Department of Pharmacology and Pharmacotherapy, Toxicology Section,University of Pecs, Medical School, Pecs, Hungary

    ABSTRACT

    The environmentally prevalent arsenate (AsV) is reduced in the body to the much more toxic arsenite (AsIII). Recently, we have demonstrated that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the reduction of AsV in the presence of glutathione, yet the role of GAPDH in AsV reduction in vivo is unknown. Therefore, we examined the effect of (S)--cholorhydrin (ACH), which forms a GAPDH-inhibitory metabolite, on the reduction of AsV in rats. These studies confirmed the in vitro role of GAPDH as an AsV reductase, inasmuch as 3 h after administration of ACH (100 or 200 mg/kg, ip) to rats both the cytosolic GAPDH activity and the AsV-reducing activity dramatically fell in the liver, moderately decreased in the kidneys, and remained unchanged in the muscle. Moreover, the AsV-reducing activity closely correlated with the GAPDH activity in the hepatic cytosols of control and ACH-treated rats. Two confounding effects of ACH (i.e., a slight fall in hepatic glutathione levels and a rise in urinary AsV excretion) prompted us to examine its influence on the disposition of injected AsV (50 μmol/kg, iv) in rats with ligated bile duct as well as in rats with ligated bile duct and renal pedicles. These experiments demonstrated that the hepatic retention of AsV significantly increased, and the combined levels of AsV metabolites (i.e., AsIII plus methylated arsenicals) in the liver decreased in response to ACH; however, ACH failed to delay the disappearance of AsV from the blood of rats with blocked excretory routes. Thus, the GAPDH inactivator ACH inhibits AsV reduction by the liver, but not by the whole body, probably because the impaired hepatic reduction is compensated for by hepatic and extrahepatic AsV-reducing mechanisms spared by ACH. It is most likely that ACH inhibits hepatic AsV reduction predominantly by inactivating GAPDH in the liver; however, a slight ACH-induced glutathione depletion may also contribute. While this study seems to support the conclusion that GAPDH in the liver is involved in AsV reduction in rats, confirmation of the in vivo role of GAPDH as an AsV reductase is desirable.

    Key Words: arsenate; reduction; glyceraldehyde-3-phosphate dehydrogenase; -chlorohydrin; glutathione.

    INTRODUCTION

    Arsenic is a common element in the environment with high toxicological importance. Chronic arsenic exposure can cause skin lesions, vascular disease, and cancer (Goering et al., 1999; Hughes, 2002; Rossman, 2003). The primary source of human arsenic exposure is contaminated drinking water, in which the prevalent compound is arsenate (AsV). Owing to the close structural similarity to inorganic phosphate (Pi), AsV can replace Pi in transport processes and enzymatic reactions (Csanaky and Gregus, 2001; Dixon, 1997; Ginsburg and Lotspeich, 1963), thereby impairing cellular metabolism. On the other hand, AsV can be reduced to arsenite (AsIII), which is much more toxic due to its facile covalent reactivity with thiols, particularly dithiols (Knowles and Benson, 1983; Thomas et al., 2001). Subsequent metabolism of arsenite yields mono- and dimethylated metabolites, among which the pentavalent ones are relatively nontoxic, but the trivalent ones are highly toxic (Petrick et al., 2001; Rossman, 2003; Thomas et al., 2001). Reduction of AsV to AsIII, as the first step in its metabolism, is therefore decisive for not only the fate of arsenic in the body, but also its toxicity and carcinogenicity.

    Although AsV can be reduced to AsIII enzymatically, in vivo relevance of such enzymes in mammals has not been demonstrated. Rat liver mitochondria take up and reduce AsV to AsIII (Nemeti and Gregus, 2002a), though their contribution to the reduction of AsV in vivo is unclear. Purine nucleoside phosphorylase (PNP), a ubiquitous cytosolic enzyme, is also capable of reducing AsV in the presence of its substrate and a dithiol (Gregus and Nemeti, 2002; Radabaugh et al., 2002), but its role in the reduction of AsV in vivo is apparently insignificant (Nemeti et al., 2003; Patterson et al., 2003). Recently, human red blood cells (RBC) have been found to possess a PNP-independent AsV reductase activity, which appears to be linked to the glycolytic pathway and depends on the availabilities of glutathione (GSH) and NAD (Nemeti and Gregus, 2004). Further characterization of this activity in RBC lysate and rat liver cytosol has pointed to two functionally linked glycolytic enzymes as AsV reductase candidates, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (Nemeti and Gregus, 2005), of which GAPDH has been demonstrated to be endowed with AsV reductase activity (Gregus and Nemeti, 2005). It has also been shown that, while GAPDH is solely responsible for the PNP-independent AsV reductase activity in human erythrocytes, it is partially responsible for such AsV reductase activity in rat liver cytosol.

