Reduction of Arsenate to Arsenite by Human Erythrocyte Lysate and Rat Liver Cytosol – Characterization of a Glutathione- and NAD-Dependent A
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《毒物学科学杂志》
Department of Pharmacology and Pharmacotherapy, Toxicology Section, University of Pécs, Medical School, Pécs, Hungary
ABSTRACT
Reduction of arsenate (AsV) to the more toxic arsenite (AsIII) is of high toxicological importance, yet in vivo relevant enzymes involved have not been identified. Purine nucleoside phosphorylase (PNP) is an efficient AsV reductase in vitro, but its role in AsV reduction is irrelevant in vivo. Intact human red blood cells (RBC) possess an AsV reductase activity that is PNP-independent, diminished by depletion of glutathione (GSH), enhanced by oxidants of erythrocytic NAD(P)H, and possibly linked to the lower part of the glycolytic pathway. In order to characterize this PNP-independent AsV reductase activity further, we examined the effects of GSH, inorganic phosphate, some inhibitors of glucose metabolism, glycolytic substrates, and pyridine, as well as adenine nucleotides on AsV reduction in lysed RBC and rat liver cytosol in the presence of BCX-1777, a PNP inhibitor. In hemolysate, GSH enhanced AsV reduction in a concentration-dependent manner, whereas phosphate inhibited it. Glycolytic substrates, especially fructose-1,6-bisphosphate and phosphoglyceric acids, improved AsV reductase activity. NAD, especially together with these substrates, strongly increased AsIII formation, whereas NADH strongly inhibited it. NADP and adenine nucleotides diminished, while 2-phosphoglycollate, which increases the breakdown of the RBC-specific compound 2,3-bisphosphoglycerate to 3-phosphoglycerate, doubled the AsV reductase activity. Although AsV reduction by the liver cytosol responded similarly to GSH, NAD, and glycolytic substrates as in the hemolysate, it was barely influenced by NADH, was diminished by 2-phosphoglycollate, and was stimulated by NADP. Collectively, hemolysate and rat liver cytosol possess a PNP-independent AsV reductase activity. This enzymatic activity requires GSH, NAD, and glycolytic substrates, and purportedly involves one or both of the two functionally linked glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. In addition, the data presented here suggest that yet another PNP-independent AsV reductase resides in the hepatic cytosol. Although this latter enzyme remains unknown, identification of the AsV reductase depending on GSH, NAD, and glycolytic substrates is presented in the following paper.
Key Words: arsenate; arsenite; reduction; glutathione; glycolysis; NAD.
INTRODUCTION
Arsenic is a long-known poison and a well-recognized environmental toxicant. Chronic arsenic exposure causes skin lesions, vascular disease, and cancer (Goering et al., 1999; Hindmarsh, 2000; Hughes, 2002; Rossman, 2003). The naturally prevalent form, the pentavalent inorganic arsenate (AsV), enters the body typically as a drinking water contaminant. Because of the close structural similarity to inorganic phosphate (Pi), AsV may replace Pi in transport processes (including cellular uptake) and enzymatic reactions (Csanaky and Gregus, 2001; Dixon, 1997; Ginsburg and Lotspeich, 1963), thereby interfering with cellular metabolism. Alternatively, AsV may be reduced to the much more toxic trivalent arsenite (AsIII) by hitherto unidentified cellular enzyme(s) (Thomas et al., 2001). Subsequently, mono- and dimethylated metabolites are formed, among which the trivalent ones are highly toxic, whereas the pentavalent ones are relatively atoxic (Petrick et al., 2001; Rossman, 2003; Thomas et al., 2001). The first step of AsV metabolism, its reduction to AsIII, therefore is not only important in governing the fate of arsenic in the body, but as a toxification step, may also determine its toxicity and carcinogenicity.
Despite the intensive research on the biochemistry of AsV reduction, the metabolic pathways and enzymes involved in it are still unknown. The recent finding that purine nucleoside phosphorylase (PNP) is capable of reducing AsV to AsIII, provided its nucleoside substrate (e.g., inosine) and a dithiol (e.g., dithiothreitol) are present simultaneously (Gregus and Németi, 2002; Radabaugh et al., 2002), promised a better understanding of the biochemical background concerning this important toxification process. However, the testing of PNP for such a role in human erythrocytes and rats in vivo has led to the conclusion that PNP does not contribute to the reduction of AsV significantly (Németi et al., 2003). Studies with cultured human keratinocytes also failed to support that PNP contributes to reduction of AsV in these cells (Patterson et al., 2003). In addition, AsV reduction catalyzed by PNP is not supported by glutathione (GSH), the most important small thiol molecule in cells, although reduction of AsV is apparently GSH-dependent in mouse embryo cells (Bertolero et al., 1987) and in rats (Csanaky and Gregus, 2005; Gyurasics et al., 1991). Experiments carried out on human red blood cells (RBC) have revealed that erythrocytes also possess a PNP-independent AsV reductase activity (Németi and Gregus, 2004). This activity appears GSH-dependent and is responsible for the most of the basal AsV reduction (i.e., without specific stimuli). Moreover, compounds that promote oxidation of NAD(P)H, thereby increasing cellular NAD(P) content, can trigger this PNP-independent AsV reduction, and importantly, reduction of AsV in erythrocytes is apparently coupled to the glycolytic pathway.
The aim of the present paper has been to ascertain that this PNP-independent but GSH- and NAD-dependent AsV-reducing activity is also present in human RBC lysate and in rat liver cytosol, and to characterize it by investigating the effects of GSH, some inhibitors of the glucose metabolism, as well as the glycolytic substrates, and pyridine and adenine nucleotides (i.e., NAD(P)/NAD(P)H and ADP/ATP, respectively). In order to exclude the role of PNP in erythrocytic and cytosolic AsV reduction, all these experiments were carried out in the presence of the PNP inhibitor BCX-1777 (Bantia and Kilpatrick, 2004; Bantia et al., 2001; Bzowska et al., 2000), which can completely inhibit the AsV-reducing activity of this enzyme (Gregus and Németi, 2002). Characterization of this PNP-independent AsV reductase activity greatly assisted us in identifying a glycolytic enzyme that can reduce AsV to AsIII, as presented in the adjoining paper (Gregus and Németi, 2005).
MATERIALS AND METHODS
Chemicals.
BCX-1777 (also called Immucillin-H) was a generous gift from BioCryst Pharmaceuticals (Birmingham, AL). D-gluconic acid sodium salt, N-(2-hydroxyethyl)-piperazine-N'-(2-ethanesulfonic acid) (HEPES), phosphoglyceric phosphokinase (PGK) from baker's yeast, fructose-1,6-bisphosphate (tetra)cyclohexylammonium salt, D,L-glyceraldehyde-3-phosphate diethyl acetal monobarium salt, 2,3-bisphosphoglyceric acid (penta)cyclohexylammonium salt, 3-phosphoglyceric acid disodium salt, 2-phosphoglyceric acid sodium salt, phosphoenolpyruvic acid sodium salt, N-acetylglucosamine (NAGA), and dehydroepiandrosterone were from Sigma. Bicinchoninic acid disodium salt was from Fluka. Glucose, glucose-6-phosphate barium salt, sodium pyruvate, reduced glutathione, NAD, NADH, NADP, NADPH, AMP, ADP, and ATP were from Reanal Ltd. (Budapest, Hungary). 2-Phosphoglycollate (2-PGly) was obtained from Roche as part of the 2,3-bisphosphoglycerate assay kit. The sources of arsenic compounds, and chemicals used in arsenic speciation have been given elsewhere (Csanaky et al., 2003; Németi and Gregus, 2002). All other chemicals were of the highest purity commercially available.
Preparation of human RBC and assay of AsV reduction by the hemolysate.
This research was approved by the Regional Scientific Research Ethics Committee of the University of Pécs, Center for Medical and Health Sciences. Blood (approximately 5 ml) was collected from healthy human volunteers after informed consent into heparinized VacutainerTM tubes. The blood was immediately centrifuged at 1000 x g, 4°C for 10 min, and the plasma and buffy coat were discarded. The pelleted RBC were resuspended in an equal volume of ice-cold buffer containing 250 mM sucrose, 25 mM HEPES, 5 mM MgCl2, and 2 mM EGTA, pH 7.4 (designated as sucrose buffer). 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 in a 50% RBC suspension. The RBC suspension was kept in ice until use for assaying AsV reduction within 3 h.
To assay PNP-independent AsV reduction by RBC lysate, erythrocyte (pre)incubations were carried out in sucrose buffer at 37°C in a final incubation volume of 0.3 ml. First, erythrocytes (50 μl packed cells) were preincubated for 5 min with 20 μM BCX-1777 (to inhibit PNP), 0.067% Nonidet P-40 (a detergent, to lyse RBC), and when indicated, 2 U glucose oxidase. Glucose oxidase was used to deplete endogenous glucose and glucose-derived glycolytic substrates, when AsV reduction was tested in the presence of specific exogenous glycolytic substrates. After preincubation, the incubation was started by adding GSH (typically 6 mM), the test compounds, followed immediately by AsV (50 μM), and was continued for 2.5 min, if otherwise not specified. The incubation was 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. However, 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, 1973), and which displaced thiol-bound AsIII, but did not oxidize the released AsIII. The incubates thus treated were stored at –80°C until analysis for AsIII and AsV. AsV reductase activity was expressed as nmol formed AsIII per minute and ml packed RBC.
Preparation of rat liver cytosol and assay of AsV reduction by the cytosol.
