testing the Role of Purine Nucleoside Phosphorylase

Balázs Németi, Iván Csanaky and Zoltán Gregus1

Department of Pharmacology and Pharmacotherapy, University of Pécs, Medical School, Szigeti út 12, H-7624 Pécs, Hungary

Received March 3, 2003; accepted April 11, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reduction of the pentavalent arsenate (AsV) to the thiol-reactive arsenite (AsIII) toxifies this environmentally prevalent form of arsenic, yet its biochemical mechanism in mammals is incompletely understood. Purine nucleoside phosphorylase (PNP) has been shown recently to function as an AsV reductase in vitro, provided its substrate (inosine or guanosine) and an appropriate dithiol (e.g., dithiothreitol, DTT) were present. It was of interest to know if this ubiquitous enzyme played a significant role in reduction of AsV to AsIII in vivo. Two approaches were used to test this. First, it was determined if compounds that influenced AsV reduction by purified PNP (i.e., nucleosides, thiols, and PNP inhibitors) would similarly affect reduction of AsV by human erythrocytes. Erythrocytes were incubated with AsV, and the formed AsIII was quantified by HPLC-hydride generation-atomic fluorescence spectrometry. The red blood cells reduced AsV at a considerable rate, which could be enhanced by inosine or inosine plus DTT. These stimulated AsIII formation rates were PNP-dependent, as PNP inhibitors strongly inhibited them. In contrast, PNP inhibitors had little if any inhibitory effect on AsIII formation in the absence of exogenous inosine, indicating that this basal rate of AsV reduction is PNP-independent. Second, the role of PNP in reduction of AsV in vivo was also assessed by investigating the effect of the PNP inhibitor BCX-1777 on the biotransformation of AsV in control and DTT-treated rats with cannulated bile duct and ligated renal pedicles. Although it abolished hepatic PNP activity, BCX-1777 influenced neither the biliary excretion of AsIII and monomethylarsonous acid, nor the tissue concentration of AsV and its metabolites in either group of AsV-injected rats. Thus, despite its in vitro activity, PNP does not appear to play a significant role in AsV reduction in human erythrocytes and in rats in vivo. Further research should clarify the in vivo relevant mechanisms of AsV reduction in mammals.

Key Words: arsenate; arsenite; purine nucleoside phosphorylase; PNP inhibitor; reduction; inosine; erythrocyte.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Arsenic is a well-known environmental poison that may cause skin lesions, vascular disease, and cancer upon prolonged exposure (Goering et al., 1999Go; Hindmarsh, 2000Go; Hughes, 2002Go). The pentavalent arsenate (AsV), being the predominant form in nature, enters the body mainly via consumption of contaminated drinking water. In the body, AsV may be eliminated by urinary excretion (Vahter, 1983Go) or taken up by the cells through their phosphate transporters (Csanaky and Gregus, 2001Go; Ginsburg and Lotspeich, 1963Go). In the cell, AsV may interfere with cellular metabolism by replacing inorganic phosphate (Pi) in enzymatic reactions (Dixon, 1997Go). Alternatively, AsV may be reduced to the trivalent arsenite (AsIII) (Thomas et al., 2001Go), which is much more toxic because of its thiol-reactivity (Knowles and Benson, 1983Go). AsIII is then metabolized further, yielding mono- and dimethylated metabolites, among which the pentavalent ones are relatively atoxic whereas the trivalent ones are even more toxic than AsIII (Petrick et al., 2001Go; Thomas et al., 2001Go). Being the first step of AsV metabolism, reduction of AsV to AsIII is not only a toxification step, but may also primarily underlie the fate, toxicity, and carcinogenicity of arsenic.

On searching for the biochemical mechanisms of AsV reduction, it has recently been discovered that liver mitochondria (Németi and Gregus, 2002aGo) and cytosol (Németi and Gregus, 2002bGo; Radabaugh and Aposhian, 2000Go) can reduce AsV to AsIII. Although the attempts to identify the mitochondrial AsV reductase have failed, the cytosolic enzyme responsible for the thiol-dependent AsV reduction has been identified as purine nucleoside phosphorylase (PNP, EC 2.4.2.1) (Gregus and Németi, 2002Go; Radabaugh et al., 2002Go). Being involved in the salvage of nucleobases (Parks and Agarwal, 1972Go), PNP catalyzes the phosphorolytic cleavage of 6-oxopurine nucleosides (i.e., inosine and guanosine) into ribose-1-phosphate and the corresponding base (i.e., hypoxanthine and guanine, respectively) using Pi. Instead of Pi, PNP can also use AsV. Provided its nucleoside substrate, such as inosine, and an appropriate thiol, such as dithiothreitol (DTT) or dihydrolipoic acid are present, the enzyme is capable of reducing AsV to AsIII in vitro (Gregus and Németi, 2002Go; Radabaugh et al., 2002Go). The PNP inhibitors CI-1000 and BCX-1777 (Bzowska et al., 2000Go) as well as Pi inhibited reduction of AsV by purified PNP or rat liver cytosol in the presence of DTT and inosine (Gregus and Németi, 2002Go; Németi and Gregus, 2002bGo).