    The goal of the present study was to test the hypothesis that GAPDH contributes to the reduction of AsV in vivo. For this purpose, we used (S)--chlorohydrin (ACH) as an experimental tool. ACH is purportedly converted by cellular enzymes to 3-chlorolactaldehyde, a structural analogue of glyceraldehyde-3-phosphate, which inhibits GAPDH (Stevenson and Jones, 1985). It has been demonstrated that treatment of rats with ACH decreases the activity of GAPDH in tissues (Jelks and Miller, 2001; Stevenson and Jones, 1985). Therefore, we decided to test the effect of ACH administered to rats in relatively large but well-tolerated doses on the GAPDH activity, as well as on the GSH-and NAD-dependent (PNP-independent) AsV reductase activity in various tissues. After finding that 3 h after its administration ACH markedly decreased both the GAPDH and AsV-reducing activities in the liver of rats, further studies were designed to determine whether pretreatment with ACH impairs reduction of the injected AsV in rats. For this purpose, separate groups of control and ACH-pretreated rats were anesthetized, injected intravenously with AsV, and then subjected to collection of blood, urine, and tissues in order to assess the ACH-induced changes in both the elimination of the injected AsV and the formation of AsV metabolites, namely AsIII, monomethylarsonic acid (MMAsV), monomethylarsonous acid (MMAsIII), and dimethylarsinic acid (DMAsV). As explained later, the results emerging as this study progressed have dictated the use of experimentally manipulated control and ACH-pretreated animals for following the disposition of AsV, first in bile duct-ligated (BDL) rats and then in bile duct- and renal pedicle-ligated (BDRPL) rats. AsV and its metabolites in urine, blood, and some other tissues of such animals were quantified by high performance liquid chromatography–hydride generation–atomic fluorescence spectrometry (HPLC-HG-AFS).

    MATERIALS AND METHODS

    Chemicals.

    BCX-1777 (also called Immucillin-H) was a generous gift from BioCryst Pharmaceuticals (Birmingham, AL). N-(2-hydroxyethyl)-piperazine-N'-(2-ethanesulfonic acid) (HEPES), phosphoglyceric phosphokinase from baker's yeast, fructose-1,6-bisphosphate (tetra)cyclohexylammonium salt, D,L-glyceraldehyde-3-phosphate diethyl acetal monobarium salt, 3-phosphoglyceric acid disodium salt, and glutathione reductase from baker's yeast were from Sigma. Bicinchoninic acid disodium salt and S-(+)-3-chloro-1,2-propanediol (S-(+)--chlorohydrin, ACH) were from Fluka. Reduced glutathione, disodium hydrogen arsenate (AsV), NAD, NADPH, and ATP were from Reanal Ltd. (Budapest, Hungary). The sources of chemicals used in arsenic speciation as well as in GSH and NPSH assays have been given elsewhere (Csanaky et al., 2003; Nemeti and Gregus, 2002a). All other chemicals were of the highest purity commercially available.

    Animals.

    Male Wistar rats weighing 250–270 g were obtained from the SPF breeding house of the University of Pecs (Hungary). The animals were kept in polypropylene cages (type III D, Charles River, Hungary) with a stainless steel grill lid, on sterile bedding (type Lignocell, Charles River, Hungary), and in rooms with 12-h light/dark cycle, 22–25°C temperature, 55–65% relative air humidity. They were provided with rodent lab chow (type VRF1, Charles River, Hungary) and tap water ad libitum. Rats were selected for the studies because this species is superior to other common laboratory animal species in AsV-metabolizing capacity (Csanaky and Gregus, 2002). All procedures were carried out on animals according to the Hungarian Animals Act (Scientific Procedures, 1998), and the study was approved by the Ethics Committee on Animal Research of the University of Pecs.

    Testing the effect of ACH on the activities of GAPDH and AsV reductase in rats.

    In order to test the effect of ACH on GAPDH and AsV reductase activities as well as the GSH or nonprotein thiol (NPSH) levels in tissues, rats were injected with ACH (100 or 200 mg/kg, ip) or saline (3 ml/kg, ip). It is important to note that we dosed the rats with (S)--chlorohydrin and not with the racemic (R,S)--chlorohydrin, as the latter contains the nephrotoxic (R)-isomer, whereas the (S)-isomer (which forms the GAPDH-inactivating metabolite) is devoid of such an effect (Jones and Cooper, 1999). Three h later liver, kidney, and muscle samples were removed, rinsed with ice-cold saline, and weighed. The liver and kidney samples were homogenized in three volumes of sucrose buffer (containing 250 mM sucrose, 25 mM HEPES, 5 mM MgCl2, 2 mM EGTA, pH 7.4), using a glass homogenization tube, first with a looser and then a tighter motor-driven Teflon pestle. The muscle was homogenized in three volumes of sucrose buffer, first with a blade homogenizer (Ultra-Turrax, Janke & Kunkel Gmbh, Staufen, Germany), then with the tighter motor-driven Teflon pestle in a glass homogenization tube. For measuring GSH or NPSH, 100 μl homogenate was transferred into a microfuge tube containing 300 μl 0.4 M perchloric acid. The samples were mixed thoroughly and kept at –80°C until assaying the GSH or NPSH content. The remaining homogenate was then centrifuged at 4°C, 10,000 x g for 20 min to obtain the postmitochondrial supernatant, which was then centrifuged in a Sorvall ultracentrifuge at 4°C, 100,000 x g for 75 min. The resultant supernatant, corresponding to the cytosolic fraction, was stored in aliquots at –80°C until assaying for GAPDH and AsV reductase activities. The protein concentration of the cytosol preparations was determined by the bicinchoninic acid method according to Brown et al. (1989).