Male Wistar rats kept under standardized conditions and weighing 250–270 g were obtained from the SPF breeding house of the University of Pécs (Hungary). 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 Pécs.
The livers of the rats were quickly removed, rinsed with ice-cold saline, and homogenized in three volumes of sucrose buffer, using a glass homogenization tube with first a looser then a tighter motor-driven Teflon pestle. The homogenate was 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 divided into aliquots and stored at –80°C until assaying its AsV reductase activity. The protein concentration of the cytosol preparations was determined by the bicinchoninic acid method according to Brown et al. (1989).
To assay cytosolic AsV reduction, cytosol (pre)incubations were carried out in sucrose buffer at 37°C in a final incubation volume of 0.3 ml. First, cytosol (5 mg protein/ml) was preincubated for 5 min with 20 μM BCX-1777 (to inhibit PNP) and when indicated, 2 U glucose oxidase. Thereafter, the incubation was started by adding GSH (typically 10 mM), the test compounds, followed immediately by AsV (50 μM), and was continued for 2.5 min, if otherwise not specified. The incubations were stopped by successive addition of 100 μl of 50 mM CdSO4 solution and 100 μl of 1.5 M perchloric acid solution containing 50 mM HgCl2. The incubates were stored at –80°C until analysis for AsIII and AsV. AsV reductase activity was expressed as pmol formed AsIII per minute and mg protein.
Arsenic analysis.
The incubates, having been subjected to protein precipitation, were centrifuged at 10,000 x g, 4°C for 10 min. AsIII and AsV in the resultant supernatant were separated and quantified by HPLC-hydride generation-atomic fluorescence spectrometry, the details of which have been given elsewhere (Gregus et al., 2000; Németi et al., 2003).
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 Glutathione, Phosphate, and Some Inhibitors of Glucose Metabolism on AsV Reduction by Human RBC Lysate and Rat Liver Cytosol
Because reduction of AsV by intact RBC appeared GSH-dependent (Németi and Gregus, 2004), it was of interest to determine whether AsV-reducing activity in hemolysate and cytosol is supported by GSH. Figure 1 demonstrates that GSH enhanced formation of AsIII from AsV in a concentration-dependent manner. At concentrations of 2, 4, 6, and 10 mM, GSH increased AsV reduction by the hemolysate approximately 5, 7, 11, and 22 fold, respectively, whereas at concentrations 2.5, 5, and 10 mM GSH geared up cytosolic AsIII formation 4.5, 7.5, and 10 fold, respectively. It is important to note that less than 1% of AsIII was formed when GSH at similar concentrations was incubated with AsV in the absence of hemolysate or cytosol than in the presence of these enzyme sources for 2.5 min.
Formation of AsIII from AsV in intact RBC is strongly inhibited by inorganic phosphate (Pi) and is modulated by erythrocytic glycolytic activity (Németi and Gregus, 2004). Therefore, we tested the effects of Pi and some compounds that inhibit certain enzymes of glucose metabolism, namely N-acetyl glucosamine (NAGA, a hexokinase inhibitor), fluoride (inhibits enolase), and dehydroepiandrosterone (DHEA, an inhibitor of glucose-6-phosphate dehydrogenase). Phosphate exhibited concentration-dependent inhibitory effect on AsV reduction by both hemolysate and cytosol (Fig. 2, top and bottom, respectively). However, the inhibition of AsIII formation caused by Pi appeared stronger in hemolysate than in cytosol. For example, 1 mM Pi diminished AsV reduction by more than 60% during 2.5 min and more than 50% during 30 min in hemolysate, but influenced it insignificantly in cytosol during either 2.5 or 30 min. Even at a concentration as high as 5 mM, Pi decreased cytosolic AsIII formation by only 58% and 44% during 2.5 and 30 min, respectively. The inhibitors of hexokinase and glucose-6-phosphate dehydrogenase (i.e., NAGA and DHEA, respectively) affected AsV reduction by neither lysed human RBC nor rat liver cytosol (Fig. 2, top and bottom, respectively) irrespective of the duration of incubation. In contrast, the enolase inhibitor fluoride inhibited the hemolysate-catalyzed AsIII formation by 60%, when the incubation lasted 30 min, but not at all when it lasted 2.5 min only. Fluoride was also ineffective in influencing AsV reduction by cytosol irrespective of the incubation time.
Effects of Glycolytic Substrates on AsV Reduction by Human RBC Lysate and Rat Liver Cytosol
The ubiquity of the glycolytic pathway and its apparent importance in AsIII formation from AsV in intact RBC prompted us to investigate the effects of the glycolytic intermediates on AsV reduction in both hemolysate and cytosol. In these studies, RBC lysate or cytosol was preincubated with glucose oxidase (except when glucose was the compound tested) to deplete endogenous glucose and the substrates derived from it, lest the endogenous compounds should alter the effect of the exogenous one being tested.
In lysed RBC, the "upper" glycolytic substrates glucose and fructose-1,6-bisphosphate (Fruc-1,6-BP) exhibited slight stimulatory effect on AsV reduction, whereas glucose-6-phosphate (Gluc-6-P) produced no influence at all (Fig. 3). Of the "lower" glycolytic substrates, 3-phosphoglycerate (3-PGA), 2-phosphoglycerate (2-PGA), phosphoenolpyruvate (PEP), and pyruvate strongly enhanced AsIII formation, whereas 2,3-bisphosphoglycerate (2,3-BPG) and, surprisingly, glyceraldehyde-3-phosphate (Ga-3-P) were found moderate inhibitors, causing approximately 40% diminution in AsIII formation. The responsiveness of the cytosolic AsV reduction to these glycolytic intermediates exhibited both similarities and differences. Like in hemolysate, Fruc-1,6-BP and phosphoglycerates (i.e., 3-PGA and 2-PGA) increased AsIII formation (Fig. 3). Unlike in the RBC lysate, Ga-3-P enhanced AsV reduction, but PEP and pyruvate failed to do so.
Effects of NAD and NADH on AsV Reduction by Human RBC Lysate and Rat Liver Cytosol in the Absence or Presence of Glycolytic Substrates
Because glycolysis is regulated by the ratio of NAD to NADH (i.e., it is accelerated by NAD and decelerated by NADH), we tested the effects of these pyridine nucleotides on AsV reduction both in the absence and in the presence of one of the above-mentioned glycolytic substrates.
In hemolysate, NAD strongly increased AsIII formation either alone or together with any of the substrates (Fig. 4, top), compared to incubations with no pyridine nucleotide added. The increment in AsV reduction brought about by NAD was most marked in the presence of Ga-3-P, when NAD increased AsIII formation almost 7 fold. In the presence of the substrates, which supported AsV reduction well alone (i.e., Fruc-1,6-BP, 3-PGA, 2-PGA, PEP, and pyruvate), NAD also exhibited marked stimulatory effect on AsIII formation (3–4.5 fold). In contrast to NAD, NADH strongly inhibited the AsV-reducing activity of RBC lysate even in the presence of substrates, which facilitated AsV reduction alone (Fig. 4, top).
In the incubations with rat liver cytosol, the effect of NAD on AsIII formation from AsV appeared very similar to that observed in hemolysate (Fig. 4, bottom), as NAD greatly enhanced the reduction of AsV both in the absence and in the presence of one of the glycolytic substrates. The most prominent stimulation was found in incubates with Fruc-1,6-BP plus NAD or with 2-PGA plus NAD (15-fold and 12-fold increase, respectively). In contrast to NAD, the effect of NADH on AsV reduction by cytosol differed from that observed in hemolysate. This reduced pyridine nucleotide-inhibited cytosolic AsIII formation significantly only in the absence of exogenous glycolytic substrates and in the presence of glucose-6-phosphate. In the presence of other substrates, NADH failed to diminish the reduction of AsV and, in combination with Fruc-1,6-BP, even markedly facilitated it.
In order to further characterize the influence of NAD and NADH on AsV reduction by RBC lysate and cytosol, we determined the time courses of their effects on AsIII formation in the presence of Fruc-1,6-BP or 2-PGA, glycolytic substrates supporting AsV reduction the most out of those involved in the upper and lower parts of glycolysis, respectively.
In hemolysate, the formation of AsIII from AsV in the presence of Fruc-1,6-BP alone was rapid initially (Fig. 5, left), but became slower later. When Fruc-1,6-BP and NAD were present simultaneously, the amount of the formed AsIII was much larger, and it increased linearly with time, exhibiting only a slight decline from linearity at later time points. NADH inhibited AsV reduction by approximately 67% throughout the 30-min period. The time courses of AsV reduction in the presence of 2-PGA (Fig. 5, right) were clearly different, at least on two points, from those observed in the presence of Fruc-1,6-BP. First, the time course of 2-PGA-supported AsV reduction both with and without NAD supplementation markedly declined from linearity past the initial 2.5–5 min, and AsIII formation almost stopped past 15 min. Second and more importantly, the 2-PGA-supported AsV reduction was inhibited by NADH only in the initial few minutes; later it surged, and by 15 min the amount of AsIII formed in the presence NADH became similar to that produced in the presence of NAD, exceeding 3-fold the quantity of AsIII formed in the absence of added pyridine nucleotides.
