Glutathione, Ascorbate, and Cellular Protection1

ICANCÃ•RRESEARCH(SUPPL.)54, Glutathione, Ascorbate, and Cellular Protection1

Alton Meister Department of Biochemistry, Cornell University Medical College, New York, New York 10021

Introduction a-tocopherol in its reduced form, either by a direct reaction or by a pathway involving ascorbate (10-17). Glutathione. which has the Glutathione. a tripeptide thiol present in virtually all animal cells, is important function of maintaining the reducing milieu of cells, is synthesized within many cells from its constituent amino acids (glu tamate, cystcine, glycine); these "nonessential" amino acids can be undoubtedly involved in the reduction of many cellular components; e.g., other tocopherols and ÃŸ-carotenearc apparently also maintained synthesized within the body and are also obtained from the diet. by glutathione-mediated reactions (e.g.. RÃ©f.18). Glutathione is also synthesized by tumors, some of which (notably An interesting aspect of glutathione metabolism and function re drug- and radiation-resistant tumors) exhibit high cellular levels of lates to drug-resistant and radiation-resistant tumors that have high glutathione and high capacity for the synthesis of glutathione. We levels of glutathione or exhibit high capacity for glutathione synthesis. reported at previous conferences in this series on the increase of Such tumors have a greater requirement for glutathione than do many cellular radiosensitivity that occurs after administration of an inhibitor normal tissues and this provides a promising chemotherapeutic ap of glutathione synthesis (1, 2) and on the effects of modulation of proach, which is considered below. glutathione metabolism (3, 4). These and related topics (5, 6) will be summarized here with emphasis on some current developments. Biochemistry of Glutathione: Enzymology and Transport Glutathione is probably the most important cellular antioxidant. Phenomena Interestingly, Fahey and Sundquist (7) found strong evidence for an evolutionary link between glutathione and aerobic eukaryotic metab Fig. 1 gives some of the biochemical transformations of glutathi olism; the findings indicate that glutathione evolved as a molecule that one, which is synthesized in two steps from glutamate, cystcine, and protects cells against oxygen toxicity. Although there is currently glycine (Reactions / and 2). Metabolic utilization of glutathione much interest in the hypothesis that oxidative phenomena may lead to follows several pathways including reactions catalyzed by the gluta thione S-transferases (mercapturate pathway). Glutathione is a sub a variety of pathological states, and that antioxidants may play a strate of the glutathione peroxidases which destroy hydrogen peroxide significant protective role, the important role that glutathione plays in and organic peroxides. The glutathione disulfide formed is reduced to the protection of cells has sometimes been insufficiently appreciated. glutathione in an NADPH-mediated reaction (Fig. 2). Glutathione not Cells that are deprived of glutathione typically suffer severe oxidative only provides reducing power needed for the conversion of dehy damage associated with mitochondria! degeneration. Analogous ef droascorbate to ascorbate, but also for the conversion of ribonucle- fects are not always found when there is a deficiency of certain other otides to deoxyribonucleotides and for a variety of thiol-disulfide cellular components that are thought to act as antioxidants. It has long interconversions; glutathione is therefore important for the synthesis been known that the antioxidant ascorbate is required in the diet of and repair of DNA, and for the folding of newly synthesized proteins. humans and certain other animals such as the guinea pig (but not by The utilization of glutathione (Fig. 1; y-glutamyl cycle) is initiated many other animals, including some commonly used in laboratory extracellularly by the actions of y-glutamyl transpeptidase and dipep- experiments; e.g., mice, rats, rabbits). The ascorbate deficiency syn tidase; these enzymes are bound to the outside of cell membranes. The drome, scurvy, which is associated with oxidative inactivation of transpeptidase acts on glutathione. glutathione disulfide and glutathi certain enzymes, can be prevented in humans by administration of as one 5-conjugates. The reactions catalyzed by y-glutamyl transpepti little as 10 mg/day of ascorbate. The officially recommended daily dase take place in the presence of amino acids and lead to the dose of ascorbate for humans is 30-100 mg/day (depending upon the formation of y-glutamyl amino acids (19). Cystine is the most active country); although much larger doses of ascorbate than this are taken amino acid acceptor of the y-glutamyl group (20); other neutral amino by some individuals, it is estimated that a substantial proportion of the acids such as methionine and glutamine are also good acceptors (21). human population takes in relatively small amounts. The question as y-Glutamyl amino acids are transported and become substrates of to whether larger doses of ascorbate and also of other "antioxidants" y-glutamyl cyclotransferase, which converts y-glutamyl amino acids would have beneficial effects has often been discussed but remains into 5-oxoproline and the corresponding free amino acids (22-25). unsettled. 5-Oxoproline is converted to glutamate in the reaction catalyzed by Experimental findings summarized here that are relevant to this 5-oxoprolinase (26, 27). Of the several reactions of the y-glutamyl question include: (a) the observation that glutathione deficiency in cycle, three require ATP, which is split to ADP and PÂ¡(Reactions /, animals that are unable to synthesize ascorbate (newborn rats, guinea 2 and 6). pigs) is lethal and that death can be prevented by giving high doses of It is notable that y-glutamyl transpeptidase is mainly extracellularly ascorbate; and (b) the onset of scurvy in guinea pigs that are fed a diet located, whereas glutathione is found principally within cells. Many deficient in ascorbate is substantially delayed by giving glutathione cells normally export glutathione. An early observation that led to the monoethyl ester, a glutathione delivery agent (6, 8). discovery of such transport was the finding of marked glutathionuria Various questions about the functions of putative antioxidant com and glutathionemia after administration of inhibitors of y-glutamyl pounds need to be considered in relation to the functions of cellular transpeptidase to experimental animals (28-30). Interestingly, the glutathione. As discussed here, one such function, shown in vivo (9), urine of animals given such inhibitors contains cysteine and y-glu- is to reduce dehydroascorbate to ascorbate. Glutathione also keeps tamylcysteine moieties as well as glutathione. Patients who are defi cient in transpeptidase show similar findings (30). The physiological 1 Presented at the 4th International Conference on Anticarcinogenesis & Radiation function of y-glutamyl transpeptidase is thus closely connected with Protection. April 1H-23, 1993, Baltimore. MD. The research described here that was carried out in the author's laboratory was supported in part by NIH Grant 2 R37 DK12034 the metabolism and transport of glutathione. When y-glutamyl from the United States Public Health Service. transpeptidase is markedly decreased, there is a substantial loss of 1969s

