TY - JOUR
AU - Gregus, Zoltán
AB - Abstract After finding that rat liver mitochondria reduce arsenate (AsV) to the more toxic arsenite (AsIII), it was of interest to know if other cell fractions also carried out this process. Postmitochondrial supernatant (PMSN), isolated from rat liver, reduced AsV to AsIII only in the presence of a thiol. Dithiothreitol (DTT) supported the reduction much more effectively than glutathione. Separation of PMSN into microsome and cytosol revealed that the AsV reducing activity resided in the cytosol. AsV-like oxyanions, e.g., phosphate (Pi) and vanadate, as well as mercurial thiol reagents inhibited the cytosolic AsV reducing activity, indicating the involvement of a Pi-utilizing SH enzyme. On searching for a reduction partner, it was found unexpectedly that oxidized pyridine nucleotides (NAD+ or NADP+), but not their reduced forms, increased AsIII formation. Some other purine nucleotide derivatives (e.g., AMP, GMP), but not pyrimidine nucleotides, also increased the rate 2–3-fold. Examination of the effect of nucleosides and nucleobases on AsV reduction yielded dramatic results: purine nucleosides (inosine or guanosine) increased the reduction 80–100-fold, whereas purine bases (hypoxanthine or guanine) decreased it 80–90%. Although the retentate obtained by ultrafiltration of cytosol was almost inactive, its AsV reductase activity could be regained by adding the filtrate or inosine or guanosine to the retentate, indicating that endogenous purine nucleosides were essential for AsV reduction by the cytosol. The hepatic cytosol of mice, hamsters, guinea pigs, and rabbits also exhibited AsV reductase activities in the presence of DTT, which were dramatically enhanced by inosine. Thus, the hepatic cytosol of all tested species can reduce AsV to AsIII. The reduction requires the presence of an appropriate thiol as well as a purine nucleoside (inosine or guanosine) and is inhibited by thiol reagents, the AsV analogue phosphate, and purine bases. Characterization of this AsV reductase activity led us to identification of a cytosolic AsV reductase, which is presented in the accompanying paper. arsenate, arsenite, reduction, thiols, inosine, guanosine, hypoxanthine, guanine, cytosol Arsenic is a widespread environmental toxicant that may cause neuropathy, skin lesions, vascular disease, and cancer upon prolonged exposure (Goering et al., 1999; Hindmarsh, 2000). It enters the body mainly via consumption of contaminated drinking water containing typically the pentavalent arsenate (AsV). In the body, AsV may be subjected to elimination by urinary excretion (Vahter, 1983, 1994) or cellular uptake via their phosphate transporters (Csanaky and Gregus, 2001; Huang and Lee, 1996). AsV may interfere with cellular metabolism by replacing inorganic phosphate (Pi) in enzymatic reactions (Dixon, 1997), including oxidative phosphorylation in mitochondria (Crane and Lipmann, 1953; Ter Welle and Slater, 1967). Alternatively, AsV may undergo reduction to arsenite (AsIII) (Thomas et al., 2001), which is much more toxic than AsV due to its thiol-reactivity (Knowles and Benson, 1983). AsIII is a precursor of mono- and dimethylated arsenic metabolites, including the comparatively atoxic pentavalent monomethylarsonic and dimethylarsinic acids, and the trivalent monomethylarsonous and dimethylarsinous acids, which are even more toxic than AsIII (Petrick, et al., 2001; Thomas et al., 2001). Thus, reduction of AsV to AsIII is the first step in the metabolic fate of AsV, and has a decisive role in determining both the elimination and the toxicity of AsV. Therefore, it is imperative to explore the mechanisms that underlie in vivo reduction of AsV to AsIII. Although AsV can be reduced to AsIII chemically by thiols in large excess (Delnomdedieu et al., 1993; Scott et al., 1993), AsV reduction is most likely an enzymatic process in vivo. AsV reductases in microorganisms have been identified (Ji and Silver, 1992; Krafft and Macy, 1998; Mukhopadhyay et al., 2000), but the identity and intracellular distribution of such enzymes in mammals have not been determined precisely. It has recently been shown that mitochondria isolated from rat liver are capable of reducing AsV to AsIII in a manner dependent on both the functional and structural integrity of these organelles Radabaugh and Aposhian (2002). However, mitochondrial AsV reductase has defied identification, because solubilized mitochondria have lost their reducing activity. Radabaugh and Aposhian (2000) have found AsV reductase activity in human liver cytosol that has been partially purified and characterized. They demonstrated that dithiothreitol (DTT), but not glutathione (GSH), supported the reducing activity well, and that the cytosol contains an unknown heat-stable cofactor with less than 3 kDa molecular mass, which was essential for reduction of AsV. Erythrocytes also reduce AsV (Delnomdedieu et al., 1995), but the participating enzyme has not been identified. The objective of the present study was to determine whether the postmitochondrial cell fractions of rat liver also contained AsV reductase activity, and if they did, to characterize this activity with the ultimate goal of identifying the enzyme involved. This paper demonstrates extensive characterization of the cytosolic AsV reductase activity in rat liver, and the presence of this activity in the liver of other laboratory animal species. This work has indeed led us to successful identification of a known enzyme capable of functioning as AsV reductase, as described in the accompanying article (Gregus and Németi, 2002). MATERIALS AND METHODS Chemicals. ADP, AMP, ATP, 1-chloro-2,4-dinitrobenzene, p-chloromercuribenzoic