Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
ICANCÕR RESEARCH (SUPPL.) 54, Glutathione, Ascorbate, and Cellular Protection1 Alton Meister Department of Biochemistry, Cornell University Medical College, New York, New York 10021 Introduction Glutathione. a tripeptide thiol present in virtually all animal cells, is synthesized within many cells from its constituent amino acids (glu tamate, cystcine, glycine); these "nonessential" amino acids can be synthesized within the body and are also obtained from the diet. Glutathione is also synthesized by tumors, some of which (notably drug- and radiation-resistant tumors) exhibit high cellular levels of glutathione and high capacity for the synthesis of glutathione. We reported at previous conferences in this series on the increase of cellular radiosensitivity that occurs after administration of an inhibitor of glutathione synthesis (1, 2) and on the effects of modulation of glutathione metabolism (3, 4). These and related topics (5, 6) will be summarized here with emphasis on some current developments. Glutathione is probably the most important cellular antioxidant. Interestingly, Fahey and Sundquist (7) found strong evidence for an evolutionary link between glutathione and aerobic eukaryotic metab olism; the findings indicate that glutathione evolved as a molecule that protects cells against oxygen toxicity. Although there is currently much interest in the hypothesis that oxidative phenomena may lead to a variety of pathological states, and that antioxidants may play a significant protective role, the important role that glutathione plays in the protection of cells has sometimes been insufficiently appreciated. Cells that are deprived of glutathione typically suffer severe oxidative damage associated with mitochondria! degeneration. Analogous ef fects are not always found when there is a deficiency of certain other cellular components that are thought to act as antioxidants. It has long been known that the antioxidant ascorbate is required in the diet of humans and certain other animals such as the guinea pig (but not by many other animals, including some commonly used in laboratory experiments; e.g., mice, rats, rabbits). The ascorbate deficiency syn drome, scurvy, which is associated with oxidative inactivation of certain enzymes, can be prevented in humans by administration of as little as 10 mg/day of ascorbate. The officially recommended daily dose of ascorbate for humans is 30-100 mg/day (depending upon the a-tocopherol in its reduced form, either by a direct reaction or by a pathway involving ascorbate (10-17). Glutathione. which has the important function of maintaining the reducing milieu of cells, is undoubtedly involved in the reduction of many cellular components; e.g., other tocopherols and ß-carotenearc apparently also maintained by glutathione-mediated reactions (e.g.. Réf.18). An interesting aspect of glutathione metabolism and function re lates to drug-resistant and radiation-resistant tumors that have high levels of glutathione or exhibit high capacity for glutathione synthesis. Such tumors have a greater requirement for glutathione than do many normal tissues and this provides a promising chemotherapeutic ap proach, which is considered below. Biochemistry of Glutathione: Enzymology and Transport Phenomena Fig. 1 gives some of the biochemical transformations of glutathi one, which is synthesized in two steps from glutamate, cystcine, and glycine (Reactions / and 2). Metabolic utilization of glutathione follows several pathways including reactions catalyzed by the gluta thione S-transferases (mercapturate pathway). Glutathione is a sub strate of the glutathione peroxidases which destroy hydrogen peroxide and organic peroxides. The glutathione disulfide formed is reduced to glutathione in an NADPH-mediated reaction (Fig. 2). Glutathione not only provides reducing power needed for the conversion of dehy droascorbate to ascorbate, but also for the conversion of ribonucleotides to deoxyribonucleotides and for a variety of thiol-disulfide interconversions; glutathione is therefore important for the synthesis and repair of DNA, and for the folding of newly synthesized proteins. The utilization of glutathione (Fig. 1; y-glutamyl cycle) is initiated extracellularly by the actions of y-glutamyl transpeptidase and dipeptidase; these enzymes are bound to the outside of cell membranes. The transpeptidase acts on glutathione. glutathione disulfide and glutathi one 5-conjugates. The reactions catalyzed by y-glutamyl transpepti dase take place in the presence of amino acids and lead to 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 P¡(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-gluis 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 the metabolism and transport of glutathione. When y-glutamyl carried out in the author's laboratory was supported in part by NIH Grant 2 R37 DK12034 from the United States Public Health Service. transpeptidase is markedly decreased, there is a substantial loss of 1969s Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1994 American Association for Cancer Research. GLUTATHIONE, ASCORBATE, AND CELLULAR OXIDATION-REDUCTION PATHWAYS PROTECTION bile. Plasma glutathione is used by many tissues, e.g., kidney, lung, and brain. Glutathione itself is not significantly transported into most of the cells of these tissues but is broken down by membrane-bound y-glutamyl transpeptidase and dipeptidase; the products of breakdown GSSG 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 reducÃ-asedeficiency, glucose-6-phosphate dehydrogenase deficiency, deficiencies of y-glutamylcysteine synthetase and of glutathione synthetase) (34), and from experimental 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 5-oxoproline. Glutathione levels are markedly reduced in some of 2HJ MERCAPTURATE PATHWAY à )/ Glutamate Y-Glu-AA these patients, but in none of them is glutathione completely absent. Experimental production of glutathione deficiency has been at tempted by administration of compounds that react with glutathione (e.g., diethyl maléate,phorone) and oxidizing agents (e.g., diamide and t-butyl hydroperoxide). However, as discussed elsewhere (5), such nonspecific agents have major limitations. Thus far, the most useful approach to production of glutathione deficiency has been treatment with specific inhibitors of y-glutamylcysteine synthetase, the enzyme that catalyzes the first and rate-limiting step of glutathione synthesis. 