    The GSH concentration of the perchloric acid–treated liver and muscle homogenates was determined according to the method of Tietze (1969), using glutathione reductase. However, we measured the concentration of NPSH according to Sedlak and Lindsay (1968) in the kidney homogenates, because the high -glutamyl transpeptidase activity in the kidneys degrades the renal GSH during homogenization. The GSH and NPSH concentrations were expressed as μmol per g wet tissue.

    The cytosolic GAPDH activity was assayed spectrophotometrically based on the decrease of NADH concentration (0.25 mM) during the GAPDH-limited conversion of 3-phosphoglycerate (3-PGA) to glyceraldehyde-3-phosphate (Ga-3-P) in the presence of excess phosphoglycerate kinase and ATP, as described earlier (Gregus and Nemeti, 2005).

    The cytosolic AsV reductase activity was assayed at 37°C in sucrose buffer in a final volume of 0.3 ml with 5 mg/ml cytosolic protein concentration under four conditions, i.e., without addition of NAD and a glycolytic substrate or with addition of NAD plus fructose-1,6-bisphosphate (Fruc-1,6-BP), or Ga-3-P, or 3-PGA, which are glycolytic substrates. In order to inactivate PNP and deplete glucose and glucose-derived substrates, the cytosol was preincubated with BCX-1777 (20 μM) and glucose oxidase (2 U) for 5 min. Thereafter, GSH (10 mM), a glycolytic substrate (1 mM) or buffer, NAD (1 mM) or buffer were added in rapid succession followed immediately by addition of AsV (50 μM). Then the incubation was started and continued for 5 min when NAD and glycolytic substrates were absent, but only for 2.5 min when NAD and a glycolytic substrate were present. The incubation was terminated by sequential addition of 100 μl 50 mM CdSO4 solution followed by 100 μl 1.5 M perchloric acid solution containing 50 mM HgCl2. The rationale for this procedure has been given elsewhere (Nemeti and Gregus, 2004). The incubates thus treated were stored at –80°C until arsenic analysis. AsV reductase activity was expressed as the amount of AsIII formed per minute and mg cytosolic protein.

    Testing the effect of ACH on the disposition of AsV in rats.

    In order to test the effect of ACH on reduction of AsV in vivo, two different experiments were carried out. In both, the rats were pretreated intraperitoneally with ACH (100 mg/kg or 200 mg/kg) or saline (3 ml/kg), 3 h before AsV administration. Immediately before injection of AsV, the rats in the first experiment were subjected to bile duct ligation (BDL-rats), whereas the rats in the second experiment underwent both bile duct and renal pedicle ligation (BDRPL-rats). The rationale of this design is given in the Results section.

    The rats to be subjected to bile duct ligation only were hydrated by gavage of 30 ml/kg saline containing 10 mM potassium chloride, to induce urine production, and anesthetized by ip injection of a mixture of fentanyl, midazolam, and droperidol (0.045, 4.5, and 5.5 mg/kg, respectively). Subsequently, the right carotid artery was cannulated, and the urinary bladder was exteriorized. Three h after pretreatment with ACH or saline, the rats were administered 3 ml/kg 10% mannitol in saline via the carotid cannula to promote urine flow. Subsequently, the bile duct was ligated, and AsV (50 μmol/kg, iv) was injected. After AsV administration, the urine was collected in 20-min periods into preweighed 1.5-ml microcentrifuge tubes for 60 min. To obtain urine, the urinary bladder was gently compressed manually. To maintain urine flow at rates of 130–180 μl/kg/min, 10% mannitol in saline was infused via the carotid cannula at a rate of 9 ml/kg/h. Throughout the experiment, the body temperature of the rats was maintained at 37°C, and additional doses of the anesthetic mix were injected in every 20–30 min. The volumes of urine samples were measured gravimetrically, taking 1.0 as specific gravity. Urinary excretion rates of arsenic compounds were calculated as the products of their concentration in urine and the urinary flow. Simultaneously with urine collection, blood samples were withdrawn from the carotid artery at 0, 5, 15, 30, 45, and 60 minutes after AsV administration into heparinized 1.5-ml microfuge tubes. At 60 min after injection of AsV, the rats were heparinized and thoroughly exsanguinated, and their liver, heart, thigh muscle, and one kidney were removed and immediately processed for arsenic analysis.

    The experiments carried out on BDRPL-rats differed from those done on BDL-rats in the following details. These rats were not hydrated, and obviously, urine was not collected. Instead of the kidney, the brain was removed from these animals at the end of experiments to determine the concentrations of the arsenicals.

    Arsenic analysis.

    Arsenic in the incubates and the ex vivo biological samples was speciated and quantified by high performance liquid chromatography–hydride generation–atomic fluorescence spectrometry (HPLC-HG-AFS). The incubates originating from the AsV reductase assays and having been subjected to protein precipitation were centrifuged at 10,000 x g, 4°C for 10 min. AsIII and AsV in the resultant supernatants were separated on a strong anion exchange guard column and analytical column (both Hamilton PRP X-100) and eluted isocratically with 60 mM sodium phosphate buffer (pH 5.75). The details of this analysis have been given elsewhere (Gregus et al., 2000; Nemeti et al., 2003).