acid, dicumarol, dithiothreitol, 2,3-meso-dimercaptosuccinic acid, glyceraldehyde-3-phosphate, GDP, GMP, GTP, hypoxanthine, inosine, mersalyl, N-acetylcysteine, and sodium o-vanadate were from Sigma (St. Louis, MO). Sodium selenate, sodium selenite, S-adenosylmethionine, S-adenosylhomocysteine, 2,3-dimercapto-1-propanol, and t-butylhydroperoxide were from Aldrich Chemical. Sodium arsenate, sodium sulfite, zinc(II)-chloride, manganese(II)-chloride, calcium chloride, adenine, adenosine, guanine, guanosine, NAD+, NADH, NADP+, NADPH, uracil, uridine, UMP, cytosine, cytidine, CMP, TRIS, Triton X-100, reduced glutathione, cysteine, 2-mercaptoethanol, and potassium chromate were purchased from Reanal (Hungary). Sodium arsenite was from Carlo Erba, magnesium chloride from Merck, cysteamine from Serva, BCNU (BiCNU®) from Bristol-Myers-Squibb, bicinchoninic acid sodium salt from Fluka, and acyclovir (Herpesin®) from LaChema (Czech Republic). Adenosine dialdehyde (periodate-oxidized adenosine) was synthesized and quantified as described (Gregus et al., 2000). Aurothioglucose was generously provided by Schering-Plough (Kenilworth, NJ), whereas 2,3-dimercapto-1-propanesulfonic acid was provided by Heyl (Berlin, Germany). Reagents for arsenic analysis are specified in (Gregus et al. 2000). All other chemicals used were of the highest purity available. Animals. CFLP mice (33–36 g) were from Charles River (Budapest, Hungary); male Wistar rats (270–320 g), English shorthair guinea pigs (400–450 g), and Syrian golden hamsters (80–100 g) were from the breeding house of the University of Pécs (Hungary). New Zealand white rabbits (1.8–2.5 kg) were from a local rabbit farm and were maintained at our animal facility. The animals were kept at 22–25°C room temperature, 55–65% relative air humidity, on a 12-h light/dark cycle, and provided with tap water and rabbit or rodent lab chow ad libitum. Isolation of postmitochondrial supernatant, microsomes, and cytosol. The livers were quickly removed, rinsed with ice-cold isotonic saline, weighed, and homogenized in 2 volumes of ice-cold 150 mM KCl–50 mM TRIS (pH 7.0 at room temperature), using a glass homogenization tube first with a looser, then a tighter motor-driven Teflon pestle. The homogenate was centrifuged at 4°C, 10,000 × g for 20 min to obtain the postmitochondrial supernatant (PMSN). In order to separate microsomes and cytosol, the PMSN was centrifuged at 4°C, 100,000 × g for 1 h. The supernatant containing the cytosolic fraction was decanted, whereas the pellet containing the microsomal fraction was resuspended in 150 mM KCl–10 mM EDTA, pH 7.4, and was subjected to the ultracentrifugation step again. The resultant pellet was resuspended in 250 mM sucrose. The postmitochondrial supernatant, cytosol, and microsomes were stored in aliquots at –80° until use. Such storage for up to 3 months did not change AsV reductase activity significantly. The protein concentrations of these preparations were determined using the bicinchoninic acid method, as described by Gregus et al. 1989). Ultrafiltration of cytosol. Five hundred μl rat liver cytosol (30 mg protein/ml) was filtered through a Microcon-30 membrane filter (molecular weight cut-off 30,000) at 4°C, 12,500 × g for 1 h. The filtrate was saved and diluted corresponding to 5 mg/ml unfiltered cytosol. The retentate was resuspended in 150 mM KCl–50 mM TRIS, pH 7.6, and recentrifuged under identical conditions. This time the filtrate was discarded and the retentate was washed once more. The final retentate was diluted to correspond to 5 mg/ml unfiltered cytosol. The filtrate and the retentate were stored in aliquots at –80°C until use. Assay of AsV reduction. PMSN, microsomes, and cytosol (protein concentrations of 10 mg/ml, 10 mg/ml, 5 mg/ml, respectively) were incubated for 10, 30, and 10 min, respectively, at 37°C in 150 mM KCl–50 mM TRIS (pH 7.6 at room temperature) with 25 μM AsV. If otherwise not indicated, incubations were started by successive addition of 0.5 mM DTT and protein, and were stopped by addition of mersalyl at a final concentration of 20 mM, and 3 volumes of ice-cold deoxygenated methanol 15 s later. Pilot experiments clarified that mersalyl effectively displaced AsIII from the DTT and proteins. After centrifuging the methanolic incubates, the supernatant was used for analysis of AsIII and AsV. Under the assay conditions, formation of AsIII was linear with respect to incubation time and protein concentration. Arsenic speciation. AsIII and AsV in the methanolic incubates were separated and quantified by HPLC (hydride generation) atomic fluorescence spectrometry according to Gomez-Ariza et al. (1998), as described in detail by Gregus et al. (2000). However, after ascertaining that the incubates contained no other AsV metabolites besides AsIII, we used isocratic rather than gradient elution routinely with 60 mM potassium phosphate buffer (pH 5.75) as an eluent, at 1 ml/min flow rate. Statistics. Significance between means was tested using one-way ANOVA followed by Duncan’s test or Student’s t-test. SPSS 8.0 for Windows (SPSS Inc.) was used for statistical analysis. RESULTS AsV reducing activity of rat liver PMSN resides in the cytosol. In order to determine if extramitochondrial AsV reductase is present in rat liver, and if it is, where it is localized, the AsV reductase activities of PMSN, microsomes, and cytosol were quantified. In the absence of exogenous thiol compound, the PMSN did not reduce AsV at all (data not shown). The PMSN was capable of reducing AsV to AsIII at a low rate in the presence of 5 mM GSH and at a much higher rate in the presence of 0.5 mM DTT (Fig. 1). The membrane solubilizing detergent Triton X-100 enhanced AsIII formation