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 which y-glutamyl phosphate is an intermediate (35). Studies with various substrates and substrate analogues led to mapping of the Fig. 1. Overall pathway of glutathione (GSH) metabolism. /. y-glutamylcysteine synthetase; 2, glulathione synthetase; 3, y-glutamyl transpeptidase; 4, cysteinyl glycine hydrolases; 5, y-glutamyl cyclotransfera.se; 6, 5-oxoprolinase; 7, glutathione 5-transferases; 8, transport and reduction of y-Glu-(Cys)2; 9, see Fig. 2. Reactions 1, 2, and 6 involve cleavage of ATP to ADP and P,. -GSSG NADPH, H Deoxyribonucleottd NADP 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 reducÃ-ase. 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 transpeptidation reaction is split extracellularly to form cysteine and glycine; this reaction may also occur intracellularly after transport of the dipeptide. y-Glutamyl transpeptidase and cysteinylglycine dipeptidase activity thus function in the recovery of cysteine moieties that are necessary for the functioning of a recycling pathway involving syn thesis and export of glutathione (33). Glutathione is exported by the liver to the blood plasma and to the CyS CyS - CySH-Gly Fig. 3. "Salvage" pathway. Glutathione (GSH), synthesized intracellularly (reactions / and 2), is exported (3) and reacts with y-glutamyl transpeptidase and extracellular cystine (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 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 pathway (31). A block at step 4 (reaction catalyzed by y-glutamyl transpeptidase) leads to accumulation of glutathione and to urinary excretion of glutathione and cysteine and y-glutamylcysteine moieties (see text). 1970s Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1994 American Association for Cancer Research. OLUTATHIONE, ASCORBATE. active site ofglutamine synthetase and to design of selective inhibitors of glutamine synthetase (a-ethyl methionine sulfoximine) and of y-glutamylcysteine synthetase [prothionine sulfoximine, BSO,2 and others (35-40)]. BSO and related compounds have very little or virtually no effect on glutamine synthetase but inhibit y-glutamylcys teine synthetase very effectively. BSO, now commercially available, has been widely used in in vitro experiments and in vivo. The effects of glutathione deficiency induced by administration of BSO have been extensively examined (see Ref. 5) and include the following: 1. Glutathione deficiency sensitizes cells to the effects of radiation, oxidative reactions, and to various toxic compounds. These effects have been applied in chemotherapy and radiation systems (5, 41-44). Tumor cell resistance associated with overproduction of glutathione is reversed by treatment with BSO in animal models and is currently being tested clinically (45). Depletion of glutathione by treatment with buthionine sulfoximine sensitizes cells to the toxic effects of heavy metals (46, 47), nitrogen mustards (48, 49), radiation (1, 2, 5), cisplatin (50), cyclophosphamide (51, 52), morphine (53), compounds that produce oxidative cytolysis (54), and others (55). 2. Glutathione deficiency leads to oxidative stress in many tissues (5). Mitochondria! and associated cell damage is found in mice treated with BSO. Mitochondria do not synthesize glutathione but obtain it by transport from the cytosol. Several tissues of adult mice are affected by administration of BSO, but in newborn rats and guinea pigs more extensive damage is found and there is early mortality due to multiorgan failure (56). Glutathione deficiency in these experimental sys tems serves as a model of endogenously produced oxidative stress. Mortality and tissue damage are significantly decreased by adminis tration of glutathione esters or of ascorbate (5, 9). 3. Deficiency of glutathione leads to decreased reduction of dehydroascorbate to ascorbate in vivo. This is observed in newborn rats and guinea pigs, animals that cannot synthesize ascorbate, and also in adult mice, which can (6). In adult mice, glutathione deficiency leads to induction of ascorbate synthesis in the liver (57), and this explains why BSO has less damaging effects on adult mice than it does on newborn rats and guinea pigs. 4. Glutathione deficiency in newborn rats and mice leads to for mation of ocular cataracts (9, 58, 59). Cataracts have also been found in some patients with inherited glutathione disulfidc reducÃ-asedefi ciency. 