The time courses of the cytosolic AsV reduction in the presence of Fruc-1,6-BP or 2-PGA and with or without NAD/NADH appeared slightly different from those described above for AsV reduction by lysed human RBC. With Fruc-1,6-BP, the major difference was that NADH, which inhibited hemolysate-mediated AsV reduction, enhanced the reduction of AsV by rat liver cytosol from the earliest time point, albeit much less than NAD did (Fig. 6, left). With 2-PGA as a glycolytic substrate, an apparent quantitative, rather than qualitative, difference is noted in the effect of NADH on erythrocytic and cytosolic reduction of AsV. Like in the hemolysate, NADH initially inhibited, later increased AsIII formation from AsV. However, the increment in AsIII production caused by NADH remained far less than that caused by NAD (Fig. 6, right), unlike in the hemolysate (Fig. 5, right).
Effects of NADP and NADPH on AsV Reduction by Human RBC Lysate and Rat Liver Cytosol in the Absence or Presence of Glycolytic Substrates
In erythrocytes, but not in hepatocytes, the only NADP-consuming and NADPH-producing route is the pentose phosphate pathway, which is under strict regulation by NADP/NADPH ratio. In order to assess the contribution of this metabolic pathway to the reduction of AsV to AsIII, RBC lysate and rat liver cytosol were incubated with NADP or NADPH in the absence or in the presence of one of the glycolytic metabolites.
In contrast to NAD, NADP did not increase AsIII formation significantly by the hemolysate either in the absence or the presence of glycolytic substrates (Fig. 7, top); moreover, NADP even inhibited AsV reduction in the presence of glucose and Gluc-6-P. NADPH was found to diminish AsIII formation in the absence of glycolytic substrates and in the presence of the upper ones (i.e., from glucose to Fruc-1,6-BP), Ga-3-P, and 2,3-BPG. In the presence of phosphoglycerates, PEP or pyruvate, NADPH did not influence AsV reduction significantly (Fig. 7, top).
In cytosolic incubations, NADP enhanced AsIII formation by 1.7–3.8 fold both in the absence of added glycolytic substrate and in the presence of every substrate tested (Fig. 7, bottom), though the extent of this enhancement was far below that caused by NAD (Fig. 4, bottom). Interestingly, NADPH also stimulated AsIII formation 1.6–2 fold in the presence of Fruc-1,6-BP, 3-PGA, 2-PGA, PEP, and pyruvate, whereas it was without significant effect with no substrate, glucose, Gluc-6-P, Ga-3-P, or 2,3-BPG added to the incubations.
Effects of ADP and ATP on AsV Reduction by Human RBC Lysate and Rat Liver Cytosol in the Absence or Presence of Glycolytic Substrates
ATP and ADP play key roles in cellular metabolism, including glycolysis. It was therefore of interest to determine if these nucleotides affected the reduction of AsV. In hemolysate, both ADP and ATP were inhibitors of AsIII formation in the presence of phosphoglycerates, PEP, or pyruvate (Fig. 8, top). In addition, ADP significantly diminished AsV reduction in the absence of substrates and in the presence of glucose or Fruc-1,6-BP. In cytosol incubations, both ADP and ATP decreased AsIII formation from AsV in the presence of phosphoglycerates, and ADP, but not ATP, diminished it in the presence of Fruc-1,6-BP and Ga-3-P (Fig. 8, bottom).
Effects of Compounds Influencing 2,3-BPG Metabolism on AsV Reduction in Human RBC Lysate and Rat Liver Cytosol
2,3-BPG is present in the erythrocytes at exceptionally high concentration (approximately 7 mM; Mulquiney et al., 1999), but only in traces in the liver. Its enzymatic degradation yields 3-PGA, a glycolytic substrate. This conversion of 2,3-BPG can be triggered by 2-phosphoglycollate (2-PGly) or inhibited by sulfate. The effects of these compounds on erythrocytic and cytosolic AsV reduction was tested in order to assess the role of 2,3-BPG degradation in AsV reduction. As demonstrated in Figure 9, 2-PGly doubled AsIII formation in hemolysate, but significantly inhibited it in cytosol. Sulfate diminished AsV reduction in RBC lysate minimally, but significantly in the cytosol.
DISCUSSION
AsV Reduction in the Hemolysate: Stimulation by GSH and NAD (but Not NADP) and Inhibition by Phosphate
The previous work (Németi and Gregus, 2004) presented circumstantial evidence that intact human erythrocytes with their PNP inactivated reduce AsV to AsIII in a GSH- and NAD(P)-dependent manner, because the GSH depletor diethylmaleate impaired, whereas compounds known to oxidize NAD(P)H to NAD(P) (e.g., pyruvate, ferricyanide, methylene blue, nitrite, tert-butylhydroperoxide, dehydroascorbate, and 4-dimethylaminophenol) enhanced formation of AsIII from AsV by the RBC. In the present work, first we sought for direct evidence that the PNP-independent erythrocytic AsV reduction requires GSH and a pyridine nucleotide (NAD or NADP) by examining how supplementation of the hemolysate with these compounds alters the AsV reductase activity.
The findings that the PNP-independent AsV-reducing activity of the human RBC lysate is negligible in the absence of added GSH and that GSH increases it in a concentration dependent manner (Fig. 1) directly demonstrate that this enzymatic activity (unlike that of PNP) requires GSH, probably as a reducing partner. In addition, this observation suggests that the enzyme(s) involved contain(s) critical thiol groups, similarly to AsV reductases identified in microorganisms (Rosen, 2002) as well as to PNP found to work as an AsV reductase in the hepatic cytosol of experimental animals (Gregus and Németi, 2002) and humans (Radabaugh et al., 2002). Furthermore, the relevance of this finding is underlined by the fact that reduction of AsV to AsIII in vivo is also GSH dependent (Csanaky and Gregus, 2005).
A most significant observation of this work is that NAD (Fig. 4, top), but not NADP (Fig. 7, top), supports the AsV reductase activity of the RBC lysate. This information clarified that the effect of various NADH and NADPH oxidants to promote AsV reduction in intact RBC (Németi and Gregus, 2004) is based on increased formation of NAD (not NADP), and was also instrumental in the forthcoming identification of the AsV reductase as a NAD-dependent enzyme.
Inorganic phosphate exerted a strong inhibitory effect on AsV reduction by intact erythrocytes (Németi and Gregus, 2004), partly because AsV and Pi compete for the chloride-bicarbonate membrane transporter that mediates their uptake into these cells. The observation that Pi also inhibits, albeit to a much lesser extent, the formation of AsIII by the hemolysate (Fig. 2) indicates that Pi interferes with the enzymatic reduction of AsIII as well. This raises the possibility that the enzyme, which catalyzes the reduction of AsV, contains a phosphate-binding site, which may also accommodate AsV.
AsV Reduction in the Hemolysate: Association with the Glycolysis (Not the PP-Pathway) within the Section from GAPDH to Enolase
It is well known that NAD accelerates, whereas NADH decelerates the glycolytic flux (Tilton et al., 1991). Therefore, the observation that NAD increases, whereas NADH decreases the rate of the hemolysate-catalyzed AsV reduction both in the absence of added glycolytic substrates and in their presence (Fig. 4, top) confirms our suggestion that reduction of AsV to AsIII in human erythrocytes is coupled to glycolysis. In contrast, the following three pieces of evidence indicate that the pentose phosphate pathway (PP-pathway), the other major route for glucose metabolism in erythrocytes (Fig. 10), is not directly involved. First, glucose depletion of intact RBC by incubation with pyruvate, which deprives the PP-pathway of substrate supply, did not decrease, but rather increased AsV reduction (Németi and Gregus, 2004). Second, supplementation of the hemolysate with NADP, that should facilitate the PP-pathway, failed to increase AsIII formation from AsV (Fig. 7, top). Third, dehydroepiandrosterone (DHEA), an inhibitor of the key PP-pathway enzyme, glucose-6-phosphate dehydrogenase (Levy, 1963), barely influenced AsV reduction in hemolysate (Fig. 2, top).
Intact RBC pretreated with pyruvate (which depleted glucose and enriched NAD in the cells, depriving the upper part of the glycolysis from substrate, but permitted the lower part to utilize 2,3-bisphosphoglycerate) exhibited a rapid AsV reduction, which was even further increased by the enolase inhibitor fluoride (Németi and Gregus, 2004). Based largely on this observation, it was proposed that the enzyme responsible for reduction of AsV should lie in the part of the glycolytic pathway from glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to enolase. Enlisted below are findings obtained from the present work that corroborate, or at least are compatible with, this tentative conclusion.
As shown in Fig. 4 (top), NAD stimulated most effectively the formation of AsIII from AsV in the presence of Ga-3-P, followed by Fruc-1,6-BP (which breaks down to Ga-3-P to feed GAPDH) and 3-PGA as well as 2-PGA (which are substrates for the enzymes between GAPDH and enolase) (Fig. 10).
The findings that N-acetylglucosamine (NAGA) had little influence on AsV reduction in hemolysate (Fig. 2), whereas 2-phosphoglycollate (2-PGly) doubled it (Fig. 9) are also compatible with the hypothesis that enzymes between GAPDH and enolase may catalyze the reduction of AsV. As an inhibitor of hexokinase (Cardenas et al., 1984), NAGA should little affect the substrate supply to this part of the glycolysis in erythrocytes, because in RBC this part can also be fueled by 2,3-BPG (Fig. 10). This latter compound is present in RBC at exceptionally high concentration (7 mM, Mulquiney et al., 1999), and can be dephosphorylated to yield 3-PGA (Fig. 10), a glycolytic substrate that can strongly enhance the AsV reductase activity of the hemolysate (Fig. 3). The main enzyme that dephosphorylates 2,3-BPG is the RBC-specific bisphosphoglycerate synthase/phosphatase (BPGS/P), the phosphatase activity of which is augmented by 2-PGly (Fothergill-Gilmore and Watson, 1989). Therefore, 2-PGly stimulated reduction of AsV, most likely by activating the BPGS/P to dephosphorylate 2,3-BPG, thereby feeding 3-PGA into the glycolysis.