by PMSN significantly (p < 0.05) only when DTT was present. Separation of the PMSN into microsomes and cytosol revealed that the microsomal fraction did not contain any AsV reductase activity, whereas the cytosol reduced AsV effectively. The cytosolic AsIII formation was not enhanced by Triton X-100 (Fig. 1). Effect of mono- and dithiols on cytosolic AsV reduction. Because the cytosolic formation of AsIII from AsV required the presence of some thiol compound, a number of thiols were tested for their ability to enhance the reduction rate. Of the monothiols, each tested at a concentration of 5 mM, GSH and N-acteylcysteine induced AsIII formation moderately (Fig. 2). Cysteine and cysteamine were twice as effective as GSH, whereas 2-mercaptoethanol enhanced it markedly (12-fold compared to GSH). The effect of D-penicillamine was negligible. Of the dithiols, each tested at a concentration of 0.5 mM, DTT and DMP appeared as effective as 5 mM 2-mercaptoethanol (Fig. 2). Dimercapto-propanesulfonic acid exhibited approximately 60% effect compared to DTT, whereas dimercaptosuccinic acid had a negligible effect (approximately 3% of DTT). Because DTT was among the thiols that supported cytosolic reduction of AsV most effectively, further assays of cytosolic AsV reductase were carried out in the presence of 0.5 mM DTT. Effect of inorganic ions on the cytosolic AsV reduction. In order to determine if inorganic ions are able to modify the cytosolic AsV reducing activity, we tested the effects of oxyanions, some of which structurally resemble AsV, as well as some cations, some of which markedly affected reduction of AsV in mitochondria (Németi and Gregus, 2002). Of the oxyanions, it was found that Pi (a structural analogue of AsV) inhibited the AsIII formation in a concentration-dependent manner; however, this inhibitory effect was far from complete, even in 1 mM concentration (Table 1). o-Vanadate (another structural analogue of AsV) was more potent as an inhibitor of AsV reduction, causing an 80% decrease in AsIII formation at a concentration of 0.1 mM. At 1 mM, sulfate and sulfite did not affect the cytosolic AsIII formation significantly, whereas chromate and selenite strongly diminished it. Selenite exhibited a highly potent inhibitory effect, completely abolishing the cytosolic AsV reductase activity at a concentration as low as 25 μM (Table 1). With the exception of zinc, none of the tested cations affected the cytosolic AsV reduction significantly. Interestingly, zinc, at concentrations of 25 and 100 μM, enhanced the AsV reduction, whereas at higher concentration it failed to stimulate AsIII formation (Table 2). Effect of reductase inhibitors and thiol reagents on the cytosolic AsV reduction. The role of some reductase enzymes was tested in cytosolic AsV reduction by using their inhibitor compounds. Dicumarol, BCNU, DNCB, and aurothioglucose, which are inhibitors of NAD(P)H-quinone reductase, glutathione reductase and thioredoxin reductase, respectively, did not decrease the cytosolic reduction of AsV significantly (Table 3). In contrast, non-specific SH reagents (mersalyl and p-chloromercuribenzoic acid) diminished the cytosolic AsIII formation significantly. Effect of nucleotides, nucleosides and nucleobases on the cytosolic reduction of AsV. On searching for the reduction partner physiologically present in the cytosol, the effect of reduced pyridine nucleotides (i.e., NADH, NADPH) was tested. Neither NADH nor NADPH enhanced the cytosolic AsV reductase activity (Fig. 3). Surprisingly, however, oxidized pyridine nucleotides (i.e., NAD+ and NADP+) increased the AsIII formation; NAD+ even doubled it. Therefore a number of nucleotide compounds were tested. Of these, S-adenosylhomocysteine, AMP, and GMP significantly increased the formation of AsIII (Fig. 3), whereas the others did not influence it significantly. The common feature of the nucleotides, which increased AsIII formation, is their ability to yield purine nucleosides (i.e., adenosine, inosine, or guanosine) during their incubation with cytosol (see Discussion). Therefore, the effects of nucleosides and nucleobases were tested. It was found that pyrimidine nucleosides (cytidine and uridine) did not influence the cytosolic AsIII formation significantly (Fig. 4). In striking contrast, the purine nucleoside adenosine increased reduction of AsV 7-fold, and inosine as well as guanosine enhanced it as much as 80–100-fold (Fig. 4). On testing the effect of nucleobases, it was found that cytosine, uracil, or adenine did not affect the cytosolic AsV reduction significantly, whereas guanine (the base contained in guanosine) and hypoxanthine (the base contained in inosine) decreased it by approximately 80% and 90%, respectively (Fig. 4). We also tested 2 synthetic nucleoside derivatives at 1 mM concentration; the known methylation inhibitor periodate oxidized adenosine (also called adenosine dialdehyde) did not influence the cytosolic AsIII formation significantly, whereas the antiviral drug acyclovir (also called acycloguanosine) caused a 75% inhibition (data not shown). Effect of Pi on the inosine-stimulated cytosolic AsV reduction. In order to test whether Pi also inhibits the inosine-stimulated cytosolic AsV reduction, the concentration-dependent effect of Pi on AsIII formation was examined in the presence of 1 mM inosine. As depicted in Figure 5, which contains, in the insert, the double reciprocal (Lineweaver-Burk) plots of the results, Pi diminished AsIII formation in a concentration-dependent but not clearly competitive manner. Because some inhibitory hypoxanthine might be formed from inosine during the incubation, we tested the effect of xanthine oxidase, which can oxidize