OXIDATION-REDUCTION PATHWAYS bile. Plasma glutathione is used by many tissues, e.g., kidney, lung, and brain. Glutathione itself is not significantly transported into most GSSG of the cells of these tissues but is broken down by membrane-bound y-glutamyl transpeptidase and dipeptidase; the products of breakdown are transported and utilized for glutathione synthesis. This is an important pathway of glutathione metabolism.

Effects of Glutathione Deficiency

Information relevant to this topic has come from observations on human mutants that have decreased levels of glutathione (e.g., pa tients with glutathione disulfide reducÃasedeficiency, glucose-6-phosphate dehydrogenase deficiency, deficiencies of y-glutamylcysteine 2HJ synthetase and of glutathione synthetase) (34), and from experimental MERCAPTURATE Ã )/ Glutamate PATHWAY studies on animals in which glutathione deficiency was produced (5). Humans deficient in glutathione may exhibit increased tendency to hemolysis, cataracts, and central nervous system abnormalities. In one condition (glutathione synthetase deficiency) there is a secondary metabolic acidosis, often life-threatening, due to overproduction of Y-Glu-AA 5-oxoproline. Glutathione levels are markedly reduced in some of these patients, but in none of them is glutathione completely absent. Experimental production of glutathione deficiency has been at Fig. 1. Overall pathway of glutathione (GSH) metabolism. /. y-glutamylcysteine tempted by administration of compounds that react with glutathione synthetase; 2, glulathione synthetase; 3, y-glutamyl transpeptidase; 4, cysteinyl glycine hydrolases; 5, y-glutamyl cyclotransfera.se; 6, 5-oxoprolinase; 7, glutathione 5-trans- (e.g., diethyl malÃ©ate,phorone) and oxidizing agents (e.g., diamide ferases; 8, transport and reduction of y-Glu-(Cys)2; 9, see Fig. 2. Reactions 1, 2, and 6 and t-butyl hydroperoxide). However, as discussed elsewhere (5), involve cleavage of ATP to ADP and P,. such nonspecific agents have major limitations. Thus far, the most useful approach to production of glutathione deficiency has been -GSSG treatment with specific inhibitors of y-glutamylcysteine synthetase, the enzyme that catalyzes the first and rate-limiting step of glutathione synthesis. Deoxyribonucleottd NADPH, H The discovery that methionine sulfoximine inhibits y-glutamylcys teine synthetase followed from previous studies which showed that this enzyme, like glutamine synthetase, involves a mechanism in NADP which y-glutamyl phosphate is an intermediate (35). Studies with various substrates and substrate analogues led to mapping of the