The analysis of the time course of AsV reduction by RBC lysate under specific conditions (Fig. 5) also indicates that NAD and one or more glycolytic enzyme(s) starting from GAPDH are critical in formation of AsIII from AsV. In the presence of Fruc-1,6-BP plus NAD, AsV reduction was maintained almost steadily at a high rate throughout an extended period (Fig. 5, left). The following considerations explain this phenomenon. Fruc-1,6-BP is readily cleaved into Ga-3-P, which is then oxidized by the NAD-supported GAPDH while forming NADH (Fig. 10). The 1,3-bisphosphoglycerate (1,3-BPG) produced by GAPDH is then converted into pyruvate, which is finally reduced to lactate by lactate dehydrogenase (LDH). This final reductive step consumes the GAPDH-produced NADH and converts it back to NAD, thereby steadily maintaining NAD supply for GAPDH (Fig. 10) and eliminating the AsV reduction inhibitor NADH. Under this condition, the abundant substrate supply for the glycolytic enzymes downward from GAPDH together with the high NAD concentration maintain the flux in this section of the glycolysis as well as the simultaneous reduction of AsV at high rates. In contrast, when Fruc-1,6-BP was added with NADH to the hemolysate, the AsIII formation rates were very low (Fig. 5, left). Under these conditions, Fruc-1,6-BP cannot be converted into pyruvate, because the high NADH concentration favors substrate flow in the opposite direction (i.e., "upward" from GAPDH). In the lack of pyruvate, NADH cannot be oxidized into NAD. Therefore, at high NADH concentration and with Fruc-1,6-BP as the substrate, the flux through the lower glycolytic section was purportedly slow, and so was the simultaneous reduction of AsV (Fig. 5, left). These considerations are also helpful in rationalizing why fluoride, an enolase inhibitor (Wang and Himoe, 1974; Warburg and Christian, 1941–1942) decreased AsIII formation from AsV by the RBC lysate after a prolonged incubation, but not after a short one (Fig. 2, top). Interruption of the glycolytic flux at enolase prevents reoxidation of the GAPDH-produced NADH to NAD by LDH, causing accumulation of NADH and depletion of NAD over time, and in turn, deceleration of AsV reduction.
When supplementing the hemolysate with 2-PGA plus NAD, the AsV reduction was very rapid initially but slowed down soon (Fig. 5, right). Under this condition, the high NAD concentration bought about two consequences. First, it prevented substrates originating from 2-PGA to reach enzymes above GAPDH, because this would have required NADH (Fig. 10). Second, it prevented pyruvate produced from 2-PGA from being reduced to lactate. These two factors finally resulted in equilibration of substrates derived from 2-PGA among the enzymes of the lower part of the glycolytic pathway. The time course of AsIII formation under this condition (i.e., very rapid initially, but slow later) most likely reflects the flux through these enzymes in order to reach equilibrium. In contrast, when the hemolysate was supplemented with 2-PGA plus NADH, the 2-PGA-derived substrates could, at least in part, be converted into pyruvate, which in turn was reduced to lactate by LDH, with simultaneous oxidation of NADH to NAD. Initially, reduction of AsV was inhibited because NADH concentration was high. Later, however, as the concentration of NADH declined and that of the NAD increased, the formation of AsIII from AsV accelerated, causing the peculiar time course of AsV reduction depicted in Figure 5 (right).
AsV Reduction in the Hemolysate and Hepatic Cytosol: Similarities and Differences
This work demonstrates that a PNP-independent but GSH- and NAD-dependent and phosphate-inhibited AsV-reducing activity is also present in the cytosolic fraction of the rat liver, suggesting that AsV is reduced, at least in part, by the same biochemical process as in intact RBC. However, we point out below a number of dissimilarities in the responsiveness of AsV reduction by the rat liver cytosol and human hemolysate to various compounds.
NADH strongly inhibited AsIII formation by hemolysate in the presence of every substrate (Fig. 4, top), whereas in rat liver cytosol NADH was inhibitory only without added substrate or in the presence of glucose-6-phosphate and was even stimulatory in the presence of Fruc-1,6-BP (Fig. 4, bottom). To understand this difference, one has to realize that liver cells have a much more extensive and complex metabolism than RBC do. Besides LDH, hepatocytes contain a number of other cytosolic enzymes that can oxidize NADH to NAD (e.g., malate dehydrogenase, glycerol phosphate dehydrogenase, -hydroxybutyrate dehydrogenase). The rapid oxidation in liver cytosol of the AsV reduction inhibitor NADH to the AsV reduction stimulator NAD might explain the rather moderate inhibitory effect of NADH on the cytosolic AsV reduction. As to Fruc-1,6-BP, this metabolite is readily cleaved into Ga-3-P and dihydroxyacetone phosphate (DHAP) in both RBC and the liver (Fig. 10). However, in the liver cytosol (but not in the hemolysate) DHAP is substrate for glycerol phosphate dehydrogenase, which reduces it into glycerol phosphate, while consuming NADH and producing NAD. NAD thus produced can then enhance oxidation of Ga-3-P and subsequent formation of lower glycolytic substrates, as well as the simultaneous reduction of AsV. This might be the reason why NADH permanently inhibited the hemolysate-catalyzed AsIII formation from AsV in the presence on Fruc-1,6-BP (Fig. 5, left), but stimulated the cytosol-catalyzed process from the very beginning (Fig. 6, left). Similar reasons may account for the difference in the effect of fluoride on AsV reduction, i.e., inhibition in the hemolysate (Fig. 2, top), but not in the cytosol (Fig. 2, bottom). As explained above, by blocking enolase, fluoride can prevent reoxidation of NADH by LDH in the lysed erythrocytes, with the NADH eventually causing inhibition of AsV reduction. In the liver extract, however, NADH may also be reoxidized by other enzymes mentioned above, which are not sensitive to fluoride.
Fundamental differences could be observed in the effect of triphosphopyridine nucleotides (i.e., NADP and NADPH) on the AsV reductase activities of the hemolysate and cytosol. While these nucleotides slightly inhibited the hemolysate-catalyzed AsV reduction (Fig. 7, top), they significantly augmented the cytosol-catalyzed process (Fig. 7, bottom). This finding raises the possibility that the liver might contain a cytosolic NADP-dependent AsV reductase activity in addition to the NAD-dependent one. This novel activity should depend on the presence of NADP, rather than NADPH, because NADP increased AsIII formation from AsV consistently more than NADPH. The following work (Gregus and Németi, 2005) presents further circumstantial evidence for another PNP-independent AsV reductase activity in the hepatic cytosol.
Inorganic phosphate much less effectively inhibited the cytosolic than the hemolysate-catalyzed AsV reduction (Fig. 2). Because the cytosolic AsV reduction was significant even at a Pi concentration as high as 5 mM (Fig. 2), it might be suggested that Pi at physiologically relevant concentrations in the hepatocytes (approximately 0.5 mM; Iles et al., 1985) permits AsV reduction by the hepatic cytosolic enzyme(s). In addition, based on the lower sensitivity of hepatic AsV reduction to Pi one might speculate that the liver contains another cytosolic AsV reductase (besides the NAD-dependent one present in both RBC and hepatocytes) that is practically unresponsive to Pi.
While 2-PGly significantly enhanced AsV reduction by the hemolysate, it failed to do so in rat liver cytosol (Fig. 9). This difference can be explained readily by the facts that 2-PGly increases enzymatic dephosphorylation of 2,3-BPG to 3-PGA (that supports AsV reduction) and that 2,3-BPG is present only in traces in cells other than RBC, including hepatocytes. It remains to be clarified, however, why sulfate that minimally decreases the hemolysate-catalyzed AsV reduction inhibits the hepatic activity significantly (Fig. 9).
There are a number of observations in this work, e.g., the inhibitory effects of 2,3-BPG and Ga-3-P (Fig. 3) as well as ATP and ADP (Fig. 8) on AsIII formation by hemolysate and/or the hepatic cytosol, which would deserve interpretation. Because findings presented in the adjoining work (Gregus and Németi, 2005) aid this interpretation, these observations will be discussed there.
In summary, this work demonstrates that a PNP-independent AsV reductase activity is present not only in human erythrocytes but also in rat liver cytosol. This enzymatic activity is stimulated by NAD, many glycolytic substrates, and GSH, the latter suggesting involvement of thiol enzymes. The glycolytic enzymes whose substrates support AsV reduction most effectively are GAPDH, PGK, and phosphoglycerate mutase (PGM); these might mediate the reduction of AsV. Out of these, only GAPDH and PGK possess reactive thiol groups (Nagradova and Schmalhausen, 1998; Vas and Csanády, 1987), making them the most likely candidates as glycolytic enzyme(s) catalyzing AsV reduction. In addition, this study raises the possibility that yet another PNP-independent AsV reductase resides in the hepatic cytosol that can utilize NADP. While this enzyme remains to be found, identification of the AsV reductase dependent on NAD, glycolytic substrate, and GSH is presented in the following paper (Gregus and Németi, 2005).
NOTES
The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.
