Abstract: The findings of the accompanying paper (Nemeti and Gregus, Toxicol. Sci. 70, 4-12) indicate that the arsenate (As(V)) reductase activity of rat liver cytosol is due to an SH enzyme that uses phosphate (or its analogue, arsenate, As(V)) and a purine nucleoside (guanosine or inosine) as substrates. Purine nucleoside phosphorylase (PNP) is such an enzyme. It catalyzes the phosphorolytic cleavage of 6-oxopurine nucleosides according to the following scheme: guanosine (or inosine) + phosphate - guanine (or hypoxanthine) + ribose-1-phosphate. Therefore, we have tested the hypothesis that PNP is responsible for the thiol- and purine nucleoside-dependent reduction of As(V) to As(III) by rat liver cytosol. As(III) formed from As(V) was quantified by HPLC-hydride generation-atomic fluorescence spectrometry analysis of the deproteinized incubates. The following findings support the conclusion that PNP reduces As(V) to As(III), using As(V) instead of phosphate in the reaction above: (1) Specific PNP inhibitors (CI-1000, BCX-1777) at a concentration of 1 muM completely inhibited cytosolic As(V) reductase activity. (2) During anion-exchange chromatography of cytosolic proteins, PNP activity perfectly coeluted with the As(V) reductase activity, suggesting that both activities belong to the same protein. (3) PNP purified from calf spleen catalyzed reduction of As(V) to As(III) in the presence of dithiothreitol (DTT) and a 6-oxopurine nucleoside (guanosine or inosine). (4) As(V) reductase activity of purified PNP, like the cytosolic As(V) reductase activity, was inhibited by phosphate (a substrate of PNP alternative to As(V)), guanine and hypoxanthine (products of PNP favoring the reverse reaction), mercurial thiol reagents (nonspecific inhibitors of PNP), as well as CI-1000 and BCX-1777 (specific PNP inhibitors). Thus, PNP appears to be responsible for the As(V) reductase activity of rat liver cytosol in the presence of DTT. Further research should clarify the mechanism and the in vivo significance of PNP-catalyzed reduction of As(V) to As(III).

Abstract: Arsenate (As(V)) is biotransformed into the more toxic arsenite (As(III)) and monomethylarsonous acid (MMAs(III)), but it is unknown how to decrease production of these harmful metabolites. We investigated the effects of foscarnet and fosfomycin, drugs interacting with the phosphate transporter, on biotransformation of As(V), an analog of inorganic phosphate. The effects of entacapone, an inhibitor of catechol-O-methyl transferase (COMT), and nitrous oxide, an inactivator of methylcobalamin, were also tested on the formation of MMAs(III) from As(III) in order to clarify the role of COMT and methylcobalamin in biomethylation of As(III). Arsenic in bile and urine of control and treated rats receiving As(V) or As(III) was speciated by HPLC-HG-AFS. In As(V)-injected rats, foscarnet, but not fosfomycin, increased the urinary excretion of As(V) and decreased the biliary and urinary excretion of As(III) as well as biliary excretion of MMAs(III). In As(III)-injected rats, however, foscarnet failed to influence the excretion of As(III) and its metabolites, suggesting that this drug inhibits the hepatic uptake and renal reabsorption of As(V), thereby decreasing formation of As(III) and MMAs(III) from As(V). Entacapone or nitrous oxide pretreatment slightly or not at all influenced the biliary excretion of MMAs(III) and urinary excretion of dimethylarsinic acid (DMAs(V)) in As(III)-injected rats. In contrast, periodate-oxidized adenosine, an inhibitor of S-adenosylmethionine-dependent methyltransferases, nearly abolished appearance of methylated arsenic metabolites in bile and urine. Thus, foscarnet facilitates urinary clearance of As(V) and decreases formation of toxic As(III) and MMAs(III), indicating that this drug may be used to promote elimination and counter toxification of As(V). Because entacapone and nitrous oxide influenced the excretion of MMAs(III) and DMAs(V) negligibly, neither COMT nor methylcobalamin appears to be involved in arsenic methylation in rats.

