Pharmac. Ther. Vol.47, pp. 359-370, 1990 Printed in Great Britain.All rights reserved

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Specialist Subject Editor: K. D. "FEW

ROLE OF THE GLUTATHIONE-GLUTATHIONE PEROXIDASE CYCLE IN THE CYTOTOXICITY OF THE ANTICANCER QUINONES JAMES H. DOROSHOW, STEVEN AKMAN, FONG-FONG CHU a n d SXEVEN ESWORTHY Department of Medical Oncology and Therapeutics Research, City of Hope National Medical Center, Duarte, CA 91010, U.S.A. Abstract--Recent studies have suggested that the selenoenzymeglutathione peroxidase, in the presence of reducing equivalents from the tripeptide glutathione, is responsible for detoxifying hydrogen peroxide and lipid hydroperoxides generated as a consequence of the cyclic reduction and oxidation of quinonecontaining anticancer agents including doxorubicin, daunorubicin, mitomycin C, diaziquone, and menadione. Alterations in the intracellular levelsof glutathione peroxidase or glutathione can significantly affect the activity of these drugs against human tumor cells and the expression of their normal tissue toxicity, especially with respect to the heart. Furthermore, augmentation of the glutathione peroxidase pathway appears to render certain human tumor cells relatively resistant to the anticancer quinones; therefore, the glutathione peroxidase system may, at least in part, modulate certain forms of acquired drug resistance in man. Thus, the glutathione peroxidase cycle appears to play a central role in maintaining intracellular peroxide homeostasis during quinone-induced oxidative stress.

CONTENTS 1. 2. 3. 4.

Oxygen Radical Production by Anticancer Quinones Sites of Drug-lnduced Oxygen Radical Toxicity The Glutathione-Glutathione Peroxidase Cycle Role of the Glutathione-Glutathione Peroxidase Cycle in the Antitumor Activity of the Anticancer Quinones 5. Role of the Glutathione~Glutathione Peroxidase Cycle in Tumor Cell Resistance to the Anticancer Quinones 6. Role of the Glutathione~Glutathione Peroxidase Cycle in the Cardiac Toxicity of the Anticancer Quinones 7. Summary Acknowledgements References

1. OXYGEN RADICAL PRODUCTION BY ANTICANCER QUINONES Quinone-containing drugs, including the benzanthraquinones doxorubicin and daunorubicin; the benzoquinone [2,5-bis(1-aziridinyl)-3,6-dioxo-1,4-cyclohexadiene-1,4-diyl]bis[carbamicacid] diethyl ester, diaziquone (AZQ); and mitomycin C are active antitumor agents that are frequently employed in the treatment of hematologic malignancies as well as carcinomas of the breast, lung, and ovary (Young et al., 1981; Bender et al., 1983; Crooke and Bradner, 1976). These drugs are electrochemically active; and recent evidence has related both their antitumor activity and normal tissue toxicity, especially the chronic cardiac toxicity of the anthracycline antibiotics (Young et al., 1981; Bachur et al., 1978; Doroshow, 1983b; Rajagopalan et al., 1988), to their participation in cyclical oxidation-reduction reactions which produce a flux of reactive oxygen species wr 47~3~:

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after drug administration (Bachur et al., 1979; Doroshow, 1986a, b; Sinha et al., 1987b). The first section of this chapter will review the evidence supporting the oxidative metabolism of the anticancer quinones in vitro and in vivo. In subsequent sections, the role of the glutathione-glutathione peroxidase cycle in mediating the antitumor activity and cardiac toxicity of these drugs will be evaluated. The benzanthraquinones doxorubicin and daunorubicin are easily reduced by one electron to their semiquinone free radical intermediates in an electrochemical cell. Land and colleagues, using pulse radiolysis, observed a reduction potential for doxorubicin of - 0 . 3 2 V versus a saturated calomel electrode (Land et al., 1983). Lown et al. examined a series of anthracyclines by cyclic voltammetry; the half-wave potential of doxorubicin in aqueous solution was - 0 . 5 8 V (Lown et al., 1979). The presence of the quinone moiety is of critical importance for the oxidation-reduction (redox) properties of the anthra-