hypoxanthine to xanthine and uric acid. Indeed, addition of xanthine oxidase into the incubation mixture doubled AsIII formation (Fig. 5, open symbols) compared to AsIII formation in the absence of xanthine oxidase (Fig. 5, closed symbols). Effect of ultrafiltration of the cytosol. To investigate if small molecules play a role in cytosolic reduction of AsV, we tested the effect of ultrafiltration. After filtrating the cytosol through a Microcon-30 membrane filter, both the resultant retentate and the filtrate became almost devoid of AsV reductase activity (Fig. 6). Nevertheless, the AsV reductase activity was regained upon recombination of the filtrate and the retentate. It is noteworthy that the reductase activity of the retentate was also regained, when 1 mM adenosine, guanosine, or inosine was added to the retentate (Fig. 6); moreover, the purine nucleosides at this concentration increased the activity of the retentate well above that of the unfiltered cytosol. Inosine-supported AsV reductase activity in the hepatic cytosol of other species. We tested if the hepatic cytosol of species other than the rat also contained thiol-dependent AsV reductase activity that could be augmented by inosine. All species tested exhibited such activity in the liver. On comparing the AsV reducing activities in liver cytosol of the 5 species in the presence of 0.5 mM DTT, it was found that rat liver cytosol contained the highest AsV reductase activity, followed by guinea pig and mouse, whereas the activity in the hepatic cytosol of the hamster and the rabbit was low (Fig. 7). Addition of inosine dramatically enhanced the DTT-supported hepatic cytosolic AsV reductase activity in each species tested. The inosine-stimulated AsV reducing activities of rat, mouse, guinea pig, and hamster liver cytosols were rather similar (Fig. 7), whereas that of the rabbit liver cytosol appeared much lower. DISCUSSION This work demonstrates that not only rat liver mitochondria (Németi and Gregus, 2002) but also the hepatic postmitochondrial cell fraction is capable of reducing AsV to the much more toxic AsIII. The enzyme responsible for AsV reductase activity was found to be restricted to the cytosolic fraction. Nevertheless, this soluble enzyme apparently becomes adsorbed to or enclosed into the microsomal membranes during preparation of PMSN, which probably accounts for the increase in AsV reductase activity of the PMSN upon membrane solubilization. The AsV reductase activity present in rat liver cytosol, similarly to that found in human liver cytosol (Radabaugh and Aposhian, 2000), requires an appropriate thiol-containing molecule for function. It appears that thiols and dithiols with no ionizable group other than the thiol moiety (e.g., dithiothreitol, dimercaptopropanol, and 2-mercaptoethanol) are especially suitable for supporting AsV reduction, whereas the charged thiol compounds, especially the dianionic ones (e.g., dimercaptosuccinate, glutathione) are less suitable. This might indicate that strong ionic charge hinders interaction of the thiol compounds with the cytosolic AsV reductase, although other factors, e.g., the pK of the SH group and presence of protonizable amino group, most likely also influence the effectiveness of the thiol compound in its partnership with the enzyme. The necessity of a thiol compound for the AsV reducing activity, together with the sensitivity of this activity to mercurial sulfhydryl reagents, suggest that the cytosolic AsV reductase contains functionally important SH group(s). Oxyanions that are structurally related to AsV, such as Pi and o-vanadate, inhibited reduction of AsV in a concentration-dependent fashion, suggesting that the AsV reductase possesses a binding site that can accommodate not only AsV, but also Pi and vanadate. However, vanadate was a stronger inhibitor of AsV reduction than Pi. Vanadate not only inhibits enzymes that catalyze phosphoryl transfer reactions (Gresser and Tracey, 1990), but also reacts with DTT to form long-lived complexes under neutral conditions in aqueous medium (Paul and Tracey, 1997). Thus, decreased availability of DTT for the AsV reductase could also contribute to the inhibitory effect of vanadate on AsIII formation from AsV. A similar mechanism may, in part, underlie the inhibitory effect of chromate, which can also react with DTT (Connett and Wetterhahn, 1985). Of the tested oxyanions unrelated to AsV, selenate weakly, whereas selenite strongly, inhibited the reduction of AsV by rat liver cytosol. Selenite reacts with SH groups (Ganther, 1986) and thus might inactivate the cytosolic AsV reductase. Selenate is first reduced to selenite (Ganther, 1986), which may explain its weaker inhibitory effect. It seems unlikely that selenite inhibited the AsV reductase activity significantly by consuming DTT during the assay, because the concentration of DTT exceeded the inhibitory concentration of selenite 50-fold. The finding that inhibitors (i.e., dicumarol, BCNU, aurothioglucose) of known cytosolic reductase enzymes (NAD(P)H-quinone oxidoreductase, glutathione reductase, or thioredoxin reductase) failed to affect the cytosolic reduction of AsV significantly, excluded these enzymes as candidate AsV reductases. It is also unlikely that the cytosolic AsV reductase could be another NADH- or NADPH-utilizing enzyme, because neither NADH nor NADPH supported the cytosolic reduction of AsV. The paradoxical observation that the oxidized pyridine nucleotides (NAD+ or NADP+), but not their reduced counterparts, enhanced reduction of AsV, greatly facilitated our progress in characterizing and finally identifying the AsV reductase. First, this finding prompted us to test the role in AsV reduction of