ACKNOWLEDGMENTS
This publication is based on a work supported by the Hungarian National Scientific Research Fund (OTKA) and the Hungarian Ministry of Health. The authors wish to thank Mónika Agyaki and István Schweibert for their excellent assistance in the experimental work.
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ABSTRACT
Reduction of arsenate (AsV) to the more toxic arsenite (AsIII) is of high toxicological importance, yet in vivo relevant enzymes involved have not been identified. Purine nucleoside phosphorylase (PNP) is an efficient AsV reductase in vitro, but its role in AsV reduction is irrelevant in vivo. Intact human red blood cells (RBC) possess an AsV reductase activity that is PNP-independent, diminished by depletion of glutathione (GSH), enhanced by oxidants of erythrocytic NAD(P)H, and possibly linked to the lower part of the glycolytic pathway. In order to characterize this PNP-independent AsV reductase activity further, we examined the effects of GSH, inorganic phosphate, some inhibitors of glucose metabolism, glycolytic substrates, and pyridine, as well as adenine nucleotides on AsV reduction in lysed RBC and rat liver cytosol in the presence of BCX-1777, a PNP inhibitor. In hemolysate, GSH enhanced AsV reduction in a concentration-dependent manner, whereas phosphate inhibited it. Glycolytic substrates, especially fructose-1,6-bisphosphate and phosphoglyceric acids, improved AsV reductase activity. NAD, especially together with these substrates, strongly increased AsIII formation, whereas NADH strongly inhibited it. NADP and adenine nucleotides diminished, while 2-phosphoglycollate, which increases the breakdown of the RBC-specific compound 2,3-bisphosphoglycerate to 3-phosphoglycerate, doubled the AsV reductase activity. Although AsV reduction by the liver cytosol responded similarly to GSH, NAD, and glycolytic substrates as in the hemolysate, it was barely influenced by NADH, was diminished by 2-phosphoglycollate, and was stimulated by NADP. Collectively, hemolysate and rat liver cytosol possess a PNP-independent AsV reductase activity. This enzymatic activity requires GSH, NAD, and glycolytic substrates, and purportedly involves one or both of the two functionally linked glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. In addition, the data presented here suggest that yet another PNP-independent AsV reductase resides in the hepatic cytosol. Although this latter enzyme remains unknown, identification of the AsV reductase depending on GSH, NAD, and glycolytic substrates is presented in the following paper.
Key Words: arsenate; arsenite; reduction; glutathione; glycolysis; NAD.
INTRODUCTION
Arsenic is a long-known poison and a well-recognized environmental toxicant. Chronic arsenic exposure causes skin lesions, vascular disease, and cancer (Goering et al., 1999; Hindmarsh, 2000; Hughes, 2002; Rossman, 2003). The naturally prevalent form, the pentavalent inorganic arsenate (AsV), enters the body typically as a drinking water contaminant. Because of the close structural similarity to inorganic phosphate (Pi), AsV may replace Pi in transport processes (including cellular uptake) and enzymatic reactions (Csanaky and Gregus, 2001; Dixon, 1997; Ginsburg and Lotspeich, 1963), thereby interfering with cellular metabolism. Alternatively, AsV may be reduced to the much more toxic trivalent arsenite (AsIII) by hitherto unidentified cellular enzyme(s) (Thomas et al., 2001). Subsequently, mono- and dimethylated metabolites are formed, among which the trivalent ones are highly toxic, whereas the pentavalent ones are relatively atoxic (Petrick et al., 2001; Rossman, 2003; Thomas et al., 2001). The first step of AsV metabolism, its reduction to AsIII, therefore is not only important in governing the fate of arsenic in the body, but as a toxification step, may also determine its toxicity and carcinogenicity.
Despite the intensive research on the biochemistry of AsV reduction, the metabolic pathways and enzymes involved in it are still unknown. The recent finding that purine nucleoside phosphorylase (PNP) is capable of reducing AsV to AsIII, provided its nucleoside substrate (e.g., inosine) and a dithiol (e.g., dithiothreitol) are present simultaneously (Gregus and Németi, 2002; Radabaugh et al., 2002), promised a better understanding of the biochemical background concerning this important toxification process. However, the testing of PNP for such a role in human erythrocytes and rats in vivo has led to the conclusion that PNP does not contribute to the reduction of AsV significantly (Németi et al., 2003). Studies with cultured human keratinocytes also failed to support that PNP contributes to reduction of AsV in these cells (Patterson et al., 2003). In addition, AsV reduction catalyzed by PNP is not supported by glutathione (GSH), the most important small thiol molecule in cells, although reduction of AsV is apparently GSH-dependent in mouse embryo cells (Bertolero et al., 1987) and in rats (Csanaky and Gregus, 2005; Gyurasics et al., 1991). Experiments carried out on human red blood cells (RBC) have revealed that erythrocytes also possess a PNP-independent AsV reductase activity (Németi and Gregus, 2004). This activity appears GSH-dependent and is responsible for the most of the basal AsV reduction (i.e., without specific stimuli). Moreover, compounds that promote oxidation of NAD(P)H, thereby increasing cellular NAD(P) content, can trigger this PNP-independent AsV reduction, and importantly, reduction of AsV in erythrocytes is apparently coupled to the glycolytic pathway.
The aim of the present paper has been to ascertain that this PNP-independent but GSH- and NAD-dependent AsV-reducing activity is also present in human RBC lysate and in rat liver cytosol, and to characterize it by investigating the effects of GSH, some inhibitors of the glucose metabolism, as well as the glycolytic substrates, and pyridine and adenine nucleotides (i.e., NAD(P)/NAD(P)H and ADP/ATP, respectively). In order to exclude the role of PNP in erythrocytic and cytosolic AsV reduction, all these experiments were carried out in the presence of the PNP inhibitor BCX-1777 (Bantia and Kilpatrick, 2004; Bantia et al., 2001; Bzowska et al., 2000), which can completely inhibit the AsV-reducing activity of this enzyme (Gregus and Németi, 2002). Characterization of this PNP-independent AsV reductase activity greatly assisted us in identifying a glycolytic enzyme that can reduce AsV to AsIII, as presented in the adjoining paper (Gregus and Németi, 2005).
MATERIALS AND METHODS
Chemicals.
BCX-1777 (also called Immucillin-H) was a generous gift from BioCryst Pharmaceuticals (Birmingham, AL). D-gluconic acid sodium salt, N-(2-hydroxyethyl)-piperazine-N'-(2-ethanesulfonic acid) (HEPES), phosphoglyceric phosphokinase (PGK) from baker's yeast, fructose-1,6-bisphosphate (tetra)cyclohexylammonium salt, D,L-glyceraldehyde-3-phosphate diethyl acetal monobarium salt, 2,3-bisphosphoglyceric acid (penta)cyclohexylammonium salt, 3-phosphoglyceric acid disodium salt, 2-phosphoglyceric acid sodium salt, phosphoenolpyruvic acid sodium salt, N-acetylglucosamine (NAGA), and dehydroepiandrosterone were from Sigma. Bicinchoninic acid disodium salt was from Fluka. Glucose, glucose-6-phosphate barium salt, sodium pyruvate, reduced glutathione, NAD, NADH, NADP, NADPH, AMP, ADP, and ATP were from Reanal Ltd. (Budapest, Hungary). 2-Phosphoglycollate (2-PGly) was obtained from Roche as part of the 2,3-bisphosphoglycerate assay kit. The sources of arsenic compounds, and chemicals used in arsenic speciation have been given elsewhere (Csanaky et al., 2003; Németi and Gregus, 2002). All other chemicals were of the highest purity commercially available.
Preparation of human RBC and assay of AsV reduction by the hemolysate.
This research was approved by the Regional Scientific Research Ethics Committee of the University of Pécs, Center for Medical and Health Sciences. Blood (approximately 5 ml) was collected from healthy human volunteers after informed consent into heparinized VacutainerTM tubes. The blood was immediately centrifuged at 1000 x g, 4°C for 10 min, and the plasma and buffy coat were discarded. The pelleted RBC were resuspended in an equal volume of ice-cold buffer containing 250 mM sucrose, 25 mM HEPES, 5 mM MgCl2, and 2 mM EGTA, pH 7.4 (designated as sucrose buffer). 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 in a 50% RBC suspension. The RBC suspension was kept in ice until use for assaying AsV reduction within 3 h.
To assay PNP-independent AsV reduction by RBC lysate, erythrocyte (pre)incubations were carried out in sucrose buffer at 37°C in a final incubation volume of 0.3 ml. First, erythrocytes (50 μl packed cells) were preincubated for 5 min with 20 μM BCX-1777 (to inhibit PNP), 0.067% Nonidet P-40 (a detergent, to lyse RBC), and when indicated, 2 U glucose oxidase. Glucose oxidase was used to deplete endogenous glucose and glucose-derived glycolytic substrates, when AsV reduction was tested in the presence of specific exogenous glycolytic substrates. After preincubation, the incubation was started by adding GSH (typically 6 mM), the test compounds, followed immediately by AsV (50 μM), and was continued for 2.5 min, if otherwise not specified. The incubation was 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. However, 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, 1973), and which displaced thiol-bound AsIII, but did not oxidize the released AsIII. The incubates thus treated were stored at –80°C until analysis for AsIII and AsV. AsV reductase activity was expressed as nmol formed AsIII per minute and ml packed RBC.
Preparation of rat liver cytosol and assay of AsV reduction by the cytosol.
Male Wistar rats kept under standardized conditions and weighing 250–270 g were obtained from the SPF breeding house of the University of Pécs (Hungary). 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 Pécs.