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cycline antibiotics; the anthracycline analogue 5iminodaunorubicin, in which an imine group is substituted for the C5 carbonyl, requires - 0 . 0 8 V more for one electron reduction (Lown et al., 1979). Furthermore, once reduced, this anthracycline becomes substantially more, rather than less, difficult to oxidize in the presence of molecular oxygen, and thus does not participate in active redox cycling (Lown et al., 1979). In contrast, the benzoquinone, AZQ, is more readily reduced electrochemically than doxorubicin ( - 0 . 1 6 V versus - 0 . 2 8 V) (Svingen and Powis, 1981: Gutierrez et al., 1984). AZQ can also be reduced non-enzymatically to its semiquinone by reducing agents such as L-cysteine, NADPH, or L-ascorbate at a faster rate than doxorubicin (Gutierrez et al., 1984, 1985; Szmigiero and Kohn, 1984; Gutierrez, 1988). Fully reduced AZQ, like the hydroquinone moiety of doxorubicin, is also capable of autooxidizing to its semiquinone (Nguyen et al., 1988; Pietronigro et al., 1979). Under specific experimental conditions in vitro, doxorubicin can participate in redox reactions as an oxidant rather than a reductant. Doxorubicin may form a specific complex with Fe 3+ which is itself capable of transferring electrons to the metal that has been bound (Sugioka and Nakano, 1982; Zweier, 1983; Gutteridge, 1984; Gianni et al., 1985; Zweier et al., 1986). The proposed mechanism of the doxorubicin-mediated reduction of Fe 3~ involves the oxidation of the ketol side chain of doxorubicin at C9, producing a semiquinone drug intermediate, followed by electron transfer to the Fe 3+ (Gianni et al., 1985, 1988; Zweier et al., 1986). Doxorubicin can also serve as a proton donor to such basic species as the superoxide anion in an aprotic environment (Nakazawa et al., 1985). The major oxidant for the doxorubicin semiquinone in aerobic solution, however, is molecular oxygen. As discussed below, all of the redox activities of the anthracycline antibiotics may be important for their cytotoxic effects. The suggestion that the participation of anticancer quinones in redox reactions might contribute to their cytotoxicity was initially given impetus by the observation that the clinically important quinone-containing anticancer agents served as excellent substrates for one-electron reduction by flavoproteins in various subcellular fractions (Handa and Sato, 1975; Bachur et al., 1977, 1978; Thayer, 1977; Pan et al., 1981). In the presence of liver microsomes, as well as cardiac sarcosomes, benzanthraquinones (such as the anthyracycline antibiotics), N-heterocyclic congeners (e.g. streptonigrin), and benzoquinones (AZQ) stimulate N A D P H oxidation (Doroshow and Hochstein, 1982). The K m values for oxyradical production with either NADPH or the anticancer quinones in liver microsomal systems range from 40~400/~M. N A D P H cytochrome P-450 reductase has been identified as the principal microsomal enzyme responsible for the one-electron reduction of the anticancer quinones (Bachur et al., 1979; Doroshow, 1983a). It has also been demonstrated that doxorubicin stimulates the oxidation of N A D H as well as N A D P H by other, NADH-dependent flavin dehydrogenases including xanthine oxidase (Pan and Bachur, 1980) and mitochondrial N A D H dehydrogenase (Thayer, 1977; Doroshow, 1983b). The ease of the enzymatic re-

duction of the anthracyclines correlates well with their one-electron reduction potential (Lown et al., 1979). Anthracyclines which do not contain the quinone moiety, and thus are difficult to reduce electrochemically (such as 5-iminodaunorubicin), do not stimulate N A D H or NADPH oxidation (Doroshow, 1983a; Peters et al., 1986). In addition to microsomes and mitochondria, subcellular fractions of both mammalian tumor cells and myocytes that have been identified as sites of quinone drug reduction include the cytoplasm (Doroshow, 1983a) and nuclei (Bachur et al., 1978, 1982). The consequences of quinone-stimulated NAD(P)H oxidation have been studied in detail. Oneelectron reduction of doxorubicin or daunorubicin leads to the cleavage of the glycosidic bond between the chromophore of doxorubicin and its daunosamine sugar, producing the doxorubicin or daunorubicin aglycone (Bachur, 1979). Quinone reduction is also associated with the appearance of a paramagnetic species that has been identified as a semiquinone free radical intermediate (Bachur et al., 1977; Kalyanaraman et al., 1980). Semiquinone free radicals of half-reduced AZQ, mitomycin C, and streptonigrin have also been demonstrated (Lown, 1983; Gutierrez et al., 1983, 1984; Pan et al., 1984). In an aqueous environment under aerobic conditions, however, the benzanthrasemiquinone radical is difficult to detect because it reacts rapidly with molecular oxygen to form the superoxide anion (Goodman and Hochstein, 1977; Bachur, 1979; Kalyanaraman et al., 1980). Doxorubicin and related anticancer quinones have been described as 'electron shuttles', capable of catalytically enhancing the flow of electrons from NAD(P)H to molecular oxygen (Bachur et al., 1978; Doroshow and Hochstein, 1982). Although the superoxide anion thus formed is not highly reactive in a protonated aqueous environment, it is rapidly converted to hydrogen peroxide by the enzyme superoxide dismutase with a second order rate constant of ~-2 x 109tool ~sec ~ (Klug-Roth et al., 1973; Fielder et al., 1974). Hydrogen peroxide may be reduced further by transition metals, such as Fe 2+, to form the hydroxyl free radical and the hydroxide anion (the Fenton reaction) (Koppenol, 1983). Superoxide anion can also function as a reducing agent capable of maintaining iron that has been oxidized by reaction with hydrogen peroxide in the fully reduced, Fe z+ , state. This so called 'iron-catalyzed Haber Weiss reaction' allows electrons to pass from the relatively slowly reactive superoxide anion through hydrogen peroxide to create a highly reactive oxidizing species with the chemical characteristics of the hydroxyl radical (Czapski, 1984). The hydroxyl free radical is amongst the most potent oxidants known with a rate constant of -~10a°mol l sec -~ (Czapski, 1984). Thus, under aerobic conditions, anticancer quinones may be cyclically reduced and oxidized while electrons flow from NAD(P)H or hypoxanthine through superoxide to the hydroxyl radical. Furthermore, under conditions of relative hypoxia, semiquinone free radical species may directly reduce hydrogen peroxide to the hydroxyl radical (Winterbourn, 1981; Gutteridge et al., 1984). Complexes of Fe 3+ and an anthracycline antibiotic in the presence of chemical, rather than enzymatic,

