Pharmac. Ther. Vol. 49, pp. 125-132, 1991 Printed in Great Britain. All rights reserved

0163-7258/91 $0.00+0.50 © 1991 PergamonPresspie

Specialist Subject Editor: K. D. TEW

HISTORICAL ASPECTS OF G L U T A T H I O N E A N D C A N C E R CHEMOTHERAPY P. MISTRY and K. R. HARRAP Drug Development Section, The Institute of Cancer Research, Cotswold Road, Sutton, Surrey SM2 5NG, U.K.

Al~tract--This presentation sets in historical context the impact of glutathione and its metabolism upon the efficacy of several anticancer drugs. The basic biochemisty of the tripeptide is reviewed briefly, highlighting its role in oxido-reduction and in the ~,-glutamyl cycle. The ability of selective modulators of glutathione metabolism, such as buthionine sulfoximine, as adjuncts to chemotherapy is also discussed. CONTENTS I. Introduction 2. Biochemical Background 2.1. Intracellular GSH 2.2. Glutathione synthesis 2.3. 7-Glutamyl transpeptidase (~,-GT) 2.4. Glutathione-S-transferases (GST) 2.5. Glutathione oxido-reduction 3. Modulation of Anticancer Drugs by GSH 3.1. Alkylating agents and nitrosoureas 3.2. Anthracyclines 3.3. Platinum drugs 4. The Role of GSH in the Activation and Uptake of Anticancer Drugs 5. Summary Acknowledgements References

I. I N T R O D U C T I O N Glutathione (4/.L.glutamyl_L.cysteinyl_glycine;GSH) is ubiquitous in nature. It was first isolated from yeast in 1888 by De Rey-Pailhade, who coined the term 'philothion' for the impure isolate (De Rey-Pailhade, 1888a,b). Its isolation and crystallization in pure form is attributed to Hopkins in 1921 (Hopkins, 1921). The correct molecular structure was suggested, variously, by Hopkins (1929), Kendall et al. (1929), Pirie and Pinhey (1929) and confirmed by chemical synthesis by Harington and Mead in 1935. From the time of its discovery, Hopkins recognized that the reduction of oxidized glutathione (GSSG) by tissues and reoxidation of GSH by molecular oxygen might confer for this new substance an important role in hydrogen and electron transfer (Hopkins, 1921; Hopkins and Dixon, 1922). However, it became apparent that the oxidation of GSH in liver preparations represented only a small fraction of their total respiratory capacity (Hopkins and Elliott, 1931), the latter being designated subsequently as the major responsibility of the cytochrome system. None the less, the facile redox cycling of glutathione remains today pivotal to the important role played by this molecule in pharmacology, toxicology and radi-

125 125 125 126 126 126 127 127 127 128 128 129 129 130 130

ation biology. It is now known that glutathione participates directly or indirectly in a number of functions important to mammalian cells, including maintenance of membrane integrity, optimal transport of amino acids, maintenance of enzyme activity and biological protection through the detoxification of xenobiotics and free radicals (Meister and Anderson, 1983; Larsson et al., 1983). The multifunctional properties of GSH and its metabolism can impact upon the etticacy of anticancer drugs, the historical aspects of which we review below. For other reviews on this subject see Arrick and Nathan (1984), Russo et aL (1986a) and Wolf et al. (1987).

2. BIOCHEMICAL B A C K G R O U N D Readers are referred to the reviews by Kosower and Kosower (1978), Reed and Beatty (1980) and Meister and Anderson (1983) for a more comprehensive treatment of this subject. 2.1. INTRACELLULARGSH GSH has a reactive thiol group, pK~ 9.65, whilst the ~-glutamyl bond renders the molecule resistant to 125

126

P. MISTRYand K. R. HARRAP

intracellular peptidases. Intracellular GSH exists mainly in three dynamically interchangeable forms-as reduced GSH, as oxidized GSSG and as mixed disulfides with either low or high molecular weight thiols. GSH thiol-ethers and esters contribute also to the overall GSH pool. Under normal steady-state conditions, reduced GSH is the major form, present at mM concentrations: GSSG produced by the action of glutathione peroxidase (GPO), thiotransferases, or nonenzymically, is present at concentrations which rarely exceed 1% of GSH due to the activity of glutathione reductase and the effiux of GSSG from the cell to serve the 7-glutamyl cycle. However, under acute oxidative stress, levels of GSSG can increase dramatically as these pathways become saturated. The intracellular concentrations of mixed disulfides are not known precisely, with reported values ranging from ! to 50% of the total cellular GSH pool; of this the GSS-protein disulfide appears to be the major form. Intracellular GSH undergoes continuous turnover, with synthesis, degradation and irreversible loss contributing to its overall half-life, which can differ markedly in various tissues, ranging from several days in the erythrocyte to less than one hour in the kidney. The major determinants of the intracellular steady state concentration of GSH are (i) synthesis, (ii) cellular efflux and 7-glutamyl transpeptidase-initiated degradation, (iii) GSH oxido-reduction and (iv) conjugation of electrophilic moieties.

cysteine synthetase and is inhibited nonallosterically by GSH; the second is catalyzed by giutathione synthetase (Fig. 1, after Meister and Anderson, 1983). The selective inhibition of ?-glutamyl cysteine synthetase by buthionine sulfoximine (BSO), has been used extensively over the last few years to deplete intracellular GSH to determine the importance of this thiol in antitumour drug action; see below (Hamilton et al., 1985; Meister, 1983; Russo et al., 1986a). 2.3. y-GLUTAMYLTRANSPEPTIDASE(~/-GT) The degradation of GSH, GSSG, as well as S-substituted GSH, is initiated by this membrane bound enzyme, which is localized on the external surface of the plasma membrane and depends on the ett~ux of relevant substrates from cells. The enzyme can either hydrolyze GSH or transfer the ~-glutamyl moiety to various acceptor amino acids or dipeptides, and forms part of the ),-glutamyl cycle involved in cellular turnover of GSH (Fig. I). Thus an increase in y-GT content of a particular cell may result in an overall increase in the intracellular concentration of GSH as a result of an increase in the levels of the constituent amino acids. The transport of intracellular GSH acts as a source of plasma GSH and GSSG and functions in the interorgan transfer of cysteine sulfur. In addition, the transport of GSH may also have a role in protecting the cell membrane by maintaining the integrity of essential thiol groups (Kosower and Kosower, 1983).