V-GLU CYCLE

Fig. 2. Oxidation reduction pathways. These reactions involve those catalyzed by glutathione transhydrogenases, glutathione peroxidases (Se-containing and others), and glutathione disulfidc reducÃase.

cysteine moieties. The pathway (illustrated in Fig. 3), which was first elucidated in studies on the kidney, serves as a recovery system for cysteine moieties (31). Thus, exported glutathione interacts with cystine and y-glutamyl transpeptidase to produce y-glutamylcystine which is transported. High levels of extracellular glutathione inhibit such transport (32). y-Glutamylcystine is reduced intracellularly to yield cysteine and y-glutamylcysteine, both of which are used for the synthesis of glutathione. The cysteinylglycine formed in the transpep- CyS CyS - CySH-Gly tidation reaction is split extracellularly to form cysteine and glycine; Fig. 3. "Salvage" pathway. Glutathione (GSH), synthesized intracellularly (reactions / this reaction may also occur intracellularly after transport of the and 2), is exported (3) and reacts with y-glutamyl transpeptidase and extracellular cystine dipeptide. y-Glutamyl transpeptidase and cysteinylglycine dipepti- (4) to produce y-glutamylcystine, which is transported (5) and reduced intracellularly (7) to form cysteine and y-glutamylcysteine. Transport of y-glutamylcystine is inhibited by dase activity thus function in the recovery of cysteine moieties that are extracellular GSH (6). Experiments in which y-glutamylcystine was selectively labeled with 15S on the internal (Sf) or external (S*) S atom gave results consistent with this necessary for the functioning of a recycling pathway involving syn pathway (31). A block at step 4 (reaction catalyzed by y-glutamyl transpeptidase) leads to thesis and export of glutathione (33). accumulation of glutathione and to urinary excretion of glutathione and cysteine and Glutathione is exported by the liver to the blood plasma and to the y-glutamylcysteine moieties (see text). 1970s