Glutathione peroxidase and quinone cytotoxicity reduction may also reduce molecular oxygen to superoxide. Doxorubicin forms a high-affinity chelate with Fe 3+ (May et al., 1980) which has been studied in detail by Myers, Zweier, and colleagues (Myers et al., 1982, 1987; Eliot et al., 1984; Gianni et al., 1985; Zweier et al., 1986). Molecules of Fe 3+ apparently bind the doxorubicin chromophore at the C11 and C12 phenolic and quinone oxygens (Muindi et al., 1985). Electrons originating from the ketol-containing side chain at C9 can reduce the bound Fe 3+ to Fe z+ which will, in turn, reduce molecular oxygen to form the superoxide anion, and subsequently, the hydroxyl radical (Zweier et al., 1986; Gianni et al., 1988). Other anthracyclines which do not possess the ketol side chain, such as daunorubicin, require thiols or other reducing species to generate reactive oxygen intermediates from the drug-iron complex. The daunorubicin analog, 5-iminodaunorubicin, which avidly binds Fe 3+, is barely capable of reducing the bound iron by an intramolecular electron donation, and thus produces significantly less superoxide and hydroxyl radical than doxorubicin (Myers et al., 1987). Because of the potent oxidizing power of the hydroxyl radical (Cohen, 1978; Ward et al., 1983), recent studies have examined the ability of various anticancer quinones to generate this oxidant in subcellular fractions and in whole cells. Komiyama and associates, using electron spin resonance spectroscopy, demonstrated the formation of an hydroxyl radical spin adduct after treatment of purified NADPH cytochrome P-450 reductase with doxorubicin, related anthracyclines, and mitomycin C (Komiyama et al., 1982). Our laboratory, as well as several others, has observed hydroxyl radical production by either spin trapping techniques or the liberation of methane from dimethyl sulfoxide with doxorubicin-treated cardiac submitochondrial particles, purified cardiac mitochondrial N A D H dehydrogenase, and cardiac sarcotubular membranes (Doroshow and Davies, 1986; Doroshow, 1983b, 1988). More recently, we have found that doxorubicin, mitomycin C and AZQ all stimulated hydroxyl radical production from intact Ehrlich ascites carcinoma cells at the same time that several oxygen radical scavengers protected these cells from the cytotoxicity of each of the anticancer quinones (Doroshow, 1986b). Similar findings for the human MCF-7 breast cancer line have also been reported (Doroshow, 1986a). Sinha and colleagues have observed doxorubicin-stimulated hydroxyl radical production using spin trapping techniques in intact MCF-7 cells and in subcellular fractions; moreover, a doxorubicin-resistant MCF-7 subline produced significantly less hydroxyl radical than the parental line, suggesting that hydroxyl radical production and tumor cell cytotoxicity are related (Sinha et al., 1987a, b, 1989). Recently, Cervantes et al. observed protection from doxorubicin toxicity by hydroxyl radical scavengers in human ovarian carcinoma cells in t~itro, both in wild-type cells and in a multidrugresistant variant (Cervantes et al., 1988). In contrast, Keizer and associates did not observe protection from doxorubicin toxicity in a Chinese hamster ovary cell variant resistant to high concentrations of molecular oxygen through the over-expression of catalase

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and superoxide dismutase (Keizer et al., 1988); however, the wild-type cells studied by these investigators already contained relatively high levels of antioxidant enzymes. A significant, and to date unresolved, question pertaining to the stimulation of hydroxyl radical production in vivo by the anticancer quinones relates to the availability of transition metals (particularly iron) in an intracellular form capable of participating in drug-enhanced oxygen radical formation. Mammalian cells routinely package iron intracellularly as ferritin macromolecules which store the metal within a protein shell unavailable under normal circumstances for reaction with the aqueous environment of the cytoplasm (Aisen and Listowsky, 1980). A few recent studies have begun to reveal how reduced iron might become available to stimulate intracellular hydroxyl radical formation. Demant has observed that doxorubicin can abstract iron directly from ferritin in solution (Demant, 1984); however, given the plasma pharmacokinetics of doxorubicin (in which micromolar drug concentrations are available for usually less than 1-3 hr), the time course involved (hours rather than minutes) makes direct abstraction of iron appear to be an unlikely event in vivo. However, Thomas and Aust as well as Winterbourn and colleagues have recently demonstrated enzymatically-induced iron release by the redox cycling herbicide paraquat and by doxorubicin (Thomas and Aust, 1986; Vile and Winterbourn, 1988; Monteiro et al., 1989); these studies confirm the findings of Biemond et al. that superoxide can act as a reducing agent to liberate iron from the ferritin shell (Biemond et al., 1984). Definitive evidence that such reactions occur intracellularly, however, is not available and the validity of this mechanism must be inferred from the numerous investigations demonstrating protection from the cardiac toxicity of doxorubicin by ICRF187 (which spontaneously hydrolyzes to form the potent iron chelator ICRF-198) and from the data of Rajagopalan et al. who observed suppression of doxorubicin-stimulated hydroxyl radical production in rat heart by ICRF-187 (Herman and Ferrans, 1981; Herman et al., 1988; Speyer et al., 1988a, b; Rajagopalan et al., 1988).

2. SITES OF D R U G - I N D U C E D OXYGEN RADICAL TOXICITY The hydroxyl radical is a strong oxidizing agent and electrophile. It attacks unsaturated bonds in lipids to produce lipid alkyl or alkylhydroperoxy free radicals. In the presence of oxygen, free radical chain reactions can propagate to produce extensive membrane lipid peroxidation (Tien et al., 1982). Myers et al. initially observed that the administration of doxorubicin to mice resulted in cardiac malondialdehyde production which could be abrogated by pretreatment with ~-tocopherol (Myers et al., 1977). Doxorubicin-stimulated membrane lipid peroxidation has subsequently been confirmed in heart and liver microsomes (Goodman and Hochstein, 1977; Mimnaugh et al., 1982, 1983), and in mitochondria (Julicher et al., 1986) and isolated nuclei (Mimnaugh et al., 1985). Oxygen radical scavengers inhibit lipid peroxi-