The aim of the present work was to determine whether PNP played a role in AsV reduction also in vivo. For this purpose, two experimental approaches were used. First, we tested whether compounds (e.g., nucleosides, thiols, PNP inhibitors) influenced the AsV reduction by human red blood cells (RBC) in the same way that they had influenced reduction of AsV catalyzed by purified PNP. The following considerations justify the use of RBC for this purpose: (1) AsV readily enters RBC (Kenney and Kaplan, 1988Go); (2) rabbit and rat erythrocytes reduce AsV partially (Delnomdedieu et al., 1994Go; Winski and Carter, 1995Go); (3) PNP is found in erythrocytes, and its activity is higher in human RBC than in the erythrocytes of most animals (Parks and Agarwal, 1972Go); (4) RBC take up inosine (substrate for PNP, Zachara et al., 1981Go) and PNP inhibitors (Bantia, personal communication); and (5) RBC are devoid of mitochondria, precluding the contribution of these organelles to the reduction of AsV in erythrocytes.

Second, we assessed the role of PNP in reduction of AsV in vivo by investigating the effect of the specific and potent PNP inhibitor BCX-1777 on the biotransformation of AsV in anesthetized, bile duct-cannulated and renal pedicle-ligated rats. Rats readily reduce AsV to AsIII, which is further biotransformed into monomethylarsonic acid (MMAsV), monomethylarsonous acid (MMAsIII), and dimethylarsinic acid (DMAsV) (Csanaky and Gregus, 2002Go). The two trivalent metabolites in this pathway, i.e., AsIII and MMAsIII, are readily excreted into bile (Csanaky and Gregus, 2002Go; Gregus et al., 2000Go). Therefore, if BCX-1777 indeed inhibits PNP in rats, and if PNP is involved in reduction of AsV in rats, then pretreatment with BCX-1777 should significantly decrease the quantities of AsIII and MMAsIII appearing in bile, and the concentrations of all AsV metabolites remaining in the tissues, while it should increase the retention of AsV in the blood and tissues. This hypothesis was tested in both control rats and rats receiving DTT, an activator of PNP-catalyzed AsV reduction.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
BCX-1777 (also called Immucillin-H) and CI-1000 (also called PD 141955) were generous gifts from BioCryst Pharmaceuticals (Birmingham, AL) and Pfizer (Ann Arbor, MI), respectively. D-gluconic acid sodium salt was obtained from Sigma, Nonidet-P40 from Aldrich, and Triton X-100 from Reanal (Budapest, Hungary). The sources of thiol compounds, nucleosides, arsenic compounds, and chemicals used in arsenic speciation have been given elsewhere (Csanaky et al., 2003Go; Németi and Gregus, 2002bGo). All other chemicals were of the highest purity commercially available.

RBC experiments.
This research was approved by the Regional Scientific Research Ethics Committee of the University of Pécs, Center for Medical and Health Sciences. Whole blood (approximately 5 ml) was obtained from healthy human volunteers after informed consent into heparinized VacutainerTM tubes. The blood was immediately centrifuged at 1,000 x g at 4°C for 10 min, and the plasma and buffy coat were discarded. In order to maximize the AsV-reducing activity of RBC, the erythrocyte suspension was prepared and assayed in a buffer free of Pi, lest exogenous Pi should antagonize uptake and/or reduction of AsV. The pelleted RBC were resuspended in an equal volume of ice-cold buffer containing 150 mM sodium gluconate, 10 mM HEPES, and 5 mM glucose, pH 7.4. This RBC suspension was then centrifuged under the same conditions as previously, followed by removal of the supernatant. This washing procedure was repeated once more. After the final centrifugation, the pellet was measured gravimetrically, and then resuspended in an equal volume of ice-cold buffer resulting a 50% RBC suspension. The RBC suspension was kept in ice until use for assaying AsV reduction within 3 h.

In order to determine AsV-reducing activity of intact RBC, the RBC were incubated with 50 µM AsV at 37°C in a total volume of 300 µl of buffer used for washing. The duration of incubation was 30 min, except in the combined presence of a PNP substrate (typically inosine) and a thiol (typically DTT), when it lasted for 10 min. Incubations were started by adding RBC (30 µl from 50% suspension) and were stopped by successive addition of 100 µl of 25 mM CdSO4 solution containing 1% Triton X-100, and 100 µl of 1.5 M perchloric acid solution containing 25 mM HgCl2. For assaying AsV reduction by hemolyzed RBC, the same conditions were used, except the incubations were started by adding 100 µl of 50% RBC suspension into the incubation medium that contained a detergent (Nonidet-P40 at a final concentration of 0.067%), and were stopped by successive addition of 100 µl of 25 mM CdSO4 solution and 100 µl of 1.5 M perchloric acid solution containing 25 mM HgCl2. Pilot experiments clarified that Hg2+ ions effectively displaced thiol-bound AsIII even in strongly acidic environment. Nevertheless, Hg2+ ions oxidized the formed AsIII when applied at neutral pH, but not in acid. Therefore, we added Cd2+ first, which binds to thiol groups at neutral but not at acidic pH (Fuhr and Rabenstein, 1973Go), and which displaced thiol-bound AsIII, but did not oxidize the released AsIII. The incubates were stored at -80°C until analysis. Before analysis, the incubates were centrifuged at 10,000 x g at 4°C for 10 min, and the resultant supernatant was applied to high performance liquid chromatography–hydride generation–atomic fluorescence spectrometry (HPLC-HG-AFS) to separate and quantify AsIII and AsV. AsV reductase activity was expressed as nmol formed AsIII per minute and ml packed RBC.