    Preparation of urine, blood, and other tissue samples for arsenic analysis by HPLC-HG-AFS has been described (Csanaky and Gregus, 2003). AsV and its metabolites were separated on Hamilton PRP X-100 guard and analytical columns, using gradient elution with an eluent containing NH4H2PO4–NH4NO3. The details of this analytical procedure have been published (Csanaky et al., 2003). Quantification of arsenic compounds was based on peak areas of samples and authentic standards (AsIII, DMAsV, MMAsV, AsV) whose detection limits have been given (Csanaky and Gregus, 2001). Because pure MMAsIII is not available commercially, MMAsIII was quantified based on the MMAsIII peak area in the sample and the AsIII peak area in the standard (Gregus et al., 2000).

    Statistics.

    Data were analyzed using one-way ANOVA followed by Duncan's test or Students' t-test, with p < 0.05 as the level of significance.

    RESULTS

    Effects of ACH on the Activity of GAPDH and the Concentration of GSH or NPSH in Tissues of Rats

    In order to ascertain that ACH diminishes GAPDH activity in tissues and to determine the magnitude of its effect, the GAPDH activity in the cytosolic fraction of the liver, kidney, and muscle was assayed 3 h after injecting rats with saline or ACH (100 or 200 mg/kg, ip). Figure 1 demonstrates that ACH at 100- and 200-mg/kg doses dramatically diminished the GAPDH activity in the liver (by 85 and 93%, respectively), but only slightly decreased it in the kidney (by 19 and 24%, respectively). The GAPDH activity in the cytosolic fraction of the muscle of rats was not influenced by ACH pretreatment.

    Because GSH plays an important role in the reduction of AsV, it was important to know if ACH pretreatment changed the GSH or NPSH concentrations in tissues. As depicted in Figure 2, ACH at doses 100 and 200 mg/kg decreased the hepatic GSH concentration by 15 and 23%, respectively, whereas the GSH concentration in the muscle and the NPSH concentration in the kidneys were unaffected.

    Effects of ACH on the Cytosolic AsV Reductase Activities in the Tissues of Rats

    It was important to know if the treatment of rats with ACH influenced the cytosolic AsV reductase activities similarly to the GAPDH activities. The AsV reductase activities in hepatic, renal, and muscle cytosols were assayed under four conditions, i.e., in the absence of exogenous NAD and glycolytic substrate, in the presence of added NAD plus Fruc-1,6-BP, NAD plus Ga-3-P, or NAD plus 3-PGA. The assays were carried out after preincubation of the cytosol with BCX-1777 and glucose oxidase, in order to inhibit PNP and to deplete endogenous glucose and glucose-derived glycolytic metabolites.

    AsV reductase activity in the hepatic cytosol not supplemented with NAD and a glycolytic substrate was decreased insignificantly by pretreatment of rats with ACH at the lower dose, but moderately (–35%) at the higher dose (Table 1). In contrast, when the hepatic cytosol was supplemented with exogenous NAD and a glycolytic substrate (Fruc-1,6-BP, Ga-3-P, or 3-PGA) to activate GAPDH and increase AsIII formation rates several fold, ACH given to rats at 100- or 200-mg/kg doses diminished the formation of AsIII from AsV by as much as 75% or 90%, respectively (Table 1), compared to the cytosolic AsV reductase activities of the liver of saline-pretreated rats.

    In the renal cytosol not supplemented with NAD and a glycolytic substrate, ACH pretreatment did not affect the AsV reductase activity (Table 1). However, in the combined presence of NAD and a glycolytic substrate, ACH decreased AsV reduction moderately, i.e., by 40–45% at the most (Table 1).

    Pretreatment of rats with ACH did not influence the AsV reductase activity in the muscle cytosol, either in the absence of exogenous NAD and glycolytic substrate (Table 1) or in their presence, compared to the saline-pretreated controls.

    Because both the GAPDH and AsV reductase activities in the hepatic cytosol were markedly diminished by pretreating rats with ACH, we analyzed if there was a correlation between the GAPDH activity and the AsV reductase activities of the cytosols prepared from the liver of control and ACH-treated rats. We found that the basal AsV reductase activity (i.e., measured in the absence of exogenous NAD and glycolytic substrate) correlated with the GAPDH activity rather poorly (Fig. 3, top left), as r2 was only 0.3. In contrast, the AsV reductase activity exhibited a clear linear correlation with the GAPDH activity, when the former was stimulated by the combined presence of NAD and Fruc-1,6-BP (Fig. 3, top right) or Ga-3-P (Fig. 3, bottom left) or 3-PGA (Fig. 3, bottom right).

    Effects of ACH on the Disposition of AsV in Bile Duct-Ligated Rats

    In order to assess the contribution of GAPDH to the in vivo reduction of AsV, we tested the effects of ACH pretreatment on the disposition of AsV in rats. We carried out these experiments on rats whose bile duct was ligated (BDL-rats), because ACH, especially at the higher dose, slightly decreased the GSH content of the liver (Fig. 2), and because shortage of GSH impairs the biliary excretion of trivalent arsenic (Csanaky and Gregus, 2005; Gyurasics et al., 1991) that could mask the effect of ACH on the in vivo reduction of AsV. In control and ACH-treated BDL-rats, we followed the disappearance of the iv-injected AsV from blood, the appearance of AsV metabolites in blood, and excretion of AsV and its metabolites in urine for 60 min. At this time, we quantified the concentrations of these arsenicals in the tissues.