glyceraldehyde-3-phosphate dehydrogenase (GA3PDH), which is a cytosolic NAD+-utilizing enzyme with a functionally important thiol group and Pi binding site, which can also accommodate AsV (Byers et al., 1979). However, neither purified GA3PDH produced AsIII from AsV when incubated with AsV, with or without glyceraldehyde-3-phosphate, NAD+, phosphoglycerate kinase, Mg2+-ADP, and DTT; nor purified GA3PDH fortified the AsV reductase activity of cytosol when added to this fraction (data not shown). Second, the aforementioned paradoxical observation prompted us to test a number of other nucleotides as well. These studies revealed that S-adenosylhomocysteine, AMP, and GMP also increased the reduction of AsV. It is important to note that all nucleotides that increased the AsV reductase activity of the cytosol can be cleaved into nucleosides by cytosolic enzymes. For example, NAD+ is hydrolyzed to ADP-ribose by NAD+-glycohydrolase, and then to adenosine by nucleotide pyrophosphatase. S-Adenosylhomocysteine is cleaved into adenosine by S-adenosylhomocysteine hydrolase, whereas AMP and GMP are converted into adenosine and guanosine, respectively, by 5‘-nucleotidase. Because the above-mentioned hydrolytic reactions may take place in the cytosol during incubations for assaying AsV reductase activity, possibly resulting in formation of nucleosides in the incubation medium from the above-mentioned nucleotides, we tested the effect of nucleosides on the cytosolic reduction of AsV. The AsV reduction was not affected or was only barely enhanced by pyrimidine nucleosides, but was increased dramatically by purine nucleosides. Of the latter, 6-oxopurine nucleosides (inosine and guanosine) were especially potent, whereas adenosine (a 6-aminopurine nucleoside) was much less potent than inosine or guanosine. This observation suggested that, during the AsV reductase assay, adenosine might be converted by the cytosolic adenosine deaminase into inosine, a highly potent activator of the AsV reductase. The stimulatory effect of zinc on cytosolic AsV reductase activity may also be attributed to increased formation of inosine, because this metal may activate adenosine deaminase, a zinc-dependent enzyme (Wilson et al., 1991), and thus may enhance conversion of endogenous adenosine to inosine, which is much more potent than adenosine in enhancing AsV reductase activity. While 6-oxopurine nucleosides strongly activated the cytosolic AsV reduction, 6-oxopurine bases (hypoxanthine or guanine) markedly inhibited this process. Collectively, these observations suggested that the cytosolic enzyme catalyzing the reduction of AsV might use 6-oxopurine nucleosides, and form 6-oxopurine nucleobases as inhibitory products during the reduction of AsV. The cytosolic thiol-dependent, inosine-activated AsV reductase activity was also present in the livers of mice, guinea pigs, hamsters, and rabbits, indicating that this cytosolic AsV reductase is found in all small laboratory animals used in arsenic research. In the human liver cytosol, the AsV reductase activity required (a) cofactor(s) less than 3 kDa in size, which could be removed by ultrafiltration (Radabaugh and Aposhian, 2000). Ultrafiltration of rat liver cytosol also yielded a retentate with almost complete lack of AsV reductase activity. The activity of the retentate was regained not only by its recombination with the filtrate but also by adding purine nucleosides (adenosine, guanosine or inosine) to the retentate, strongly suggesting that human and rat liver enzymes are similar, and that the cofactor necessary for cytosolic AsV reductase may represent a mixture of endogenous purine nucleosides normally present in the hepatic cytosol. After finding that inosine was an activator of the AsV reductase, it was of interest to reexamine the inhibitory effect of Pi on the inosine-activated enzyme and to determine if the inhibition is competitive. Pi also inhibited the reduction of AsV in the presence of inosine in a concentration-dependent fashion; however, the inhibition pattern was not clearly competitive, suggesting that other factors might confound the effect of Pi. Indeed, addition of xanthine oxidase, which can oxidize hypoxanthine to xanthine and uric acid, markedly decreased the inhibitory effect of Pi. This indicates that hypoxanthine, which had been found to be inhibitory, was produced during the AsV reductase assay. These observations confirm the tentative conclusion that the enzyme reducing AsV to AsIII is a Pi-utilizing enzyme that can convert inosine to hypoxanthine. In addition, the inhibitory effect of Pi questions the role of this cytosolic AsV reductase as an intracellular enzyme capable of converting AsV into AsIII. However, at the intracellular concentration of free Pi in the liver (0.5 mM; Iles et al., 1985) the inhibition caused by Pi was far from complete, suggesting that the intracellular Pi level permits this cytosolic AsV reductase to contribute to the cellular reduction of AsV. In summary, the present work has characterized the cytosolic AsV reductase activity and allowed us to make the following observations and putative conclusions: The cytosolic AsV reductase activity requires the presence of an appropriate thiol compound, and is impaired by thiol-reagents, therefore the participating enzyme most likely possesses SH groups critical for function. Pi inhibits the reducing activity; therefore, the catalyzing enzyme probably utilizes Pi. 6-Oxopurine nucleosides enhance the AsV reductase activity enormously, and therefore, the AsV reductase probably also accepts 6-oxopurine nucleosides as substrates. 