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1994 American Association for Cancer Research. OLUTATHIONE, ASCORBATE. AND CELLULAR PROTECTION active site ofglutamine synthetase and to design of selective inhibitors these agents might make tumor cells more sensitive to chemotherapy of glutamine synthetase (a-ethyl methionine sulfoximine) and of and to radiation treatment (41 ). The potential usefulness of BSO in the y-glutamylcysteine synthetase [prothionine sulfoximine, BSO,2 and sensitization of cells to radiation was first directly shown in studies on others (35-40)]. BSO and related compounds have very little or several human lymphoid cell lines (1, 2). Cells that had 4-5% of the virtually no effect on glutamine synthetase but inhibit y-glutamylcys control levels of glutathione were found to be much more sensitive teine synthetase very effectively. BSO, now commercially available, than the controls to the effects of y-irradiation. It was found that has been widely used in in vitro experiments and in vivo. The effects treatment with BSO of mice bearing B16 melanomas sensitizes this of glutathione deficiency induced by administration of BSO have been radio-resistant tumor to radiation (61, 62). Treatment with BSO alone extensively examined (see Ref. 5) and include the following: did not affect tumor growth, but treatment with BSO and radiation led 1. Glutathione deficiency sensitizes cells to the effects of radiation, to significant decrease in the size of the tumor and to an increase in the oxidative reactions, and to various toxic compounds. These effects longevity of the tumor-bearing mice. It was also found that treatment have been applied in chemotherapy and radiation systems (5, 41-44). of resistant Icukemias with BSO led to sensitization of the tumors to Tumor cell resistance associated with overproduction of glutathione is phenylalanine mustard (48. 49). In studies with mice bearing such reversed by treatment with BSO in animal models and is currently resistant tumors, i.p. infusion of BSO led to sensitization of the tumors being tested clinically (45). Depletion of glutathione by treatment with and to an increase in the life span of the treated animals. Additional buthionine sulfoximine sensitizes cells to the toxic effects of heavy studies on the relationship between glutathione levels and the expres metals (46, 47), nitrogen mustards (48, 49), radiation (1, 2, 5), sion of primary drug resistance and cross-resistance in human ovarian cisplatin (50), cyclophosphamide (51, 52), morphine (53), compounds cancer cell lines, together with studies in which an i.p. model of that produce oxidative cytolysis (54), and others (55). human ovarian cancer was developed in nude mice, were of impor 2. Glutathione deficiency leads to oxidative stress in many tissues tance because they led to a clinical trial of BSO which is now in (5). Mitochondria! and associated cell damage is found in mice treated progress (44, 45). In these studies it was also found that resistance of with BSO. Mitochondria do not synthesize glutathione but obtain it by the tumors to phenylalanine mustard is associated with resistance to transport from the cytosol. Several tissues of adult mice are affected other drugs such as Adriamycin. Of interest, these drug-resistant cells by administration of BSO, but in newborn rats and guinea pigs more are also resistant to radiation. It seems probable that at least one type extensive damage is found and there is early mortality due to multi- of multidrug resistance is associated with overproduction of glutathi organ failure (56). Glutathione deficiency in these experimental sys one; however, other cellular mechanisms can also lead to drug resis tems serves as a model of endogenously produced oxidative stress. tance. Much attention has been given to multidrug resistance associ Mortality and tissue damage are significantly decreased by adminis ated with a novel membrane glycoprotein; this type of resistance is tration of glutathione esters or of ascorbate (5, 9). associated with decreased accumulation within the tumor of a number 3. Deficiency of glutathione leads to decreased reduction of dehy- of structurally unrelated drugs. However, this P-glycoprotein-related droascorbate to ascorbate in vivo. This is observed in newborn rats and system does not appear to confer radiation resistance. guinea pigs, animals that cannot synthesize ascorbate, and also in Cellular levels of glutathione may determine the degree of drug adult mice, which can (6). In adult mice, glutathione deficiency leads resistance or radiation resistance of a particular tumor, but the capac to induction of ascorbate synthesis in the liver (57), and this explains ity of a tumor cell to synthesize glutathione may also be an important why BSO has less damaging effects on adult mice than it does on factor in resistance. The ability of a cell to synthesize glutathione newborn rats and guinea pigs. rapidly in response to a stress may be as important or perhaps more 4. Glutathione deficiency in newborn rats and mice leads to for important than the initial cellular level of glutathione. This idea is mation of ocular cataracts (9, 58, 59). Cataracts have also been found supported by model studies on a strain of Escherichia coli enriched in in some patients with inherited glutathione disulfidc reducÃasedefi its content of y-glutamylcysteine synthetase and glutathione syn ciency. thetase by recombinant DNA techniques (63). Recent studies on 5. Treatment of peripheral blood mononuclear cells with BSO was tumors that are resistant and sensitive to Adriamycin are consistent found to markedly inhibit their proliferation (4), and later work has with this idea (5, 64). confirmed that glutathione deficiency decreases lectin-induced prolif Studies on human ovarian tumor cell lines that are resistant to eration of lymphocytes (60). Although glutathione is required for cisplatin showed that cellular glutathione levels are greatly proliferation, the mechanism of its function in this system is still increased (13- to 50-fold) as compared with the sensitive cells of unsettled. origin (65). The cell lines examined exhibited up to 1000-fold increases in resistance to cisplatin. Cisplatin resistance was Application of Glutathione Depletion to the Treatment of associated with increased expression of mRNAs for y- Tumors: Sensitization of Tumors to Chemotherapy glutamylcysteine synthetase and y-glutamyl transpeptidasc and with and to Radiation increased activities of these enzymes. Thus, y-glutamylcysteine synthetase and y-glutamyl transpeptidase appear to contribute to the Early studies showed that tumor cells that are resistant to alkylating development of cisplatin resistance. It is notable that there was a agents have increased levels of nonprotein thiol, later shown to be significant increase in the levels of both subunits of y- glutathione (see Ref. 5). The resistance of leukemic cells to phenyl- glutamylcysteine synthetase. The heavy subunit (Mr 72,614) of this alanine mustard was found not to be related to an effect on uptake or enzyme (66) contains the binding sites for the substrates (ATP, efflux of the mustard, but to be closely related to the cellular level of glutamate, cysteine) and is feedback inhibited by glutathione (67). glutathione (48, 49). The resistant cells converted phenylalanine mus However, the light subunit (Mr 30,548) of this enzyme is required tard to a nontoxic compound in a glutathione-dependent dehydrochlo- for optimal activity and for physiologically appropriate feedback rination reaction. inhibition (68). It seems therefore to function in a regulatory After development of the amino acid sulfoximine inhibitors of manner. y-glutamylcysteinc synthetase, it was suggested that treatment with Because the effects of glutathione deficiency induced in experi mental animals by administration of BSO can be reversed to a sig ~ The abbreviation used is: BSO. buthionine sulfoximine. nificant extent by administration of large amounts of ascorbate (sec 1971s