2.2. GLUTATHIONESYNTHESIS The synthesis of GSH from its constituent amino acids occurs as a result of two ATP-requiring steps. The first step is catalyzed by the enzyme 7-glutamyl

2.4. GLUTATHIONE-S-TRANSFERASES(GST) This important pathway involves conjugation reactions which play a major role in cellular defence

,~nY~ ,~_q FEI]UCTION ~ I0 ~

| ~

mmoxtm~g

(ELECTF~ILE} X ~

~GATION )~

~"'~

TR~i--3

CY~_GLY . . - - -~

GLUTAT'HIONE

I,B,TZI~E) Y

.4K.

" mmu~mil

'6--/

I '~ CYSH-GLY- ~

7-GLU~ '~YSH

SI~It'TASE!

X I ,I,

/ CYS-X\ 31aTRaNSPB=TI~ ¢EIYL~8 P-GLU-CYS-X N-AC-CYS-X

GLU ~

4

i

~,~ 5-OXOPROLINE

aTP

AA

FIG. I. Summary of glutathione metabolism: Adapted from Meister and Anderson (1983). Reaction h ?-GLU-CYSH synthetase; reaction 2: GSH synthetase; reactions 3 and 3a: glutamyl transpeptidase; reaction 4: y-glutamyl cyclotransferase; reaction 5: 5-oxoprolinase; reactions 6 and 6a: dipeptidase; reaction 7: GSH S-transferases; reaction 8: N-acetylase; reaction 9: GSH peroxidase; reaction 10: transhydrogenases; reaction I 1: GSSG reductase; reaction 12: oxidation of GSH by 02; Conversion of GSH to GSSG is also mediated by free radicals.

Glutathione and cancer chemotherapy against toxic substances and in the further metabolism of certain endogenous compounds. Conjugation may occur spontaneously or enzymatically, via glutathione-S-transferases, with a wide variety of electrophilic substances, including clinically useful antineoplastic agents. GSTs are a family of isoenzymes which have different but overlapping substrate specificities and are individual products of separate genes (Mannervik et al., 1990). They are present in virtually all human tissues and are elevated in some tumor cells (Lewis et al., 1989; Mannervik, 1985). The conjugation of a particular electrophilic substance may thus depend upon the intracellular concentration of GSH and the presence of an appropriate GST isoenzyme. 2.5. GLUTATHIONEOXIDO-REDUCTION GSH fulfils an important role in cellular defence mechanisms against hydrogen peroxide, organic peroxides and free radicals, produced as a consequence of aerobic metabolism (Meister and Anderson, 1983; Reed and Beatty, 1980). The reduction of free radicals is nonenzymatic, whereas the reduction of peroxides is catalyzed by selenium-containing GSH peroxidase (GPO) and by other proteins which also exhibit GST activity. The metabolism of these reactive intermediates is associated with GSH oxidation and the majority of the GSSG produced is reduced by glutathione reductase (GR), a NADPH dependent enzyme. Hence, cell survival in the presence of these reactive intermediates is dependent on the capacity of the GSH oxidation-reduction cycle, that is, on the intracellular concentration of GSH, GR, GPO/GST, as well as the ability to generate NADPH via the pentose phosphate shunt (Kappus, 1986; Reed and Beatty, 1980).

3. MODULATION OF ANTICANCER DRUGS BY GSH From the above brief outline of GSH metabolism it is evident that this thiol and its associated enzymes might influence antineoplastic drug action in both normal and tumor tissues in a variety of ways, for example: (i) by reduction of drug cytotoxicity (increase in resistance), by detoxification of electrophilic species or of reactive intermediates. GSH may also reduce drug cytotoxicity by increasing repair of critical DNA lesions since it is utilized in the synthesis of DNA precursors (deoxyribonucleoside triphosphates) and by DNA repair enzymes (Holmgren, 1979; Lai et al., 1989; Luthman et al., 1979); (ii) GSH and its dependent enzymes may increase the activity of anticancer agents by enhancing the formation of active species; (iii) GSH may promote drug uptake; (iv) alterations in GSH turnover due to drug therapy or drug modulators may influence the action of subsequent or concurrent therapeutic agents; (v) the overall efficacy of drugs might be altered depending on whether these changes occur in normal or tumor tissue. Below we describe examples of antitumor drugs where activity is influenced by GSH and GSHdependent enzymes and discuss the implications of this on drug efficacy. JIlT 4 q - l . 2 - - 1

127

3.1. ALKYLATING AGENTSAND N1TROSOUREAS

GSH has long been implicated in the mechanism of resistance of tumor cells to electrophilic antitumor agents such as alkylating agents, which are susceptible to nucleophilic attack by sulfhydryl groups. In the early 1950s the toxicity of the nitrogen mustard mechlorethamine (methyl-bis-(chloroethyl)-amine) was shown to be decreased by both cysteine (Brandt and Griffin, 1951) and cysteamine (Peczenik, 1953). Subsequently, both of these sulfhydryl compounds were demonstrated to increase the levels of intracellular glutathione (Meister and Anderson, 1983; Revesz and Modig, 1965). Hence the detoxification may have been mediated by glutathione synthesis rather than by a direct interaction of the nitrogen mustard with cysteine or cysteamine. In addition, Hirono (1960) reported a correlation between intracellular nonprotein sulfhydryl groups (NPSH) and resistance to mechiorethamine and its N-oxide in murine tumor cells, whilst Calcutt and Connors (1963) demonstrated a correlation between sensitivity to merophan, an isomer of melphalan, and the ratio of protein sulfhydryls to NPSH in murine tumors. Others measured GSH levels in alkylating agent resistant cells and observed correlations between elevated GSH and alkylating agent resistance (Ewtoo et al., 1981; Hirono, 1961; Suzukake et al., 1983). Parenthetically, Endresen and colleagues drew attention to the high levels of the thiol-rich protein, metallothionein, in cultured cells exhibiting acquired resistance to chlorambucii (Endresen et al., 1983). Additional studies with LI210 murine leukemia cells (Suzukake et al., 1982), CHO cells (Begleiter et al., 1983), human ovarian (Green et aL, 1984) and medulloblastoma cells (Skapek et al., 1988) have also demonstrated that resistance to the alkylating agents melphalan and chlorambucil may be mediated by elevated GSH and/or GST levels. Furthermore, sensitization of these cells to alkylating agents was achieved following selective inhibition of GSH synthesis by BSO. Moreover, in the ovarian and human medulloblastoma cell lines, the increase in cytotoxicity of melphalan following BSO pretreatment was greater in the sensitive than in the resistant line. These results suggested that BSO may act as a sensitizer, as well as a modifier of acquired resistance, to alkylating agents such as melphalan. Sensitization to alkylating agents by BSO pretreatment has also been reported both /n vitro and /n vivo using ovarian and medulloblastoma xenografts (Andrews et al., 1985; Hamilton et al., 1985; Ozois et al., 1987; Skapek et al., 1988). Moreover, the administration of BSO did not potentiate the toxicity of melphalan in nontumor bearing animals. Acrolein, a major metabolite of cyclophosphamide is metabolized via a GST isoenzyme to 3-hydroxypropylmercapturic acid, and excreted (Chasseaud, 1979). The elevated GSH concentration in CHO cells possessing acquired resistance to alkylating agents has been attributed to an increase in ~-GT levels (Lewis et al., 1988b). Chlorambucil-resistant CHO (Robson et al., 1987) and Walker 256 rat mammary carcinoma cells (Wang and Tew, 1985; Bullet et al., 1987) have also been reported to possess increased ct-GST activity. It is also worth noting that the

