ToxSci Advance Access originally published online on December 12, 2008
Toxicological Sciences 2009 107(2):309-311; doi:10.1093/toxsci/kfn257
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Published by Oxford University Press 2008.
Unraveling Arsenic—Glutathione Connections
Pharmacokinetics Branch, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, MD B143-1, U.S. Environmental Protection Agency, 109 Alexander Drive, Research Triangle Park, North Carolina 27711
1 For correspondence via fax: 919-541-1937. E-mail: thomas.david{at}epa.gov.
Received December 3, 2008; accepted December 5, 2008
The paper by Muniz Ortiz et al. (2009)
in this issue of Toxicological Sciences extends a long line of research on the role of glutathione (GSH) in the metabolism and toxicity of arsenic (As). These investigators used Drosophila as their model organism and the tools of classical and molecular genetics to investigate the genetic basis of variation in sensitivity to the toxic effects of inorganic As. By measuring eclosion (egg hatching) rates for different Drosophila strains cultured on arsenite-containing medium, they identified strains that differed manyfold in sensitivity to As. Using As-sensitive or -resistant strains, they showed an As-resistant phenotype to be related to the genotype for glutathione synthetase (GS). The GS gene encodes the enzyme that catalyzes the second and final step in a pathway that produces GSH from its constituent amino acids. Armed with this information, they used RNA interference to show that altered expression of the GS gene affects sensitivity to As in cultured cells and in flies. Taken in sum, these results emphasize the potential importance of GS genotype that determines the capacity to produce GSH in determining the phenotype for sensitivity to the toxic and carcinogenic effects of As.
To appreciate the significance of this research, it is useful to consider the context in which this research developed, our current knowledge of the linkages between As and GSH metabolism, and the prospects for future research to elucidate this relation.
LOOKING BACKWARD
The intertwining of the history of the tripeptide GSH (glutamylcysteinylglycine) and As began shortly after the discovery of GSH by Hopkins and coworkers in 1922. It was soon recognized that the cysteinyl moiety in GSH bound trivalent arsenicals and that GSH could protect cells and organisms against the toxic effects associated with As exposure. Research throughout the 1920s and 1930s on GSH-As interactions laid the groundwork for development of an antidote to the arsenical war gas Lewisite (chlorovinyldichloroarsine). In an intensive effort starting in 1939, British scientists used their fundamental understanding of the chemistry of interactions between trivalent As and GSH, a monothiol, to guide development of a safe and effective antidote for Lewisite. The dithiol compound British anti-Lewisite (2,3-dimercaptopropanol) was the product of this early attempt at rational drug design (Ord and Stocken, 2000).
LOOKING FOR LINKAGES
How are metabolism of As and GSH intertwined at the molecular level? Interest in the role of GSH in As toxicity has been renewed during the past two decades as investigators have focused on understanding the molecular processes that underlie the actions of As as a toxicant and a carcinogen. Because methylated metabolites of inorganic As have been recognized to be critical species that exert unique toxic and carcinogenic effects, emphasis has also been placed on understanding the role of GSH in the metabolism of inorganic As.
Current understanding of some interactions of As and GSH is summarized in Figure 1. The nexus of the interaction between GSH and As is the use of GSH as a reductant in the reaction that converts pentavalent As to trivalent As. The function of GSH as reductant of pentavalent As has been characterized in chemically defined systems and in intact cells (Delnomdedieu et al., 1994; Scott et al., 1993). Trivalent As formed in this reduction reaction is quickly complexed by GSH; hence, GSH-dependent reduction and complexation are inextricably linked in cells. Complexes of GSH with trivalent arsenicals (arsenite and methylarsonous acid) have been detected in biological systems (Kala et al., 2004; Suzuki et al., 2001). Glutathione disulfide (GSSG) generated from GSH during reduction of pentavalent As is reduced to GSH in a reaction catalyzed by glutathione reductase (GR). Notably, arsenite, methylarsonous acid and dimethylarsinous acid, and the complexes of GSH with arsenite and methylarsonous acid are inhibitors of GR (Styblo et al., 1997). Hence, there is a negative feedback loop involving reduction and complexation of trivalent As by GSH and the modulation of GR activity by trivalent arsenicals and their complexes with GSH. Altered regeneration of GSH from GSSG due to inhibition of GR may affect the intracellular GSH:GSSG ratio. The resulting shift in cellular redox status may be one of the perturbations that underlies actions of arsenicals as toxicants and carcinogens.
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GSH-dependent reduction of pentavalent As also produces species containing trivalent As that are preferred substrates for As (+3 oxidation state) methyltransferase (As3mt). This cytosolic enzyme catalyzes both S-adenosylmethionine (AdoMet)–dependent production of mono-, di-, and trimethylated arsenicals that contain pentavalent As and the reaction in which pentavalent As in methylated products is reduced to trivalency (Thomas et al., 2007
). Although the activity of As3mt requires a physiological dithiol-containing reductant (e.g., thioredoxin [Tx]), its activity is not dependent on the presence of GSH. GSH does not substitute for dithiol reductants in As3mt-catalyzed reactions; however, its presence in in vitro assays containing recombinant As3mt, Tx, AdoMet, and arsenite has an overall stimulatory effect on the formation of mono- and dimethylated arsenicals (Waters et al., 2004). By comparison, the addition of GSSG to this in vitro assay system lowers the As3mt-catalyzed rate of formation of methylated products from arsenite (W. Xing, B. Adain and D. J. Thomas, unpublished data). Recent work with recombinant As3mt and biotinylated GSH or GSSG shows that each peptide binds to the enzyme, suggesting a molecular basis for regulation of catalytic activity (P. Seales and D. J. Thomas, unpublished data). Thus, one could postulate that the oxidation of GSH to GSSG that accompanies the reduction of pentavalent As would alter the GSH:GSSG ratio so that the activity of As3mt would be altered.