below), it is relevant to consider the ascorbate status of patients treated Table 1 Effects of glutathiurie deficiency

with BSO (see Ref. 69). It is possible that administration of large EffectDeath, pigs-f-arats Guinea mice000+M0-+IIII+ doses of ascorbate to such patients would negate the desired effects of 4-6daysCell ++

BSO administration. 'LiverKidneyLungBrainLensSkeletaldamage ++ On the Essentiality of Glutathione: Mitochondria! Function ++ +++ + When BSO is administered to rats or mice there is rapid decline of 0+ 0II the glutathione levels of the liver and kidney to an apparent limiting muscleJejunum, value of 15-20% of the total glutathione initially present. Further colonHeartStomachLymphocytes" treatment with BSO leads to additional and gradual decline of cellular 0Ãœas glutathione. This biphasic decrease in the cellular glutathione level led +,deathMainly cell damage or to further investigations which showed that a substantial fraction of indicated.Adult cellular glutathione is sequestered in the mitochondria. BSO is not mitochondrial.Newborn transported significantly into mitochondria (70). However, mitochon dria were found to lack the enzymes required for glutathione synthe sis; therefore, the failure of BSO to enter mitochondria is not relevant, early mortality. The lethal effects of glutathione deficiency in animals but the absence of the synthetases from mitochondria showed that such as newborn rats and guinea pigs (which do not synthesize mitochondrial glutathione must arise from the cytosol (71). Studies on ascorbate) appear to be related to multiorgan failure involving mito isolated rat liver mitochondria indicate that mitochondrial glutathione chondrial and other damage in liver, kidney, and lung (56). In liver homeostasis is regulated by a multicomponent transport system which there is focal necrosis: in kidney, there is proximal tubular damage; appears to explain the remarkable ability of mitochondria to take up and in lung, there is lamellar body degeneration. The lungs of adult and to retain glutathione (72). Evidence was found for two transport mice showed damage, although less than in newborn rats. ers with apparent Km values of 60 Â¡JLMand5.4 mM. Extramitochondrial The mitochondrial and other cell damage seen in newborn animals glutathione promotes mitochondrial uptake and exchange, and the and in adults were prevented by administration of glutathione esters or intermembranous space appears to function as a recovery zone that by administration of ascorbate (see below). facilitates efficient cycling of matrix glutathione. Decreased levels of Function of Glutathione in the Reduction of Dehydroascorbate glutathione produced by administration of BSO decrease the net export of glutathione from mitochondria to the cytosol. That the net A function of glutathione in the reduction of dehydroascorbate was efflux of glutathione from mitochondria is very slow when there are suspected by early investigators. Borsook et al. (74) concluded that low levels of extramitochondrial glutathione is consistent with a glutathione is involved in the reduction of dehydroascorbate by ani mechanism that conserves mitochondrial glutathione during periods mal tissues, but Guzman-Barron. another early pioneer in this field, of cytosolic glutathione depletion. considered this unlikely (75). Hopkins and Morgan (76) studied this A significant fraction of the oxygen utilized by mitochondria (about reaction in plants. The reduction of dehydroascorbate to ascorbate was 2-5%) is converted, apparently through Superoxide, to hydrogen examined by a number of investigators in in vitro animal systems (see peroxide (73). When glutathione levels are greatly decreased, hydro Ref. 6), and purified preparations of glutaredoxin and protein disulfide gen peroxide accumulates, and this leads to extensive mitochondrial isomerase were found to exhibit substantial glutathione-dependent damage. Other antioxidants may be involved in the protection of dehydroascorbate reducÃaseactivity (77). mitochondria, but glutathione appears to be the principal functional Convincing evidence linking glutathione to the reduction of dehy one. Mitochondria do not contain catalase and are therefore largely, if droascorbate in vivo has recently been obtained (6, 9). In the first of not entirely, dependent upon glutathione and glutathione peroxidases. these studies, newborn rats treated with BSO were found to have Electron microscopy has revealed that mitochondrial damage is an marked depletion of tissue (liver, kidney, lung, brain, eye) ascorbate. important consequence of glutathione deficiency in many tissues. Both the levels of ascorbate and total ascorbate (ascorbate plus dehy These effects, which are produced without application of external droascorbate) were decreased (6). It is of interest that when ascorbate stress, develop after glutathione is depleted by administration of BSO was also given to these newborn rats, the levels of glutathione in the (5). Not only mitochondria but other types of cellular damage were tissues and in their mitochondria were increased significantly, indi found, including nuclear effects, and in the lungs, effects on the cating that ascorbate can spare glutathione. Findings closely similar to lamellar bodies. There appears to be a relationship between the extent those made on newborn rats were made in adult guinea pigs (78). In of mitochondrial depletion of glutathione and cellular damage, as this species also, tissue damage and early mortality due to glutathione estimated by determinations of citrate synthetase and electron micros deficiency are greatly decreased or prevented by administration of copy. In studies on adult mice, degeneration of skeletal muscle was ascorbate. Treatment with ascorbate spares mitochondrial glutathione found when the mitochondrial glutathione levels were decreased to as found also in newborn rats (9). about 20% of the controls. Similarly, mitochondrial and lamellar body Although glutathione deficiency is lethal to newborn rats and damage in lung type II cells were found when the mitochondrial guinea pigs, adult mice are able to survive because they can synthesize glutathione levels were about 21% of the controls. Jejunal mucosal ascorbate. Treatment of adult mice with BSO actually leads to an damage was found with mitochondrial glutathione levels of about initial increase of the ascorbate level in the liver (57). Within 4 h after 13% of the controls. In newborn rats, cataracts appeared when the lens BSO administration, the level of ascorbate in the liver increases about mitochondrial glutathione levels were about 20% of the controls. 2-fold and then decreases with concomitant accumulation of dehy A summary of the effects of glutathione deficiency induced by droascorbate. In other tissues, the ascorbate levels decreased and the administration of BSO is given in Table 1. In adult mice, prolonged levels of dehydroascorbate increased. Therefore, an early effect of treatment with BSO did not produce cellular damage in the liver, glutathione deficiency in adult mice appears to be an induction of heart, or kidneys. In both newborn rats and guinea pigs, treatment with ascorbate synthesis in the liver. Such induction does not occur in BSO led to death within 4-6 days, whereas adult mice did not exhibit newborn rats or in guinea pigs, findings consistent with the view that 1972s

