Pharmac. Ther. Vol. 51, pp. 155-194, 1991 Printed in Great Britain. All rights reserved

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GLUTATHIONE DEFICIENCY PRODUCED BY INHIBITION OF ITS SYNTHESIS, AND ITS REVERSAL; APPLICATIONS IN RESEARCH AND THERAPY ALTON MEISTER Department of Biochemistry, Cornell University Medical College, 1300 York Avenue, New York, N Y 10021, U.S.A. Abstract--Glutathione, which is synthesized within cells, is a component of a pathway that uses NADPH to provide cells with their reducing milieu. This is essential for (a) maintenance of the thiols of proteins (and other compounds) and of antioxidants (e.g. ascorbate, ct-tocopherol), (b) reduction of ribonucleotides to form the deoxyribonucleotide precursors of DNA, and (c) protection against oxidative damage, free radical damage, and other types of toxicity. Glutathione interacts with a wide variety of drugs. Despite its many and varied cellular functions, it is possible to achieve therapeutically useful modulations of glutathione metabolism. This article emphasizes an approach in which the synthesis of glutathione is selectively inhibited in vivo leading to glutathione deficiency. This is achieved through use of transition-state inactivators of 7-glutamylcysteine synthetase, the enzyme that catalyzes the first and rate-limiting step of glutathione synthesis. The effects of marked glutathione deficiency, thus produced in the absence of applied stress, include cellular damage associated with severe mitochondrial degeneration in a number of tissues. Such glutathione deficiency is not prevented or reversed by giving glutathione. The cellular utilization of GSH involves its extracellular degradation, uptake of products, and intracellular synthesis of GSH. This is a normal pathway by which cysteine moieties are taken up by cells. Glutathione deficiency induced by inhibition of its synthesis may be prevented or reversed by administration of glutathione esters which, in contrast to glutathione, are readily transported into cells and hydrolyzed to form glutathione intracellularly. Research derived from this model has led to several potentially useful therapeutic approaches, one of which is currently in clinical trial. Thus, certain tumors, including those that exhibit resistance to several drugs and to radiation, are sensitized to these modalities by selective inhibition of glutathione synthesis. An alternative interpretation is suggested which is based on the concept that some resistant tumors have high capacity for glutathione synthesis and that such increased capacity may be as significant or more significant in promoting the resistance of some tumors than the cellular levels of glutathione. Therapeutic approaches are proposed in which normal cells may be selectively protected against toxic antitumor agents and radiation by cysteine- and glutathione-delivery compounds. Current studies suggest that research on other modulations of glutathione metabolism and transport would be of interest.

CONTENTS 1. 2. 3. 4.

Introduction Enzymology and Metabolism of Glutathione (Overview) Transport of Glutathione Glutathione Deficiency 4.1. Inborn defects of synthesis 4.2. Experimental production of glutathione deficiency 4.2.1. Nonspecific agents 4.2.2. Amino acid sulfoximines 4.3. Mitochondrial glutathione 4.4. Glutathione deficiency and its reversal by administration of GSH esters 4.4.1. General findings 4.4.2. Glutatbione deficiency in mice 4.4.3. Glutathione deficiency in newborn rats and mice 4.4.4. Conclusions 5. Glutathione Depletion and Tumor Therapy 5.1. General (and brief historical) considerations 5.2. Sensitization of tumors to chemotherapy and radiation by treatment with buthionine sulfoximine

Abbreviations: GSH, glutathione; GSSG, glutathione disulfide. 155

156 157 158 161 161 162 162 162 164 165 165 167 170 172 175 175 176

156

A. MEISTER 5.3. Cellular levels of GSH versus cellular capacity for GSH synthesis as determinants of resistance and sensitivity to radiation and drugs 6. Therapy Based on Increase of Cellular Glutathione 6.1. General considerations 6.2. Cysteine delivery 6.3. Glutathione delivery Acknowledgements References 1. I N T R O D U C T I O N