For determining the effectiveness of BCX-1777 and CI-1000 as PNP inhibitors in intact RBC, these compounds at concentrations of 0, 2.5, 5, 10, 20, or 40 µM were incubated with RBC for 5 min under the same conditions used for assaying AsV reduction. Thereafter, the incubates were immediately centrifuged for 30 sec at 10,000 x g, and the supernatant was carefully removed. The pelleted RBC were washed by resuspending them in 200 µl ice-cold buffer and centrifuging the suspension again. After discarding the supernatant, the RBC pellet was hemolyzed in 135 µl buffer containing 1% Triton X-100. The resulting hemolysate was diluted 10-fold, and its PNP activity was assayed within an h.

Animal experiments.
Male Wistar rats weighing 250–270 g, obtained from the specific pathogen-free (SPF) breeding house of the University of Pécs (Hungary), were used. Conditions for keeping animals have been given in Németi and Gregus (2002b)Go. All procedures were carried out according to the Hungarian Animals Act (Scientific Procedures, 1998), and the study was in agreement with the rules of the Ethics Committee on Animal Research of the University of Pécs.

In order to test the in vivo effectiveness of BCX-1777 as an inhibitor of PNP in liver, rats were anesthetized with urethane (1.2 g/kg) injected ip (5 ml/kg), and their body temperature was maintained at 37°C by means of heating radiators. Through a median abdominal incision, the renal pedicles were ligated, and the animals were injected with BCX-1777 (50 µmol/kg, iv) in saline (2 ml/kg, iv) through their left saphenous vein. Control animals were given saline (2 ml/kg, iv). Fifteen min after injection of BCX-1777 or saline, the liver was perfused through the portal vein with 50 ml ice-cold isotonic saline to clear the blood and the blood-borne drug from the liver. The liver was then quickly removed and homogenized in three volumes of 100 mM potassium phosphate buffer (pH 7.5), using a motor-driven Teflon pestle and glass homogenization tube. The homogenate was centrifuged at 4°C, 10,000 x g for 20 min. The resultant postmitochondrial supernatant was kept on ice until assaying PNP activity within three h.

For determining the in vivo effect of BCX-1777 on metabolism of AsV, rats were anesthetized with urethane and injected with saline (2 ml/kg, iv) or BCX-1777 (50 µmol/kg, iv) after ligation of their renal pedicles as described above. Thereafter, the common bile duct was cannulated with the shaft of a 23-gauge needle attached to a PE-50 tubing (Clay Adams, Parsippany, NY). Fifteen min after administration of saline or BCX-1777, AsV (50 µmol/kg, iv) was injected. DTT (300 µmol/kg, iv) was administered to some rats 2 min before AsV. After injection of AsV, bile was collected in 20-min periods for 60 min into pre-weighed 1.5-ml microcentrifuge tubes immersed into ice. The volumes of bile samples were measured gravimetrically, taking 1.0 as specific gravity. Biliary excretion rates of arsenic compounds were calculated as the products of their concentration in bile and the biliary flow. 60 min after AsV administration, the rats were exsanguinated, blood was collected into heparinized tubes, and heart, liver, and muscle samples were removed.

Arsenic analysis.
AsIII and AsV in the deproteinized RBC incubates were separated and quantified by HPLC-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) according to Gomez-Ariza et al. (1998)Go, as described in detail by Gregus et al. (2000)Go. However, after ascertaining that the incubates contained no other AsV metabolites besides AsIII, we used isocratic rather than gradient elution routinely, with 60 mM sodium phosphate buffer (pH 5.75) as an eluent at 1.1 ml/min flow rate.

Bile and tissue samples were immediately prepared for arsenic speciation as described in detail (Csanaky et al., 2003Go). Gradient HPLC-HG-AFS procedures were used for speciation and quantification of arsenic in the bile (Gregus et al., 2000Go) and tissues (Csanaky et al., 2003Go).

PNP assay.
PNP activity of rat liver postmitochondrial supernatant (diluted 100-fold) and human RBC hemolysate was assayed according to the method of Kalckar (1947)Go, as described in detail by Gregus and Németi (2002)Go. This assay measures the formation of uric acid from inosine in the presence of excess Pi and xanthine oxidase.

Statistics.
SPSS 8.0 for Windows (SPSS, Inc.) was used for statistical analysis. Data were analyzed using one-way ANOVA followed by Duncan’s test and Student’s t-test, with p < 0.05 as the level of significance.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Role of PNP in AsV Reduction by RBC
Effect of compounds facilitating PNP-catalyzed AsV reduction.
Washed human RBC were capable of reducing AsV to AsIII at a rate of 1.45 ± 0.15 nmol per minute and ml packed RBC (Fig. 1Go, top). Since PNP is known to catalyze AsIII formation from AsV in the presence of appropriate nucleoside substrate (i.e., inosine or guanosine) and DTT, we tested whether nucleosides affected the observed AsIII formation. It was found that, in the absence of an exogenous thiol, purine nucleosides (i.e., inosine, guanosine, or adenosine) increased AsIII formation approximately 2.5-fold (Fig. 1Go, top), whereas pyrimidine nucleosides (i.e., uridine or cytidine) did not influence it. In the presence of DTT, purine nucleosides appeared even more powerful in enhancing AsV reduction (Fig. 1Go, bottom), with inosine being the most effective, as it increased AsIII formation more than sixfold. Pyrimidine nucleosides were ineffective also in the presence of DTT.