5. Treatment of peripheral blood mononuclear cells with BSO was found to markedly inhibit their proliferation (4), and later work has confirmed that glutathione deficiency decreases lectin-induced prolif eration of lymphocytes (60). Although glutathione is required for proliferation, the mechanism of its function in this system is still unsettled. AND CELLULAR PROTECTION these agents might make tumor cells more sensitive to chemotherapy and to radiation treatment (41 ). The potential usefulness of BSO in the sensitization of cells to radiation was first directly shown in studies on several human lymphoid cell lines (1, 2). Cells that had 4-5% of the control levels of glutathione were found to be much more sensitive than the controls to the effects of y-irradiation. It was found that treatment with BSO of mice bearing B16 melanomas sensitizes this radio-resistant tumor to radiation (61, 62). Treatment with BSO alone did not affect tumor growth, but treatment with BSO and radiation led to significant decrease in the size of the tumor and to an increase in the longevity of the tumor-bearing mice. It was also found that treatment of resistant Icukemias with BSO led to sensitization of the tumors to phenylalanine mustard (48. 49). In studies with mice bearing such resistant tumors, i.p. infusion of BSO led to sensitization of the tumors and to an increase in the life span of the treated animals. Additional studies on the relationship between glutathione levels and the expres sion of primary drug resistance and cross-resistance in human ovarian cancer cell lines, together with studies in which an i.p. model of human ovarian cancer was developed in nude mice, were of impor tance because they led to a clinical trial of BSO which is now in progress (44, 45). In these studies it was also found that resistance of the tumors to phenylalanine mustard is associated with resistance to other drugs such as Adriamycin. Of interest, these drug-resistant cells are also resistant to radiation. It seems probable that at least one type of multidrug resistance is associated with overproduction of glutathi one; however, other cellular mechanisms can also lead to drug resis tance. Much attention has been given to multidrug resistance associ ated with a novel membrane glycoprotein; this type of resistance is associated with decreased accumulation within the tumor of a number of structurally unrelated drugs. However, this P-glycoprotein-related system does not appear to confer radiation resistance. Cellular levels of glutathione may determine the degree of drug resistance or radiation resistance of a particular tumor, but the capac ity of a tumor cell to synthesize glutathione may also be an important factor in resistance. The ability of a cell to synthesize glutathione rapidly in response to a stress may be as important or perhaps more important than the initial cellular level of glutathione. This idea is supported by model studies on a strain of Escherichia coli enriched in its content of y-glutamylcysteine synthetase and glutathione syn thetase by recombinant DNA techniques (63). Recent studies on tumors that are resistant and sensitive to Adriamycin are consistent with this idea (5, 64). Studies on human ovarian tumor cell lines that are resistant to cisplatin showed that cellular glutathione levels are greatly increased (13- to 50-fold) as compared with the sensitive cells of origin (65). The cell lines examined exhibited up to 1000-fold increases in resistance to cisplatin. Cisplatin resistance was associated with increased expression of mRNAs for yApplication of Glutathione Depletion to the Treatment of glutamylcysteine synthetase and y-glutamyl transpeptidasc and with Tumors: Sensitization of Tumors to Chemotherapy increased activities of these enzymes. Thus, y-glutamylcysteine and to Radiation 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 yglutathione (see Ref. 5). The resistance of leukemic cells to phenylglutamylcysteine 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 dehydrochlofor 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 Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1994 American Association for Cancer Research. OLUTATHIONE. ASCORBATE, below), it is relevant to consider the ascorbate status of patients treated with BSO (see Ref. 69). It is possible that administration of large doses of ascorbate to such patients would negate the desired effects of BSO administration. AND CELLULAR PROTECTION Table 1 Effects of glutathiurie deficiency EffectDeath, treatment with BSO leads to additional and gradual decline of cellular glutathione. This biphasic decrease in the cellular glutathione level led to further investigations which showed that a substantial fraction of cellular glutathione is sequestered in the mitochondria. BSO is not 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, but the absence of the synthetases from mitochondria showed that mitochondrial glutathione must arise from the cytosol (71). Studies on isolated rat liver mitochondria indicate that mitochondrial glutathione homeostasis is regulated by a multicomponent transport system which appears to explain the remarkable ability of mitochondria to take up and to retain glutathione (72). Evidence was found for two transport ers with apparent Km values of 60 ¡JLM and 5.4 mM. Extramitochondrial glutathione promotes mitochondrial uptake and exchange, and the intermembranous space appears to function as a recovery zone that facilitates efficient cycling of matrix glutathione. Decreased levels of glutathione produced by administration of BSO decrease the net export of glutathione from mitochondria to the cytosol. That the net efflux of glutathione from mitochondria is very slow when there are low levels of extramitochondrial glutathione is consistent with a mechanism that conserves mitochondrial glutathione during periods of cytosolic glutathione depletion. A significant fraction of the oxygen utilized by mitochondria (about 2-5%) is converted, apparently through Superoxide, to hydrogen peroxide (73). When glutathione levels are greatly decreased, hydro gen peroxide