6-Oxopurine nucleobases strongly diminish the reducing activity, therefore the catalyzing enzyme may produce 6-oxopurine nucleobases. It is most likely that the cytosolic AsV reductase activity characterized in this work and identified in the accompanying paper (Gregus and Németi, 2002) is identical to the enzyme recently found in human liver cytosol (Radabaugh and Aposhian, 2000) that is activated by DTT and requires cofactor(s) smaller in size than 3 kDa, probably representing purine nucleosides. TABLE 1 Effect of Some Oxyanions on the Formation of Arsenite from Arsenate by Rat Liver Cytosol Anions  Concentration (μM)  AsIII formation  Note. Rat liver cytosol (5 mg protein/ml) was preincubated at 37°C with one of the anions in 150 mM KCl–50 mM TRIS (pH 7.6) for 5 min. Then the AsV reductase assay was started by successive addition of 0.5 mM DTT and 25 μM AsV, and the incubation was continued for 10 min. AsIII formation is given in pmol/min/mg protein; values are means ± SEM of 4 incubations with liver cytosols prepared from different rats.  *Significant difference (p < 0.05) from AsIII formation in the absence of tested oxyanions.  None  —  42.55 ± 3.98  Phosphate        250  34.18 ± 2.72    500  25.62 ± 1.63*    750  19.42 ± 0.47*    1000  14.59 ± 0.09*  o-Vanadate        25  24.34 ± 1.50*    100  9.67 ± 0.87*    250  4.94 ± 0.63*    1000  1.41 ± 0.35*  Chromate        100  28.72 ± 4.31*    1000  6.09 ± 1.80*  Sulfate  1000  45.94 ± 6.83  Sulfite  1000  45.76 ± 7.72  Selenate        250  33.76 ± 2.98    1000  4.91 ± 0.71*  Selenite        2.5  40.81 ± 5.62    10  8.82 ± 1.32*    25  0.00 ± 0.00*  Anions  Concentration (μM)  AsIII formation  Note. Rat liver cytosol (5 mg protein/ml) was preincubated at 37°C with one of the anions in 150 mM KCl–50 mM TRIS (pH 7.6) for 5 min. Then the AsV reductase assay was started by successive addition of 0.5 mM DTT and 25 μM AsV, and the incubation was continued for 10 min. AsIII formation is given in pmol/min/mg protein; values are means ± SEM of 4 incubations with liver cytosols prepared from different rats.  *Significant difference (p < 0.05) from AsIII formation in the absence of tested oxyanions.  None  —  42.55 ± 3.98  Phosphate        250  34.18 ± 2.72    500  25.62 ± 1.63*    750  19.42 ± 0.47*    1000  14.59 ± 0.09*  o-Vanadate        25  24.34 ± 1.50*    100  9.67 ± 0.87*    250  4.94 ± 0.63*    1000  1.41 ± 0.35*  Chromate        100  28.72 ± 4.31*    1000  6.09 ± 1.80*  Sulfate  1000  45.94 ± 6.83  Sulfite  1000  45.76 ± 7.72  Selenate        250  33.76 ± 2.98    1000  4.91 ± 0.71*  Selenite        2.5  40.81 ± 5.62    10  8.82 ± 1.32*    25  0.00 ± 0.00*  View Large TABLE 2 Effect of Some Cations on the Formation of Arsenite from Arsenate by Rat Liver Cytosol Cation  Concentration (μM)  AsIII formation  Note. Rat liver cytosol (5 mg protein/ml) was preincubated at 37°C with one of the cations in 150 mM KCl–50 mM TRIS (pH 7.6 at room temperature) for 5 min. Then the AsV reductase assay was started by successive addition of 0.5 mM DTT and 25 μM AsV, and the incubation was continued for 10 min. AsIII formation is given in pmol/min/mg protein; values are means ± SEM of 3 incubations with liver cytosols prepared from different rats.  *Significant difference (p < 0.05) from AsIII formation in the absence of the tested cations.  None  —  45.36 ± 3.23  Zn2+        10  53.82 ± 6.05    25  65.38 ± 9.95*    100  74.83 ± 12.1*    250  56.48 ± 5.24  Mn2+        25  41.63 ± 3.79    250  39.75 ± 5.35  Ca2+        25  47.17 ± 9.40    250  51.68 ± 9.80  Mg2+        25  50.80 ± 8.96    250  42.84 ± 9.07  Cation  Concentration (μM)  AsIII formation  Note. Rat liver cytosol (5 mg protein/ml) was preincubated at 37°C with one of the cations in 150 mM KCl–50 mM TRIS (pH 7.6 at room temperature) for 5 min. Then the AsV reductase assay was started by successive addition of 0.5 mM DTT and 25 μM AsV, and the incubation was continued for 10 min. AsIII formation is given in pmol/min/mg protein; values are means ± SEM of 3 incubations with liver cytosols prepared from different rats.  *Significant difference (p < 0.05) from AsIII formation in the absence of the tested cations.  None  —  45.36 ± 3.23  Zn2+        10  53.82 ± 6.05    25  65.38 ± 9.95*    100  74.83 ± 12.1*    250  56.48 ± 5.24  Mn2+        25  41.63 ± 3.79    250  39.75 ± 5.35  Ca2+        25  47.17 ± 9.40    250  51.68 ± 9.80  Mg2+        25  50.80 ± 8.96    250  42.84 ± 9.07  View Large TABLE 3 Effect of Some Reductase Inhibitors and Thiol Reagents on the Formation of Arsenite from Arsenate by Rat Liver Cytosol Compound  Target protein  Concentration (μM)  AsIII formation  Note. Rat liver cytosol (5 mg protein/ml) was preincubated at 37°C with one of the inhibitor compounds in 150 mM KCl–50 mM TRIS (pH 7.6) for 5 min. Then the AsV reductase assay was started by successive addition of 0.5 mM DTT and 25 μM AsV, and the incubation was continued for 10 min. In order to prevent the thiol reagents from reacting with DTT during the assay, these reagents were eliminated by adding 2 mM glutathione to the preincubation mixture 1 min before starting the assay. AsIII formation is given in pmol/min/mg protein; values are means ± SEM of 4 incubations with liver cytosols prepared from different rats.  *Significant difference (p < 0.05) from AsIII formation in the absence of test compounds.  