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FIG. 1. Effect of nucleosides on reduction of AsV by RBC. Washed human RBC (15 µl packed cells/300 µl) were incubated with 50 µM AsV at 37°C in 150 mM sodium gluconate, 10 mM HEPES, and 5 mM glucose (pH 7.4) containing one of the nucleosides (1 mM) with or without DTT (0.5 mM). The incubation was started by adding the RBC suspension, and was stopped by successive addition of 25 mM CdSO4 containing 1% Triton X-100, and 1.5 M PCA containing 25 mM HgCl2, both 1/3 volumes of the incubate. The incubation lasted for 30 min in the absence of DTT (top) and for 10 min in the presence of DTT (bottom). Bars represent means ± SEM of four incubations with RBC prepared from different individuals. Asterisks indicate significant difference (p < 0.05) from the AsIII formation observed in the absence of added nucleosides. Ino, inosine; Guo, guanosine; Ado, adenosine; Uri, uridine; Cyti, cytidine.

 
Because the PNP-catalyzed AsV reduction requires the presence of a thiol, a number of thiol compounds were tested for their capability to enhance AsIII formation by RBC. In the absence of exogenous inosine, none of the dithiols (each tested at a concentration of 0.5 mM) appeared effective in amplifying AsIII formation (Fig. 2Go, top); moreover, dimercaptopropanesulfonic acid (DMPS) even decreased it significantly. Of the monothiols (each tested at a concentration of 1 mM), cysteine doubled AsIII formation, while the others were ineffective. In the presence of inosine, the dithiol DTT and DMP increased AsIII formation by 3.5-fold and DMPS by twofold, whereas DMSA did not affect it significantly (Fig. 2Go, bottom). Of the monothiols, 2-mercaptoethanol enhanced AsV reduction almost twofold, but the others had no significant effect.



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FIG. 2. Effect of thiol compounds on reduction of AsV by RBC. Human RBC were incubated with AsV as described under Figure 1Go in the presence of one of the thiols (monothiols 1 mM, dithiols 0.5 mM) with or without inosine (1 mM). The incubation lasted for 30 min in the absence of inosine (top) and for 10 min in the presence of inosine (bottom). Bars represent means ± SEM of 4 incubations with RBC prepared from different individuals. Asterisks indicate significant difference (p < 0.05) from the AsIII formation observed in the absence of added thiols. DTT, dithiothreitol; DMP, dimercaptopropanol; DMPS, dimercaptopropane-sulfonic acid; DMSA, dimercaptosuccinic acid; GSH, glutathione; CYS, cysteine; NAC, N-acetylcysteine; CYA, cysteamine; PNA, D-penicillamine; 2-ME, 2-mercaptoethanol.

 
In order to clarify whether AsV-reducing activity of RBC remained functional upon hemolysis, RBC were incubated in the presence of Nonidet P40, a nonionic detergent. Upon hemolysis RBC lost more than 98% of their AsV-reducing activity present in intact cells, as the solubilized RBC produced 0.022 nmol/min/ml packed cell AsIII. By adding thiols to the hemolysate, the activity could be regained partially (Fig. 3Go, top). In the absence of exogenous inosine, the dithiol DTT, DMP, and DMPS increased AsIII formation by 16, 20, and 13-fold, respectively, whereas DMSA was ineffective. Of the monothiols, all except N-acetylcysteine enhanced AsV reduction significantly, although less than 10-fold. In the presence of 1 mM inosine, the solubilized RBC formed AsIII from AsV at a rate of 0.42 nmol/min/ml packed RBC. DTT, DMP, and DMPS increased this rate by 150, 185, and 92-fold (Fig. 3Go, bottom), respectively, whereas DMSA exhibited a weak effect. In the presence of inosine, none of the monothiols influenced AsIII formation by solubilized RBC significantly.



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FIG. 3. Effect of thiol compounds on reduction of AsV by solubilized RBC. Human RBC (50 µl packed cells/300 µl) were incubated with 50 µM AsV at 37°C in 150 mM sodium gluconate, 10 mM HEPES, 5 mM glucose (pH 7.4) containing 0.067% Nonidet-P40, and with or without inosine (1 mM) and one of the thiols (monothiols 1 mM, dithiols 0.5 mM). The incubation was stopped by successive addition of 1/3 incubate volumes of 25 mM CdSO4 solution and 1.5 M PCA solution containing 25 mM HgCl2. The incubation lasted for 30 min in the absence of inosine (top) and for 10 min in the presence of inosine (bottom). Bars represent means ± SEM of four incubations with RBC prepared from different individuals. Asterisks indicate significant difference (p < 0.05) from the AsIII formation observed in the absence of added thiols. DTT, dithiothreitol; DMP, dimercaptopropanol; DMPS, dimercaptopropanesulfonic acid; DMSA, dimercaptosuccinic acid; GSH, glutathione; CYS, L-cysteine; NAC, N-acetylcysteine; CYA, cysteamine; PNA, D-penicillamine; 2-ME, 2-mercaptoethanol.