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dation mediated by doxorubicin and related anthracyclines (Myers et al., 1977). 5-Iminodaunorubicin, which does not generate a flux of reactive oxygen metabolites (Doroshow, 1983a, b; Davies et al., 1983), also does not stimulate lipid peroxidation (Mimnaugh et al., 1982, 1983). Moreover, 5iminodaunorubicin actually inhibits doxorubicinmediated lipid peroxidation, probably by binding adventitious iron (Myers et al., 1987). These data support the suggestion that doxorubicin-related lipid peroxidation is mediated by the flux of reactive oxygen species generated after drug treatment. Because of the protean manifestations of peroxidative membrane injury, including altered mitochondrial electron flow and energy production (Neri et al., 1984; Marcillat et al., 1989), as well as disruption of mitochondrial and microsomal calcium homeostasis (Harris and Doroshow, 1985), lipid membrane injury produced by the anticancer quinones could play an important role in drug-induced cytotoxicity. Another major target of quinone drug-stimulated oxygen radical attack is cellular DNA. Oxygen radicals are a well-known cause of oxidation of nucleic acid bases and sugars (Teebor et al., 1988), leading to DNA strand scission (Berlin and Haseltine, 1981), and mutations (Levin et al., 1982). Quinone-containing drugs require activation to their semiquinone free radical in order to detect mutagenicity in the Ames assay (Chesis et al., 1984); and the anticancer quinones, including doxorubicin and mitomycin C, are known to be mutagenic in this system (Westerdorf et al., 1984). Schwartz originally described strand breaks in tumor cell DNA caused by doxorubicin and daunorubicin (Schwartz, 1975). Subsequently, Lown and colleagues demonstrated strand scission by redox cycling anthracyclines and mitomycin C in purified DNA preparations which was stimulated by reducing agents and inhibited by antioxidant enzymes and hydroxyl radical scavengers and which correlated well with the redox capabilities of the drugs (Lown et al., 1977, 1978, 1979, 1982; Lown and Weir, 1978; Peters et al., 1986). Other laboratories have found that the hydroxyl radical scavenger thiourea, as well as extracellular antioxidant enzymes such as catalase and superoxide dismutase, can decrease single strand breaks in the DNA of the L1210 murine leukemia (Pommier et al., 1983; Potmesil et al., 1984). These data suggest that under certain experimental situations single-strand scission of DNA by the anticancer quinones may occur via oxygen radical intermediates as well as through an interaction with topoisomerase II. It is clear, however, that the relative concentrations of anthracycline and DNA are major determinants of the degree of strand scission that can be demonstrated in ~'itro. Deoxyribose degradation by doxorubicin and mitomycin C has also been shown to involve drug semiquinone intermediates as well as the hydroxyl free radical (Bates and Winterbourn, 1982; Gutteridge et al., 1984). Finally, Myers and coworkers have demonstrated that the iron bound to doxorubicin can form a ternary complex with DNA which is capable of cleaving the DNA in the presence of thiol reducing agents (Eliot et al., 1984). Electronspin resonance studies have shown that the irondoxorubicin-DNA complex can also reduce molecu-

lar oxygen to the hydroxyl radical, which may, itself, be responsible for the observed DNA cleavage. In summary, considerable evidence exists to indicate that anticancer quinones stimulate reactive oxygen production in mammalian cells, and that these reactive species may be responsible for some of the toxic effects of these drugs. In the remainder of this review, the role of the glutathione-glutathione peroxidase cycle as a defense against drug-induced oxidative stress will be examined.

3. THE G L U T A T H I O N E - G L U T A T H I O N E PEROXIDASE CYCLE Major advances have been made in our understanding of glutathione (GSH) metabolism over the past 15yr (Meister, 1983). Briefly, the tripeptide glutathione (7-glutamylcysteinylglycine) is synthesized de novo chiefly in the liver, and then translocated to plasma. Plasma glutathione serves as a source for the constituent amino acids (principally cysteine) for glutathione synthesis in peripheral organs (Meister, 1983). Cysteine-glycine enters cells after cleavage of glutamate by a membrane-bound transpeptidase. Cysteine, cleaved from cysteineglycine by dipeptidases, may be coupled to glutamate by the enzyme 7-glutamyl cysteine synthetase. To complete the glutathione synthetic cycle, glycine is coupled to glutamate-cysteine by the enzyme glutathione synthetase. The abundance of glutathione in mammalian cells is extraordinary, reaching millimolar concentrations in the liver; ordinarily, > 98% of the glutathione in the cell remains in its reduced form. Glutathione may be turned over by translocation outside the cell; by formation of thioethers or mixed disulfides; by reactions used to conjugate xenobiotics; or by oxidation by free radicals or peroxides to the disulfide, GSSG. The peroxide-mediated oxidation of GSH is catalyzed by selenium-dependent glutathione peroxidases (GSHPx) and by selenium-independent glutathioneS-transferases (Wendel, 1980). Recent studies from several laboratories, including our own, have demonstrated that the mammalian glutathione peroxidases consist of a family of proteins with at least three and perhaps as many as five members that each possess a high degree of homology around the catalytic active site. The best-studied member of this group is the ubiquitous cytosolic enzyme of 88,000 Da that is especially abundant in the liver, kidney and in erythrocytes (Wendel, 1980). Although principally cytoplasmic, some activity is present in the mitochondrial compartment. Another selenium-dependent glutathione peroxidase has been discovered and isolated from plasma (Avissar et al., 1989). We have obtained a partial amino acid sequence of the human plasma glutathione peroxidase which appears to be homologous to, yet distinct from, the cytosolic enzyme (unpublished data). Two more glutathione peroxidase-like cDNA clones have been isolated from cells originating from human liver (Chu et al., 1988; Dunn et al., 1989) although their protein products have not yet been identified. Additionally, a monomeric glutathione peroxidase has been purified from rat liver and from porcine liver and heart (Ursini et al., 1985;