    Figure 4 demonstrates the time courses of blood concentrations of AsV and its metabolites in BDL-rats pretreated with ACH. At the dose of 100 mg/kg, ACH did not influence the disappearance of AsV from the blood. However, ACH at 200 mg/kg increased the decay of AsV in the blood, as from 15 min onward the blood concentration of AsV in the ACH-dosed rats was significantly lower than in the controls (Fig. 4, top left). AsIII appeared in blood as early as 5 min after AsV injection, and its concentration reached a plateau at 15 min (Fig. 4, bottom left). The lower ACH dose did not alter the blood concentration versus time curve of AsIII significantly, whereas the higher ACH dose resulted in an approximately 30% fall in AsIII levels from 15 min after AsV administration. The concentration of the blood-borne methylated AsV metabolites (i.e., MMAsV, MMAsIII, and DMAsV) increased gradually from 15 min. ACH at the lower dose tended to diminish the concentrations of MMAsV (Fig. 4, top right) and MMAsIII (Fig. 4, middle right), whereas at the higher dose, it significantly decreased the blood concentrations of both monomethylated AsV metabolites in BDL-rats from 15 min after AsV administration, as compared to the saline-treated controls. The concentration of DMAsV was unaffected by 100 mg/kg ACH, but it was significantly diminished by 200 mg/kg ACH at 15 and 30 min after injection of AsV, but not later.

    The time course for the urinary excretion rate of AsV and AsIII in control and ACH-treated BDL-rats is shown in Figure 5. Unexpectedly, ACH given to rats at 200-mg/kg dose markedly enhanced the renal excretion of AsV (Fig. 5, top), whereas the lower dose barely affected it. The urinary flow was also significantly increased by the higher ACH dose (Fig. 5, bottom). In contrast, the urinary excretion of AsIII was diminished markedly in rats given 200 mg/kg ACH, but only moderately in those receiving 100 mg/kg ACH (Fig. 5, middle).

    The tissue concentrations of AsV and its metabolites in BDL-rats pretreated with saline or ACH (100 or 200 mg/kg) 60 min after AsV administration are illustrated in Figure 6. In the liver of rats treated with ACH at either dose, the concentration of AsV increased significantly compared to saline-pretreated controls (Fig. 6, top left). ACH did not change the concentration of AsIII significantly at either dose, whereas it tended to decrease the levels of the methylated arsenicals (i.e., MMAsV, MMAsIII, and DMAsV) at the lower dose, and significantly diminished them at the higher dose. In the kidneys, the concentration of AsV was decreased by ACH pretreatment only at the 200-mg/kg dose (Fig. 6, bottom left), whereas the level of AsIII was significantly lowered by ACH at both doses. The concentrations of the monomethylated AsV metabolites remained unchanged, but the DMAsV level decreased as a result of ACH pretreatment at the higher dose. In the heart of BDL-rats, the higher ACH dose significantly diminished the concentrations of AsV, AsIII, and MMAsV and tended to decrease that of MMAsIII and DMAsV (Fig. 6, top right), while the lower dose had no influence on the concentrations of AsV and its metabolites. In the muscle, ACH given to rats at the higher dose lowered the concentrations of AsV, AsIII, MMAsIII, and DMAsV and was apt to diminish that of MMAsV (Fig. 6, bottom right), whereas at the lower dose, ACH did not affect the concentrations of AsV and its metabolites.

    Effects of ACH on the Disposition of AsV in Bile Duct- and Renal Pedicle-Ligated Rats

    Because ACH pretreatment significantly increased urinary excretion of AsV (Fig. 5, top) and this unexpected effect could confound an inhibitory effect of ACH on AsV reduction in vivo, we repeated these experiments on another three groups of rats with not only their bile duct but also their renal pedicles ligated (BDRPL-rats). In control and ACH-treated BDRPL-rats, we followed the disappearance of the iv-injected AsV from blood, as well as the appearance of AsV metabolites in blood for 60 min. At this time, we quantified the concentrations of the arsenicals in the tissues.

    Figure 7 depicts the time courses of the blood concentrations of AsV and its metabolites. The concentration of AsV tended to increase as a result of ACH dosing, though the increases were not significant statistically (Fig. 7, top left). The blood concentration of AsIII was also barely affected. In contrast, the gradual accumulation of MMAsV (Fig. 7, top right) and MMAsIII (Fig. 7, middle right) in the blood was markedly decreased by pretreatment with ACH at either dose from 15 min after AsV administration. The rise in the concentration of the late metabolite DMAsV tended to be delayed, and by 60 min after injection of AsV, its concentration became significantly lower in ACH-dosed rats as compared to controls.

    The tissue concentrations of AsV and its metabolites in BDRPL-rats pretreated with saline or ACH (100 or 200 mg/kg) 60 min after AsV administration are shown in Figure 8. In the liver, ACH pretreatment at both doses greatly increased the concentration of AsV (Fig. 8, top left), but failed to change that of AsIII. The hepatic concentration of MMAsIII fell markedly, while that of DMAsV diminished moderately but significantly in response to ACH. The ACH-induced diminution in hepatic MMAsV levels was not significant statistically. In the brain, ACH did not influence the concentrations of AsV and its metabolites (Fig. 8, bottom left), except for AsIII, which was slightly increased by only the lower ACH dose. In the heart, ACH pretreatment did not cause any significant alteration in the concentrations of AsV and its metabolites (Fig. 8, top right). Interestingly, the muscle concentration of AsIII was significantly elevated by both ACH doses (Fig. 8, bottom right). The levels of AsV, MMAsV, and MMAsIII were not affected, while the DMAsV concentration was slightly decreased by the higher ACH dose.