 
Effect of compounds inhibiting PNP-catalyzed AsV reduction.
In order to clarify the role of PNP in reducing AsV to AsIII by RBC, a number of conditions known to inhibit PNP-catalyzed AsV reduction were tested. It was determined first whether potent and specific PNP inhibitors incubated for 5 min with washed human RBC could inactivate PNP in these cells. It was found that BCX-1777 almost completely abolished PNP activity in intact RBC even at a concentration as low as 2.5 µM (Fig. 4Go), whereas CI-1000 appeared fairly ineffective even at a concentration as high as 40 µM. It is important to realize, however, that the degree of PNP inhibition thus measured could easily underestimate that manifested in the incubated RBC, because the inhibitor could, at least partly, dissociate from the PNP during preparation of the incubated RBC for the PNP assay and during the assay.



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FIG. 4. Effect of PNP inhibitors on PNP activity in erythrocytes. RBC were first incubated for 5 min with increasing concentrations of either BCX-1777 or CI-1000 then pelleted, washed, solubilized, and diluted as described in Materials and Methods. It is important to realize that the degree of PNP inhibition thus measured could be an underestimation of that manifested in the incubated RBC, because the inhibitor could, at least partly, dissociate from the PNP during preparation of the incubated RBC for the PNP assay and during the assay. Symbols represent means ± SEM of two incubations with RBC prepared from different individuals.

 
The effects of BCX-1777 and CI-1000 (0, 2.5, 5, 10, 20, and 40 µM) on AsV reduction by intact RBC were tested under four conditions, i.e., in the absence of both exogenous inosine and DTT, in the presence of either DTT or inosine, and in the presence of both of these compounds that activate PNP-catalyzed AsV reduction (Fig. 5Go). It was found that neither PNP inhibitor decreased significantly the AsV reduction by RBC not supplemented with DTT and inosine (Fig. 5Go, top left). In the presence of DTT, BCX-1777 at each concentration tested decreased AsV reduction significantly, albeit only by 23–30% (Fig. 5Go, top right), whereas inhibitory effect of CI-1000 was statistically significant only at concentrations of 20 µM or higher. In the presence of inosine, BCX-1777 markedly inhibited the inosine-stimulated AsIII formation at each tested concentration (Fig. 5Go, bottom left), whereas CI-1000 was clearly less effective. When the AsV-reducing activity of RBC was stimulated by addition of both inosine and DTT, the PNP inhibitors were even more effective in decreasing AsIII formation: BCX-1777 and CI-1000 lowered the inosine, and DTT-stimulated AsV reductase activity of RBC to 11% and 16%, respectively (Fig. 5Go, bottom right). Nevertheless, even in the presence of 40 µM BCX-1777, an AsIII forming activity of approximately 1 nmol/min/ml packed RBC remained.



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FIG. 5. Effect of PNP inhibitors on reduction of AsV by RBC. Human RBC were incubated with AsV as described under Fig. 1Go in the presence of either PNP inhibitor (BCX-1777 or CI-1000) at the indicated concentrations under four different conditions: in the absence or presence of both DTT (0.5 mM) and inosine (1 mM), and in the presence of DTT or inosine. The incubation lasted for 30 min, but only for 10 min when both inosine and DTT were present. Symbols represent means ± SEM of four incubations with RBC prepared from different individuals. Asterisks indicate significant difference (p < 0.05) from the AsIII formation observed in the absence of added PNP inhibitor (i.e., at 0 µM concentration).

 
It was of interest to know whether the extremely high AsV-reducing activity of the hemolysate in the presence of both inosine and DTT (Fig. 3Go, bottom) was lowered by conditions that were known to inhibit AsV reduction catalyzed by purified PNP. Inorganic phosphate at 5 mM concentration diminished AsV reduction by hemolyzed RBC by 95% (Table 1Go). At 20 µM concentration, BCX-1777 decreased AsIII formation by more than 99%, whereas CI-1000 appeared less effective, as it inhibited AsV reduction by 96% (Table 1Go).


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TABLE 1 Effect of Conditions Known to Influence Purine Nucleoside Phosphorylase-Catalyzed Arsenate Reduction on Reduction of Arsenate by Solubilized Erythrocytes
 
Role of PNP in AsV Reduction in Rats
Inhibition of PNP by BCX-1777 in vivo.
To determine if BCX-1777 inhibited PNP in vivo, we assayed the hepatic PNP activity 15 min after injecting anesthetized rats with BCX-1777 (50 µmol/kg iv). These studies were performed on rats with ligated renal pedicles in order to prevent the rapid elimination of BCX-1777 via the urine (S. Bantia, personal communication). It was found that the PNP activity in the hepatic postmitochondrial supernatants of control rats receiving saline (the vehicle for BCX-1777) was 22.5 ± 2.05 units/g (n = 3), whereas the hepatic PNP activity was undetectable in the BCX-1777-treated animals (n = 3).

Effect of BCX-1777 on the biliary excretion and tissue concentrations of AsV and its metabolites.
In order to clarify the role of PNP in AsV metabolism in vivo, we examined the biliary excretion and tissue concentration of AsV and its metabolites in anesthetized, renal pedicle-ligated rats injected with BCX-1777 (50 µmol/kg, iv) 15 min prior to administration of AsV. These studies were carried out not only on rats receiving AsV with or without BCX-1777, but also on rats that were additionally injected with DTT (300 µmol/kg, iv) 2 min before administration of AsV. Thus, the effect of the PNP inhibitor was studied on control rats (given only AsV), and also on rats dosed with DTT, the activator of PNP-catalyzed AsV reduction.