Glutathione peroxidase and quinone cytotoxicity Duan et al., 1988). This monomeric glutathione peroxidase exhibits substantial phospholipid hydroperoxidase activity against membrane lipid peroxidation (Thomas et al., 1990). The originallydescribed intracellular activity is found in essentially all mammalian cells and has recently been cloned (Yoshimura et al., 1988). The cytoplasmic enzyme has also been expressed in human MCF-7 breast cancer cells (Chu et al., 1989). Glutathione peroxidases are capable of catalyzing the reduction of hydrogen peroxide or lipid hydroperoxides to water or lipid alcohols, respectively, using GSH as the reductant. The oxidized glutathione resulting from the breakdown of peroxide intermediates is converted to the fully active tripeptide by the action of glutathione reductase (GSSGRed), which requires reducing equivalents from NADPH. Thus, a cyclical detoxification pathway exists to reduce peroxides via the oxidation of NADPH in the cytoplasm of all cells, the gluthione-glutathione peroxidase cycle: 2GSH + ROOH ....

GSHPx . . . . .

> GSSG + H20 + ROH

GSSG + NADPH + H ÷ ---

GSSGRed . . . .

> 2GSH + NADP ÷

Studies by Cohen and Hochstein in the erythrocyte originally established that the glutathioneglutathione peroxidase cycle was of critical importance for the detoxification of low levels of intracellular peroxides, and thus played the critical role in defending the erythrocyte from drug-induced oxidative stress (Cohen and Hochstein, 1963). Only over the past few years has the importance of this detoxification pathway been investigated in other cell types; as will be outlined below, this pathway appears to play an essential role in the defense of the heart and of some tumor cell types against an oxygen radical cascade launched by exposure to the anticancer quinones.

4. ROLE OF THE G L U T A T H I O N E G L U T A T H I O N E PEROXIDASE CYCLE IN THE A N T I T U M O R ACTIVITY OF THE ANTICANCER QUINONES Over the past five years investigators from several laboratories have examined the role of each of the components of the glutathione-glutathione peroxidase cycle in the cytotoxicity of quinone-containing antitumor agents for mammalian cells, including various tumor cell lines, both in vitro and in vivo. Since GSH is the critical reductant needed for the glutathione peroxidase cycle, recent studies have evaluated the effect of manipulating intracellular glutathione levels on the cytotoxicity of anticancer quinones. Using the 7-glutamylcysteine synthetase inhibitor L-buthionine sulfoximine at concentrations which could deplete reduced glutathione levels intra° cellularly without decreasing tumor cell viability, the cytotoxicity of doxorubicin can be significantly increased in Chinese hamster V79 cells (Russo and Mitchell, 1985), in buccal mucosa carcinoma cells

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(Lee et al., 1988), and in the P815 mouse mastocytoma (Arrick et al., 1983). It is important to note, however, that a substantial (>75%) decrease in intracellular GSH content is generally required before a significant increase in tumor cell cytotoxicity can be demonstrated; this suggests that because of the cyclical oxidation and reduction of GSH by the glutathione peroxidase cycle, glutathione molecules can be efficiently recycled to reduce hydrogen peroxide or lipid hydroperoxides and that the absolute intracellular concentration of GSH must be markedly depleted before hydroperoxide metabolism is adversely affected. In related experiments, attempts have been made to increase intracellular GSH levels (or related thiols) to examine whether an overabundance of intracellular thiols could diminish the cytotoxicity of anticancer quinones. Russo and colleagues showed that stimulation of GSH synthesis with 2-oxothiazolidine4-carboxylate could increase the doxorubicin concentration required for fifty percent inhibition of tumor cell growth by a modest degree (Russo and Mitchell, 1985). Although a complete understanding of the mechanism(s) by which intracellular thiols alter the cytoxicity of anticancer quinones remains to be elucidated, it seems clear that enhancing intracellular thiol levels, either by pretreatment with thiol reducing agents such as N-acetyl cysteine or thiourea, or by over-expression of metal|othioneins, can markedly decrease, and in some cases eliminate, the cytotoxic effects of doxorubicin, mitomycin C, AZQ and related compounds (Pommier et ai., 1983; Doroshow, 1986a, b; Kelley et al., 1988). It is tempting to speculate that these exogenous thiols provide a pool of reducing equivalents capable of supplementing the antioxidant properties of the glutathione peroxidase cycle and, in some cases, supplying alternative targets for peroxidatic attack or alternative substrates for intracellular peroxidases. The regeneration of reduced glutathione for the glutathione peroxidase cycle requires the presence of an adequate supply of reduced pyridine nucleotides and the enzyme glutathione reductase. Studies from Reed's laboratory demonstrated the importance of an intact glutathione-glutathione peroxidase cycle for the cytotoxicity of doxorubicin in isolated rat hepatocytes (Babson et al., 1981; Reed, 1986). Using the nitrosourea BCNU to inhibit liver cell glutathione reductase activity, these investigators showed that cells pretreated with non-toxic concentrations of BCNU demonstrated a marked decrease in viability upon exposure to doxorubicin, and that this was accompanied by a concurrent increase in membrane lipid peroxidation. The changes in both cytotoxicity and membrane peroxidation were significantly decreased by the co-administration of the free radical scavenger ~-tocopherol. Furthermore, treatment of hepatocytes with diethylmaleate (which can bind covalently to GSH but seldom decreases GSH levels below 75% of control) did not increase the cytotoxicity of doxorubicin. These experiments support the hypothesis that normal activity of the enzymes which maintain glutathione in its reduced form are the critical constituents of the glutathione peroxidase cycle. It is unfortunate that these studies cannot be duplicated unambiguously in mammalian tumor