None  —  —  40.69 ± 7.13  Dicumarol  NAD(P)H-quinone reductase  10  38.21 ± 3.98      100  34.91 ± 4.40  BCNU  Glutathione reductase  100  34.41 ± 3.16      1000  32.67 ± 4.71  DNCB  Thioredoxin reductase  25  35.16 ± 3.70      250  41.08 ± 5.73  Aurothioglucose  Thioredoxin reductase  100  57.50 ± 6.91      1000  33.02 ± 3.27  Mersalyl  SH enzymes  1000  14.42 ± 2.02*  PCMB  SH enzymes  1000  16.44 ± 2.92*  Compound  Target protein  Concentration (μM)  AsIII formation  Note. Rat liver cytosol (5 mg protein/ml) was preincubated at 37°C with one of the inhibitor compounds in 150 mM KCl–50 mM TRIS (pH 7.6) for 5 min. Then the AsV reductase assay was started by successive addition of 0.5 mM DTT and 25 μM AsV, and the incubation was continued for 10 min. In order to prevent the thiol reagents from reacting with DTT during the assay, these reagents were eliminated by adding 2 mM glutathione to the preincubation mixture 1 min before starting the assay. AsIII formation is given in pmol/min/mg protein; values are means ± SEM of 4 incubations with liver cytosols prepared from different rats.  *Significant difference (p < 0.05) from AsIII formation in the absence of test compounds.  None  —  —  40.69 ± 7.13  Dicumarol  NAD(P)H-quinone reductase  10  38.21 ± 3.98      100  34.91 ± 4.40  BCNU  Glutathione reductase  100  34.41 ± 3.16      1000  32.67 ± 4.71  DNCB  Thioredoxin reductase  25  35.16 ± 3.70      250  41.08 ± 5.73  Aurothioglucose  Thioredoxin reductase  100  57.50 ± 6.91      1000  33.02 ± 3.27  Mersalyl  SH enzymes  1000  14.42 ± 2.02*  PCMB  SH enzymes  1000  16.44 ± 2.92*  View Large FIG. 1. View largeDownload slide Reduction of AsV by rat liver postmitochondrial supernatant (PMSN), microsomes, and cytosol. PMSN (10 mg protein/ml), microsomes (10 mg protein/ml) or cytosol (5 mg protein/ml) was incubated at 37°C in 150 mM KCl–50 mM TRIS (pH 7.6) for 10 min with 25 μM AsV in the presence of 5 mM GSH or 0.5 mM DTT and with or without 0.2% Triton X-100. Bars represent means ± SEM of 5 incubations, each with liver fractions prepared from different rats. FIG. 1. View largeDownload slide Reduction of AsV by rat liver postmitochondrial supernatant (PMSN), microsomes, and cytosol. PMSN (10 mg protein/ml), microsomes (10 mg protein/ml) or cytosol (5 mg protein/ml) was incubated at 37°C in 150 mM KCl–50 mM TRIS (pH 7.6) for 10 min with 25 μM AsV in the presence of 5 mM GSH or 0.5 mM DTT and with or without 0.2% Triton X-100. Bars represent means ± SEM of 5 incubations, each with liver fractions prepared from different rats. FIG. 2. View largeDownload slide Effect of thiols on cytosolic AsV reduction. Rat liver cytosol (5 mg protein/ml) was incubated at 37°C in 150 mM KCl–50 mM TRIS (pH 7.6) for 10 min with 25 μM AsV in the presence of different monothiols (5 mM) or dithiols (0.5 mM). Bars represent means ± SEM of 5 incubations, each with liver cytosols prepared from different rats. AsIII formation in the presence of any thiol was significantly larger (p < 0.05) than in the absence of an exogenous thiol, which was zero. (GSH, reduced glutathione; CYS, cysteine; NAC, N-acetylcysteine; CA, cysteamine; PA, D-penicillamine; 2-ME, 2-mercaptoethanol; DTT, dithiothreitol; DMP, dimercaptopropanol; DMPS, dimercapto-propanesulfonic acid; DMSA, dimercaptosuccinic acid) FIG. 2. View largeDownload slide Effect of thiols on cytosolic AsV reduction. Rat liver cytosol (5 mg protein/ml) was incubated at 37°C in 150 mM KCl–50 mM TRIS (pH 7.6) for 10 min with 25 μM AsV in the presence of different monothiols (5 mM) or dithiols (0.5 mM). Bars represent means ± SEM of 5 incubations, each with liver cytosols prepared from different rats. AsIII formation in the presence of any thiol was significantly larger (p < 0.05) than in the absence of an exogenous thiol, which was zero. (GSH, reduced glutathione; CYS, cysteine; NAC, N-acetylcysteine; CA, cysteamine; PA, D-penicillamine; 2-ME, 2-mercaptoethanol; DTT, dithiothreitol; DMP, dimercaptopropanol; DMPS, dimercapto-propanesulfonic acid; DMSA, dimercaptosuccinic acid) FIG. 3. View largeDownload slide Effect of nucleotide compounds on cytosolic reduction of AsV. Rat liver cytosol (5 mg protein/ml) was preincubated at 37°C in 150 mM KCl–50 mM TRIS (pH 7.6) for 5 min with one of the nucleotides (1 mM). Then DTT (0.5 mM) and AsV (25 μM) were added successively and the incubation was continued for 10 min. Bars represent means ± SEM of 3 incubations with liver cytosols prepared from different rats. SAME, S-adenosylmethionine; SAHC, S-adenosylhomocysteine. *Significant difference (p < 0.05) from AsIII formation observed in the absence of any of the nucleotide compounds. FIG. 3. View largeDownload slide Effect of nucleotide compounds on cytosolic reduction of AsV. Rat liver cytosol (5 mg protein/ml) was preincubated at 37°C in 150 mM KCl–50 mM TRIS (pH 7.6) for 5 min with one of the nucleotides (1 mM). Then DTT (0.5 mM) and AsV (25 μM) were added successively and the incubation was continued for 10 min. Bars represent means ± SEM of 3 incubations with liver cytosols prepared from different rats. SAME, S-adenosylmethionine; SAHC, S-adenosylhomocysteine. *Significant difference (p < 0.05) from AsIII formation observed in the absence of any of the nucleotide compounds. FIG. 4. View largeDownload slide Effect of nucleosides and nucleobases on cytosolic reduction of AsV. Rat liver cytosol was preincubated at 37°C in 150 mM KCl–50 mM TRIS (pH 7.6) for 5 min with one of the nucleosides (left, 0.1 mg protein/ml) or bases (right, 5 mg protein/ml). Then DTT (0.5 mM) and AsV (25 μM) were added successively, and the incubation was continued for 10 min. Bars represent means ± SEM of 3 incubations with liver cytosols prepared from different rats. *Significant difference (p < 0.05) from AsIII formation observed in the absence of added nucleoside or nucleobase. FIG. 4. View largeDownload slide Effect of nucleosides and nucleobases on cytosolic reduction of AsV. Rat liver cytosol was preincubated at 37°C in 150 mM KCl–50 mM TRIS (pH 7.6) for 5 min with one of the nucleosides (left, 0.1 mg protein/ml) or bases (right, 5 mg protein/ml). Then DTT (0.5 mM) and AsV (25 μM) were added successively, and the