128

P. MISTRVand K. R. HARRAP

resistance of Yoshida sarcoma cells to cyclophosphamide, and to phosphoramide mustard, was associated with a six-fold increase in GST activity (McGown and Fox, 1986). Furthermore, in CHO cells, gene amplification was shown to be responsible for the elevated GST activity and this was associated also with an approximately 5-fold increase in GSTmediated peroxidase activity (Lewis et al., 1988b; Robson et al., 1987). Hence, gene amplification leading to overexpression of GST isoenzymes may be involved in the mechanisms of cross-resistance between alkylating agents and compounds such as doxorubicin (adriamycin) whose cytotoxic action is dependent on the generation of reactive species (Hamilton et al., 1985; Lewis et al., 1988b; Robson et al., 1987). Nitrosoureas are also able to modulate GSH metabolism. Notably, the carbamoylating product of nitrosurea breakdown is a potent inhibitor of glutathione reductase (Babson and Reed, 1978). Further, reduced glutathione reductase activity has been correlated with enhanced nitrosourea cytotoxicity (Tew and Wang, 1982; Tew ez al., 1985). Both GSH and GST levels were elevated in nitrosurea (and mustine) refractory rat gliosarcoma cells (Evans et al., 1987). 3.2. ANTHRACYCLINES

The generation of semiquinones or oxyradicals by redox cycling of the quinone moiety of doxorubicin is believed to contribute both to the antitumor and cardiotoxic activity of this and related anthracyclines (Myers, 1986; Powis, 1987). Therefore detoxification (resistance) to anthracyclines could arise from an increase in intracellular GSH concentration or from elevated levels of antioxidant enzymes such as GPO. Indeed, levels of GSH have been correlated with the cytotoxic activity of doxorubicin in a number of tumor cells and normal tissues over the last few years (Babson e! al., 1981; Hamilton et al., 1985; Kramer et al., 1988; Lee and Siemann, 1989; Lee et al., 1988). Depletion of cellular GSH with BSO has been shown to increase doxorubicin toxicity in both sensitive and resistant human ovarian (Hamilton et al., 1985) and buccal mucosal (Lee et al., 1988) cells. However, other studies with P388 and Ll210 leukemia cells have suggested that GSH concentration does not play a focal role in doxorubicin cytotoxicity (Ramu ez aL, 1984; Romine and Kessel, 1986). The discrepancy in these results may be explained by recent reports (Babson et al., 1981; Kramer et al., 1988; Lee et al., 1988) which suggest that the cell's ability to redoxcycle GSH, that is, the area under the concentration x time curve (AUC) of intracellular GSH, may be more important than its absolute initial levels. Furthermore, the results of Kramer et al. (1988) have suggested that the MDR phenotype may include alterations in the GSH redox cycle in addition to decreased drug accumulation and increased production of Pl70 glycoprotein. These studies also showed that a similar dual mechanism of resistance may occur in previously untreated human colorectal cancer, a tumor which is inherently refractory to chemotherapy. The intracellular metabolism of GSH also appears to be important in the dose-limiting cardiotoxicity of anthracyclines. Reduction of GSH,

by pretreatment with diethyl maleate, increased the lethality of doxorubicin (Olsen et al., 1983), while administration of a-tocopherol (Myers et al., 1976), cysteamine (Freeman et al., 1980) and N-acetylcysteine (Doroshow et al., 1982), protected against doxorubicin toxicity without interfering with its antitumor activity, thus increasing the therapeutic index. Interestingly, GST has been found to be 45-fold elevated in an adriamycin-resistant subline of MCF-7 cells which express the multidrug resistance phenotype. In addition, transfection of GST genes into recipient cells has been shown to protect against anthracycline and other free radical generating anticancer drug toxicities (Puchalski and Fahl, 1990; Nakagawa ez al., 1990). The associated peroxidase activity would act to reduce oxygen-radical-determined cytotoxicities (Bachur et al., 1979; Batist et al., 1986; Holleran ez al., 1986). The activity of other antitumor drugs, such as mitomycin C, streptonigrin and lapachol, which are believed to act through the production of free radicals, may be similarly modulated by cellular GSH metabolism (Arrick and Nathan, 1984; Kappus, 1986). 3.3. PLATINUMDRUGS A majority of evidence suggests that the primary cytotoxic target of platinum drugs is DNA and that interference with platinum-DNA adduct formation, or promotion of its repair, may lead to reduction in the cytotoxic action of such drugs. Platinum is known to react avidly with sulfur iigands, hence it has been postulated that elevated cellular GSH may reduce the cytotoxicity of cisplatin and other platinum drugs. Elevated GSH levels have been reported in a number of cisplatin resistant human and murine tumor models (Eastman et al., 1988; Hamilton et al., 1985; Lewis et al., 1988a; Richon et al., 1987; Teicher et al., 1987). In one study, Lewis et al. (1988a) found that, relative to a sensitive ovarian cell line (PE01), GSH and GSH-dependent enzymes were higher in a resistant (PE04) line established from the same patient after relapse on combined cisplatin, chlorambucil, and 5-fluorouracil therapy. Moreover, Russo et al. (1986b) have demonstrated that elevation of GSH in human lung fibroblasts by pretreatment with oxothiazolidine-4-carboxylate (OTZ) reduces cisplatin sensitivity. Interestingly, no changes in GSH levels or cisplatin cytotoxicity were observed in a human lung adenocarcinoma line (A549) after OTZ pretreatment. Hence it may be possible to alter cisplatin efficacy by manipulating the concentration of GSH in normal tissues using OTZ. Hosking et al. (1990) have reported a significant correlation between GSH coneentration and sensitivity to cisplatin, in a range of tumor cell lines. A positive correlation between cisplatin cytotoxicity and selenium-independent GPO activity was also reported in these lines. In addition, we have observed a significant correlation, in eight human ovarian carcinoma cell lines, between GSH concentration and ICs0 values for carboplatin (r = 0.83) and CHIP (r = 0.91) as well as for cisplatin (r = 0.93); the correlation for tetraplatin was not as significant (r = 0.73) (Mistry et al., 1990). Conflicting results have been reported on the effects of GSH depletion, by BSO pretreatment, on