    Effects of ACH on the Hepatic Concentrations of AsV and Its Metabolites As Well As on the Metabolite/AsV Ratio in Bile Duct-Ligated Rats and in Bile Duct- and Renal Pedicle-Ligated Rats

    Because ACH pretreatment exerted the most marked inhibition on both GAPDH and AsV reductase activities in the hepatic cytosol (Fig. 1 and Table 1, respectively), we compared the concentrations of AsV, its metabolites (i.e., the sum of AsIII, MMAsV, MMAsIII, and DMAsV), and the metabolite/AsV ratio in both BDL-rats and BDRPL-rats pretreated with ACH and injected with AsV 3 h later. In both groups of rats, ACH pretreatment increased the hepatic AsV concentration more than two-fold (Fig. 9, top), compared to saline-pretreated controls. The metabolite concentration decreased by 100 mg/kg ACH insignificantly, but by almost 50% after administration of 200 mg/kg ACH in both groups of rats (Fig. 9, middle). In contrast, the metabolite to AsV ratio was strongly lowered by ACH pretreatment. The lower ACH dose decreased the ratio by approximately 65%, whereas the higher dose by almost 80% in both BDL-rats and BDRPL-rats (Fig. 9, bottom).

    DISCUSSION

    This study on rats treated with ACH, a chemical known to be dehydrogenated in vivo to the GAPDH-inhibitory metabolite (S)-3-chloro-lactaldehyde (Jelks and Miller, 2001; Stevenson and Jones, 1985), presents two new pieces of evidence that GAPDH functions as a cytosolic AsV reductase in vitro (Gregus and Nemeti, 2005; Nemeti and Gregus, 2005). First, ACH treatment affected the GAPDH and the AsV reductase activities in the cytosols of various tissues similarly: it decreased both markedly in the liver, it diminished both moderately in the kidney, and it failed to influence either one in the muscle (Fig. 1 and Table 1). Second, when the hepatic cytosols from control and ACH-pretreated rats were simultaneously assayed for both GAPDH and AsV reductase activities under conditions of abundant substrate supply for GAPDH, the AsV reductase activity closely correlated with the GAPDH activity (Fig. 3). However, the AsV reductase activity did not correlate closely with the GAPDH activity in the tissues of saline-treated control rats. For example, while GAPDH activities in the liver, kidney, and muscle were 0.74, 0.88, and 4.62 U/mg protein, respectively (Fig. 1), the AsV reductase activities in these tissues in the presence of NAD and Fruc-1,6-BP were 413, 221, and 1046 pmol/min/mg protein (Table 1). This finding might suggest that the glycolytic and AsV reductase activities of GAPDH in the cytosol originating from various tissues are influenced differentially by some tissue-related factors.

    The major question this study attempted to answer was, however, whether or not GAPDH functions as an AsV reductase also in vivo. The importance of clarifying this point is attested by the example of PNP, which works very efficiently as an AsV reductase under in vitro conditions (Gregus and Nemeti, 2002; Radabaugh et al., 2002), yet probing its role in reduction of AsV to AsIII in rats has yielded negative results (Nemeti et al., 2003). Nevertheless, while both PNP and GAPDH require a small thiol compound to catalyze AsV reduction, only the AsV-reducing activity of GAPDH (and not of PNP) is supported by the physiologically important and abundant GSH (Gregus and Nemeti, 2002, 2005; Nemeti and Gregus, 2002b, 2005), a feature making the in vivo involvement of this glycolytic enzyme in AsV reduction more feasible. To experimentally test such an involvement, we studied the disposition of AsV in rats pretreated with ACH that when given to rats caused partial inactivation of GADPH, decreasing not only its classical glycolytic activity (Fig. 1) but also its AsV-reducing activity (Table 1). It was assumed that contribution of GAPDH to AsV reduction in vivo could be confirmed if ACH treatment delayed the elimination of AsV and impaired the formation of AsV metabolites (i.e., AsIII, MMAsV, MMAsIII, and DMAsV). However, during the course of this study we recognized two effects of ACH that could make a potential ACH-induced impairment in AsV reduction in rats difficult to uncover: (1) a moderate decrease in hepatic GSH levels (Fig. 2) and (2) a considerable increase in the urinary excretion of AsV (Fig. 5). In order to minimize the confounding influence of the former (which would compromise the hepatobiliary transport of the trivalent metabolites of AsV), we tested the effects of ACH on AsV disposition in BDL-rats, and to eliminate the influence of the latter (which would diminish the availability of AsV for reduction), we carried out the experiments also on BDRPL-rats. Comparing the ACH-induced alterations observed in BDL-rats with those in BDRPL-rats assists us in sorting out the responses attributable to ACH-induced increase in urinary AsV excretion and the changes that may result from impaired reduction of AsV.