178 179 179 180 182 185 185

catalyze the synthesis of GSH were first recognized by Bloch (1949). The reaction catalyzed by 7-glutamylGlutathione (L- 7 - glutamyl-L-cysteinyl-glycine; cyclotransferase was elucidated in 1956 (Connell GSH), an essential tripeptide found in virtually all and Hanes, 1956). Discovery of 5-oxoprolinase animal cells, has been the subject of much research stimulated the idea of the ~,-glutamyl cycle (Oriowski since its discovery by de Rey-Pailhade (1888a, b), and and Meister, 1970; Van Der Werf et al., 1971; especially after its structural elucidation about a half Meister, 1973). The enzymes involved have been isolated in highly purified forms and their properties century later. The earliest work on this compound showed that it reacts spontaneously with elemental and mechanisms of action have been studied sulfur, a property that led de Rey-Pailhade to name (Meister, 1978b, 1989b). These developments in it philothion (from the Greek words for love and enzymology have led to detailed understanding of sulfur). Later the molecule itself was found to contain the synthesis and utilization of GSH and have made sulfur and the term glutathione was proposed by it possible to construct a chart of the metabolism of Hopkins (1921) for what he initially thought to be GSH and to relate the actions of these enzymes to a dipeptide containing glutamate and cysteine. particular physiological functions. It is now widely understood that GSH is an Hopkins found that GSH accounts for "practically the whole of the nonprotein organically bound sulfur important component of a pathway that uses of the cell". He recognized that cells contain "pre- N A D P H to provide cells with their reducing milieu. dominantly the reduced form", and that the "chief Such reducing power is used for maintenance of the significance of the occurrence of cysteine in the thiol groups of intracellular proteins and other dipeptide, rather than free, lies in the fact that it is molecules, e.g. cysteine, dihydrolipoate, coenzyme A, and of antioxidant molecules such as ascorbate and thereby protected from metabolic breakdown". -tocopherol. It is used for reduction of ribonucleoThese conclusions have been confirmed and explained by subsequent studies on the enzymology of tides to form the deoxyribonucleoside triphosphate GSH (Meister, 1989a). Hopkins and collaborators precursors of DNA. Through its reactions, GSH proposed that GSH (which he and others recognized protects cells against oxidative and other types of as a tripeptide by 1930), functions as a reducing agent damage which may arise from compounds of enthat maintains enzymes in an active state, and that dogenous and exogenous sources. As first appreciated GSH reduces other compounds such as dehydro- by Hopkins, GSH serves as a storage form and also ascorbate (Hopkins and Morgan, 1936, 1938). as a transport form of cysteine moieties, and thus Glyoxalase, an enzyme activity that converts protects the thiol of cysteine from otherwise rapid methylglyoxal to D-lactate, was independently discov- metabolic utilization. GSH is synthesized within ered by Neuberg (1913) and Dakin and Dudley many types of cells. Its stability within the cell is (1913a, b) and was later found to require GSH as a favored by its lack of susceptibility to ~-glutamylcofactor (Lohmann, 1932). GSH is also a coenzyme cyclotransferase and intracellular peptidases and by for a number of other enzymes. 7-Glutamyl trans- the presence of substantial GSSG reductase activity. peptidase, initially recognized as an 'antiglyoxylase' Its export from cells functions to protect cell factor (Dakin and Dudley, 1913c), was later shown to membranes from oxidative damage and leads to catalyze amino acid-stimulated breakdown of GSH interorgan and intraorgan transfer of cysteine (Hanes et al., 1950). Mercapturic acids, first identified moieties. 7-Glutamyl transpeptidase functions in the as urinary excretion products after administration of cellular recovery of cysteine moieties. halogenated benzenes to animals (Baumann and This report emphasizes studies in which GSH Preusse, 1879; Jaffe, 1879), were later shown to be deficiency has been produced by selective inhibition formed by the action o f G S H S-transferases followed of ~-glutamylcysteine synthetase. This method for by other reactions in which GSH adducts are metab- depleting GSH, which does not involve application of olized (Booth et aL, 1961). GSSG reductase, found stress, has significant advantages over those that initially in plants (Mapson and Goddard, 1951; Conn involve use of nonspecific agents such as t-butyl and Vennesland, 1951), is now known to be as widely hydroperoxide and diethylmaleate. Some of the distributed as GSH. GSH peroxidase was discovered effects of cellular GSH deficiency thus produced have in erythrocytes (Mills, 1957); later it was found to been studied. When cellular GSH is decreased in this contain selenium, and several GSH peroxidases, some way the normal physiological endogenous formation lacking selenium, have been found. The enzymes that of reactive oxygen species is largely unopposed and

Glutathione deficiency OXIDATION-REDUCTION

157

GSSG~ -~

it'

-,-,s / 7

\\\

(~ / / ooo..oonuc,.o--..I t

I,~

Transhydrogenases/

R~uctase

I~J ~

/

~, radicals

/

/

/ " ~ NADP+

,, o,o "hGlu-Cys-Gly~ 2--Cys'0; MERCAPTURATE

\ (~

....