accumulates, and this leads to extensive mitochondrial damage. Other antioxidants may be involved in the protection of mitochondria, but glutathione appears to be the principal functional one. Mitochondria do not contain catalase and are therefore largely, if not entirely, dependent upon glutathione and glutathione peroxidases. Electron microscopy has revealed that mitochondrial damage is an important consequence of glutathione deficiency in many tissues. These effects, which are produced without application of external stress, develop after glutathione is depleted by administration of BSO (5). Not only mitochondria but other types of cellular damage were found, including nuclear effects, and in the lungs, effects on the lamellar bodies. There appears to be a relationship between the extent of mitochondrial depletion of glutathione and cellular damage, as estimated by determinations of citrate synthetase and electron micros copy. In studies on adult mice, degeneration of skeletal muscle was found when the mitochondrial glutathione levels were decreased to about 20% of the controls. Similarly, mitochondrial and lamellar body damage in lung type II cells were found when the mitochondrial glutathione levels were about 21% of the controls. Jejunal mucosal damage was found with mitochondrial glutathione levels of about 13% of the controls. In newborn rats, cataracts appeared when the lens mitochondrial glutathione levels were about 20% of the controls. A summary of the effects of glutathione deficiency induced by administration of BSO is given in Table 1. In adult mice, prolonged treatment with BSO did not produce cellular damage in the liver, heart, or kidneys. In both newborn rats and guinea pigs, treatment with BSO led to death within 4-6 days, whereas adult mice did not exhibit Guinea mice000+M0-+IIII+ 'LiverKidneyLungBrainLensSkeletal damage ++ ++ ++ + 0+ 0II On the Essentiality of Glutathione: Mitochondria! Function When BSO is administered to rats or mice there is rapid decline of the glutathione levels of the liver and kidney to an apparent limiting value of 15-20% of the total glutathione initially present. Further pigs-f-a rats ++ daysCell 4-6 + muscleJejunum, colonHeartStomachLymphocytes" 0Ãœas +, cell damage or deathMainly indicated.Adult mitochondrial.Newborn early mortality. The lethal effects of glutathione deficiency in animals such as newborn rats and guinea pigs (which do not synthesize ascorbate) appear to be related to multiorgan failure involving mito chondrial and other damage in liver, kidney, and lung (56). In liver there is focal necrosis: in kidney, there is proximal tubular damage; and in lung, there is lamellar body degeneration. The lungs of adult mice showed damage, although less than in newborn rats. The mitochondrial and other cell damage seen in newborn animals and in adults were prevented by administration of glutathione esters or by administration of ascorbate (see below). Function of Glutathione in the Reduction of Dehydroascorbate A function of glutathione in the reduction of dehydroascorbate was suspected by early investigators. Borsook et al. (74) concluded that glutathione is involved in the reduction of dehydroascorbate by ani mal tissues, but Guzman-Barron. another early pioneer in this field, considered this unlikely (75). Hopkins and Morgan (76) studied this reaction in plants. The reduction of dehydroascorbate to ascorbate was examined by a number of investigators in in vitro animal systems (see Ref. 6), and purified preparations of glutaredoxin and protein disulfide isomerase were found to exhibit substantial glutathione-dependent dehydroascorbate reducÃ-aseactivity (77). Convincing evidence linking glutathione to the reduction of dehy droascorbate in vivo has recently been obtained (6, 9). In the first of these studies, newborn rats treated with BSO were found to have marked depletion of tissue (liver, kidney, lung, brain, eye) ascorbate. Both the levels of ascorbate and total ascorbate (ascorbate plus dehy droascorbate) were decreased (6). It is of interest that when ascorbate was also given to these newborn rats, the levels of glutathione in the tissues and in their mitochondria were increased significantly, indi cating that ascorbate can spare glutathione. Findings closely similar to those made on newborn rats were made in adult guinea pigs (78). In this species also, tissue damage and early mortality due to glutathione deficiency are greatly decreased or prevented by administration of ascorbate. Treatment with ascorbate spares mitochondrial glutathione as found also in newborn rats (9). Although glutathione deficiency is lethal to newborn rats and guinea pigs, adult mice are able to survive because they can synthesize ascorbate. Treatment of adult mice with BSO actually leads to an initial increase of the ascorbate level in the liver (57). Within 4 h after BSO administration, the level of ascorbate in the liver increases about 2-fold and then decreases with concomitant accumulation of dehy droascorbate. In other tissues, the ascorbate levels decreased and the levels of dehydroascorbate increased. Therefore, an early effect of glutathione deficiency in adult mice appears to be an induction of ascorbate synthesis in the liver. Such induction does not occur in newborn rats or in guinea pigs, findings consistent with the view that 1972s Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1994 American Association for Cancer Research. OLUTATHIONE, ASCORBATE. AND CELLULAR PROTECTION GLU+CySH GLY >- GSSG —-GLUTAREDOXIN "" DEHYDROASCORBATE ASCORBATE H202 BREAKDOWN Fig. 4. Relationships between glutathione and ascorbatc (from Ref. l>). these animals do not synthesize