Glutathione and cancer chemotherapy platinum drug activity. The depletion of GSH in human ovarian carcinoma cells increased cisplatin cytotoxicity in both sensitive and drug resistant variants (Hamilton et al., 1985). The enhanced sensitivity in these ovarian ceils after BSO pretreatment may have been due to inhibition of DNA repair, together with reduced inactivation of reactive platinum species by GSH (Lai et al., 1989). These authors have suggested that an inhibition of repair of cisplatin-DNA adducts after BSO pretreatment may be due to both a direct effect on DNA repair enzymes, through GSH depletion, and an indirect effect via reduction of the deoxyribonucleoside triphosphate pools. GSH is essential for the synthesis of these DNA precursors from ribonucleoside diphosphates by ribonucleotide reductase through a system involving glutaredoxin, NADPH, and GSH reductase (Hoimgren, 1979; Luthman et al., 1979). Pretreatment of CHO and mouse sarcoma cells with BSO in vitro substantially enhanced the cytotoxicity of a PtlV compound CHIP, but had much less effect on two PtlI compounds, cisplatin and carboplatin (Smith and Brock, 1988). We have also observed greater enhancement of PtlV (tetraplatin and CHIP) than PtlI (cisplatin and carboplatin) drug cytotoxicity in one sensitive (CHI) and two resistant (SKOV-3 and HX/62) human ovarian carcinoma cell lines, after BSO mediated GSH depletion (Mistry et al., 1990). Cisplatin cytotoxicity was also enhanced by BSO treatment in LI210 murine leukemia cells with primary resistance to melphalan and cross-resistance to cisplatin but, not in LI210 cells with acquired resistance to cisplatin (Andrews et al., 1986). Exposure to BSO also failed to reverse cisplatin resistance, but not melphalan cross-resistance, in some ovarian cells (Andrews et al., 1985). However, subsequent studies with these ovarian cells showed that cisplatin sensitivity could be partially restored by prolonged exposure to BSO (Andrews et al., 1988). Hence the authors suggest that profound depletion of GSH may be required to enhance cisplatin cytotoxicity, and that the mechanistic interaction of cisplatin with intracellular GSH is different from that of melphalan. Both increased and unaltered GST activities have been reported in cisplatin-resistant human and murine tumor models (De Graeff et al., 1988; Hamilton et al., 1985; Lewis et al., 1988a; Teicher et al., 1987), but as yet no direct proof of the involvement of this family of enzymes in forming G S H platinum drug adducts has been provided. Although the exact role of GSH and GSH-dependent enzymes in modulating platinum drug action remains to be determined, it appears that it may be possible to improve the clinical efficacy of these anticancer agents, particularly the PtlV compounds, by manipulating tissue GSH concentrations. Parenthetically, the putative role of metallothioneins in cisplatin resistance has received attention. Although cisplatin can interact with a metallothionein, it is an exceptionally poor inducer of the protein and this mechanism is unlikely to be favored in acquired cisplatin resistance (Bakka et al., 1981; Zalazowski et al., 1984). In general it would appear that the induction of metallothionein in response to cisplatin challenge is modest and cannot account quantitatively for the degree of acquired resistance

129

observed (Andrews et al., 1987). Similarly, there is no clear relationship between the level of metallothioneins induced (by various metals) in kidney and liver and consequent reduction in cisplatin toxicity (Naganuma et al., 1985). Nonetheless it is clear that the mRNA for this protein is induced during the development of acquired cisplatin resistance in vitro (Kelley and Lazo, 1987). It would appear that the contribution of metallothionein to acquired cisplatin resistance requires further evaluation.

4. THE ROLE OF GSH IN THE ACTIVATION AND UPTAKE OF ANTICANCER DRUGS Glutathione may play a role in mediating bleomycin cytotoxicity. The clinical formulation comprises a mixture of two closely related glycopeptides, bleomycin A2 and bleomycin B2 and is free of copper (Ishizuka et al., 1967; Lazo et al., 1987; Umezawa et al., 1966). In vivo it is known to chelate various metal ions, in particular iron and copper (Dabrowiak, 1980; Takahashi et al., 1977). The antitumor activity of bleomycin is thought to depend upon the cleavage of DNA by formation of reactive oxygen species such as hydroxyl radicals (Sausville et al., 1978; Solaiman et al., 1979, Suzuki et al., 1969). It has been suggested that an Fe(II)-bleomycin complex may act as a redox catalyst in the reduction of 02 to such active species and that GSH provides the reducing equivalent (Antholine et aL, 198 I; Solaiman et al., 1979). However, the involvement of reducing agents other than GSH cannot be ruled out (Russo et al., 1984). Neocarzinostatin (NCS), whose antitumor activity is also dependent upon DNA cleavage by free radical generation is thought to require both reduction by cellular thiols and electron transfer to oxygen for its activation (Hatayama and Goldberg, 1980; Kappen and Goldberg, 1978). GSH is probably the major cellular reducing agent inolved, since its depletion reduces the cytotoxic action of NCS (DeGraff et al., 1985). GSH has also been implicated in the transport of methotrexate (MTX). Leszczynska and Pfaff (1982) showed that MTX uptake by rat hepatocytes could be accelerated by the addition of GSH to the medium. They postulated that GSH-induced alteration of the plasma membrane -S-S-/-SH group redox status was involved in the control of MTX permeability. In addition, coadministration of GSH, in its reduced, but not oxidized form, with rifamycin SV in rodents was demonstrated to alter the tissue distribution and pharmacokinetics of the antibiotic (Leszczynska, 1980). Hence it may be possible to improve the efficacy of certain drugs by coadministration with GSH.