/y-GLUTAMYL

c,c.

\ ~ /

Z;'--..

J~,~" /

"

Q -Y

Feedback

io,,0,:,on ,

yt" \ o,s-,

~(~ N-Ac-Cys-X

¢ ,.~,u-~,

.,¢,-,°~,

Q'~

5.Oxoproline ~ ~,TP ''~

"

AA FIG. 1. Outline of the biochemistry of GSH. AA, amino acids; X, compounds that react with GSH to form conjugates. (1) 7-glutamylcysteine synthetase; (2) GSH synthetase; (3) ~,-glutamyltranspeptidase; (4) dipeptidases; (5) ?-glutamylcyclotransferase; (6) 5-oxoprolinase; (7) GSH S-transferases; (8) N-acetyltransferase; (9) GSH peroxidases (Se-containing and non-Se-containing); (10) GSH thiol transferases such as glutaredoxin and protein disulfide isomerase; (11) reaction of free radicals with GSH; (12) glutathione disulfide (GSSG) reductase; (13)transport of ?-Glu-(Cys)2; (14) GSH functions as a coenzyme for formaldehyde dehydrogenase, maleylactoaeetate isomerase, glyoxalase, prostaglandin endoperoxidase isomerases, and dichlorodiphenyltrichloroethane (DDT)-dehydrochlorinase and similar enzymes. In the glyoxalase reaction, the hemimercaptal formed nonenzymatically by reaction of methylglyoxal and GSH is converted by glyoxalase I to S-lactyl-GSH, which is split by glyoxalase II to D-lactate and GSH. In the formaldehyde dehydrogenase reaction, S-formyl GSH is formed (GSH+ HCHO+NAD +) and hydrolyzed to formate and GSH. Modified from Meister, 1988b.

cellular damage, such as that produced in mitochondria, occurs. GSH deficiency is not readily remedied by administration of GSH, which is not effectively transported into cells. However, GSH deficiency can be prevented or reversed by administration of GSH esters which serve as efficient transport vehicles for GSH. Thus, an experimental model is available for the study of the functions of GSH in which GSH can be selectively depleted by inhibition of its synthesis, and restored by giving GSH esters. Research derived from this model has suggested potentially useful therapeutic approaches several of which are considered here.

2. E N Z Y M O L O G Y A N D METABOLISM O F G L U T A T H I O N E (OVERVIEW) The scheme given in Fig. 1 provides an overview of the metabolism and functions of GSH. It includes the reactions of the v-glutamyl cycle, which account for

the synthesis and breakdown of GSH, and other reactions, such as those involving interconversion of GSH and GSSG, and those leading to formation of S-conjugates. (For recent reviews of GSH metabolism and function, see Dolphin et al., 1989, Taniguchi et al., 1989, Larsson et al., 1983; Meister and Anderson, 1983; Meister, 1989c.) GSH is synthesized by the consecutive actions of ~,-glutamylcysteine synthetase and GSH synthetase. Both reactions involve formation of enzyme-bound acyl phosphate intermediates, v-Glutamylcysteine synthetase normally functions at substantially less than maximal rate due to feedback inhibition by GSH. Intracellular GSH is utilized by several enzymes including the GSH transhydrogenases and GSH peroxidases; the GSSG formed in these reactions is converted to GSH by GSSG reductase, which utilizes NADPH. Conversion of GSH to various S-substituted adducts is catalyzed by the GSH S-transferases. GSH forms conjugates with compounds of endogenous origin such as leukotriene A and estrogens.

158

A. MEISTER

\

l

GS.SG~"-~ I /

~"~bG ~ H ~

GSH /

~

T-GLU-(CYSI =

-GLU-(CYS)z

CYSH-GLY (CYS)=

FIG. 2. Metabolism and transport of GSH and 7-glutamylcystine by the kidney. (1) Synthesis of 7-glutamylcysteine from glutamate and cysteine by 7-glutamylcysteine synthetase; (2) synthesis of GSH from ~,-glutamylcysteine and glycine by GSH synthetase; (3) reduction of GSSG by GSSG reductase; (4) reduction of 7-glutamylcystine by transhydrogenation with GSH; (5) feedback inhibition of 7-glutamylcysteine synthetase by GSH; (6) transport of GSH out of cell; (7) transport of y-glutamylcystine into cell; (8) formation of ?-glutamylcystine from GSH and cystine by ~,-glutamyl transpeptidase; (9) inhibition of transport of ?-glutamylcystine by high levels of extracellular GSH. 7Glutamylcyst(e)ine is also a substrate of 7-glutamylcyclotransferase which converts it to cyst(e)ine and 5-oxoproline. Reprinted from Anderson and Meister, 1983.

