ICANCÕRRESEARCH(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 ß-carotenearc apparently also maintained synthesized within the body and are also obtained from the diet. by glutathione-mediated reactions (e.g.. Réf.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 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-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 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1994 American Association for Cancer Research. GLUTATHIONE, ASCORBATE, AND CELLULAR PROTECTION 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 reducíasedeficiency, glucose-6-phos- phate dehydrogenase deficiency, deficiencies of y-glutamylcysteine 2HJ synthetase and of glutathione synthetase) (34), and from experimental MERCAPTURATE à )/ 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 maléate,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