5. SUMMARY GSH appears to fulfil an unequivocal role in detoxifying radical intermediates and electrophilic metabolites derived from both normal metabolism and anticancer drugs (predominantly those which embrace alkylating or radical-dependent cytotoxic mechanisms). The availability of agents such as BSO,

P. MISTRY and K. R. HARRAP

130

capable of selectively modifying intracellular G S H levels, have been powerful tools in deciding the relevance of G S H as a resistance determinant. Such compounds may also have selective value when used in binary combination with established anticancer drugs. In this respect, the outcome of on-going clinical studies is awaited eagerly. G S H would appear to be important also in promoting the uptake of other drugs, such as methotrexate, where carrier-mediated incorporation and subsequent metabolism to polyglutamated species has been demonstrated as an important component of cytotoxicity. In such instances, G S H may well be exerting an essential role in the maintenance of membrane function and integrity. Our knowledge of this simple, yet still fascinating molecule has accrued over a century of investigation. Of particular importance to cancer chemotherapy have been the initial observations of Gowland Hopkins concerning the putative role of G S H in oxido-reduction, still relevant today, and the more recent discovery of the 7-glutamyl cycle and its modulatory influence on the efficacy of cytotoxic drugs. Acknowledgements--This work was supported by grants to

The Institute of Cancer Research: Royal Cancer Hospital from the Cancer Research Campaign and the Medical Research Council. The authors are grateful to Mrs Ann Ford for her excellent preparation of the manuscript.

REFERENCES ANDREWS,P. A., MURPHY, M. P. and HOWELL,S. B. (1985) Differential potentiation of alkylating and platinating agent cytotoxicity in human ovarian carcinoma cells by glutathione depletion. Cancer Res. 45: 6250-6253. ANDREWS,P. A., MURPHY, M. P. and HOWELL,S. B. (1986) Differential sensitization of human ovarian carcinoma and mouse LI210 cells to cisplatin and melphalan by glutathione depletion. Molec. Pharmac. 30: 643-650. ANDREWS,P. A., MURPHY,M. P. and HOWELL,S. B. (1987) Metallothionein-mediated cisplatin resistance in human ovarian carcinoma cells. Cancer Chemother. Pharmac. 19: 149-154. ANDREWS, P. A., SCHEIFER, i . A., MURPHY, M. P. and HOWELL, S. B. (1988) Enhanced potentiation of cisplatin cytoxicity in human ovarian carcinoma cells by prolonged glutathione depletion. Chem.-Biol. Interact. 65: 51-58. ANTHOLINE, W. E., PETERING,D. H., SARYUAN,L. A. and BROWN, C. E. (1981) Interactions among iron II bleomycin, Lewis bases, and DNA. Proc. ham. Acad. Sci. U.S.A. 78: 7517-7520. ARRICK, B. A. and NATHAN, L. F. (1984) Glutathione metabolism as a determinant of therapeutic efficacy: A review. Cancer Res. 44: 4224-4232. BABSON,J. R. and REED,D. J. (1978) Inactivation of glutathione reductase by 2-chloroethylnitrosourea derived isocyanates. Biochem. biophys. Res. Commun. 83: 754--762. BABSON,J. R., ABELL,N. S. and REED,D. J. (1981) Protective role of the glutathione redox cycle against adriamycin mediated toxicity in isolated hepatocytes. Biochem. Pharmac. 30: 2299-2304. BACHUR, N. R., GORDON, S. L., GEE, M. V. and KON, H. (1979) NADPH cytochrome P-450 reductase activation of quinone anticancer agents to free radicals. Proc. hath. Acad. Sci. U.S.A. 76: 954--957. BAKKA, A., ENDRESEN, L., JOHNSEN, A. B. S., EDMINSON, P. D. and RUGSIAD, H. E. (1981) Resistance against cis-dichlorodiammineplatinum in cultured cells with a

high content of metallothionein. Toxic. appl. Pharmac. 61: 215-226. BATIST,G., TUPULE,A., SINttA, B. K., KATrd, A. G., MYERS, C. E. and COWA~, K. H. (1986) Over-expression of a novel anionic glutathione transferase in multidrug resistant human breast cancer cells. J. biol. Chem. 261: 15544-15549. BEGLEITER,A., GROVER, J., FROESE,E. and GOLDENmmG, G. J. (1983) Membrane transport, sulfhydryl levels and DNA cross-linking in Chinese hamster ovary cell mutants sensitive and resistant to melphalan. Biochem. Pharmac. 132: 293-300. BRANDT, E. L. and GmFFtN, A. C. (1951) Reduction of toxicity of nitrogen mustards by cysteine. Cancer 4: 1030-1035. BULLER, A. L., CLAPPER, M. L. and TEW, K. D. (1987) Glutathione S-transferases in nitrogen mustard-resistant and -sensitive cell lines. Molec. Pharmac. 31: 575-578. CALCUTT,G. and CONNOP,S, T. A. 0963) Tumor sulfhydryl levels and sensitivity to the nitrogen mustard merophan. Biochem. Pharmac. 12: 839-845. C'MASSEAUD,L. F. (1979) The role of glutathione and glutathione-S-transferase in the metabolism of chemical carcinogens and other electrophilic agents. Adv. Cancer Res. 29: 176-199. DAaROWlAK, J. C. (1980) The coordination chemistry of bleomycin: A review. J. inorg. Biochem. 13: 317-337. DE GRAEFF,A., SLEaOS,R. J. C. and RODENHUm,S. (1988) Resistance to cisplatin and analogues: Mechanisms and potential clinical implications. Cancer Chemother. Pharmac. 22: 325-332. DEGRAFF, W. G., Russo, A. and MITC~LL, J. B. (1985) Glutathione depletion greatly reduces neocarzinostatin cytotoxicity in chinese hamster V79 cells. J. biol. Chem. 260: 8312-8315. DE REY-PAILHADE, J. (1888a) Sur un corps d'origine organique hydrog6nant le soufre :i froid. Compt. Rend. Acad. Sci. 106: 1683-1684. DE REY-PAmHADE,J. (1888b) Novelle recherches physiologyque sur la substance organique hydrog6nant le soufre :i froid. Compt. Rend. Acad. Sci. 107: 43-44. DOROSHOW,J. H., LOCKER,G. Y., IvmM, I. and MvEgs, C. E. (1982) Prevention of doxorubicin cardiac toxicity in the mouse by N-acetylcysteine. J. din. Invest. 68.' 1053-1064. EASTMAN, A., SCHULTE,N., SHEIBANI, N. and SORENSON, C. M. (1988) Mechanisms of resistance to platinum drugs. In: Proc. 5th Int. Syrup. Platinum and Other Metal Coordination Compounds in Cancer Chemother., pp. 178-196, NICOLINI, M. (ed.) Martinus Nijhoff, Boston. ENDRES£N, L., BAKKA, A. and RUGSTAD, H. E. (1983) Increased resistance to chlorambucil in cultured cells with a high concentration of cytoplasmic metallothionein. Cancer Res. 43: 2918-2926. EVANS,C. G., BODELL,W. J., TOKUDA,K., JOANE-SE12ER,P. and SMITH,M. T. (1987) Glutathione and related enzymes in rat brain tumour cell resistance to 1,3-bis(2-chloroethyl)-l-nitrosourea and nitrogen mustard. Cancer Res. 47: 2525-2530. EWTOO,H. L., HIKENS,J. H. and SHARMA,C. D. (1981) Role of glutathione in the metabolism dependent toxicity and chemotherapy of cyclophosphamide. Cancer Res. 41: 3584--3591. FREEMAN,R. W., MACDONALD,J. S., OLSON,R. D., BoEgra, R. C., OATES,J. A. and HARmSON,R. D. (1980) Effect of sulphydryl-containing compounds on the antitumour effects of adriamycin. Toxic. appl. Pharmac. 54: 168-175. GP.J~N, J. A., VISTtCA,D. T., YOUNG,R. C., HAMILTON,T. C., ROGAN, A. M. and OZOLS, R. F. (1984) Potentiation of melphalan eytotoxicity in human ovarian cancer cell lines by $1utathione depletion. Cancer Res. 44:5427-5431. HAMILTON,T. C., WINKLER, M. A., LOUSlE,K. G., BATISr, G., BL,-ml~s, B. C., TSL~O, T., GROTZINGER, K. R.,