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cells. Since BCNU is the only readily available inhibitor of glutathione reductase and since enzyme inhibition occurs at a drug concentration which is itself cytotoxic and produces significant inhibition of DNA synthesis in rapidly dividing cells, it has proven difficult to examine the role of glutathione reductase in the tumor cell cytotoxicity of doxorubicin and other quinone-containing anticancer agents. The role of the peroxide detoxifying enzyme glutathione peroxidase in the expression of anticancer quinone tumor cell toxicity has only recently begun to be investigated. The importance of the seleniumcontaining cytoplasmic enzyme in the regulation of intracellular peroxide homeostasis was evaluated initially by Nathan and colleagues (Nathan et al., 1981). These investigators found that the cytoplasmic glutathione peroxidase activity of murine leukemia cells could be decreased by 50-65% by serial, intraperitoneal passage of the cells in selenium-deficient mice. The cytotoxicity of hydrogen peroxide generated by activated macrophages in their murine tumor system was increased when the glutathione peroxidase activity of these cells was significantly decreased. We have shown previously that treatment of both Ehrlich ascites carcinoma cells and human breast cancer cells with Ebselen (2-phenyl-l-2-benzisoselenazol-3(2H)-one; a synthetic plasma membrane-permeable chemical with glutathione peroxidase activity) significantly decreases the cytoxicity of doxorubicin, mitomycin C, and AZQ (Doroshow, 1986a, b). Furthermore, recent studies from our laboratory have demonstrated that the cytoplasmic glutathione peroxidase activity of human MCF-7 breast cancer cells can be increased by up to 10-15-fold after introduction of the purified protein into these cells by a mechanical technique (McNeil et al., 1984; Doroshow et al., 1989). Under conditions of enhanced glutathione peroxidase activity, the ICs0 concentration for doxorubicin in MCF-7 cells is increased 3-fold. Relative insensitivity to mitomycin C and menadione (2-methyl-l,4-naphthoquinone) also occurs in tumor cells with increased glutathione peroxidase activity; on the other hand, no effect on the cytotoxicity of 5-iminodaunorubicin could be demonstrated in glutathione peroxidase-loaded MCF-7 cells, as might be expected for an anthracycline analogue which does not generate reactive oxygen species (Doroshow, 1983a; Doroshow et al., 1989). In summary, these studies suggest that at least for mammalian tumor cells in vitro, the glutathioneglutathione peroxidase cycle can play an important role in the cytotoxic effect of the anticancer quinones. Whether cell killing is modified at the level of DNA cleavage, calcium homeostasis, or mitochondrial electron transport by alterations in this oxidant detoxification pathway remains a focus for current investigation.

5. ROLE OF THE G L U T A T H I O N E G L U T A T H I O N E PEROXIDASE CYCLE IN T U M O R CELL RESISTANCE TO THE A N T I C A N C E R QUINONES Because of their broad spectrum of antineoplastic activity (Young et al., 1981; Bender et al., 1983;

Crooke and Bradner, 1976), the elucidation o1" the mechanism(s) of acquired and intrinsic resistance to the therapeutic effects of anticancer quinones is one of the most important current issues in oncologic pharmacology. Several recent studies suggest that the glutathione-glutathione peroxidase cycle may play an important role in both natural and acquired drug resistance. Batist and colleagues (Batist et al., 1986) initially demonstrated that an MCF-7 cell variant that was selected by the development of resistance to stepwise escalations in doxorubicin concentration in vitro, expressed an -~ 10-fold increase in glutathione peroxidase activity (as well as an anionic glutathioneS-transferase). Further studies by Sinha et al. have shown that over-expression of glutathione peroxidase activity in these cells can decrease doxorubicinrelated hydroxyl radical production in subcellular fractions (Sinha et al., 1989). We have recently found that the increased glutathione peroxidase specific activity in these cells can be explained by an increase in glutathione peroxidase mRNA (Akman et al., 1990), rather than by amplification of the gene for the cytoplasmic enzyme. In light of recent data indicating that human colonic carcinomas appear to over-express both reduced glutathione and glutathione peroxidase activity compared to adjacent non-cancerous mucosa (Mekhail-Ishak et al., 1989), as well as a recent report demonstrating increased glutathione peroxidase activity in human lung carcinomas, changes in the glutathione peroxidase cycle might be related to the intrinsic insensitivity of anticancer quinones in these diseases (Carmichael et al., 1988). In addition to changes in glutathione peroxidase activity in drug-resistant tumor cells, several laboratories have recently shown that depletion of intracellular reduced glutathione pools can at least partially reverse anthracycline resistance. This has occurred even in resistant cells possessing the typical multiple drug resistance phenotype which do not demonstrate major differences in glutathione or glutathione peroxidase levels compared to their parental line; these cells include a doxorubicin-resistant human ovarian carcinoma (Hamilton et al., 1985), a human myelogenous leukemia (Lutzky et al., 1989), as well as a human breast cancer line (Kramer et al., 1988; Dusre et al., 1989). In summary, although the precise mechanisms involved in oxidative tumor cell injury by the anticancer quinones continue to be investigated, sufficient data currently exist to support the possibility that quinone-induced reactive oxygen formation contributes to the ability of these drugs to kill malignant cells, and that the glutathione-glutathione peroxidase cycle is one of the major pathways involved in the defense of malignant cells against drug-induced reactive oxygen formation.