ascorbate. Thus, the findings summa rized in Table 1 appear to be closely connected with the ability of the experimental animals to synthesize ascorbate. However, glutathione deficiency in adult mice is not fully compensated by increased ascor bate synthesis. Thus, electron microscopy of the lungs of adult mice treated with BSO showed substantial damage to type II cell lamellar bodies (79). There was also decreased formation of intraulvcolar tubular myelin, which is secreted by the lamellar bodies. Phosphatidylcholine. the main constituent of lungs surfactant, is synthesized within the lamellar bodies and is secreted as a protein complex into the alveolar subphase where it is transformed into tubular myelin. Markedly decreased levels of phosphatidylcholine were found in the lungs of adult mice and in the bronchoalveolar fluid after treatment with BSO. Simultaneous treatment with ascorbate prevented the de crease in phosphatidylcholine levels in the lung as well as cellular damage (80). The reactions given in Fig. 4 appear to account for the finding that ascorbate can spare glutathione. That glutathione can spare ascorbate was shown in studies on guinea pigs given a diet deficient in ascorbate (8). These animals typically develop scurvy after 14-20 days. How ever, when treated with glutathione monoethyl ester, the onset of scurvy was significantly delayed (to at least 40 days). Modulation of Glutathione Metabolism As discussed above, tumors that require high levels of cellular glutathione or high cellular capacity for glutathione synthesis are placed at a disadvantage by treatments that decrease cellular synthesis of glutathione. On the other hand, most normal cells have a large excess of glutathione. As suggested previously (3), tumors that lack or have low levels of catalase might be successfully treated with BSO alone. It is of interest that BSO exhibits a high degree of inhibitory activity against melanoma-derived cell lines (81-83). In one study, seven human melanoma cell lines were found to be sensitive to BSO and it was suggested that BSO may be an effective agent for mela noma. Although no simple relationship between glutathione metabo lism and sensitivity to BSO was recognized in human melanoma cells (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 dopaquinone), or as a consequence of increased oxidative phenomena asso ciated with melanin synthesis. Melanin formation may increase the 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. Cellular glutathione levels may be increased by administration of compounds that serve as precursors of cysteine, such as A'-acetyl-Lcysteine and L-2-oxothiazolidine-4-carboxylate (5, 42, 84). Utilization of the latter compound requires the activity of 5-oxoprolinase which converts i.-2-oxothiazolidine-4-carboxylate to i.-cysteine. Certain ex perimental tumors seem to lack or to be markedly deficient in 5-oxo 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 this compound to increase glutathione synthesis, which tumors would 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 compounds (31). This approach has the advantage of bypassing the feedback-inhibited step of glutathione synthesis catalyzed by y-glutamylcysteine synthetase. Glutathione levels may also be in creased by administration of glutathione esters, which (in contrast to glutathione) are well transported into many types of cells and split to form glutathione (85-87). Although modulation of glutathione metabolism has largely fo cused on efforts to decrease or to increase cellular glutathione levels, other modulations have been considered (3, 4). For example, inhibi tion of y-glutamyl transpeptidase would be expected to interrupt the "salvage" pathway of glutathione synthesis (Fig. 3). Since certain tumors have increased levels of y-glutamyl transpeptidase. it might be thought that this enzyme would be u promising target for chemother apy. Patients with inborn deficiency of y-glutamyl transpeptidase excrete large amounts of glutathione, y-glutamylcysteine, and cys teine moieties in their urine, and analogous effects have been observed in experimental animals treated with inhibitors of y-glutamyl transpeptidase (see above). Compounds currently available for inhi bition of y-glutamyl transpeptidase include a combination of serine and borate, but rather high concentrations of these are needed for effective inhibition. Acivicin is a more effective inhibitor, but this compound is nonspecific and inhibits a number of glutamine amidotransferases. Acivicin has been tested as an anticancer compound apparently without success. Other compounds that inhibit y-glutamyl transpeptidase include 6-diazo-5-oxo-i.-norlcucine and azaserine. An other potentially effective approach is administration of y-glutamyl amino acids, which compete with the natural substrates and thus produce glutathionuria (see Ref. 88). References 1. Meister. A. Glutathione metabolism and transport. In: O. F. Nygaard and M. G. Simic (eds.), Radioprotectors and Anticarcinogens. pp. 121-151. New York: Academic Press, 1983. 2. Dethmers, J. K.. and Meister. A. Glutathione export hy human lymphoid cells: depletion of glutathione by inhibition of its synthesis decreases export and increases sensitivity to irrauiauon. pian. Acad. /\cau. ^ci. u;>/\. 78: in: 7492-7496, m**¿— /HVO, 1981. ivni. sensitivilv irradiation. rroc. Proc. Nati. Sci. USA, 3. Meister, A. Modulation of glulathione levels and metabolism, in: P. Cerutti, O. F. Nygaard and M. G. Simic (eds.). Anticarcinogenesis and Radiation Protection, pp. 1973s Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1994 American Association for Cancer Research. OLUTATHIONE, ASCORBATE. AND CELLULAR 361-372. New York: Plenum Publishing Corp., 1988. 