Glutathione and cancer chemotherapy McKoY, W. M., YOUNO, R. C. and OZOLS, R. F. (1985) Augmentation of adriamycin, melphalan and cisplatin cytotoxieity in drug-resistant and -sensitive human ovarian cancer cell line by buthionine sulfoximine mediated (3SH depletion. Biochem. Pharmac. 34: 2583-2586. HARINGrON, C. H. and MEAD,T. H. (1935) The synthesis of glutathione. Biochem. J. 29: 1602-1611. HATAYA~O,, T. and GOLDSERG, I. H. (1980) Deoxyribonucleic acid sugar damage in the action of neocarzinostatin. Biochemistry 19: 5890-5898. HIRONO, I. (1960) Non-protein sulphydryl group in the original strain and sub-line of the ascites tumour resistant to alkylating reagents, Nature 186: 1059-1060. HmONO, I. (1961) Mechanism of natural and acquired resistance to methyl-bis-(beta-chloroethyl)amine-N-oxide in ascites tumours. Gann 52: 39-48. HOLLERAN, W. M., DE GREGOPaO, M. V., GANAPATHI, R., WILBUR, J. R. and MACHER, B. A. (1986) Characterisation of cellular lipids in doxorubicin-sensitive and -resistant P388 mouse leukaemia cells. Cancer Chemother. Pharmac. 17:11-15. HOLMGREN, A. (1979) Glutathione-dependent synthesis of deoxyribonucleotides~purification and characterisation of glutaredoxin from Eschericia coll. J. biol. Chem. 254: 3664-3671. HOPKINS, F. G. (1921) An autoxidizable constituent of the cell. Biochem. J. 15: 286-305. HOPKINS, F. G. (1929) Glutathione: A reinvestigation. J. biol. Chem. 84: 269-320. HOPKINS, F. G. and DIXON, M. (1922) Glutathione II. A thermstable oxidation-reduction system J. biol. Chem. 54: 527-563. HOPKINS, F. G. and ELLIOT'r, K. A. C. (1931) Relation of glutathione to cell respiration with special reference to hepatic tissue. Proc. R. Soc. (Lond.) BI09: 58-88. HOSKING, L. K., WHELAN, R. D. H., SHELLARD, S. A., BEDFORD, P. and HILL, B. T. (1990) An evaluation of the role of glutathione and its associated enzymes in the expression of differential sensitivities to antitumour agents shown by a range of human tumour cell lines. Biochem. Pharmac. 40: 1833-1842. ISHIZUKA, i . , TAKAYAMA,H., TAKEUCHI,T. and UMEZAWA, H. (1967) Activity and toxicity of bleomycin. J. Antibiot., Tokyo A 20: 11-24. KAPPEN, L. S. and GOLDBERG,I. H. (1978) Activation and inactivation of neocarzinostatin-induced cleavage of DNA. Nucleic Acids Res. 5: 2959-2967. KAPPUS, H. (1986) Overview of enzyme systems involved in bioreduction of drugs and in redox cycling. Biochem. Pharmac. 35: 1-16. KELLEY,S. L. and LAZO,J. S. (1987) Metallothionein content and antineoplastic drug resistance. Proc. Am. Assoc. Cancer Res. 28: 281. KENDALL, E. C., MCKENZIE, B. F. and MASON, H. U (1929) Glutathione---l. Its preparation in crystalline form and its identification. J. biol. Chem. 84: 657-674. KOSOWER, N. S. and KOSOWER,E. M. (1978) The glutathione status of cells. Int. Rev. Cytol. 54: 109-159. KOSOWER, N. S. and KOSOWER, E. M. (1983) Glutathione and cell membrane thiol status. In: Functions of Glutathione: Biochemical, Physiological, Toxicological and Clinical Aspects, pp. 307-315, LARSSON,A., ORgXN1US,S., HOLMGREN, A. and MANNERVIK, B. (eds) Raven Press, New York. KRAMER, R. A., ZAKKER, J. and K1M, G. (1988) Role of the glutathione redox cycle in acquired and de novo multidrug resistance. Science 241: 694-697. LAI, G. M., OZOLS, R. F., YOUNG, R. C. and HAMILTON,T. C. (1989) Effect of glutathione on DNA repair in cisplatinresistant human ovarian cancer cell lines. J. hath. Cancer Inst. 81: 535-539. LARSSON, A., ORRENIUS, S., HOLMGREN,A. and MANNERVIK, B. (eds) (1983) Functions of Glutathiane: Biochemical,