6. ROLE OF THE G L U T A T H I O N E G L U T A T H I O N E PEROXIDASE CYCLE IN THE C A R D I A C TOXICITY OF THE A N T I C A N C E R QUINONES The enzymatic antioxidant capacity of the mammalian heart, especially in comparison with the activity levels of the kidney and liver, appears to be of

Glutathione peroxidase and quinone cytotoxicity limited scope. The superoxide dismutase activity of the heart is 25-30% of that in the liver; furthermore, cardiac tissue contains essentially no glutathione-S-transferase and only 1-2% of the catalase levels found in hepatocytes (Lawrence and Burk, 1978; Revis and Marusic, 1978; Doroshow et al., 1980; Wang et al., 1980; Jackson et al., 1984; Kanter et al., 1985; Tomlinson et al., 1985; Lazzarino et al., 1987). Glutathione peroxidase activities in the hearts and livers of several species including mice, rats, and dogs are comparable; however, the glutathione peroxidase activity of the rabbit heart is only 20% of that in liver (Revis and Marusic, 1978; Doroshow et al., 1980; Kanter et al., 1985). The normal level of GSH in the rat heart is ~-8#mol/mg protein with a range of from 1.5 to 4 #mol/mg in other species (Doroshow et al., 1979; Hazelton and Lang, 1980; Olson et al., 1980; Julicher et al., 1986; Lee et al., 1987; Lazzarino et al., 1987; Thayer, 1988). These levels are substantially lower than those found in the liver and are similar to the range of glutathione concentrations found to compromise the protection of hepatocytes from the cytotoxic effect of doxorubicin after exposure to BCNU (Babson et al., 1981). This suggests that maintenance of intracellular peroxide homeostasis in the heart may depend on the efficient shuttling of a limited supply of glutathione between GSH peroxidase and GSSG reductase by the glutathione-glutathione peroxidase cycle. If anthracycline redox cycling can transiently overwhelm the capacity of the heart to eliminate peroxides, in the presence of a limited concentration of superoxide dismutase, conditions could be established that would favor the production of the hydroxyl radical by way of the iron-catalyzed Haber-Weiss reaction. This possible pathophysiologic sequence is especially pertinent in light of evidence demonstrating that purified glutathione peroxidase can be inhibited by a hydroxyl radical flux (Searle and Willson, 1980). Hence, breakdown of the glutathione-glutathione peroxidase cycle by reductions in the levels of either reduced glutathione, glutathione peroxidase, or glutathione reductase would permit a markedly enhanced degree of cardiac tissue injury by an anthracycline-stimulated oxygen radical flux. Studies of anthracycline cardiac toxicity in vivo utilizing a single large dose of doxorubicin to produce heart damage have revealed a significant, 60% drop in cardiac, but not liver, glutathione peroxidase levels for up to 72hr after administration of the drug (Doroshow et al., 1980). Furthermore, pretreatment of mice with pharmacologic doses of vitamin E (as D-ct-tocopherol) prior to doxorubicin blocked the drug-related inhibition of glutathione peroxidase activity (Doroshow and Locker, 1982). The drug doses required to produce a substantial decrease in glutathione peroxidase activity are at or near the LD~0 level in the mouse (10-15 mg/kg). This may explain why other investigators using lower drug doses did not find a major change in cardiac glutathione peroxidase after acute doxorubicin treatment in the rabbit (Wang et al, 1980; Jackson et al., 1984). Interspecies variation in antioxidant defenses, as outlined above, may also play a role in these differences. If doxorubicin was administered chronically in either the

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mouse or rabbit, cumulative, potentially lethal drug doses were also required to demonstrate an approximate 40% inhibition of enzyme activity (Revis and Marusic, 1978). In this study, inhibition of cardiac glutathione peroxidase activity was accompanied by an elevation in myocardial calcium content. Several investigators have also examined the effect of anthracycline antibiotics on tissue glutathione concentrations. Glutathione levels have been observed to decline up to 48 hr after the administration of an acutely fatal dose of doxorubicin in rabbit heart and erythrocytes (Wang et al., 1980) and in mouse heart, liver, and erythrocytes (Doroshow et al., 1979; Olson et al., 1980). A similar response has been observed using a perfused rat heart model (Julicher et al., 1986). The nadir level of reduced glutathione occurs between 3-12 hr after drug treatment in the liver and heart; however, the timing of changes in erythrocyte glutathione levels has varied between studies (Olson et al., 1980; Wang et al., 1980). The magnitude of the drop in glutathione level is relatively small in liver and heart (20-30%) and has been observed to rebound completely within 24-48 hr after doxorubicin administration. In the perfused rat heart model, the drop in reduced glutathione can be accounted for, in part, by the production of GSSG (Julicher et al., 1986). These results suggest that the oxidant stress produced in the anthracycline-treated heart drives the glutathione-glutathione peroxidase cycle toward the accumulation oxidized glutathione, an indication that the peroxide detoxifying capacity of the myocyte has been exceeded. If chronic doxorubicin administration in man leads to an impairment of the glutathione peroxidase system, a transient window of vulnerability opened by the combined drop of glutathione and glutathione peroxidase may allow for substantial free radical-related cardiac membrane injury. Two means of modulating glutathione peroxidase levels in animal tissues have been employed in studies of doxorubicin cardiac toxicity. These are diet and chronic exercise. The rationale for experiments utilizing dietary manipulation is based on the fact that glutathione peroxidase has a selenocysteine residue at its active site. Cells deficient in selenium cannot complete the translation of the enzyme because the selenium is incorporated into the peptide subunit co-translationally in the form of this critical selenocysteine residue. Selenium starvation has been used in animal studies of doxorubicin toxicity to reduce tissue glutathione peroxidase levels prior to the administration of the anthracycline (Doroshow et al., 1980; Facchinetti et al., 1983; Chen et al., 1986). In the studies from our laboratory (Doroshow et al., 1980; Doroshow and Locker, 1982), the effect of selenium deprivation on both lethality and cardiac morphology after doxorubicin administration was examined. In these experiments, cardiac glutathione peroxidase activity was reduced to 18% of control level prior to doxorubicin treatment. Median survival after a dose of 15 mg/kg doxorubicin decreased from 26 to 11 days in selenium-deficient mice. Furthermore, microscopic features of anthracycline cardiac toxicity were markedly enhanced by selenium starvation. A similar increase in the morbidity of doxorubicin in selenium-deprived