Figure 6Go depicts the biliary excretion of the trivalent metabolites of AsV (i.e., AsIII and MMAsIII) in the four groups of rats, with the left panels demonstrating the time courses of excretion and the right panels indicating the 1-h cumulative excretion. As the main finding, this figure demonstrates that BCX-1777 failed to influence the biliary excretion of either AsIII or MMAsIII both in control rats (i.e., rats not given DTT) and in rats receiving DTT. As an auxiliary finding, Fig. 6Go also demonstrates that DTT strongly increased the biliary excretion of AsIII while decreasing the output of MMAsIII both in control rats and BCX-1777-treated rats.



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FIG. 6. Effects of BCX-1777 on the biliary excretion of AsV metabolites in control rats and in rats receiving DTT. Immediately after ligation of renal pedicles, the rats were injected with saline (2 ml/kg, iv) or BCX-1777 (50 µmol/kg, iv), and 15 min later with AsV (50 µmol/kg, iv). DTT (300 µmol/kg, iv) was administered 2 min before AsV. Bile was collected in 20-min periods after injection of AsV. Symbols and bars represent means ± SEM of six rats. Significant differences were found neither between the respective values of rats receiving BCX-1777 (but not DTT) and those of saline-injected controls, nor between the respective values of rats receiving BCX-1777 plus DTT and those given saline plus DTT.

 
Figure 7Go demonstrates the tissue concentrations of AsV and its metabolites in the four groups of rats 60 min after injection of AsV. Both inorganic, non-methylated arsenicals (i.e., AsIII and AsV) and methylated metabolites (MMAsIII, MMAsV, and DMAsV) were detected in the tissues of AsV-exposed rats. In rats not receiving DTT, BCX-1777 treatment resulted in moderate but statistically significant increase in the hepatic concentration of AsV (Fig. 7Go, top left), whereas the tissue concentrations of AsV metabolites remained unchanged in response to inhibition of PNP. In rats given DTT, the PNP inhibitor treatment slightly decreased AsV concentration in the heart, elevated the AsIII concentration in the blood and the liver (Fig. 7Go, bottom left), and diminished the concentration of DMAsV in each tissue (Fig. 7Go, bottom right). It was observed additionally that DTT alone increased by at least 50% the concentrations of AsIII, MMAsIII, and MMAsV in the liver, as well as the MMAsIII concentration in the blood, as compared to these concentrations in the saline-injected rats.



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FIG. 7. Effects of BCX-1777 on the blood, hepatic, heart, and muscle concentrations of AsV and its metabolites in control rats and in rats receiving DTT. Immediately after ligation of renal pedicles, the rats were injected with saline (2 ml/kg, iv) or BCX-1777 (50 µmol/kg, iv), and 15 min later with AsV (50 µmol/kg, iv). DTT (300 µmol/kg, iv) was administered 2 min before AsV. Blood and tissues were removed 60 min after injection of AsV. Bars represent means ± SEM of six animals. The cross indicates significant difference (p < 0.05) between the respective value of rats receiving BCX-1777 (but not DTT) and the saline-injected control rats, whereas asterisks indicate significant difference (p < 0.05) between the respective values of rats receiving BCX-1777 plus DTT and those given saline plus DTT.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In a manner completely inhibitable by PNP inhibitors, PNP reduces AsV to the much more toxic AsIII in vitro, provided its nucleoside substrate (inosine or guanosine) and an appropriate dithiol are present simultaneously (Gregus and Németi, 2002Go; Radabaugh et al., 2002Go). Reduction of AsV appears to take place in the course of the arsenolytic cleavage of 6-oxopurine nucleosides. It has been speculated that a CX5R motif at the Pi/AsV binding site of the enzyme may play a catalytic role in AsV reduction, just like a similar motif represents the catalytic center of some microbial AsV reductases (Gregus and Németi, 2002Go). On the basis of considerations outlined in the introduction, we tested the hypothesis that PNP played a significant role in reduction of AsV to AsIII in human RBC and in rats in vivo.

This study demonstrates that BCX-1777, one of the tested PNP inhibitors, is effective in inhibiting the physiologic catalytic activity of PNP (i.e., the phosphorolytic cleavage of inosine) in both human erythrocytes (Fig. 4Go) and in rats (described in Results). It is interesting to note, however, that while BCX-1777 almost completely inhibited the activity of the enzyme in human RBC even at a concentration as low as 2.5 µM, CI-1000, the other inhibitor, appeared quite ineffective (Fig. 4Go) despite the fact that these two compounds have comparable Ki values in the submicromolar range (Bzowska et al., 2000Go). However, the degree of PNP inhibition measured after removal of the extracellularly applied inhibitor and solubilizing the RBC does not necessarily reflect that manifested inside the erythrocytes during the incubation of RBC with the inhibitor, because the inhibitor could, at least partly, dissociate from the PNP during preparation of the incubated RBC for the PNP assay and during the assay. It is noteworthy that, although BCX-1777 and CI-1000 appear almost equipotent PNP inhibitors based on their Ki values (Bzowska et al., 2000Go), their mechanisms of action differ fundamentally. BCX-1777 mimics the structure of nucleoside substrates in their transition state formed during catalysis; therefore, the nucleosides (inosine or guanosine) cannot displace it competitively (Bzowska et al., 2000Go). In contrast, CI-1000, as a structural analogue of 6-oxopurine nucleosides, is a competitive inhibitor, being susceptible for displacement from the active site on the enzyme by nucleosides. Therefore, the apparent ineffectiveness of CI-1000 to inhibit erythrocyte PNP, as opposed to the marked inhibitory effect of BCX-1777 (Fig. 4Go), is most likely accountable for the notion that during preparation and assay of RBC, CI-1000 is easily displaced from the enzyme by inosine, whereas BCX-1777 is not.