4. Meister, A. Selective modification of glutathionc metabolism. Science (Washington DC), 220.- 471-477, 1983. 35. 5. Meister, A. Glutathione deficiency produced by inhibition of its synthesis and its reversal; applications in research and therapy. Pharmacol. Ther., 5/: 155-194, 1991. 6. Meister. A. On the antioxidant effects of ascorbic acid and glutathione. Biochem. Pharmacol., 44: 1905-1915, 1992. 7. Fahcy. R. C., and Sundquist. A. R. Evolution of glutathione metabolism. Adv. Enzymol., 64: 1-53, 1991. 8. Mârtensson,J. M., Han. J., Griffith. O. W.. and Meister, A. Glulathinne ester delays the onset of scurvy in ascorbate-deficient guinea pigs. Proc. Nail. Acad. Sci. USA. °0: 317-321. 1993. 9. Mârtensson.J. M.. and Meister. A. Glutathione deficiency decreases tissue ascorbate levels in newborn rats: Ascorbate spares glutathionc and protects. Proc. Nail. Acad. Sci. USA, 88: 4656-4660. 1991. 10. Packer. J. E.. Slater, T. F., and Wilson, R. L. Direct observation of a free radical interaction between vitamin E and vitamin C. Nature (Lond.), 278: 737-738, 1979. 11. Niki, E., Tsuchiya. J., Tanimura, R.. and Kamiya. Y. Regeneration of vitamin E from a-chromanoyl radical by glutathione and vitamin C. Chem. Lett. Jpn., 23: 789-792, 1982. 12. Reddy, C. C., Scholz. R. W.. Thomas, C. E.. and Massaro, E. J. Vitamin E dependent reduced glutathione inhibition of rat liver microsomal lipid peroxidation. Life Sci., 31: 571-576, 1982. 13. Doha, T., Burton. B. W., and Ingold. K. U. Antioxidant and co-antioxidant activity of vitamin C. The effect of vitamin C, either alone or in the presence of vitamin E or a water-soluble vitamin E analogue, upon the peroxidation of aqueous multi-lamellar phospholipid liposomes. Biochim. Biophys. Acta, 835: 298-303, 1985. 14. Lecdlc. R. A., and Aust, S. D. The effect of glutathione on the vitamin E requirement for inhibition of liver chromosomal lipid peroxidation. Lipids, 25: 241-245, 1990. 15. Graham, K. S.. Reddy, C. C., and Scholz, R. W. Reduced glulalhione effects on or-tocopherol concentration of rat liver microsomes undergoing NADPH-dcpendent lipid peroxidation. Lipids, 24: 909-914. 1989. 16. Scholich. H., Murphy. M. E.. and Sies. H. Antioxidant activity of dihydrolipoatc against microsomal lipid peroxidation and its dependence on a-tocopherol. Biochim. Biophys. Acia, IOOI: 256-261, 1989. 17. Wefers. H.. and Sics. H. The protection by ascorbate and glulathione against microstimai lipid peroxidation is dependent on vitamin E. Eur. J. Biochem.. 174: 353-357, 1988. 18. h.il.il I., and Grundy. S. M. Preservation of the endogenous antioxidants in low density lipoprotcin by ascorbate but not probucol during oxidative modification. J. Clin. Invest.. 87: 597-601, 1991. 19. Allison. R. D.. and Meister, A. Evidence thai (ranspcptidation is a significant function of y-glulamyl transpeplidase. J. Biol. Chem., 256: 2988-2992, 1981. 211. Thompson, G. A., and Meisler, A. Utilization of L-cystine by the -y-glutamyl Iranspt'ptidasc-y-glulamvl cyclotransferase pathway. Proc. Nati. Acad. Sci. USA, 72: 1985-1988, 1975. 21. Täte.S. S., and Meister. A. Interaction of y-glulamyl transpeplidase with amino acids. dipeptides, and derivatives and analogs of glutathione. J. Biol. Chem.. 249: 75937602, 1974. 22. Meister. A. On the enzymology of amino acid transport. Science (Washington DC). 180: 33-39. 1973. 23. Orlowski, M.. and Meister. A. y-GIutamyl cyclotransferase; distribution, isozymic forms, and specificity. J. Biol. Chem., 24«:2836-2844, 1973. 24. Griffith. O. W., Bridges, R. J., and Meister, A. Evidence that the y-glulamyl cycle functions in r/wj using intracellular glutathione: effects of amino acids and selective inhibition of enzymes. Proc. Nati. Acad. Sci. USA, 75: 5405-5408. 1978. 25. Taniguchi. N., and Meisler. A. y-Glulamyl cyclotransferase from rat kidney: sulfhydryl groups and isolation of a stable form of the enzyme. J. Biol. Chem., 253: 1799-1806. 1978. 26. Van der Werf. P.. Orlowski. M.. and Meisler, A. Enzymatic conversion of 5-oxo-Lproline (I-pyrrolidonc carhoxylate) to L-glutamate coupled with ATP cleavage to ADP: a reaction in the y-glutamyl cycle. Proc. Nail. Acad. Sci. USA, 68: 2982-2985. 1971. 27. Scddon. A. P.. and Meislcr. A. Trapping of an intermediale in the reaclion catalyzed by 5-oxoprolina.se. J. Biol. Chem., 267: 11538-11541, 1986. 28. Griffith, O. W.. and Meister. A. Translocalion of intracellular glulathione to mem brane-bound y-glulamyl Iranspeplidase as a discrete step in the y-glutamyl cycle; glutathionuria after inhibition of transpeptidase. Proc. Nati. Acad. Sci. USA, 76: 268-272. 1979. 29. Griffith. O. W., Novogrodsky, A., and Meister. A. Translocation of glulalhione from lymphoid cells that have markedly different y-glutamyl transpeplidase activities. Proc. Nail. Acad. Sci. USA. 76: 2249-2252, 1979. 30. Griffith. O. W.. and Meister. A. Excrelion of cysleine and y-glulamylcysleine moielies in human and experimental animai y-glutamyl transpeptidase deficiency. Proc. Nail. Acad. Sci. USA. 77: 3384-3387, 1980. 31. Anderson, M. E., and Meister, A. Transpon and direct utilization of y-glutamylcyst(e)ine for glutathione synthesis. Proc. Nati. Acad. Sci. USA. 80: 707-711, 1983. 32. Griffith. O. W.. Bridges, R. J., and Meister, A. Transport of y-glutamyl amino acids; role of glutalhione and y-glulamyl Iranspeptidase. Proc. Nati. Acad. Sci. USA. 76: 6319-6322, 1979. 33. Meister. A. Metabolism and funclion of glulathione. In: D. Dolphin, R. Poulson. and O. Avramovic (eds.). Glulathione: Chemical. Biochemical and Medical Aspects, pp. 367-374. New York: John Wiley and Sons. 1989. 