The livers of the rats were quickly removed, rinsed with ice-cold saline, and homogenized in three volumes of sucrose buffer, using a glass homogenization tube with first a looser then a tighter motor-driven Teflon pestle. The homogenate was 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 divided into aliquots and stored at –80°C until assaying its AsV reductase activity. The protein concentration of the cytosol preparations was determined by the bicinchoninic acid method according to Brown et al. (1989).
To assay cytosolic AsV reduction, cytosol (pre)incubations were carried out in sucrose buffer at 37°C in a final incubation volume of 0.3 ml. First, cytosol (5 mg protein/ml) was preincubated for 5 min with 20 μM BCX-1777 (to inhibit PNP) and when indicated, 2 U glucose oxidase. Thereafter, the incubation was started by adding GSH (typically 10 mM), the test compounds, followed immediately by AsV (50 μM), and was continued for 2.5 min, if otherwise not specified. The incubations were stopped by successive addition of 100 μl of 50 mM CdSO4 solution and 100 μl of 1.5 M perchloric acid solution containing 50 mM HgCl2. The incubates were stored at –80°C until analysis for AsIII and AsV. AsV reductase activity was expressed as pmol formed AsIII per minute and mg protein.
Arsenic analysis.
The incubates, having been subjected to protein precipitation, were centrifuged at 10,000 x g, 4°C for 10 min. AsIII and AsV in the resultant supernatant were separated and quantified by HPLC-hydride generation-atomic fluorescence spectrometry, the details of which have been given elsewhere (Gregus et al., 2000; Németi et al., 2003).
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 Glutathione, Phosphate, and Some Inhibitors of Glucose Metabolism on AsV Reduction by Human RBC Lysate and Rat Liver Cytosol
Because reduction of AsV by intact RBC appeared GSH-dependent (Németi and Gregus, 2004), it was of interest to determine whether AsV-reducing activity in hemolysate and cytosol is supported by GSH. Figure 1 demonstrates that GSH enhanced formation of AsIII from AsV in a concentration-dependent manner. At concentrations of 2, 4, 6, and 10 mM, GSH increased AsV reduction by the hemolysate approximately 5, 7, 11, and 22 fold, respectively, whereas at concentrations 2.5, 5, and 10 mM GSH geared up cytosolic AsIII formation 4.5, 7.5, and 10 fold, respectively. It is important to note that less than 1% of AsIII was formed when GSH at similar concentrations was incubated with AsV in the absence of hemolysate or cytosol than in the presence of these enzyme sources for 2.5 min.
Formation of AsIII from AsV in intact RBC is strongly inhibited by inorganic phosphate (Pi) and is modulated by erythrocytic glycolytic activity (Németi and Gregus, 2004). Therefore, we tested the effects of Pi and some compounds that inhibit certain enzymes of glucose metabolism, namely N-acetyl glucosamine (NAGA, a hexokinase inhibitor), fluoride (inhibits enolase), and dehydroepiandrosterone (DHEA, an inhibitor of glucose-6-phosphate dehydrogenase). Phosphate exhibited concentration-dependent inhibitory effect on AsV reduction by both hemolysate and cytosol (Fig. 2, top and bottom, respectively). However, the inhibition of AsIII formation caused by Pi appeared stronger in hemolysate than in cytosol. For example, 1 mM Pi diminished AsV reduction by more than 60% during 2.5 min and more than 50% during 30 min in hemolysate, but influenced it insignificantly in cytosol during either 2.5 or 30 min. Even at a concentration as high as 5 mM, Pi decreased cytosolic AsIII formation by only 58% and 44% during 2.5 and 30 min, respectively. The inhibitors of hexokinase and glucose-6-phosphate dehydrogenase (i.e., NAGA and DHEA, respectively) affected AsV reduction by neither lysed human RBC nor rat liver cytosol (Fig. 2, top and bottom, respectively) irrespective of the duration of incubation. In contrast, the enolase inhibitor fluoride inhibited the hemolysate-catalyzed AsIII formation by 60%, when the incubation lasted 30 min, but not at all when it lasted 2.5 min only. Fluoride was also ineffective in influencing AsV reduction by cytosol irrespective of the incubation time.
Effects of Glycolytic Substrates on AsV Reduction by Human RBC Lysate and Rat Liver Cytosol
The ubiquity of the glycolytic pathway and its apparent importance in AsIII formation from AsV in intact RBC prompted us to investigate the effects of the glycolytic intermediates on AsV reduction in both hemolysate and cytosol. In these studies, RBC lysate or cytosol was preincubated with glucose oxidase (except when glucose was the compound tested) to deplete endogenous glucose and the substrates derived from it, lest the endogenous compounds should alter the effect of the exogenous one being tested.
In lysed RBC, the "upper" glycolytic substrates glucose and fructose-1,6-bisphosphate (Fruc-1,6-BP) exhibited slight stimulatory effect on AsV reduction, whereas glucose-6-phosphate (Gluc-6-P) produced no influence at all (Fig. 3). Of the "lower" glycolytic substrates, 3-phosphoglycerate (3-PGA), 2-phosphoglycerate (2-PGA), phosphoenolpyruvate (PEP), and pyruvate strongly enhanced AsIII formation, whereas 2,3-bisphosphoglycerate (2,3-BPG) and, surprisingly, glyceraldehyde-3-phosphate (Ga-3-P) were found moderate inhibitors, causing approximately 40% diminution in AsIII formation. The responsiveness of the cytosolic AsV reduction to these glycolytic intermediates exhibited both similarities and differences. Like in hemolysate, Fruc-1,6-BP and phosphoglycerates (i.e., 3-PGA and 2-PGA) increased AsIII formation (Fig. 3). Unlike in the RBC lysate, Ga-3-P enhanced AsV reduction, but PEP and pyruvate failed to do so.
Effects of NAD and NADH on AsV Reduction by Human RBC Lysate and Rat Liver Cytosol in the Absence or Presence of Glycolytic Substrates
Because glycolysis is regulated by the ratio of NAD to NADH (i.e., it is accelerated by NAD and decelerated by NADH), we tested the effects of these pyridine nucleotides on AsV reduction both in the absence and in the presence of one of the above-mentioned glycolytic substrates.
In hemolysate, NAD strongly increased AsIII formation either alone or together with any of the substrates (Fig. 4, top), compared to incubations with no pyridine nucleotide added. The increment in AsV reduction brought about by NAD was most marked in the presence of Ga-3-P, when NAD increased AsIII formation almost 7 fold. In the presence of the substrates, which supported AsV reduction well alone (i.e., Fruc-1,6-BP, 3-PGA, 2-PGA, PEP, and pyruvate), NAD also exhibited marked stimulatory effect on AsIII formation (3–4.5 fold). In contrast to NAD, NADH strongly inhibited the AsV-reducing activity of RBC lysate even in the presence of substrates, which facilitated AsV reduction alone (Fig. 4, top).
In the incubations with rat liver cytosol, the effect of NAD on AsIII formation from AsV appeared very similar to that observed in hemolysate (Fig. 4, bottom), as NAD greatly enhanced the reduction of AsV both in the absence and in the presence of one of the glycolytic substrates. The most prominent stimulation was found in incubates with Fruc-1,6-BP plus NAD or with 2-PGA plus NAD (15-fold and 12-fold increase, respectively). In contrast to NAD, the effect of NADH on AsV reduction by cytosol differed from that observed in hemolysate. This reduced pyridine nucleotide-inhibited cytosolic AsIII formation significantly only in the absence of exogenous glycolytic substrates and in the presence of glucose-6-phosphate. In the presence of other substrates, NADH failed to diminish the reduction of AsV and, in combination with Fruc-1,6-BP, even markedly facilitated it.
In order to further characterize the influence of NAD and NADH on AsV reduction by RBC lysate and cytosol, we determined the time courses of their effects on AsIII formation in the presence of Fruc-1,6-BP or 2-PGA, glycolytic substrates supporting AsV reduction the most out of those involved in the upper and lower parts of glycolysis, respectively.
In hemolysate, the formation of AsIII from AsV in the presence of Fruc-1,6-BP alone was rapid initially (Fig. 5, left), but became slower later. When Fruc-1,6-BP and NAD were present simultaneously, the amount of the formed AsIII was much larger, and it increased linearly with time, exhibiting only a slight decline from linearity at later time points. NADH inhibited AsV reduction by approximately 67% throughout the 30-min period. The time courses of AsV reduction in the presence of 2-PGA (Fig. 5, right) were clearly different, at least on two points, from those observed in the presence of Fruc-1,6-BP. First, the time course of 2-PGA-supported AsV reduction both with and without NAD supplementation markedly declined from linearity past the initial 2.5–5 min, and AsIII formation almost stopped past 15 min. Second and more importantly, the 2-PGA-supported AsV reduction was inhibited by NADH only in the initial few minutes; later it surged, and by 15 min the amount of AsIII formed in the presence NADH became similar to that produced in the presence of NAD, exceeding 3-fold the quantity of AsIII formed in the absence of added pyridine nucleotides.
The time courses of the cytosolic AsV reduction in the presence of Fruc-1,6-BP or 2-PGA and with or without NAD/NADH appeared slightly different from those described above for AsV reduction by lysed human RBC. With Fruc-1,6-BP, the major difference was that NADH, which inhibited hemolysate-mediated AsV reduction, enhanced the reduction of AsV by rat liver cytosol from the earliest time point, albeit much less than NAD did (Fig. 6, left). With 2-PGA as a glycolytic substrate, an apparent quantitative, rather than qualitative, difference is noted in the effect of NADH on erythrocytic and cytosolic reduction of AsV. Like in the hemolysate, NADH initially inhibited, later increased AsIII formation from AsV. However, the increment in AsIII production caused by NADH remained far less than that caused by NAD (Fig. 6, right), unlike in the hemolysate (Fig. 5, right).