Conjugation of GSH with compounds of exogenous origin are of continuing interest in toxicological phenomena and in the metabolism of drugs. The 'mercapturic acid pathway' typically involves conversion of GSH S-conjugates to the corresponding conjugates of cysteinylglycine. These are split by dipeptidase to give the cysteine S-conjugates which may be acetylated to form mercapturic acids. A number of S-substituted cysteine and GSH derivatives undergo other chemical transformations. The breakdown of GSH occurs extracellularly and is catalyzed by 7-glutamyl transpeptidase and dipeptidases bound to the external surfaces of cell membranes. GSH is exported to the membrane bound enzymes under normal physiological conditions. Small amounts of GSSG may be transported normally, and such export increases markedly when the intracellular GSSG level is increased. Breakdown of GSH S-conjugates also requires export to the membrane bound enzymes. Thus, 7-glutamyl transpeptidase acts on exported GSH, GSSG, and GSH S-conjugates. Transpeptidation which takes place in the presence of amino acids, leads to formation of 7-glutamyl amino acids. The most active amino acid acceptor is cystine; other neutral amino acids, especially methionine and glutamine, are also active acceptors. 7-Glutamyl amino acids formed in this way are transported into the cell. Transpeptida-

tion may lead to formation of 7-glutamyl-GSH, which has been found in bile and in kidney. 7Glutamyl amino acids (but not GSH) are substrates of the intracellular enzyme 7-glutamylcyclotransferase, which converts 7-glutamyl amino acids into 5-oxoproline and the corresponding free amino acids. 5-Oxoproline is converted to glutamate in the ATPdependent reaction catalyzed by 5-oxoprolinase. Interaction of 7-glutamyl transpeptidase with GSH and cystine leads to formation of "/-glutamylcystine, which is transported and reduced intraceIlularly to form cysteine and l'-glutamylcysteine (Fig. 2). Utilization of the latter by GSH synthetase completes an alternative pathway which bypasses the step catalyzed by 7-glutamylcysteine synthetase and appears to serve as a recovery system for cysteine moieties. Cysteinylglycine may be split on the membrane, or be oxidized and split to form cystine and glycine; it and its disulfide may also be transported into the cell and hydrolyzed. The reactions of the 7-glutamyl cycle account for the synthesis and utilization of GSH and involve transport of GSH and of )'-glutamyl amino acids, thus constituting a pathway in which GSH is exported from cells and in which there is recovery by cells of the exported cysteine and other amino acid moieties.

3. T R A N S P O R T OF G L U T A T H I O N E Many types of cells normally export GSH; the major transport form is GSH rather than GSSG. An early observation that dramatically demonstrated the occurrence of such transport was the finding of marked glutathionuria after administration of inhibitors of 7-glutamyl transpeptidase to experimental animals (Griffith and Meister, 1979b; Anderson et al., 1980; Meister, 1978a). Plasma GSH levels were found to increase after administration of transpeptidase inhibitors. Thus, inhibition of GSH utilization leads to extracellular accumulation of GSH and to its urinary excretion. Not only GSH but also 7glutamylcysteine and cysteine moieties are excreted when 7-glutamyl transpeptidase inhibitors are given. These findings and others on the interaction of "l-glutamyl transpeptidase with cysteine indicate that the physiological role of 7-glutamyl transpeptidase is intimately connected with the metabolism and transport of GSH and its derivatives and metabolites (Griffith and Meister, 1979a, b, 1980; Griffith et al., 1981; Meister, 1985a, Allison and Meister, 1981; Thompson and Meister, 1975; Anderson and Meister, 1983). When GSH synthesis is inhibited (see below) the cellular level of GSH decreases because export of GSH continues in the absence of significant GSH resynthesis. That the rate of decrease of cellular GSH is similar to the rate of export indicates that there is usually little intracellular degradation of GSH. The turnover of cellular GSH is thus largely accounted for