34. Meister, A., and Larsson. A. Glulalhione synlhetase deficiency and other disorders of the y-glutamyl cycle. In: C. R. Scriver. A. L. Beaudet, W. S. Sly, and D. Valle (eds.), 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. PROTECTION The Metabolic Basis of Inheriled Disease. Ed. 6. pp. 855-868. New York: McGrawHill. 19X9. Meisler, A. A Irail of research: from glulamine synlhelase lo selective inhibition of glulalhione synlhesis. ChemTracls-Biochem. Molec. Biol.. .ÃŽ:75-106, 1992. Meister, A. Inhibition of glutamine synlhelase and y-glutamylcysteine synthctase by melhionine sulfoximine and relaled compounds. In: N. Seiler. M. J. Jung, and J. Koch-Weser (eds.). Enzyme-aclivated Irreversible Inhibitors, pp. 187-211. Amster dam: Elsevier-North Holland BiomédicalPress, 1978. Griffith, O. W., Anderson, M. E., and Meisler. A. Inhibition of glutathione biosyn thesis by prolhionine sulfoximine (S-n-propyl-homocysleine sulfoximine). a scleclive inhibitor of y-glulamylcysteinc synlhetase. J. Biol. Chem.. 254: 1205-1210, 1979. Griffith. O. W.. and Meisler. A. Polent and specific inhibition of glulalhione synthesis by huthionine sulfoximine (S-/i-butyl homocysteine sulfoximine). J. Biol. Chem., 254: 7558-7560, 1979. Griffith, O. W. Bulhionine sulfoximine and its higher homologs. J. Biol. Chem., 257: 13704-13712. 1982. Meister, A. On the biochemistry of glutalhione. in: N. Taniguchi et al. (eds.), Glutathione Centennial: Molecular Properties and Clinical Implications, pp. 3—21. New York: Academic Press, 1989. Meister, A., and Griffilh. O. W. Effects of methionine sulfoximine analogs on the synthesis of glutamine and glutalhione; possible chemotherapeutic implicalions. Cancer Treal. Rep.. 6.?: 1115-1121, 1979. Meisler. A. Novel drugs thai affect glutalhione melabolism. In: P. V. Woolley and K. D. Tew (eds.). Mechanisms of Drug Resistance in Ncoplaslic Cells, Bristol Myers Symposium No. 9. Chapter 7, pp 99-127. New York: Academic Press, 1988. Vistica, D. T., and Ahmad. S. Acquired resistance of tumor cells to i -phenylalanine mustard: implications for the design of a clinical trial involving glutathione depletion. In: N. Taniguchi et al. (eds.). Glutathione Centennial Molecular Perspectives and Clinical Implications, Chaplcr 21, pp. 301-305. New York: Academic Press. 1989. Ozols, R. F.. Hamilton. T. C. Masuda. H.. and Young. R. C. Manipulation of cellular thiols lo influence drug resistance. In: P. V. Woolley and K. D. Tew (eds.). Mech anisms of Drug Resistance in Neoplastic Cells, Bristol Myers Symposium No. 9. Chapter 19, pp. 289-305. New York: Academic Press, 1988. Hamilton. T.. O'Dwyer, P.. Young, R., Tew, K., Padavic, K., Comis, R., and Ozols. R. Phase 1 trial of bulhionine sulfoximine (BSO) plus melphalan (l-PAM) in palients with advanced cancer. Proc. A. Meet. Am. Soc. Clin. Oncol.. °:A2KI. 1990. Singhal. R. K., Anderson. M. E.. and Meister, A. Glutalhione. a first line of defense against cadmium loxicity. FASEB J., /: 220-223, 1987. Naganuma. A.. Anderson. M. E.. and Meister. A. Cellular glutalhione is a determinant of sensitivity to mercuric chloride loxicity: prevention of toxicity by giving glutalhi one monoeslcr. Biochcm. Pharmacol., 40: 693-697, 1990. Suzukake, K.. Petro, B. J.. and Vislica. D. T. Reduction in glulalhione content of 1--PAM resistant L1210 cells confers drug sensitivily. Biochem. Pharmacol.. 31: 121-124, 1982. Suzukake, K.. Vislica. B. P.. and Vislica. D. T. Dechlorination of i -phenylalanine mustard by sensitive and resistant tumor cells and its relationship to intracellular glutathione contenÃ-.Biochem. Pharmacol., 32: 165-167, 1983. Anderson, M. E.. Naganuma. A., and Meisler. A. Proteclion against cisplatin toxicily by adminislration of glutathione ester. FASEB J., 4: 3251-3255, 1990. Ishikawa. M.. Sasaki. K-L, and Takayanagi. Y. Injurious effect of huthionine sul foximine. an inhibitor of glulalhione biosynthesis, on Ihe lethality and uroloxicity of cyclophosphamide in mice. J. Pharmacol. Jap., 5/: 146-149, 1989a. Ishikawa. M.. Takayanagi. Y.. and Sasaki. K-l. Modification of cydophosphamidcinduced urotoxicily by buthionine sulfoximine and disulfiram in mice. Res. Commun. Palh. Pharmacol., 65: 265-268, 1989b. McCarlney. M. A. Effect of glutathione depiction on morphine toxicily in mice. Biochem. Pharmacol., 38: 207-209. 1989. Arrick. B. A.. Nathan. C. F.. Griffilh, O. W.. and Cohn, Z. A. Glulalhione depletion sensitizes tumor cells to oxidative cytolysis. J. Biol. Chem., 257: 1231-1237, 1982. Perez, R. P.. Hamilton. T. C.. and Ozols, R. F. Resistance to alkylaling agents and eisplatin: insighls from ovarian carcinoma model systems. Pharmacol. Ther.. 48: 19-27, 1990. Mârtensson,J. M., Jain, A., Stole, E., Frayer, W., Auld, P. A. M., and Meister. A. Inhibilion of glutathione synthesis in the newborn rat: a model of cndogenouslyproduced oxidative stress. Proc. Nati. Acad. Sci. USA, 88: 9360-9364, 1991. Mârtensson.J. M..aduli and mice. Meister. A. Glulalhione increases hepalic ascorbic acid synlhesis in Proc. Nati. Acad. deficiency Sci. USA'. 89: 11566-11568, 1992. 58. Calvin. H. L, Medvedovsky. C., and Worgul, B. V. Near-total glutalhione depletion and age-specific cataracts induced by buthionine sulfoximine in mice. Science (Wash ington DC), 233: 553-555, 1986. 59. Mârtensson,J. M., Sleinherz. R.. Jain, A., and Meisler, A. Glulathione ester prevenÃ-s buthionine sulfoximinc-induced cataracts and lens epithelial cell damage. Proc. Nati. Acad. Sci. USA, 86: 8727-8731, 1989. 60. Suthanlhiran. M., Anderson. M. E.. Sharma. V. K.. and Meisler, A. Glutalhione regulates activation-dependent DNA synthesis in highly purified normal human T lymphocytes stimulated via the CD2 and CD3 antigens. Proc. Nati. Acad. Sci. USA, 87: 3343-3347. 