Abstract: In rats exposed to arsenite (As(III)) or arsenate (As(V)), the biliary excretion of arsenic depends completely on availability of hepatic glutathione, suggesting that both As-III and As(V) are transported into bile in thiol-reactive trivalent form (Gyurasics et al. [1991], Biochem. Pharmacol. 42, 465-368). To test this hypothesis, the bile and urine of bile duct-cannulated rats injected with As(III) or As(V) (50 µmol/kg. iv) were collected periodically for 2 h and analyzed for arsenic metabolites by HPLC-hydride generation-atomic fluorescence spectrometry. Arsenic was excreted predominantly into bile in As(III)-injected rats, but the urine was the main route of excretion in As(V)-exposed rats. Injected Asm was excreted in urine practically unchanged, whereas both As(V) and As(III) appeared in urine after administration of As(V). Irrespective of the arsenical administered, the bile contained 2 main arsenic species, namely As(III) and a hitherto unidentified metabolite. Formation of this metabolite could be prevented by pretreatment of the rats with the methylation inhibitor periodate-oxidized adenosine, indicating that it is a methylated arsenic compound. This metabolite could be converted in vitro into monomethylarsonic acid (MMAs(V)) by oxidation, whereas synthetic MAAs(V) could be converted into the unknown metabolite by reduction. Consequently, this biliary metabolite of both As(III) and As(V) is monomethylarsonous acid (MMAs(III)), a long-hypothesized, but never identified, intermediate in the biotransformation of As(III) and As(V). Although MMAs(III) is thought to be formed from an oxidized precursor, rats injected with MMAs(V) did not excrete MMAs(III). in summary, the ingreanic arsenicals investigated are transported into bile exclusively in trivalent forms, namely as As(III) and MMAs(III), but are excreted in urine in both tri- and pentavalent forms. Identification of MMAs(III) is signified by the fact that this metabolite is more toxic than As(III) and As(V) and thus formation of MMAs(III) represents toxification of inorganic arsenic.

Abstract: Arsenate (As(V)) is a structural analogue of phosphate (P), yet its toxic effect is likely due to its reduction to the more toxic arsenite (As(III)), the mechanism of which is still unclear. Since mitochondria take up As(V) as they do Pi, they may reduce As(V) to As(III). To test this hypothesis isolated rat liver mitochondria were incubated with As(V), and the incubate was analyzed for As(V) and As(III) by HPLC-HG-AFS. Mitochondria rapidly reduced As(V) to As(III). Of the substrates supporting the citric acid cycle, glutamate enhanced the reduction most effectively. ADP increased, whereas AMP and ATP decreased, As(III) formation. These effects could be prevented by atractyloside. Electron transport inhibitors and uncouplers abolished As(III) formation, whereas ATP-synthase inhibitors almost completely inhibited it. Phosphate decreased As(III) formation in a concentration-dependent manner. Inhibitors of mitochondrial Pi-moving transporters abolished As(III) formation. Sulfate, sulfite, or thiosulfate that are transported by the dicarboxylate carrier caused partial inhibition. As(III) was recovered completely from the supernatant of the mitochondrial incubate, suggesting that mitochondria exported the formed As(III). Testing the effects on mitochondrial As(V) reduction of chemicals that are inhibitors or substrates of thioredoxin reductase failed to support the role of this enzyme in reduction of As(V). Depletion of mitochondrial glutathione impaired mitochondrial As(V) reducing activity but also diminished the respiratory control ratio. Upon solubilization of mitochondria, their As(V)-reducing activity was lost and was not recovered by addition of GSH and NADH or NADPH. Summarizing, mitochondria work as reactors: they take up As(V), rapidly reduce it, and export the formed As(III). Disruption of functional or structural integrity of mitochondria severely impairs biotransformation of As(V) into As(III) in this organelle. (C) 2002 Elsevier Science (USA).

Abstract: In most mammalian species, inorganic arsenicals are extensively biotransformed and excreted both in unchanged form and as metabolites. In the bile of fats receiving arsenate (As(V)) or arsenite (As(III)) we have identified monomethylarsonous acid (MMAs(III)), purportedly the most toxic metabolite of inorganic arsenic. As rats are not commonly accepted for studying arsenic metabolism, we carried out a comparative investigation on the excretion of As(V), As(III) and their metabolites in five animal species in order to determine whether they also form MMAs(III) from As(V) and As(III). Anaesthetised bile duct-cannulated rats, mice, hamsters, rabbits, and guinea pigs were injected with As(V) or As(III) (50 µmol/kg, i.v.) and their bile and urine was collected for 2 h. Arsenic in bile and urine was speciated by HPLC-hydride generation-atomic fluorescence spectrometry and the excretion rates of As(V), As(III), monomethylarsonic acid (MMAs(V)), MMAs(III) and dimethylarsinic acid (DMAs(V)) were quantified. All species injected with As(V) excreted arsenic preferentially into urine, whereas all animals receiving As(III), except rabbits, delivered more arsenic into bile than urine. Bile contained almost exclusively trivalent arsenic (i.e. As(III) and/or MMAs(III)), whereas As(V), As(III) and DMAs(V) appeared in urine. Except for guinea pigs, which do not methylate arsenic, the other species formed MMAs(III) and excreted it into bile. Having excreted as much as 8% of the dose of As(III) of As(V) in 2 h as MMAs(III), rats were by far the most efficient producers of this supertoxic metabolite. Thus, although the rat is not a good model for studying long-term arsenic disposition, this species appears especially valuable in studies on As(III) methyltransferase and in vivo formation of MMAs(III). (C) 2002 Elsevier Science Inc. All rights reserved.