Effects of NADP and NADPH on AsV Reduction by Human RBC Lysate and Rat Liver Cytosol in the Absence or Presence of Glycolytic Substrates
In erythrocytes, but not in hepatocytes, the only NADP-consuming and NADPH-producing route is the pentose phosphate pathway, which is under strict regulation by NADP/NADPH ratio. In order to assess the contribution of this metabolic pathway to the reduction of AsV to AsIII, RBC lysate and rat liver cytosol were incubated with NADP or NADPH in the absence or in the presence of one of the glycolytic metabolites.
In contrast to NAD, NADP did not increase AsIII formation significantly by the hemolysate either in the absence or the presence of glycolytic substrates (Fig. 7, top); moreover, NADP even inhibited AsV reduction in the presence of glucose and Gluc-6-P. NADPH was found to diminish AsIII formation in the absence of glycolytic substrates and in the presence of the upper ones (i.e., from glucose to Fruc-1,6-BP), Ga-3-P, and 2,3-BPG. In the presence of phosphoglycerates, PEP or pyruvate, NADPH did not influence AsV reduction significantly (Fig. 7, top).
In cytosolic incubations, NADP enhanced AsIII formation by 1.7–3.8 fold both in the absence of added glycolytic substrate and in the presence of every substrate tested (Fig. 7, bottom), though the extent of this enhancement was far below that caused by NAD (Fig. 4, bottom). Interestingly, NADPH also stimulated AsIII formation 1.6–2 fold in the presence of Fruc-1,6-BP, 3-PGA, 2-PGA, PEP, and pyruvate, whereas it was without significant effect with no substrate, glucose, Gluc-6-P, Ga-3-P, or 2,3-BPG added to the incubations.
Effects of ADP and ATP on AsV Reduction by Human RBC Lysate and Rat Liver Cytosol in the Absence or Presence of Glycolytic Substrates
ATP and ADP play key roles in cellular metabolism, including glycolysis. It was therefore of interest to determine if these nucleotides affected the reduction of AsV. In hemolysate, both ADP and ATP were inhibitors of AsIII formation in the presence of phosphoglycerates, PEP, or pyruvate (Fig. 8, top). In addition, ADP significantly diminished AsV reduction in the absence of substrates and in the presence of glucose or Fruc-1,6-BP. In cytosol incubations, both ADP and ATP decreased AsIII formation from AsV in the presence of phosphoglycerates, and ADP, but not ATP, diminished it in the presence of Fruc-1,6-BP and Ga-3-P (Fig. 8, bottom).
Effects of Compounds Influencing 2,3-BPG Metabolism on AsV Reduction in Human RBC Lysate and Rat Liver Cytosol
2,3-BPG is present in the erythrocytes at exceptionally high concentration (approximately 7 mM; Mulquiney et al., 1999), but only in traces in the liver. Its enzymatic degradation yields 3-PGA, a glycolytic substrate. This conversion of 2,3-BPG can be triggered by 2-phosphoglycollate (2-PGly) or inhibited by sulfate. The effects of these compounds on erythrocytic and cytosolic AsV reduction was tested in order to assess the role of 2,3-BPG degradation in AsV reduction. As demonstrated in Figure 9, 2-PGly doubled AsIII formation in hemolysate, but significantly inhibited it in cytosol. Sulfate diminished AsV reduction in RBC lysate minimally, but significantly in the cytosol.
DISCUSSION
AsV Reduction in the Hemolysate: Stimulation by GSH and NAD (but Not NADP) and Inhibition by Phosphate
The previous work (Németi and Gregus, 2004) presented circumstantial evidence that intact human erythrocytes with their PNP inactivated reduce AsV to AsIII in a GSH- and NAD(P)-dependent manner, because the GSH depletor diethylmaleate impaired, whereas compounds known to oxidize NAD(P)H to NAD(P) (e.g., pyruvate, ferricyanide, methylene blue, nitrite, tert-butylhydroperoxide, dehydroascorbate, and 4-dimethylaminophenol) enhanced formation of AsIII from AsV by the RBC. In the present work, first we sought for direct evidence that the PNP-independent erythrocytic AsV reduction requires GSH and a pyridine nucleotide (NAD or NADP) by examining how supplementation of the hemolysate with these compounds alters the AsV reductase activity.
The findings that the PNP-independent AsV-reducing activity of the human RBC lysate is negligible in the absence of added GSH and that GSH increases it in a concentration dependent manner (Fig. 1) directly demonstrate that this enzymatic activity (unlike that of PNP) requires GSH, probably as a reducing partner. In addition, this observation suggests that the enzyme(s) involved contain(s) critical thiol groups, similarly to AsV reductases identified in microorganisms (Rosen, 2002) as well as to PNP found to work as an AsV reductase in the hepatic cytosol of experimental animals (Gregus and Németi, 2002) and humans (Radabaugh et al., 2002). Furthermore, the relevance of this finding is underlined by the fact that reduction of AsV to AsIII in vivo is also GSH dependent (Csanaky and Gregus, 2005).
A most significant observation of this work is that NAD (Fig. 4, top), but not NADP (Fig. 7, top), supports the AsV reductase activity of the RBC lysate. This information clarified that the effect of various NADH and NADPH oxidants to promote AsV reduction in intact RBC (Németi and Gregus, 2004) is based on increased formation of NAD (not NADP), and was also instrumental in the forthcoming identification of the AsV reductase as a NAD-dependent enzyme.
Inorganic phosphate exerted a strong inhibitory effect on AsV reduction by intact erythrocytes (Németi and Gregus, 2004), partly because AsV and Pi compete for the chloride-bicarbonate membrane transporter that mediates their uptake into these cells. The observation that Pi also inhibits, albeit to a much lesser extent, the formation of AsIII by the hemolysate (Fig. 2) indicates that Pi interferes with the enzymatic reduction of AsIII as well. This raises the possibility that the enzyme, which catalyzes the reduction of AsV, contains a phosphate-binding site, which may also accommodate AsV.
AsV Reduction in the Hemolysate: Association with the Glycolysis (Not the PP-Pathway) within the Section from GAPDH to Enolase
It is well known that NAD accelerates, whereas NADH decelerates the glycolytic flux (Tilton et al., 1991). Therefore, the observation that NAD increases, whereas NADH decreases the rate of the hemolysate-catalyzed AsV reduction both in the absence of added glycolytic substrates and in their presence (Fig. 4, top) confirms our suggestion that reduction of AsV to AsIII in human erythrocytes is coupled to glycolysis. In contrast, the following three pieces of evidence indicate that the pentose phosphate pathway (PP-pathway), the other major route for glucose metabolism in erythrocytes (Fig. 10), is not directly involved. First, glucose depletion of intact RBC by incubation with pyruvate, which deprives the PP-pathway of substrate supply, did not decrease, but rather increased AsV reduction (Németi and Gregus, 2004). Second, supplementation of the hemolysate with NADP, that should facilitate the PP-pathway, failed to increase AsIII formation from AsV (Fig. 7, top). Third, dehydroepiandrosterone (DHEA), an inhibitor of the key PP-pathway enzyme, glucose-6-phosphate dehydrogenase (Levy, 1963), barely influenced AsV reduction in hemolysate (Fig. 2, top).
Intact RBC pretreated with pyruvate (which depleted glucose and enriched NAD in the cells, depriving the upper part of the glycolysis from substrate, but permitted the lower part to utilize 2,3-bisphosphoglycerate) exhibited a rapid AsV reduction, which was even further increased by the enolase inhibitor fluoride (Németi and Gregus, 2004). Based largely on this observation, it was proposed that the enzyme responsible for reduction of AsV should lie in the part of the glycolytic pathway from glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to enolase. Enlisted below are findings obtained from the present work that corroborate, or at least are compatible with, this tentative conclusion.
As shown in Fig. 4 (top), NAD stimulated most effectively the formation of AsIII from AsV in the presence of Ga-3-P, followed by Fruc-1,6-BP (which breaks down to Ga-3-P to feed GAPDH) and 3-PGA as well as 2-PGA (which are substrates for the enzymes between GAPDH and enolase) (Fig. 10).
The findings that N-acetylglucosamine (NAGA) had little influence on AsV reduction in hemolysate (Fig. 2), whereas 2-phosphoglycollate (2-PGly) doubled it (Fig. 9) are also compatible with the hypothesis that enzymes between GAPDH and enolase may catalyze the reduction of AsV. As an inhibitor of hexokinase (Cardenas et al., 1984), NAGA should little affect the substrate supply to this part of the glycolysis in erythrocytes, because in RBC this part can also be fueled by 2,3-BPG (Fig. 10). This latter compound is present in RBC at exceptionally high concentration (7 mM, Mulquiney et al., 1999), and can be dephosphorylated to yield 3-PGA (Fig. 10), a glycolytic substrate that can strongly enhance the AsV reductase activity of the hemolysate (Fig. 3). The main enzyme that dephosphorylates 2,3-BPG is the RBC-specific bisphosphoglycerate synthase/phosphatase (BPGS/P), the phosphatase activity of which is augmented by 2-PGly (Fothergill-Gilmore and Watson, 1989). Therefore, 2-PGly stimulated reduction of AsV, most likely by activating the BPGS/P to dephosphorylate 2,3-BPG, thereby feeding 3-PGA into the glycolysis.