1990. hi. Griffith. O. W. Glutalhione and cell survival. In: S. Ebraski (ed.). Cellular Regulation and Malignanl Growih, pp. 292-300. Tokyo: Japan Societies Press, Springer. 1985. 62. Meister. A. Modulation of intracellular levels of glutathionc. in: F. Valeriote and !.. Baker (eds.). Biochemical Modulation of Anticancer Agents: Experimental Clinical Approaches, pp. 245-275. Boston. MA: Marlinus Nijhoff. 1986. 63. Moore. W. R., Anderson. M. E.. Meisler, A.. Murala. K.. and Kimura. A. Increased capacity for glutathione synthesis enhances resistance to radiation in Escherichia coli: a possible model for mammalian cell protection. Proc. Nail. Acad. Sci. USA, 86: 1461-1464. 1989. 1974s Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1994 American Association for Cancer Research. OLUTATHIONE. ASCÃœRBATE. AND CELLULAR PROTECTION 64. Lee, F. Y. F.. Siemann, D. W., and Sutherland, R. M. Changes ¡ncellular glutathione content during adriamycin treatment in human ovarian cancer-a possible indicator of chemosensitivity. Br. J. Cancer, 60: 291-298, 1989. 65. Godwin, A. K., Meister, A., O'Dwyer, P. J., Hamilton, T. C., Huang, C-S., and 77. Wells, W. W., Xu. D. P., Yang, Y., and Rocque, P. A. Mammalian Ihioltransferase (glutarcdoxin) and protein disulfide isomcrase have dchydroascorbate reducÃ-aseac tivity. J. Biol. Chem., 265: 15361-15364, 1990. 78. Griffith, O. W., Han. J.. and Mârtensson.J. M. Vitamin C' protects adult guinea pigs Anderson, M. E. High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis. Proc. Nati. Acad. Sci. USA. 89: 3070-3074. 1992. Yan, N.. and Meister. A. Amino acid sequence of rat kidney y-glutamylcystcine synlhetase. J. Biol. Chem., 265: 1588-1593, 1990. Huang, C-S., Chang, L-S., Anderson, M. E., and Meister, A. Catalytic and regulatory properties of the heavy subunit of rat kidney y-glutamylcystcine synthctase. J. Biol. Chem., 26«:19675-19678, 1993. Huang, C-S., Anderson. M. E.. and Meister, A. Amino acid sequence function of the light subunit of rat kidney •y-glutamylcysteinesynthetase. J. Biol. Chem.. 268: 20578-20583, 1993. Meister, A. Depletion of glutathione in normal and malignant human cells in riva by t.-huthioninc sulfoximine: possible interaction with ascorbatc. J. Nail. Cancer Inst., 84: 1601-1602, 1992. Meister, A., and Griffith, O. W. Effects of BSO and related compounds on mitochondrial glutathione levels (Abstract). Federation Proc., 42: 2642. 1983. Griffith, O. W., and Meister, A. Origin and turnover of mitochondrial glulathionc. Proc. Nati. Acad. Sci. USA, 82: 4668-4672. 1985. Mârtensson,J. M., Lai, J. C'. K., and Meister, A. High affinity transport of glutathione against tissue damage and lethality caused by BSO-mediated glutathione depletion (Abstract 4708). FASEB J.. 5: 1991. 7°. Mârtensson,J. M., Jain. A.. Frayer. W., and Meister, A. Glutathione metabolism in the lung: inhibition of its synthesis leads to lamellar body and mitochondrial defects. Proc. Nati. Acad. Sci. USA, «6:5296-5300, 1989. 80. Jain. A.. Mârtensson,J. M., Menta, T., Krauss, A. N.. Auld, P. A. M., and Meister, A. Ascorbic acid prevents oxidative stress in glutathione-deficient mice; effects on lung type-2 cell lamellar bodies, lung surfactant, and skeletal muscle. Proc. Nati. Acad. Sci. USA, #9: 5093-5097, 1992. 81. Kable, E. P. W., Favier, D., and Parsons, P. G. Sensitivity of human melanoma cells to i.-dopa and m.-buthioninc(S,R)-sulfoximine. Cancer Res., 4V: 2327-2331, 1989. 82. Dorr, R. T., Liddil, J. D., and Sohle, M. J. Cytotoxic effects of glutathione synthesis inhibition by i -buthioninc-(SR)-sulfoximine on human and murine tumor cells. Invest. New Drugs, 4: 305-313. 1986. 83. Peinado, P., Martinez-Liarte. J. H.. del Marmol. V., Solano. F.. and Lozano. J. A. Glulalhionc depletion in mouse melanoma cells increases their sensitivity to oxidative lysis. Cancer J., 5: 348-353, 1992. 84. Williamson. J. M.. and Meister, A. Stimulation of hepatic glutathione formation by administration of l-2-oxt)thiazolidine-4-carboxylate, a 5-oxo-L-prolinase substrate. Proc. Nati. Acad. Sci. USA, 78: 936-939, 1981. 85. Puri, R. N., and Meister. A. Transport of glutathione as y-glutamylcysteinylglycyl ester, into liver and kidney. Proc. Nati. Acad. Sci. USA, KO: 5258-5260, 1983. 86. Anderson, M. E., and Meister, A. Glutathione monoesters. Anal. Biochem., 183: 16-20, 1989. 87. Levy, E. J., Anderson, M. E.. and Meister, A. Transport of glutathione diethyl ester into human cells. Proc. Nati. Acad. Sci. USA, W: 9171-9175, 1993. 88. Anderson. M. E., and Meister, A. Inhibition of y-glutamyl transpeptidase and glutathionuria produced by y-glutamyl amino acids. Proc. Nati. Acad. Sci. USA, 83: 5029-5032, 1986. 66. 67. 68. 69. 70. 71. 72. is part of a multicomponent system essential for mitochondrial function. Proc. Nad. Acad. Sci. USA, 87: 7185-7189, 1990. 73. Boveris. A., Oshino, N., and Chance, B. The cellular production of hydrogen peroxide. Biochem. J.. 128: 617-630, 1972. 74. Borsook, H., Davenport, H. W., Jeffreys, C. E. P., and Warner. R. C. The oxidation of ascorbic acid and its reduction in \-iiro and m vìvo. J. Biol. Chem.. 117: 237-279, 1937. 75. Guzman-Barron. E. Thiol groups of biological importance. Adv. Enzymol.. 11: 201-266. 1951. 76. Hopkins. F. G., and Morgan, E. J. Some relations between ascorbic acid and glutathionc. Biochem. J.. 30: 1446-1462, 1936. 1975s Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1994 American Association for Cancer Research. Glutathione, Ascorbate, and Cellular Protection Alton Meister Cancer Res 1994;54:1969s-1975s. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/54/7_Supplement/1969s.citation Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1994 American Association for Cancer Research.