Department of Pharmacology and Pharmacotherapy, University of Pécs, Medical School, Szigeti út 12, H-7624 Pécs, Hungary
Received April 5, 2002; accepted June 3, 2002
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ABSTRACT |
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Key Words: arsenate; arsenite; reduction; thiols; inosine; guanosine; hypoxanthine; guanine; cytosol.
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INTRODUCTION |
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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).
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MATERIALS AND METHODS |
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Animals.
CFLP mice (3336 g) were from Charles River (Budapest, Hungary); male Wistar rats (270320 g), English shorthair guinea pigs (400450 g), and Syrian golden hamsters (80100 g) were from the breeding house of the University of Pécs (Hungary). New Zealand white rabbits (1.82.5 kg) were from a local rabbit farm and were maintained at our animal facility. The animals were kept at 2225°C room temperature, 5565% 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 KCl50 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 x 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 x g for 1 h. The supernatant containing the cytosolic fraction was decanted, whereas the pellet containing the microsomal fraction was resuspended in 150 mM KCl10 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 x g for 1 h. The filtrate was saved and diluted corresponding to 5 mg/ml unfiltered cytosol. The retentate was resuspended in 150 mM KCl50 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 KCl50 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 Duncans test or Students t-test. SPSS 8.0 for Windows (SPSS Inc.) was used for statistical analysis.
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RESULTS |
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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).
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DISCUSSION |
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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:
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: 36-72-536-218. E-mail: zoltan.gregus{at}aok.pte.hu.
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