    It can be tentatively suggested that ACH-induced changes in AsV disposition observed in BDL-rats but not in BDRPL-rats can be ascribed to the increased renal excretion of AsV. Such alterations include the more rapid elimination of AsV from blood (Fig. 4, top left), lower concentrations of AsV in the heart and muscle (Fig. 6), and decreased levels of AsIII, as well as mono- and dimethylated arsenicals in the heart and/or the muscle (Fig. 6). All these changes were brought about by ACH injected in the larger dose, which caused a diuretic effect with an increase in urinary AsV (Fig. 5) and, secondarily, the aforementioned alterations. Increased loss of AsV into the urine (in addition to its impaired reduction) may also contribute to decreases in the formation of AsIII, its excretion into urine (Fig. 5, middle), and retention in kidney (Fig. 6), changes observed in ACH-dosed BDL-rats.

    On the other hand, responses to ACH in AsV disposition seen in both BDL-rats and BDRPL-rats or only in the latter animals may be attributed to ACH-induced changes of AsV biotransformation. For example, in both BDL- and BDRPL-rats, ACH given in either 100- or 200-mg/kg dose caused significant elevations of hepatic AsV levels, whereas 200 mg/kg ACH decreased the concentrations of methylated AsV metabolites in the blood (Figs. 4 and 7) and the liver (Figs. 6 and 8). Only in BDRPL-rats did 100 mg/kg ACH lower the levels of methylated arsenicals in the blood (Fig. 7) and the liver (Fig. 8). All these changes, and especially the increased retention of AsV in the liver (Figs. 6 and 8, top left), are compatible with the conclusion that treatment with ACH impairs hepatic reduction of AsV as well as formation of AsIII and AsIII-derived methylated metabolites; however, interference of ACH treatment with methylation of AsIII cannot be excluded. The findings presented in Figure 9, showing that the combined concentration of hepatic AsV metabolites as well as the ratio of hepatic AsV metabolites to unchanged AsV significantly decreased in response to ACH dosing in both BDL-and BDRPL-rats, also support the conclusion that the treatment inhibited the reduction of AsV in the liver. However, it remains to be explained why AsIII levels in the liver failed to decline in response to ACH administration in the face of impaired hepatic AsV reduction. One may speculate that an expected decline in AsIII concentration originating from diminished formation of AsIII from AsV may have been counterbalanced by a diminished elimination of the formed AsIII via methylation, and/or through export, and/or by more extensive binding of AsIII to hepatocellular proteins. The slight but statistically significant ACH-induced depletion of hepatic GSH (Fig. 2) might cause such changes.

    While the hepatic reduction of AsV was apparently impaired in ACH-treated rats compared to the saline-treated controls, disappearance of the injected AsV from blood was not delayed even in BDRPL-rats (Fig. 7). The relative role of liver and extrahepatic tissues in reduction of AsV in rats is unknown. Nevertheless, if the extrahepatic AsV reduction is significant, impaired reduction of AsV to AsIII in the liver caused by ACH (Table 1) might have been compensated for by AsV reduction in other tissues, which was little or not affected by ACH (Table 1). It is tempting to speculate that such a phenomenon explains the increased AsIII levels in the muscle of BDRPL-rats in response to ACH treatment (Fig. 8). It also has to be considered that ACH must have spared a fraction of AsV reduction even in the liver, as it markedly but incompletely inhibited GAPDH-mediated hepatic AsV reduction (Table 1) and probably left other AsV-reducing hepatic mechanisms intact. Such mechanisms function, for example, in the mitochondria (Nemeti and Gregus, 2002a) and the cytosol, the latter catalyzed by hitherto unidentified enzyme(s) other than PNP and GAPDH (Nemeti and Gregus, 2005). Thus, hepatic AsV reduction unrelated to GAPDH and extrahepatic AsV reduction little or not at all affected by ACH could have maintained elimination of AsV from the blood in ACH-dosed BDRPL-rats.

    Finally, this study has to be interpreted with some caution with respect to the question it aimed to address, i.e., whether GAPDH works as an AsV reductase in vivo. We demonstrated that ACH treatment of rats dramatically diminished the GAPDH activity and the GAPDH-catalyzed AsV reductase activity in the hepatic cytosol and presented circumstantial evidence that in the liver (but not in other tissues) of ACH-treated rats the in vivo reduction of AsV was impaired. Collectively, these findings suggest that GAPDH contributes to the in vivo reduction of AsV to AsIII, at least in the rat liver. Nevertheless, because ACH administration also resulted in a slight decrease in hepatic GSH level and because reduction of AsV in vivo in rats (Csanaky and Gregus, 2005) and in vitro by rat liver cytosol (Nemeti and Gregus, 2005) as well as by purified GAPDH (Gregus and Nemeti, 2005) requires GSH, the impairment of hepatic AsV reduction in ACH-dosed rats may be due in part to ACH-induced GSH depletion. Therefore, further experimentation is deemed necessary before a definitive conclusion is made that GAPDH not only works as an AsV reductase in vitro, but it also significantly contributes to reduction of AsV to AsIII in vivo.

    ACKNOWLEDGMENTS

    This publication is based on a work supported by the Hungarian National Research Fund (OTKA) and the Hungarian Ministry of Health. The authors wish to thank Monika Agyaki and István Schweibert for their excellent assistance in the experimental work.

    REFERENCES

    Brown, R. E., Jarvis, K. L., and Hyland, K. J. (1989). Protein measurement using bicinchoninic acid: Elimination of interfering substances. Anal. Biochem. 180, 136–139.

    Csanaky, I., and Gregus, Z. (2001). Effect of phosphate transporter and methylation inhibitor drugs on the disposition of arsenate and arsenite in rats. Toxicol. Sci. 63, 29–36.