The analysis of the time course of AsV reduction by RBC lysate under specific conditions (Fig. 5) also indicates that NAD and one or more glycolytic enzyme(s) starting from GAPDH are critical in formation of AsIII from AsV. In the presence of Fruc-1,6-BP plus NAD, AsV reduction was maintained almost steadily at a high rate throughout an extended period (Fig. 5, left). The following considerations explain this phenomenon. Fruc-1,6-BP is readily cleaved into Ga-3-P, which is then oxidized by the NAD-supported GAPDH while forming NADH (Fig. 10). The 1,3-bisphosphoglycerate (1,3-BPG) produced by GAPDH is then converted into pyruvate, which is finally reduced to lactate by lactate dehydrogenase (LDH). This final reductive step consumes the GAPDH-produced NADH and converts it back to NAD, thereby steadily maintaining NAD supply for GAPDH (Fig. 10) and eliminating the AsV reduction inhibitor NADH. Under this condition, the abundant substrate supply for the glycolytic enzymes downward from GAPDH together with the high NAD concentration maintain the flux in this section of the glycolysis as well as the simultaneous reduction of AsV at high rates. In contrast, when Fruc-1,6-BP was added with NADH to the hemolysate, the AsIII formation rates were very low (Fig. 5, left). Under these conditions, Fruc-1,6-BP cannot be converted into pyruvate, because the high NADH concentration favors substrate flow in the opposite direction (i.e., "upward" from GAPDH). In the lack of pyruvate, NADH cannot be oxidized into NAD. Therefore, at high NADH concentration and with Fruc-1,6-BP as the substrate, the flux through the lower glycolytic section was purportedly slow, and so was the simultaneous reduction of AsV (Fig. 5, left). These considerations are also helpful in rationalizing why fluoride, an enolase inhibitor (Wang and Himoe, 1974; Warburg and Christian, 1941–1942) decreased AsIII formation from AsV by the RBC lysate after a prolonged incubation, but not after a short one (Fig. 2, top). Interruption of the glycolytic flux at enolase prevents reoxidation of the GAPDH-produced NADH to NAD by LDH, causing accumulation of NADH and depletion of NAD over time, and in turn, deceleration of AsV reduction.
When supplementing the hemolysate with 2-PGA plus NAD, the AsV reduction was very rapid initially but slowed down soon (Fig. 5, right). Under this condition, the high NAD concentration bought about two consequences. First, it prevented substrates originating from 2-PGA to reach enzymes above GAPDH, because this would have required NADH (Fig. 10). Second, it prevented pyruvate produced from 2-PGA from being reduced to lactate. These two factors finally resulted in equilibration of substrates derived from 2-PGA among the enzymes of the lower part of the glycolytic pathway. The time course of AsIII formation under this condition (i.e., very rapid initially, but slow later) most likely reflects the flux through these enzymes in order to reach equilibrium. In contrast, when the hemolysate was supplemented with 2-PGA plus NADH, the 2-PGA-derived substrates could, at least in part, be converted into pyruvate, which in turn was reduced to lactate by LDH, with simultaneous oxidation of NADH to NAD. Initially, reduction of AsV was inhibited because NADH concentration was high. Later, however, as the concentration of NADH declined and that of the NAD increased, the formation of AsIII from AsV accelerated, causing the peculiar time course of AsV reduction depicted in Figure 5 (right).
AsV Reduction in the Hemolysate and Hepatic Cytosol: Similarities and Differences
This work demonstrates that a PNP-independent but GSH- and NAD-dependent and phosphate-inhibited AsV-reducing activity is also present in the cytosolic fraction of the rat liver, suggesting that AsV is reduced, at least in part, by the same biochemical process as in intact RBC. However, we point out below a number of dissimilarities in the responsiveness of AsV reduction by the rat liver cytosol and human hemolysate to various compounds.
NADH strongly inhibited AsIII formation by hemolysate in the presence of every substrate (Fig. 4, top), whereas in rat liver cytosol NADH was inhibitory only without added substrate or in the presence of glucose-6-phosphate and was even stimulatory in the presence of Fruc-1,6-BP (Fig. 4, bottom). To understand this difference, one has to realize that liver cells have a much more extensive and complex metabolism than RBC do. Besides LDH, hepatocytes contain a number of other cytosolic enzymes that can oxidize NADH to NAD (e.g., malate dehydrogenase, glycerol phosphate dehydrogenase, -hydroxybutyrate dehydrogenase). The rapid oxidation in liver cytosol of the AsV reduction inhibitor NADH to the AsV reduction stimulator NAD might explain the rather moderate inhibitory effect of NADH on the cytosolic AsV reduction. As to Fruc-1,6-BP, this metabolite is readily cleaved into Ga-3-P and dihydroxyacetone phosphate (DHAP) in both RBC and the liver (Fig. 10). However, in the liver cytosol (but not in the hemolysate) DHAP is substrate for glycerol phosphate dehydrogenase, which reduces it into glycerol phosphate, while consuming NADH and producing NAD. NAD thus produced can then enhance oxidation of Ga-3-P and subsequent formation of lower glycolytic substrates, as well as the simultaneous reduction of AsV. This might be the reason why NADH permanently inhibited the hemolysate-catalyzed AsIII formation from AsV in the presence on Fruc-1,6-BP (Fig. 5, left), but stimulated the cytosol-catalyzed process from the very beginning (Fig. 6, left). Similar reasons may account for the difference in the effect of fluoride on AsV reduction, i.e., inhibition in the hemolysate (Fig. 2, top), but not in the cytosol (Fig. 2, bottom). As explained above, by blocking enolase, fluoride can prevent reoxidation of NADH by LDH in the lysed erythrocytes, with the NADH eventually causing inhibition of AsV reduction. In the liver extract, however, NADH may also be reoxidized by other enzymes mentioned above, which are not sensitive to fluoride.
Fundamental differences could be observed in the effect of triphosphopyridine nucleotides (i.e., NADP and NADPH) on the AsV reductase activities of the hemolysate and cytosol. While these nucleotides slightly inhibited the hemolysate-catalyzed AsV reduction (Fig. 7, top), they significantly augmented the cytosol-catalyzed process (Fig. 7, bottom). This finding raises the possibility that the liver might contain a cytosolic NADP-dependent AsV reductase activity in addition to the NAD-dependent one. This novel activity should depend on the presence of NADP, rather than NADPH, because NADP increased AsIII formation from AsV consistently more than NADPH. The following work (Gregus and Németi, 2005) presents further circumstantial evidence for another PNP-independent AsV reductase activity in the hepatic cytosol.
Inorganic phosphate much less effectively inhibited the cytosolic than the hemolysate-catalyzed AsV reduction (Fig. 2). Because the cytosolic AsV reduction was significant even at a Pi concentration as high as 5 mM (Fig. 2), it might be suggested that Pi at physiologically relevant concentrations in the hepatocytes (approximately 0.5 mM; Iles et al., 1985) permits AsV reduction by the hepatic cytosolic enzyme(s). In addition, based on the lower sensitivity of hepatic AsV reduction to Pi one might speculate that the liver contains another cytosolic AsV reductase (besides the NAD-dependent one present in both RBC and hepatocytes) that is practically unresponsive to Pi.
While 2-PGly significantly enhanced AsV reduction by the hemolysate, it failed to do so in rat liver cytosol (Fig. 9). This difference can be explained readily by the facts that 2-PGly increases enzymatic dephosphorylation of 2,3-BPG to 3-PGA (that supports AsV reduction) and that 2,3-BPG is present only in traces in cells other than RBC, including hepatocytes. It remains to be clarified, however, why sulfate that minimally decreases the hemolysate-catalyzed AsV reduction inhibits the hepatic activity significantly (Fig. 9).
There are a number of observations in this work, e.g., the inhibitory effects of 2,3-BPG and Ga-3-P (Fig. 3) as well as ATP and ADP (Fig. 8) on AsIII formation by hemolysate and/or the hepatic cytosol, which would deserve interpretation. Because findings presented in the adjoining work (Gregus and Németi, 2005) aid this interpretation, these observations will be discussed there.
In summary, this work demonstrates that a PNP-independent AsV reductase activity is present not only in human erythrocytes but also in rat liver cytosol. This enzymatic activity is stimulated by NAD, many glycolytic substrates, and GSH, the latter suggesting involvement of thiol enzymes. The glycolytic enzymes whose substrates support AsV reduction most effectively are GAPDH, PGK, and phosphoglycerate mutase (PGM); these might mediate the reduction of AsV. Out of these, only GAPDH and PGK possess reactive thiol groups (Nagradova and Schmalhausen, 1998; Vas and Csanády, 1987), making them the most likely candidates as glycolytic enzyme(s) catalyzing AsV reduction. In addition, this study raises the possibility that yet another PNP-independent AsV reductase resides in the hepatic cytosol that can utilize NADP. While this enzyme remains to be found, identification of the AsV reductase dependent on NAD, glycolytic substrate, and GSH is presented in the following paper (Gregus and Németi, 2005).
NOTES
The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.
ACKNOWLEDGMENTS
This publication is based on a work supported by the Hungarian National Scientific Research Fund (OTKA) and the Hungarian Ministry of Health. The authors wish to thank Mónika Agyaki and István Schweibert for their excellent assistance in the experimental work.
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