incubation was continued for 10 min. Bars represent means ± SEM of 3 incubations with liver cytosols prepared from different rats. *Significant difference (p < 0.05) from AsIII formation observed in the absence of added nucleoside or nucleobase. FIG. 5. View largeDownload slide Effect of inorganic phosphate on the inosine-stimulated AsV reduction. Rat liver cytosol (0.1 mg protein/ml) was incubated at 37°C in 150 mM KCl–50 mM TRIS (pH 7.6) with 25 μM AsV for 10 min in the presence of 0.5 mM DTT as well as 0, 0.25, 0.5, or 1 mM inorganic phosphate (Pi). The assays with 1 mM Pi were also performed in the presence of xanthine oxidase (50 mU/ml) under otherwise identical conditions (open symbols). Symbols represent means ± SEM of 3 incubations with liver cytosol prepared from different rats. AsIII formation rates in the presence of Pi at any concentrations tested are significantly lower (p < 0.05) than those found in the presence of the corresponding AsV concentrations in the absence of Pi. The insert shows the double reciprocal (Lineweaver-Burk) plot of the AsV-phosphate interaction. FIG. 5. View largeDownload slide Effect of inorganic phosphate on the inosine-stimulated AsV reduction. Rat liver cytosol (0.1 mg protein/ml) was incubated at 37°C in 150 mM KCl–50 mM TRIS (pH 7.6) with 25 μM AsV for 10 min in the presence of 0.5 mM DTT as well as 0, 0.25, 0.5, or 1 mM inorganic phosphate (Pi). The assays with 1 mM Pi were also performed in the presence of xanthine oxidase (50 mU/ml) under otherwise identical conditions (open symbols). Symbols represent means ± SEM of 3 incubations with liver cytosol prepared from different rats. AsIII formation rates in the presence of Pi at any concentrations tested are significantly lower (p < 0.05) than those found in the presence of the corresponding AsV concentrations in the absence of Pi. The insert shows the double reciprocal (Lineweaver-Burk) plot of the AsV-phosphate interaction. FIG. 6. View largeDownload slide Effect of ultrafiltration on cytosolic reduction of AsV. Rat liver cytosol was ultrafiltered through a Microcon 30 membrane filter, as described in Materials and Methods, to prepare a retentate and filtrate. Cytosol (5 mg protein/ml), filtrate (corresponding to 5 mg protein/ml unfiltered cytosol) or retentate (corresponding to 5 mg protein/ml unfiltered cytosol) were incubated at 37°C in 150 mM KCl–50 mM TRIS (pH 7.6) for 10 min with 25 μM AsV in the presence of 0.5 mM DTT. When the AsV reducing activity of the retentate was also assayed in the presence of adenosine, guanosine, or inosine, these nucleosides (1 mM) were preincubated with the retentate for 5 min before starting the reaction with addition of DTT and AsV. Bars represent means ± SEM of 3 incubations with liver cytosols prepared from different rats. *Significant difference (p < 0.05) from AsIII formation by the unfiltered cytosol. #Significant difference (p < 0.05) from AsIII formation by the retentate. FIG. 6. View largeDownload slide Effect of ultrafiltration on cytosolic reduction of AsV. Rat liver cytosol was ultrafiltered through a Microcon 30 membrane filter, as described in Materials and Methods, to prepare a retentate and filtrate. Cytosol (5 mg protein/ml), filtrate (corresponding to 5 mg protein/ml unfiltered cytosol) or retentate (corresponding to 5 mg protein/ml unfiltered cytosol) were incubated at 37°C in 150 mM KCl–50 mM TRIS (pH 7.6) for 10 min with 25 μM AsV in the presence of 0.5 mM DTT. When the AsV reducing activity of the retentate was also assayed in the presence of adenosine, guanosine, or inosine, these nucleosides (1 mM) were preincubated with the retentate for 5 min before starting the reaction with addition of DTT and AsV. Bars represent means ± SEM of 3 incubations with liver cytosols prepared from different rats. *Significant difference (p < 0.05) from AsIII formation by the unfiltered cytosol. #Significant difference (p < 0.05) from AsIII formation by the retentate. FIG. 7. View largeDownload slide Reduction of AsV by hepatic cytosols from different species. Liver cytosols were incubated at 37°C in 150 mM KCl–50 mM TRIS (pH 7.6) for 10 min with 25 μM AsV in the presence of 0.5 mM DTT. The incubations were carried out without added inosine at 5 mg/ml protein concentration (top), and in the presence of 0.5 mM inosine at 0.1 mg/ml protein concentration (bottom). Bars represent means ± SEM of 3 incubations with liver cytosols prepared from different animals. FIG. 7. View largeDownload slide Reduction of AsV by hepatic cytosols from different species. Liver cytosols were incubated at 37°C in 150 mM KCl–50 mM TRIS (pH 7.6) for 10 min with 25 μM AsV in the presence of 0.5 mM DTT. The incubations were carried out without added inosine at 5 mg/ml protein concentration (top), and in the presence of 0.5 mM inosine at 0.1 mg/ml protein concentration (bottom). Bars represent means ± SEM of 3 incubations with liver cytosols prepared from different animals. This study was presented at the 41st annual meeting of the Society of Toxicology, March 2002, Nashville, Tennessee. The Abstract was published in Toxicol. Sci.66(Supp.), 2002, p. 83. 1 To whom correspondence should be addressed. Fax: 36-72-536-218. E-mail: zoltan.gregus@aok.pte.hu. This publication is based on work supported by the Hungarian National Scientific Research Fund (OTKA) and the Hungarian Ministry of Health. 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TI - Reduction of Arsenate to Arsenite in Hepatic Cytosol
JF - Toxicological Sciences
DO - 10.1093/toxsci/70.1.4
DA - 2002-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/reduction-of-arsenate-to-arsenite-in-hepatic-cytosol-KCySWR5hqT
SP - 4
EP - 12
VL - 70
IS - 1
DP - DeepDyve
ER -