This study has yielded some observations that are in apparent agreement with a role of PNP in the erythrocytic reduction of AsV. Because the AsV reduction catalyzed by PNP requires the simultaneous presence of inosine and DTT (Gregus and Németi, 2002Go), the increased AsIII formation by RBC in the presence of both inosine and DTT (Figs. 1Go and 2Go, bottom) could originate, at least partly, from the AsV reductase activity of PNP. Also in hemolysate, which almost completely lost its reducing activity, inosine plus DTT increased AsV reduction extremely, most likely through the AsV reductase activity of PNP. PNP inhibitors almost completely prevented this DTT-plus-inosine-induced increase in AsIII formation, both in intact erythrocytes (Fig. 5Go, bottom right) and in hemolysate (Table 1Go). However, the PNP substrate 6-oxopurine nucleosides (inosine and guanosine), as well as adenosine (a non-substrate, which is readily converted into inosine by adenosine deaminase), but not the pyrimidine nucleosides enhanced AsV reduction by intact RBC even when DTT was not present. Unless an endogenous thiol compound is present in RBC (that could substitute for DTT), it is unlikely that PNP contributed directly to AsIII formation in the absence of DTT, because the AsV-reducing activity of the enzyme requires the presence of a dithiol (Gregus and Németi, 2002Go). However, the inosine-stimulated reduction of AsV could also be strongly inhibited by PNP inhibitors (Fig. 5Go, bottom left), suggesting that either an endogenous DTT-like thiol compound is present in the erythrocytes, which supports the direct AsV-reducing activity of PNP, or inosine acted through PNP and enhanced AsIII formation indirectly. As to the former possibility, of the endogenous thiol compounds, only dihydrolipoic acid effectively supported the PNP-catalyzed AsV reduction (Radabaugh et al., 2002Go). Although the concentration of dihydrolipoic acid in erythrocytes is unknown, it is unlikely that it reaches the in vitro effective millimolar level. As to the indirect role of PNP in inosine-stimulated AsV reduction, phosphorolytic cleavage of nucleosides by PNP could diminish the Pi concentration in the erythrocytes, thereby increasing the possibility for AsV to reach the active site of a still unknown AsV-reducing enzyme, provided that the enzyme, like PNP, possesses a catalytically important AsV/Pi binding site. Alternatively, cleavage of nucleosides also yields ribose-1-phosphate, which may then be converted sequentially into ribose-5-phosphate, ribulose-5-phosphate, and xylulose-5-phosphate (McIntyre et al., 1989Go). These compounds may, in turn, be transformed into glycolytic intermediates (e.g., glyceraldehyde-3-phosphate or fructose-6-phosphate), thus fuelling up other metabolic pathways, which might support reduction of AsV by intact RBC. Further research is warranted to explore these hypothetic mechanisms.

Other observations seriously question the direct contribution of PNP to reduction of AsV to AsIII in erythrocytes and rats. In the absence of exogenous inosine, DTT, which activated the PNP-catalyzed AsV reduction in rat liver cytosol (Németi and Gregus, 2002bGo), exhibited little if any stimulatory effect on AsIII formation by intact RBC (Fig. 2Go, top). The rather modest inhibition of this DTT-stimulated AsV reduction by PNP inhibitors (Fig. 5Go, top right) indicates that AsIII formation from AsV in erythrocytes in the presence of DTT is largely PNP-independent. Cysteine poorly stimulated AsV reduction in rat liver cytosol (Németi and Gregus, 2002bGo); yet, it enhanced AsIII formation from AsV by intact RBC (Fig. 2Go, top). This cysteine-stimulated AsV reduction was unresponsive to PNP inhibitors (data not shown), also indicating that PNP is not involved in it. When neither inosine nor DTT was present in the incubate, both PNP inhibitors failed to diminish the reduction of AsV by RBC significantly, even at a concentration as high as 40 µM. These data collectively suggest that PNP does not contribute significantly to the reduction of AsV in intact human erythrocytes not supplemented with nucleoside substrates of PNP and an appropriate dithiol compound.

Testing the role of PNP in AsV reduction in rats using the potent PNP inhibitor drug BCX-1777 also yielded negative results, despite the fact that PNP activity became undetectable in the liver of BCX-1777–dosed rats by the time of AsV administration. If PNP had significantly contributed to reduction of AsV to AsIII in rats, BCX-1777 should have increased retention of AsV in the renal pedicle-ligated rats and should have decreased formation and biliary excretion of AsV metabolites, because reduction of AsV precedes formation of all AsV metabolites. Contrary to these expectations, BCX-1777 pretreatment caused no marked alteration in either the tissue concentrations of AsV (Fig. 7Go, top left), or in the biliary excretion of AsIII and MMAsIII (Fig. 6Go) and the tissue concentrations of these and other AsV metabolites (Fig. 7Go), when compared to saline-pretreated controls. These findings strongly suggest that PNP does not contribute to reduction of AsV to AsIII in rats, at least in the extrarenal tissues.

