Laboratory of Comparative Carcinogenesis, National Cancer Institute at NIEHS, 111 Alexander Drive, Research Triangle Park, North Carolina, 27709
ABSTRACT
The articles highlighted in this issue are this issue are "Purine Nucleoside Phosphorylase as a Cytosolic Arsenate Reductase" by Zoltán Gregus and Balázs Németi (pp. 412) and "Reduction of Arsenate to Arsenite in Hepatic Cytosol" by Balázs Németi and Zoltán Gregus (pp. 1319).
Environmental arsenic exposure is clearly a high priority issue to human populations around the world, including within the United States, and has well defined toxic and carcinogenic potential. The main source of arsenic exposure in most populations is drinking water, where inorganic forms of arsenic predominate (NRC, 1999). The inorganic forms of arsenic include the trivalent form, arsenite (AsIII), and the pentavalent form, arsenate (AsV). High levels of inorganic arsenic in contaminated drinking water can be found around the world, including Taiwan, China, Chile, Mexico, India, Bangladesh, as well as in several areas in the U.S. and Europe, and can lead to a variety of toxic manifestations (NRC, 1999
). Indeed, exposures in the United States to elevated levels of arsenic in the drinking water have been associated with an elevated risk of cancer (Lewis et al., 1999
). Inorganic arsenic in drinking water is often in the form of AsV. Environmental arsenic "contamination" is not always anthropogenic in nature, and natural deposits are very important in dictating drinking water levels (NRC, 1999
).
Although this metalloid shows well defined toxic manifestations, the mechanisms of arsenic toxicity, including carcinogenicity, are largely undefined. AsV can be easily absorbed and enters the body or cells via phosphate transport mechanisms (NRC, 1999; Csanaky and Gregus, 2001
). Upon entering the cell, AsV can interfere with cellular metabolism by replacing inorganic phosphate (Pi) in enzymatic reactions, including oxidative phosphorylation in mitochondria (NRC, 1999
). AsV can also be readily reduced to AsIII, a more toxic form of inorganic arsenic that shows high reactivity with cellular thiols. Unlike many toxic inorganics, arsenic undergoes conjugative metabolism (i.e., methylation) in cells, and this biotransformation is intimately linked to its toxicity in an as yet incompletely understood fashion. Thus, AsIII is a precursor for the various mono- and di-methylated arsenic metabolites (Thomas et al., 2001
), including methylarsonic acid (MMAV), methylarsonous acid (MMAIII), dimethylarsinic acid (DMAV), and dimethylarsinous acid (DMAIII). There is now good evidence that a methylated metabolite of arsenic may in fact be the ultimate toxic species (Petrick et al., 2000
; Styblo et al., 2000
; Thomas et al., 2001
). The reduction of AsV to AsIII is the critical first step in the metabolic fate of inorganic arsenate and has a decisive role in determining the elimination and the toxicity of inorganic arsenic. Thus, understanding the metabolism of inorganic arsenic is critical to defining the molecular mechanisms of arsenic toxicity and carcinogenicity (Goering et al., 1999
; Thomas et al., 2001
).
AsV reductase has been identified in microorganisms (Mukhopadhyay et al., 2000). However, the two highlight manuscripts, together with recent work from Aposhians group (Radabaugh and Aposhian, 2000
; Wildfang et al., 2001
; Radabaugh et al., 2002
), have identified and defined the intracellular distribution of AsV reductase in mammalian species and provide an important advance in our understanding of the metabolism of this important environmental toxicant. In experimental work that provides the foundation of the highlighted papers, Greguss group recently observed that mitochondria isolated from rat liver are capable of reducing AsV to AsIII in a manner dependent on both the functional status and structural integrity of these organelles and was sensitive to inhibitors of oxidative phosphorylation (Németi and Gregus, 2002
). Unfortunately, solubilization of mitochondria abolished their AsV reductase activity, even in the presence of cellular reductants like GSH and NADPH, precluding isolation of a mitochondrial AsV reductase (Németi and Gregus, 2002
). Thus, subcellular fractions besides mitochondria are considered for AsV reductase activity and identification in the present work. In the human liver cytosol, AsV reductase activity has recently been detected by using [73As] arsenate coupled with a HPLC system and a radioisotope detector (Radabaugh and Aposhian, 2000
). This human liver cytosolic AsV reductase has also been partially purified by DEAE-Sephasel and Sephacryl S-200 HR chromatography, and its molecular mass is approximately 72kDa (Radabaugh and Aposhian, 2000
).
The highlighted articles describe in detail the characterization of a rat cytosolic AsV reductase and the identification of this AsV reductase as a Purine Nucleoside Phosphorylase (PNP), using stepwise classical biochemical and toxicological approaches. To quantify the formation of AsIII, the authors used a sensitive HPLC method based on hydride generation-atomic fluorescence spectrometry optimized for AsIII detection that they developed earlier (Gregus et al., 2000). To extend their work on AsV reductase in mitochondria (Németi and Gregus, 2002
), the postmitochondria fraction of rat liver was used, and AsV reductase activity was clearly demonstrated. Indeed, AsV reductase activity was found in the cytosol, but it was not present in the microsomes. Because cytosolic formation of AsIII from AsV requires the presence of a thiol compound, they further tested a number of thiols for their ability to enhance the rate of AsIII reduction. Of the ten common thiols tested, 2-ME, DTT, DMP, and DMPS were very efficient in assisting AsV reduction, while GSH, which had previously been thought to be a critical thiol in AsV reduction, was ineffective. This agrees with the findings of others in human liver cytosol preparations, where GSH has little effect on AsV reduction (Radabaugh and Aposhian, 2000
). In searching for the cofactor required for AsV reduction, it was found in the highlighted work that oxidized pyridine nucleotide (NAD+ or NADP+), but not their reduced forms, increased AsIII formation. The fact that purine nucleotide derivatives, such as AMP and GMP, also increased AsV reduction led the authors of the highlighted work to examine the effects of the nucleosides inosine or guanosine on AsV reductase activity. Surprisingly, both nucleosides increased the AsV reductase activity 80100 fold, while purine bases (hypoxanthine or guanine) decreased it by 8090%. Additional experiments showed that elimination of endogenous purine nucleosides also blocked AsV reduction in the cytosol. The liver cytosols of mice, hamsters, guinea pigs and rabbits also exhibited significant AsV reductase activity, which was dramatically enhanced by inosine. The work from Aposhians group has shown that among 17 species of nonhuman primates, all show significant AsV reductase activity in liver cytosol, even though most (13) of them lack arsenic methyltransferase, an enzyme critical to subsequent arsenic conjugation (Wildfang et al., 2001
). Together these findings indicate that liver cytosolic AsV reductase is ubiquitous among mammalian species and likely plays a pivotal role in the toxicity of inorganic arsenic.