Glutathione deficiency by export and it is often possible to estimate cellular GSH turnover rates from the rates of decrease of cellular GSH after GSH synthesis is inhibited. The impressive glutathionuria that occurs after administration of ?-glutamyl transpeptidase inhibitors is analogous to the glutathionuria and glutathionemia exhibited by humans with inborn ?-glutamyl transpeptidase deficiency (Meister and Larsson, 1989). It thus appears that there is normally an appreciable flow of GSH from cells and tissues into the extracellular space and the plasma. ?-Glutamyl transpeptidase and dipeptidase activities serve importantly in the recovery of cysteine moeities by a process in which there is a recycling of cysteine moieties for membrane transport of GSH. The cellular export of GSH appears to protect the cell membrane against oxidative and other types of damage by maintaining thiol groups and other components of the membrane such as ~t-tocopherol. GSH export may also provide a way of reducing compounds in the immediate environment of the cell membrane and it may facilitate transport of certain compounds. The export of GSH to membrane bound ?-glutamyl transpeptidase, a step in the 7-glutamyl cycle, leads to ?-glutamyl amino acid formation. ?-Glutamyl amino acids are readily transported into certain cells; ?-glutamyl cystine is a major y-glutamyl amino acid product of the action of ?-glutamyl transpeptidase, and transport of this compound appears to be of significance in the cellular recovery of cysteine (Thompson and Meister, 1975; Allison and Meister, 1981; Anderson and Meister, 1983; Meister and Anderson, 1983). GSH export by the liver constitutes the major source of plasma GSH. GSH is also exported into the bile. Plasma GSH is utilized by several tissues, especially the kidney, lung, and biliary system. Relatively high levels of GSH are found in the rat hepatic vein plasma (Anderson et al., 1980), and low levels of GSH are found in renal vein plasma (Anderson et al., 1980; Bartoli et al., 1978; H/iberle et al., 1979) consistent with such interorgan GSH transport. GSH is transported from renal cells to the renal tubule where it is degraded by ?-glutamyl transpeptidase and dipeptidase. The transport of GSH from renal cells to the tubular lumen is substantial and in mouse kidney accounts for 80-90% of the total GSH turnover (Meister, 1983b). Transport of GSH from liver to plasma functions in a distribution system for cysteine moieties in which the liver plays a major role and in which tissues that have substantial ?-glutamyl transpeptidase and dipeptidase activities utilize circulating GSH for the synthesis of cellular proteins and resynthesis of GSH. A number of studies have been concerned with the mechanism of GSH export and also with the specificity of this system because GSSG and GSH conjugates are also exported. Export of GSH from human lymphoid cells was found to occur at a rate that is proportional to the intracellular GSH level (Fig. 3)

159 I

I

I

I

I

:

.~2

~

I

.

"

0

I 2 Int.racellular Glutathione, mM FIG. 3. Export of GSH by CEM cells grown in the presence of buthionine sulfoximine. Samples were removed at intervals. The cells were centrifuged and resuspended (10 7 cells per ml) in phosphate-buffered saline and assayed for intracellular GSH and the rate of GSH export. Reprinted from Dethmers and Meister, 1981. (Dethmers and Meister, 1981). Studies have also been carried out on GSH transport by renal brush border membranes (Inoue and Morino, 1985) and by rat liver canalicular membrane vesicles (Inoue et al., 1983, 1984). There is evidence for membrane potential-dependent GSH transport by renal brush border membranes. Studies on various isolated cells and membrane vesicles derived from these have led to somewhat different conclusions about the mechanism and specificity of GSH export (Deleve and Kapiowitz, 1990). This process may be different for GSH, GSSG and GSH conjugates and in different cells; further work is needed. Export of GSSG appears to function in elimination of this disulfide from the cell, and may constitute an emergency mechanism to protect the cell against the deleterious effects of GSSG, which reacts with protein thiols (and others) to form mixed disulfides. There is evidence that ATP is required for the export of GSSG and for conjugates of GSH (Beutler, 1983; Ishikawa, 1989). Many ceils export GSH under normal conditions in which there are very low levels of extracellular GSH; the reverse of this process has not been shown in vivo. However, the possibility that intact GSH may be transported into some cells cannot be excluded. Studies on several organs and cell types have provided evidence that intact GSH is not transported into cells (Hahn et al., 1978; Yoshimura et al., 1982; Jensen and Meister, 1983; Dethmers and Meister, 1981; Tsan et al., 1989). The finding that the plasma GSH concentration over the kidney decreases more than can be accounted for by glomerular filtration (H/iberle et al., 1979; Fonteles et al., 1976; Anderson et al., 1980) led to consideration of the idea that there is basolateral uptake of nonfiltered GSH (Ormstad et al., 1980; Mclntyre and Curthoys, 1980). However, a detailed study showed that there is little, if any, net basolateral transport of GSH (Abbott et al., 1984; Curthoys, 1986; Inoue et al., 1986); as discussed elsewhere, transport phenomena observed in isolated vesicle systems may not necessarily reflect phenomena