An unsettled aspect of this scheme for interactions between GSH and As are the functions and fates of As-GSH complexes. It has been suggested that complexation of arsenite with GSH is the initial step in a reaction scheme catalyzed by As3mt which produces in turn mono- and dimethylated As-GSH complexes (Hayakawa et al., 2005). Notably, this postulated scheme for sequential methylation of trivalent As-GSH complexes does not require oxidation of trivalent As to accompany donation of a methyl group from AdoMet to the arsenical substrate. Because the presence of GSH is not required for As3mt to function catalytically in As methylation reactions, As-GSH complexes cannot be exclusively used as substrates in this reaction. However, these complexes could be substrates in the cellular environment where high concentrations for GSH could favor their formation and persistence. Discerning the relative contribution of As-GSH complexes as substrates for As3mt in the cellular environment is a formidable technical problem which awaits development of better methods to characterize As-binding proteins and peptides in intact cells. The formation of As-GSH complexes may also be a step in the pathway for efflux of arsenicals from cells. It has been suggested that GSH transferases, especially glutathione transferase P1 (GSTP1), catalyze formation of As-GSH complexes which are preferred substrates for the ATP-binding cassette membrane transporters which mediate efflux from cells. For example, in vesicles prepared from a human lung cell line that overexpresses multidrug resistance protein 1 (MRP1), the efflux of arsenite depends on the presence of GSH (Leslie et al., 2004). Colocalization of GSTP1 with MRP1 could control the rate of formation of complexes and the rate of efflux. Although this is an intriguing hypothesis, there are no data concerning the kinetics of formation of As-GSH complexes in GSTP1-catalyzed reactions.
LOOKING FORWARD
The genome-based approaches used by Muniz Ortiz and associates allow evaluation of the significance of a single gene in context of an organism's response to exposure of As. These approaches are exceptionally powerful in relatively simple and well-characterized organisms like Drosophila, allowing studies of genotype-phenotype relations that would be more difficult or impossible in other organisms. Paradoxically, their findings of a critical role for GS in the fly's response to As may be valuable because it directs our attention away from the reaction catalyzed by glutamylcysteine ligase, the first and apparently rate-limiting step in GSH synthesis. Typically, the rate-limiting step in a biosynthetic pathway is assumed to be the site at which a critical influence might be exerted. Knowing that the GS genotype has an effect on response to As suggests that it would be of value to examine interindividual variation in GS gene structure and expression in humans. Translated from Drosophila to humans, these findings pose the question of whether some of the variation among individuals seen in response to chronic exposure to inorganic As be related to variation in GS gene structure and expression. As a practical matter, additional information on the relation between GS genotype and the phenotype for response to As may help to reduce some of the uncertainties in the risk assessment for this metalloid and assist regulators in their task of protecting public health.
NOTES
Disclaimer: This article has been reviewed in accordance with the policy of the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
REFERENCES
Delnomdedieu M, Basti MM, Styblo M, Otvos JD, Thomas DJ. Complexation of arsenic species in rabbit erythrocytes. Chem. Res. Toxicol. (1994) 7:621–627.[CrossRef][Web of Science][Medline]
Hayakawa T, Kobayashi Y, Cui X, Hirano S. A new metabolic pathway of arsenite: arsenic-glutathione complexes are substrates for human arsenic methyltransferase Cyt19. Arch. Toxicol. (2005) 79:183–191.[CrossRef][Web of Science][Medline]
Kala SV, Kala G, Prater CI, Sartorelli AC, Lieberman MW. Formation and urinary excretion of arsenic triglutathione and methylarsenic diglutathione. Chem. Res. Toxicol. (2004) 17:243–249.[CrossRef][Web of Science][Medline]
Leslie EM, Haimeur A, Waalkes MP. Arsenic transport by the human multidrug resistance protein 1 (MRP1/ABCC1). Evidence that a triglutathione conjugate is required. J. Biol. Chem. (2004) 279:32700–32708.
Muniz Ortiz J, Opoka R, Kane D, Cartwright IL. Investigating arsenic susceptibility from a genetic perspective in Drosophila reveals a key role for glutathione synthetase. Toxicol. Sci. (2009) (in press).
Ord MG, Stocken LA. A contribution to chemical defence in World War II. Trends Biochem. Sci. (2000) 25:253–256.[CrossRef][Web of Science][Medline]
Scott N, Hatlelid KM, MacKenzie NE, Carter DE. Reactions of arsenic(III) and arsenic(V) species with glutathione. Chem. Res. Toxicol (1993) 6:102–106.[CrossRef][Web of Science][Medline]
Styblo M, Serves SV, Cullen WR, Thomas DJ. Comparative inhibition of yeast glutathione reductase by arsenicals and arsenothiols. Chem. Res. Toxicol. (1997) 10:27–33.[CrossRef][Web of Science][Medline]
Suzuki KT, Tomita T, Ogra Y, Ohmichi M. Glutathione-conjugated arsenics in the potential hepato-enteric circulation in rats. Chem. Res. Toxicol. (2001) 14:1604–1611.[CrossRef][Web of Science][Medline]
Thomas DJ, Li J, Waters SB, Xing W, Adair BM, Drobna Z, Devesa V, Styblo M. Arsenic (+3 oxidation state) methyltransferase and the methylation of arsenicals. Exp. Biol. Med. (2007) 232:3–13.
Waters SB, Devesa V, Del Razo LM, Styblo M, Thomas DJ. Endogenous reductants support the catalytic function of recombinant rat cyt19, an arsenic methyltransferase. Chem. Res. Toxicol. (2004) 17:404–409.[CrossRef][Web of Science][Medline]
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