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mice was observed by Facchinetti (Facchinetti et al., 1983). However, using a rat model of chronic, low dose doxorubicin toxicity, Chen (Chen et al., 1986) found no effect of selenium starvation on the severity of drug-related cardiomyopathy in this species; however, the renal toxicity of doxorubicin was significantly increased in the rat by selenium deprivation. The differences in doxorubicin dose and schedule, and the relative sensitivity of the mouse heart to free radical attack compared to that of the rat may help to explain the variability observed in these experiments. While the removal of selenium from the diet appears to enhance the toxicity of doxorubicin in murine model systems, the effect of selenium supplementation on doxorubicin toxicity is somewhat difficult to assess. Several investigators have attempted to ameliorate the cardiotoxic effects of doxorubicin by the administration of acute or chronic doses of selenium. In general, these investigations have been driven by the hypothesis that selenium supplementation might enhance glutathione peroxidase levels in tissues. Unfortunately, as Levander (Levander, 1987) has shown, selenium sufficient animals do not respond to additional selenium supplementation by increasing tissue glutathione peroxidase levels. Thus, the life-extending effects of selenium supplementation in certain species and not others as demonstrated in numerous investigations by Van Vleet and colleagues (Van Vleet et al., 1977, 1980; Van Vleet and Ferrans, 1980), and the morphologic evidence of cardiac protection with supplemental dietary selenium in the rabbit model of doxorubicin cardiac toxicity recently presented by Dimitrov et al. (Dimitrov et al., 1987) are difficult to interpret on the basis of any significant change in tissue glutathione peroxidase activity. As demonstrated by Burk and associates (Burk et al., 1980) in experiments with analogues of the pulmonary toxin paraquat, which generates a significant oxidant stress in the lung, elemental selenium may itself be providing substantial antioxidant activity in these studies independent of its incorporation into the glutathione peroxidase molecule, Hence, the significant differences in selenium dose, route, and schedule amongst the investigations of Van Vleet and Dimitrov could explain the variable nature of their results. In addition to alterations in dietary selenium, exercise has been used to modulate the level of antioxidant enzymes in tissues. Kanter et al. (Kanter et al., 1985) found that vigorous exercise for ten continuous weeks in advance of the initiation of a chronic course of doxorubicin significantly ameliorated the ultrastructural features of anthracycline cardiac toxicity. Cardiac catalase, superoxide dismutase, and glutathione peroxidase levels were all significantly increased in the exercised mice compared to controls. The major change, however, was in cardiac catalase activity, which doubled prior to doxorubicin treatment in the mice exposed to chronic exercise. Glutathione peroxidase levels increased only 6%. This study supports the premise that the peroxide handling capacity of the heart is limited, and that enhancement of enzymatic peroxide detoxification, in this case by an increase in catalase activity rather than a change in glutathione peroxidase, improves

myocardial tolerance to doxorubicin. In another recent study (Quintanilha and Packer, 1983), rats exposed to chronic exercise developed a 70% increase in cardiac glutathione reductase activity. Given the low level of de novo glutathione synthesis in the heart, the activity of cardiac glutathione reductase may be very influential in determining the size of the reduced glutathione pool available for detoxification of druginduced hydrogen and lipid peroxides by glutathione peroxidase.

7. S U M M A R Y The studies reviewed here demonstrate the ability of the anticancer quinones to undergo cycles of futile oxidation and reduction in both human tumor cells and mammalian myocytes, intact cells as well as essentially all intracellular compartments. There are now sufficient data available to support the premise that the oxidative metabolism of the anticancer quinones makes a significant contribution to the cytotoxic effects of these compounds; under certain circumstances, amplification of antioxidant defenses, and in particular the glutathione-glutathione peroxidase cycle, may contribute to the resistance of human tumor cells to quinone-containing antineoplastic agents. Several important unanswered questions remain, however, regarding the role of the glutathioneglutathione peroxidase cycle in anticancer quinone toxicity. These questions include: (1) the mechanisms of peroxide disposal in mitochondrial and nuclear, rather than cytoplasmic, compartments; (2) the identification of the source(s) of catalytic transition metals in both tumor cells and the heart required for drug-induced hydroxyl radical formation; and (3) the elucidation of the principal foci for oxidative injury, i.e. the calcium pump, nucleic acids, growth factor receptors etc. In each case, future studies are required to determine the mechanisms by which the glutathione-glutathione peroxidase system controls oxidative damage in normal tissues and tumors. Acknowledgements--This work was supported by grants

from the National Cancer Institute (CA 31788 and CA 33572).

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Role of the glutathione-glutathione peroxidase cycle in the cytotoxicity of the anticancer quinones.

Recent studies have suggested that the selenoenzyme glutathione peroxidase, in the presence of reducing equivalents from the tripeptide glutathione, i...
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