The observation that cytosolic AsV reductase activity was greatly enhanced in the presence of 6-oxopurine nucleosides and was inhibited by 6-oxopurine bases, inorganic phosphate and mercurial thiol reagents, prompted Greguss group to conclude that the reductase may have functionally important thiol groups, may accept inosine or guanosine as well as Pi as substrates, and may yield hypoxanthine or guanine as products. The enzyme that fits the deduced characteristics is known as Purine Nucleoside Phosphorylase (PNP). Thus, in the second highlighted manuscript the authors test the hypothesis that PNP in rat liver cytosol could indeed work as an AsV reductase. They obtain three lines of evidence to support this hypothesis. First, specific and potent PNP inhibitors, CI-1000 and BCX-1777, completely block cytosolic AsV reductase activity. Second, anion-exchange chromatography of cytosolic proteins demonstrates that PNP activity precisely co-eluted with AsV reductase activity, suggesting that both activities belong to the same protein. Finally, purified PNP obtained from calf spleen catalyzed the ex vivo reduction of AsV to AsIII in a manner very similar to AsV reductase purified from rat liver. Thus, this thiol-dependent cytosolic AsV reductase was identified as PNPan enzyme that normally uses phosphate to cleave purine nucleosides (inosine or guanosine) into bases (hypoxanthine or guanine) and ribose-1-phosphate, but that can also act on AsV instead of phosphate to produce AsIII.
The conclusion that PNP is a cytosolic arsenate reductase was supported by a separate study by Radabaugh et al. (2002). By using ion exchange, molecular exclusion, hydroxyapatite chromagraphy, preparative isoelectric focusing, and electrophoresis, a human liver cytosolic AsV reductase was purified stepwise to obtain two bands on SDS-B-mercaptoethanol-PAGE. One of these bands was a 34 kDa protein. This band was excised from the gel and sequencing by LC-MS/MS, followed by sequence analyses against the OWL database SWISS-PROT with PIR. Based on amino acid homology and other properties, they also reached the conclusion that in human liver AsV reductase and human PNP are identical proteins (Radabaugh et al., 2002
). Thus, both this work (Radabaugh et al., 2002
) and the series of highlighted papers point toward PNP as a potentially important initial enzyme in the metabolism of inorganic arsenic, which by reducing AsV produces the much more toxic AsIII species and thereby plays a critical role in the toxicity of inorganic arsenic in mammalian systems.
From this series of work it becomes clear that the enzyme that may be responsible for first critical step in inorganic arsenic metabolism, namely AsV reduction, is a normally occurring enzyme with a clear intentional function that has little to do with arsenic metabolism. It is difficult to imagine that this nucleoside phosphorylase enzyme actually evolved to deal with arsenic in any kind of specific fashion. From this, it must be deduced that the ability of PNP to reduce certain forms of the metalloid is fortuitous rather than intentional. Thus, at least this initial step of arsenic metabolism may be mediated through a normally occurring enzyme that in all likelihood was designed for a totally different function. It would appear that arsenic is reduced through its mimicry of endogenous substances, in this case perhaps Pi, that are required for enzymatic activity in what is otherwise a nonproductive use of the PNP. If the first critical step in cellular metabolism of arsenic is through a fortuitous rather than intentional event, it is clearly possible that other aspects of arsenic metabolism may be similarly unintentional. This includes, potentially, the conjugative methylation reactions and the accompanying reductions that follow the initial AsV reduction. In this regard, there is a striking biodiversity in the activity of liver arsenic methyltransferases, and these methyltransferases can be quite active in one species but completely absent in very closely related species, as with humans and many nonhuman primates (Goering et al., 1999; Wildfang et al., 2001
). The fact that nonenzymatic methylation of arsenic can occur in some cases indicates great variability in arsenic metabolism (Goering et al., 1999
; Wildfang et al., 2001
). Thus, as this important series of work points out, arsenic metabolism may occur through enzymes that are responsible for entirely different functions, and this needs to be kept in clear focus and investigated in the future studies. In addition, it should be considered that inorganic arsenic, as part of its toxic manifestations, may place a metabolic "burden" on the cell by diverting enzymes from their normal functions. Thus, the highlighted work may contribute not only to our practical knowledge of arsenic metabolism, but also to the conceptual nature of how fortuitous toxicant metabolism may eventually affect toxicity. Nevertheless, further research is warranted to clarify the mechanism of PNP-catalyzed AsV reduction and the in vivo role of PNP in forming the toxic AsIII from the less toxic and more environmentally prevalent AsV.
NOTES
1 For correspondence via fax: (919) 541-3970. E-mail: waalkes{at}niehs.nih.gov.
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