GLU+CySH (81), further studies may reveal such a connection. Overproduction of melanin in these cells may be accompanied by increased formation of products that are detoxified by reactions involving glutathione. The process of melanin formation involves increased utilization of gluta thione, either as a participant in the synthesis of melanin (e.g., in the reaction of glutathione or of cysteine derived from it, with dopaqui- none), or as a consequence of increased oxidative phenomena asso ciated with melanin synthesis. Melanin formation may increase the GLY requirement for glutathione by the melanoma cell; decreasing gluta thione synthesis by giving BSO would thus be expected to affect the melanoma cell much more than normal cells, which have a lower requirement for glutathione. >- GSSG Cellular glutathione levels may be increased by administration of compounds that serve as precursors of cysteine, such as A'-acetyl-L- â€”-- cysteine and L-2-oxothiazolidine-4-carboxylate (5, 42, 84). Utilization GLUTAREDOXIN "" of the latter compound requires the activity of 5-oxoprolinase which converts i.-2-oxothiazolidine-4-carboxylate to i.-cysteine. Certain ex DEHYDROASCORBATE ASCORBATE perimental tumors seem to lack or to be markedly deficient in 5-oxo H202 prolinase activity, whereas most normal tissues convert this com pound effectively to L-cysteine, which is effectively used for glutathione synthesis. A combination therapy involving the use of an antitumor compound plus the oxothiazolidine has been previously suggested (3, 4); according to this strategy, normal tissues may use BREAKDOWN this compound to increase glutathione synthesis, which tumors would Fig. 4. Relationships between glutathione and ascorbatc (from Ref. l>). not be able to do, and this would be expected to increase the thera peutic effectiveness of anticancer agents. Glutathione levels may also be increased by treatment with y-glutamylcystinc and related these animals do not synthesize ascorbate. Thus, the findings summa compounds (31). This approach has the advantage of bypassing rized in Table 1 appear to be closely connected with the ability of the the feedback-inhibited step of glutathione synthesis catalyzed by experimental animals to synthesize ascorbate. However, glutathione y-glutamylcysteine synthetase. Glutathione levels may also be in deficiency in adult mice is not fully compensated by increased ascor creased by administration of glutathione esters, which (in contrast to bate synthesis. Thus, electron microscopy of the lungs of adult mice glutathione) are well transported into many types of cells and split to treated with BSO showed substantial damage to type II cell lamellar form glutathione (85-87). bodies (79). There was also decreased formation of intraulvcolar Although modulation of glutathione metabolism has largely fo tubular myelin, which is secreted by the lamellar bodies. Phosphati- cused on efforts to decrease or to increase cellular glutathione levels, dylcholine. the main constituent of lungs surfactant, is synthesized other modulations have been considered (3, 4). For example, inhibi within the lamellar bodies and is secreted as a protein complex into tion of y-glutamyl transpeptidase would be expected to interrupt the the alveolar subphase where it is transformed into tubular myelin. "salvage" pathway of glutathione synthesis (Fig. 3). Since certain Markedly decreased levels of phosphatidylcholine were found in the tumors have increased levels of y-glutamyl transpeptidase. it might be lungs of adult mice and in the bronchoalveolar fluid after treatment thought that this enzyme would be u promising target for chemother with BSO. Simultaneous treatment with ascorbate prevented the de apy. Patients with inborn deficiency of y-glutamyl transpeptidase crease in phosphatidylcholine levels in the lung as well as cellular excrete large amounts of glutathione, y-glutamylcysteine, and cys damage (80). teine moieties in their urine, and analogous effects have been observed The reactions given in Fig. 4 appear to account for the finding that in experimental animals treated with inhibitors of y-glutamyl ascorbate can spare glutathione. That glutathione can spare ascorbate transpeptidase (see above). Compounds currently available for inhi was shown in studies on guinea pigs given a diet deficient in ascorbate bition of y-glutamyl transpeptidase include a combination of serine (8). These animals typically develop scurvy after 14-20 days. How and borate, but rather high concentrations of these are needed for ever, when treated with glutathione monoethyl ester, the onset of effective inhibition. Acivicin is a more effective inhibitor, but this scurvy was significantly delayed (to at least 40 days). compound is nonspecific and inhibits a number of glutamine amido- Modulation of Glutathione Metabolism transferases. Acivicin has been tested as an anticancer compound apparently without success. Other compounds that inhibit y-glutamyl As discussed above, tumors that require high levels of cellular transpeptidase include 6-diazo-5-oxo-i.-norlcucine and azaserine. An glutathione or high cellular capacity for glutathione synthesis are other potentially effective approach is administration of y-glutamyl placed at a disadvantage by treatments that decrease cellular synthesis amino acids, which compete with the natural substrates and thus of glutathione. On the other hand, most normal cells have a large produce glutathionuria (see Ref. 88). excess of glutathione. As suggested previously (3), tumors that lack or have low levels of catalase might be successfully treated with BSO References alone. It is of interest that BSO exhibits a high degree of inhibitory 1. Meister. A. Glutathione metabolism and transport. In: O. F. Nygaard and M. G. Simic activity against melanoma-derived cell lines (81-83). In one study, (eds.), Radioprotectors and Anticarcinogens. pp. 121-151. New York: Academic Press, 1983. seven human melanoma cell lines were found to be sensitive to BSO 2. Dethmers, J. K.. and Meister. A. Glutathione export hy human lymphoid cells: and it was suggested that BSO may be an effective agent for mela depletion of glutathione by inhibition of its synthesis decreases export and increases sensitivilvsensitivity to irrauiauon.irradiation. rroc.Proc. Nati.pian. Acad./\cau. ^ci.Sci. USA,u;>/\. 78:in: 7492-7496, m**Â¿â€”/HVO,ivni. 1981. noma. Although no simple relationship between glutathione metabo 3. Meister, A. Modulation of glulathione levels and metabolism, in: P. Cerutti, O. F. lism and sensitivity to BSO was recognized in human melanoma cells Nygaard and M. G. Simic (eds.). Anticarcinogenesis and Radiation Protection, pp. 1973s

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1975s

Alton Meister

Cancer Res 1994;54:1969s-1975s.

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