131

Physiological, Toxicological and Clinical Aspects, Raven Press, New York. LAZO, J. S., SEmi, S. M. and FILDERMAN, A. E. (1987) Metabolism of bleomycin and bleomycin-like compounds. In: Metabolism and Action of Anticancer Drugs, pp. 194-210, POwlS, (3. and I~OUGH, R. A. (eds) Taylor and Francis, London. LEE, F. Y. F. and SmMANN,D. W. (1989) Isolation by flow cytometry of human ovarian tumor cell subpopulation exhibiting a high glutathione content phenotype and increased resistance to adriamycin. Int. J. Radiat. Oncol. Biol. Phys. 16: 1315-1319. LEE, F. Y. F., VESSEY, A. R. and SIEMANN, D. W. (1988) Glutathione as a determinant of cellular response to doxorubicin. NCI Monogr. 6: 211-215. LESZCZY~SKA, A. (1980) Effect of reduced glutathione ((3SH) on pharmacokinetics and distribution of rifamycin SV in rats. Biochem. Pharmac. 30: 71-76. LESZCZYNSKA, A. and PFAFF, E. (1982) Activation by reduced glutathione of methotrexate transport into isolated rat liver cells. Biochem. Pharmac. 31:1911-1918. LEWIS, A. D., HAVES, J. D. and WOLF, C. R. (1988a) (31utathione and glutathione-dependent enzymes in ovarian adenocarcinoma cell lines derived from a patient before and after the onset of drug resistance: intrinsic differences and cell cycle effects. Carcinogenesis 9: 1283-1287. LEWIS, A. D., HICKSON, I. D., ROBSON,C. N., HARRIS, A. L., HAYES, J. D., GRIFFITHS, S. A., MANSON, M. M., HALL, A. E., MOSS,J. E. and WOLF, C. R. (1988b) Amplification and increased expression of alpha class glutathione S-transferase-encoding genes associated with resistance to nitrogen mustards. Proc. nam. Acad. Sci. U.S.A. 85: 8511-8515. LEWIS, A. D., FORRESTER, L. M., HAYES, J. D., WAREING, C. J., CARMICHAEL,J., HARRIS, A. L., MOOGHEN, M. and WOLF, C. R. (1989) Glutathione S-transferase isoenzymes in human tumours and tumour derived cell lines. Br. J. Cancer 60: 327-331. LUTHMAN, M., ERIKSSON, S., HOLMGREN, A. and THELANDER, L. (1979) Glutathione-dependent hydrogen donor system for calf thymus ribonucleoside-diphosphate reductase. Proc. natn. Acad. Sci. U.S.A. 76: 2158-2162. MANNERVIK, B. (1985) The isoenzymes of glutathione transferase. Adv. Enzymol. related Areas molec. Biol. 57: 357-417. MANNERVIK, B., BOARD, P. G., BERHANE, K., BJORNESTEDT, R., CASTRO,V. M., DANIEl,SON, U. H., HAD, X.-Y., KOLM, R., OLIN, B., PRINCIPATO, G. B., RIDDERSTROM, M., STENBERG,G. and WIDERSTEN,M. (1990) Classes of glutathione transferases: Structural and catalytic properties of the enzymes. In: Glutathione-S-transferases and Drug Resistance, pp. 35-46, HAYES, J. D., PICKETT, C. B. and MANTLE, T. J. (eds) Taylor and Francis, London. McGOwN, A. T. and Fox, B. W. (1986) A proposed mechanism of resistance to cyclophosphamide and phosphoramide mustard in a Yoshida cell line /n vitro. Cancer Chemother. Pharmac. 17: 223-226. MEISTER, A. (1983) Selective modification of glutathione metabolism. Science 220: 472-477. MEISTER, A. and ANDERSON, M. E. (1983) Glutathione. A. Rev. Biochem. 52: 711-760. MISTRY, P., KELLAND, U R., ABEL, G., SIDHAR, S. and HARRAP, K. R. (1990) The relationships between glutathione, glutathione-S-transferase and platinum drug cytotoxicity in eight human ovarian carcinoma cell line. Proc. Am. Assoc. Cancer Res. 31: 368. MYERS, C. (1986) Anthracyclines. In: EORTC Cancer Chemotherapy Annals, Vol. 8, pp. 52-63, PINEDO, H. M. and CHABN'ER, B. A. (eds) Elsevier, Amsterdam. MYERS, C. E., McGuIp,.E, W. P. and YOUNG, R. (1976) Adriamycin: amelioration of toxicity by alpha-tocopherol. Cancer Treat. Rep. 60: 961-962.