    Csanaky, I., and Gregus, Z. (2002). Species variations in the biliary and urinary excretion of arsenate, arsenite and their metabolites. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 131, 355–365.

    Csanaky, I., and Gregus, Z. (2003). Effect of selenite on the disposition of arsenate and arsenite in rats. Toxicology 186, 33–50.

    Csanaky, I., and Gregus, Z. (2005). Role of glutathione in reduction of arsenate and of gamma-glutamyltranspeptidase in disposition of arsenite in rats. Toxicology 207, 91–104.

    Csanaky, I., Nemeti, B., and Gregus, Z. (2003). Dose-dependent biotransformation of arsenite in rats—not S-adenosylmethionine depletion impairs arsenic methylation at high dose. Toxicology 183, 77–91.

    Dixon, H. B. F. (1997). The biochemical action of arsonic acids, especially as phosphate analogues. Adv. Inorg. Chem. 44, 191–227.

    Ginsburg, J. M., and Lotspeich, W. D. (1963). Interrelations of arsenate and phosphate transport in the dog kidney. Am. J. Physiol. 205, 707–714.

    Goering, P. L., Aposhian, H. V., Mass, M. J., Cebrián, M., Beck, B. D., and Waalkes, M. P. (1999). The enigma of arsenic carcinogenesis: Role of metabolism. Toxicol. Sci. 49, 5–14.

    Gregus, Z., Gyurasics, á., and Csanaky, I. (2000). Biliary and urinary excretion of inorganic arsenic: Monomethylarsonous acid as a major biliary metabolite in rats. Toxicol. Sci. 56, 18–25.

    Gregus, Z., and Nemeti, B. (2002). Purine nucleoside phosphorylase as a cytosolic arsenate reductase. Toxicol. Sci. 70, 13–19.

    Gregus, Z., and Nemeti, B. (2005). The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase works as an arsenate reductase in human red blood cells and rat liver cytosol. Toxicol. Sci. 85, 859–869.

    Gyurasics, á., Varga, F., and Gregus, Z. (1991). Glutathione-dependent biliary excretion of arsenic. Biochem. Pharmacol. 42, 465–468.

    Hughes, M. F. (2002). Arsenic toxicity and potential mechanisms of action. Toxicol. Lett. 133, 1–16.

    Jelks, K. B., and Miller, M. G. (2001). -Chlorohydrin inhibits glyceraldehyde-3-phosphate dehydrogenase in multiple organs as well as in sperm. Toxicol. Sci. 62, 115–123.

    Jones, A. R., and Cooper, T. G. (1999). A re-appraisal of the post-testicular action and toxicity of chlorinated antifertility compounds. Int. J. Androl. 22, 130–138.

    Knowles, F. C., and Benson, A. A. (1983). The biochemistry of arsenic. Trends Biochem. Sci. 8, 178–180.

    Nemeti, B., Csanaky, I., and Gregus, Z. (2003). Arsenate reduction in human erythrocytes and rats—Testing the role of purine nucleoside phosphorylase. Toxicol. Sci. 74, 22–31.

    Nemeti, B., and Gregus, Z. (2002a). Mitochondria work as reactors in reducing arsenate to arsenite. Toxicol. Appl. Pharmacol. 182, 208–218.

    Nemeti, B., and Gregus, Z. (2002b). Reduction of arsenate to arsenite in hepatic cytosol. Toxicol. Sci. 70, 4–12.

    Nemeti, B., and Gregus, Z. (2004). Glutathione-dependent reduction of arsenate in human erythrocytes—a process independent of purine nucleoside phosphorylase. Toxicol. Sci. 82, 419–428.

    Nemeti, B., and Gregus, Z. (2005). 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. 85, 847–858.

    Patterson, T. J., Ngo, M., Aronov, P. A., Reznikova, T. V., Green, P. G., and Rice, R. H. (2003). Biological activity of inorganic arsenic and antimony reflects oxidation state in cultured human keratinocytes. Chem. Res. Toxicol. 16, 1624–1631.

    Petrick, J. S., Jagadish, B., Mash, E. A., and Aposhian, H. V. (2001). Monomethylarsonous acid (MMAIII) and arsenite: LD50 in hamsters and in vitro inhibition of pyruvate dehydrogenase. Chem. Res. Toxicol., 14, 651–656.

    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.

    Rossman, T. G. (2003). Mechanism of arsenic carcinogenesis: An integrated approach. Mutat. Res. 533, 37–65.

    Sedlak, J., and Lindsay, R. H. (1968). Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal. Biochem. 25, 192–205.

    Stevenson, D., and Jones, A. R. (1985). Production of (S)-3-chlorolactaldehyde from (S)--chlorohydrin by boar spermatozoa and the inhibition of glyceraldehyde-3-phosphate dehydrogenase in vitro. J. Reprod. Fert. 74, 157–165.

    Thomas, D. J., Styblo, M., and Lin, S. (2001). The cellular metabolism and systemic toxicity of arsenic. Toxicol. Appl. Pharmacol. 176, 127–144.

    Tietze, F. (1969). Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: Applications to mammalian blood and other tissues. Anal. Biochem. 27, 502–522.

查询更多arsenate相关信息在本站>>

《毒物学科学杂志》2006年3月第90卷第1期