It was thought that in vivo absence of an appropriate endogenous thiol compound, which could support the PNP-catalyzed AsV reduction, like DTT does in vitro, might account for the inability of PNP to function as an AsV reductase in rats. In order to test this hypothesis, we also investigated the effect of BCX-1777 in rats injected with DTT prior to AsV administration. The dose of this membrane-permeable dithiol compound was selected so that its concentration in the body should approach 0.5 mM, i.e., a concentration, at which DTT supports the AsV-reducing activity of PNP in vitro (Gregus and Németi, 2002Go). Compared to the DTT-receiving rats, however, the BCX-1777 plus DTT-treated rats failed to retain more AsV in tissues, and excrete less AsIII and MMAsIII in bile, or contain in their tissues less AsV metabolites with the exception of DMAsV (Figs. 6Go and 7Go). The 20–30% decline in the tissue levels of this late metabolite in DTT-treated rats in response to BCX-1777 (Fig. 7Go, bottom right) remains unclear; it might be a consequence of the not readily explained increase of hepatic concentration of AsIII (Fig. 7Go, bottom left), which is known to inhibit formation of DMAsV (Csanaky et al., 2003Go; Zakharyan et al., 1999Go). Thus, we failed to obtain experimental support to the role of PNP as an AsV reductase even in DTT-exposed rats.

This study has also produced some auxiliary findings that deserve mentioning. For example, we observed that DMPS inhibited AsIII formation by intact erythrocytes in the absence of added inosine (Fig. 2Go, top). RBC take up AsV exclusively via their Cl-/HCO3- exchanger, also called Band III protein (Kenney and Kaplan, 1988Go), which can also transport DMPS (Wildenauer et al., 1982Go). Thus, competition between AsV and DMPS for this transporter might explain the DMPS-induced inhibition of AsIII formation in erythrocytes.

Another remarkable observation is that, compared to control animals, DTT strongly increased the biliary excretion of AsIII, whereas it slightly decreased that of MMAsIII (Fig. 6Go) and elevated the hepatic concentrations of both monomethylated arsenicals (Fig. 7Go). To offer an explanation, it is to be considered that AsIII and MMAsIII form relatively stable complexes with dithiols (Knowles and Benson, 1983Go), and that conjugation with the anionic reduced glutathione (GSH) is necessary so that arsenic can be transported into bile via Mrp2 (Kala et al., 2000Go). Complexation with DTT still permits AsIII to bind to glutathione with its remaining valence and to be transported into bile. However, MMAsIII cannot bind to GSH after complexed with DTT, and therefore this complex would be retained in the liver.

In summary, despite the fact that PNP effectively catalyzes the reduction of AsV in vitro in the course of the arsenolytic cleavage of inosine or guanosine, provided an appropriate dithiol is present, the findings presented here fail to support the hypothesis that PNP contributes significantly to AsV reduction either in intact human erythrocytes or in rats both in the absence and in the presence of such dithiol. The question then arises of why PNP cannot participate in reduction of AsV in vivo, even in the presence of DTT, when it can work as an AsV reductase in vitro. Limited quantity of PNP is an unlikely factor, because PNP is abundant in human erythrocytes (Stoeckler et al., 1978Go) and is present in a relatively high concentration in rat liver, accounting for about 0.2% of soluble protein (Hoffee et al., 1978Go). It is probable, however, that the intracellular Pi, acting as a PNP substrate competitive with AsV, suppresses the AsV reductase activity of PNP. Indeed, Pi in the unbound state, at concentrations present in erythrocytes and rat liver, i.e., approximately 0.2 mM (Fornaini et al., 1985Go) and 0.5 mM (Iles et al., 1985Go), respectively, significantly but incompletely inhibited AsV reductase activity of PNP at saturating concentration of inosine (Gregus and Németi, 2002Go). Additionally, the physiological concentrations of inosine in human erythrocytes (3.5 µM, Tavazzi et al., 2000Go) and rat liver (1.6 µM, Pillwein et al., 1987Go) are rather low compared to those in the hepatic cytosolic fraction and the hemolysate, where much inosine could be formed from breakdown of adenine nucleotides. Therefore, the minimal availability of inosine, together with the presence of Pi in functioning cells, should severely limit the in vivo rate of PNP-catalyzed arsenolytic nucleoside cleavage and the coupled reduction of AsV. Thus, while previous studies (Gregus and Németi, 2002Go; Radabaugh et al., 2002Go) promised a better understanding of the biochemistry of AsV reduction after demonstrating that PNP can function as an AsV reductase in the hepatic cytosolic fraction, the findings presented here call for further research to clarify the in vivo relevant mechanism(s) for conversion of AsV to the more toxic AsIII.


    ACKNOWLEDGMENTS
 
This publication is based on work supported by the Hungarian National Scientific Research Fund (OTKA) and the Hungarian Ministry of Health. The authors wish to thank István Schweibert for his excellent assistance with analytical work.


    NOTES
 
1 To whom correspondence should be addressed. Fax: 36-72-536-218. E-mail: zoltan.gregus{at}aok.pte.hu. Back


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