132

P. MLSTRY and K. R. HAmod,

NAGANUMA, A., SOTAI-t, M., KOYAMA, Y. and IMURA, N. (1985) Protective effect of metallothionein inducing metals on lethal toxicity of cis-diamminedichloroplatinum in mice. Toxic. Lett. 24: 203-207. NAKAGAWA, K., SALIO, N., TSUCHIDA, S., SAKAI, M., TSUNOKAWA, M., YOKOTA,J., MURUMA'rsu, M., SATO, K., TERADA, M. and "FEw, K. D. (1990) Glutathione S-transferase n as determinant of drug resistance in transfectant cell lines. J. biol. Chem. 265: 429~4301. OLSEN, R. D., MACDONALD, J. S., VAN BOXTEL, C. J., BOERTH, R. C., HARBISON,R. D., SOLNIM,A. E., FREEMAN, R. W. and DATES, J. A. (1983) Regulatory role of glutathione and soluble sulfhydryl groups in the toxicity of adriamycin, d. Pharmac. exp. Ther. 215: 450-454. OZOLS, R. F., LOUIE, K. G., PLOWMAN, J., BEHRENS, B. C., FINE, R. L., DYKES, n. and HAMILTON, T. C. (1987) Enhanced melphalan cytotoxicity in human ovarian cancer in vitro and in tumor-bearing nude mice by buthionine sulfoximine depletion of glutathione. Biochem. Pharmac. 36: 147-153. PECZENIK,O. (1953) Influence of cysteinamine, methylamine and cortisone on the toxicity and activity of nitrogen mustard. Nature 172: 454-455. PmIE, N. W. and PINHEY, K. G. (1929) The titration curve of glutathione, d. biol. Chem. 84: 321-333. POWlS, G. (1987) Anthracycline metabolism and free radical formation. In: Metabolism and Action o f Anti-cancer Drugs, pp. 211-260, POwlS, G. and PROUGH, R. A. (eds) Taylor and Francis, London. PUCHALSKI, R. B. and FAHL, W. E. (1990) Expression of recombinant glutathione S-transferase n, Ya or Yb t confers resistance to alkylating agents. Proc. ham. Acad. Sci. U.S.A. 87: 2443-2447. RAMU, A., COHEN, L. and GLAUBIGER,D. (1984) Oxygen radical detoxification enzymes in doxorubicin-sensitive and resistant P388 murine leukaemia cells. Cancer Res. 44: 1976-1980. REED, D. J. and BEAr'I'Y, P. W. (1980) Biosynthesis and regulation of glutathione: Toxicological implications. In: Reviews in Biochemical Toxicology, Vol. 2, pp. 213-241, HODGSON, E., BEND, J. R. and PHILPOT, R. M. (eds) Elsevier, New York. REVESZ, L. and MODIG, H. (1965) Cysteamine-induced increase of cellular glutathione level: A new hypothesis of the radioprotective mechanism. Nature, Land. 207: 430-431. RICHON, V. M., SCHULTE, N. and EASTMAN,A. (1987) Multiple mechanisms of resistance to cis-diamminedichloro platinum (It) in routine leukaemia LI210 cells. Cancer Res. 47: 2056-2061. ROBSON, C. N., LEWIS, A. n., WOLF, C. R., HAYES, J. n., HALL, A., PROCTOR, S. J., HARRIS, A. L. and H1CKSON, I. D. (1987) Reduced levels of drug-induced DNA crosslinking in nitrogen mustard-resistant Chinese hamster ovary cells expressing elevated glutathione S-transferase activity. Cancer Res. 47: 6022--6027. ROMINE, M. T. and ~ E L , D. (1986) Intracellular glutathione as a determinant of responsiveness to antitumour drugs. Biochem. Pharmac. 35: 3323-3326. Russo, A., MITCHELL, J. B., McPHERSON, S. and FRIEDMAN, N. (1984) Alteration of bleomycin cytotoxicity by glutathione depletion or elevation. Int. J. Radiat. Onco/. Biol. Phys. 10:1675 1678. Russo, A., CARMICHAEL, J., FRIEDMAN, N., DEGRAFF, W., TOCHNER, Z., GLATSTEIN, E. and MITCHELL,J. B. (1986a) The role of intracellular glutathione in antineoplastic chemotherapy. Int. J. Radiat. Oncol. Biol. Phys. 12: 1347-1354.

Russo, A., DEGRAFF, W., FRIEDMAN,N. and MITCHELL,J. B. (1986b) Selective modulation of glutathione levels in human normal versus tumour cell and subsequent differential response to chemotherapy drugs. Cancer Res. 46: 2845-2848. SAUSVILLE, E. A., PEISACH, J. and HORWITZ, S. B. (1978) Effects of chelating agents and metal ions on the degradation of DNA bleomycin. Biochemistry 17: 2740-2745. SKAPEK, S. X., COLVIN,O. M., GRIFFITH,O. W., ELION,G. B., BIGHER, D. D. and FRIEDMAN, H. S. (1988) Enhanced melphalan cytotoxicity following buthionine sulfoximinemediated glutathione depletion in a human medulloblastoma xenograft in athymic mice. Cancer Res. 48: 2764-2767. SMITH, E. and BROCK, A. P. (1988) An in vitro study comparing the cytotoxicity of three platinum complexes with regard to the effect of thiol depletion. Br. J. Cancer 57: 548-552. SOLAIMAN,D., 1L~O,E. A., PETERING,D. H., SEALY,R. C. and ANrHIOLINE, W. E. (1979) Chemical, biochemical, and cellular properties of copper and iron bleomycins. Int. J. Radiat. Oncol. Biol. Phys. 5: 1519-1521. SUZUKAKE, K., PETRO, B. J. and VISTICA, D. T. (1982) Reduction in glutathione content of L-PAM resistant LI210 cells confers drug sensitivity. Biochem. Pharmac. 31: 121-124. SUZUKAKE, K., VISTICA, B. P. and VISTICA, D. T. (1983) Dechlorination of L-PAM by sensitive and resistant tumour cells and its relationship to intracellular glutathione content. Biochem. Pharmac. 31: 165-167. SUZUKI, H., NAGIA, K., YAMAKI, H., TANAKA, N. and UMEZAWA, H. (1969) On the mechanism of action of bleomycin: Scission of DNA strands in vitro and in vivo. J. Antibiot., Tokyo 22: 446-448. TAKAHASHI,K., YOSHIOKA,O., MATSUDA,A. and UMEZAWA, H. (1977) Intracellular reduction of cupric ion of bleomycin copper complex and transfer of cuprous ions to a cellular protein. J. Antibiotics 30: 861-869. TEICHER, B. A., HOLDEN, S. A., KELLEY, M. J., SHEA, T. C., CUCCHI, C. A., ROSOWSKY,A., HENNER, W. D. and F~I, E., I11 (1987) Characterisation of a human squamous carcinoma cell line resistant to cis-diamminedichloroplatinum 11. Cancer Res. 47: 388-393. TEw, K. D. and WANG, A. L. (1982) Selective toxicity of haloethylnitrosoureas in a carcinoma cell line resistant to bifunctional alkylating agents. Molec. Pharmac. 21: 729-738. TEw, K. D., K'fLE, G., JOHNSON, A. and WANG, A. L. (1985) Carbamoylation of glutathione reductase and changes in cellular and chromosome morphology in a rat cell line resistant to nitrogen mustards but collaterally sensitive to nitrosoureas. Cancer Res. 45: 2326--2333. UMEZAWA, H., MAEDA, K., TAKEUCHI, T. and OKAMI,Y. (1966) New antibiotics, bleomycin A and B. J. Antibiotics, Tokyo A 19: 200-209. WANG, A. L. and TEW, K. D. (1985) Increased glutathione S-transferase activity in a cell line with acquired resistance to nitrogen mustard. Cancer Treat. Rep. 69: 677~82. WOLF, C. R., LEWIS, A. D., CARMICHAEL,J., ADAMS, D. J., ALLAN, S. G. and ANSELL, D. J. (1987) The role of glutathione in determining the response of normal and tumour cells to anticancer drugs. Biochem. Soc. Trans. 15: 728-730. ZALAZOWSKI, A. J., GARVEY, J. S. and HOESCHELE, J. D. (1984) In rivo and in vitro binding of platinum to metallothionein. Archs Biochem. Biophys. 229: 246-252.