160

A. MEISTER

that occur in vivo (Abbott et al., 1984; Inoue et al., 1986). Studies on isolated renal basolateral residues led to the conclusion that GSH is transported by a sodium-ion dependent pathway (Lash and Jones, 1984). The high apparent Km value for this process, and the absence of convincing evidence that the vesicles were formed in the correct orientation need to be considered. Studies on mouse lung in vivo (M~rtensson et al., 1989a) and on isolated perfused rat lung (Berggren et al., 1984) led to the conclusion that utilization of plasma GSH by this organ is analogous to that by lymphoid cells (Jensen and Meister, 1983) and the kidney (Abbott et al., 1984) in that it involves extracellular GSH breakdown, transport of products, and intracellular synthesis of GSH. Studies in which cells in suspension isolated from kidney, intestine, and lung were protected from destruction by t-butylhydroperoxide, paraquat of menadione by adding GSH led to the conclusion (Hagen and Jones, 1989) that intact GSH is transported into the cells and that the observed protection is dependent on such GSH transport. Transport of GSH across basolateral cell membranes was thought to occur because protection was decreased by probenecid, but the observed protective effect of GSH is most reasonably explained by its extracellular breakdown, transport of products and intracellular resynthesis of GSH; this is consistent with the finding that under the same conditions the free amino acid constituents of GSH also afforded protection. In these studies, inhibitors of 7-glutamylcysteine synthetase and of 7-glutamyl transpeptidase were added, but complete inhibition of these activities would not be expected under the conditions used; the activity levels during the period of study were not reported. Although decrease of protection was found after adding 7-glutamyl glutamate and 7-glutamyl-c~aminobutyryl-glycine, effects that were attributed to inhibition of GSH transport (Lash et al., 1986; Hagen and Jones, 1989), such decrease may most likely be ascribed to inhibition of ,/-glutamyl transpeptidase (Anderson and Meister, 1986). Lash and Tokarz (1990) found that isolated distal renal tubular cells were protected against oxidative damage by addition of GSH, and that such protection was abolished by addition of inhibitors of GSH degradation and synthesis (acivicin and buthionine sulfoximine); it was therefore justifiably concluded that distal renal tubular cells do not transport intact GSH. The findings of Lash and Tokarz (1990) and earlier ones (Hagen and Jones, 1989) on proximal renal tubular cells, which led to the conclusion that intact GSH is transported into these cells, are most likely explained by the use of experimental conditions in which ,;glutamyl transpeptidase and 7-glutamylcysteine synthetase were not sufficiently inhibited. It would be expected that under conditions in which these enzyme activities are more effectively inhibited addition of GSH would not be protective. Proximal and distal

renal tubular cells thus appear to have markedly different capacities for GSH synthesis and degradation. There are several reports on the uptake of GSH from the intestinal lumen. Transport of GSH in everted sacs of rat small intestine was reported to be sodium ion-independent, inhibitable by triglycine and glycyI-L-leucine, and to have properties typical of carrier-mediated diffusion (Hunjan and Evered, 1985; Evered and Wass, 1970). Studies in which an in situ closed loop vascular perfusion system was used led to the conclusion that intact GSH is transported into the mesenteric circulation by a sodium ion-dependent process (Hagen and Jones, 1987). GSH transport in brush border membrane vesicles of rabbit small intestine treated with acivicin to inhibit 7-glutamyl transpeptidase was reported to be sodium ionindependent (Vincenzini et al., 1988). Intestinal brush border membrane vesicles from the pig were reported to transport GSH in a sodium ion-stimulated process (Linder et al., 1984). Studies in which GSH was administered orally to mice and rats showed little or no uptake into blood plasma (M~rtensson et al., 1990a; Yoshimura et al., 1982). A small increase of plasma GSH was reported after oral administration of GSH to rats (Hagen et al., 1990), in these studies, the control GSH values for jugular vein samples averaged 12 ~M and the levels found 90 min after GSH administration by gavage, were reported to be about 30/~M. However, the levels reported (Hagen et al., 1990) are well within the range expected for samples of normal rat blood plasma (20-40/IM (Anderson and Meister, 1980)). Results reported (Hagen et al., 1990) after administration of inhibitors (buthionine sulfoximine, acivicin) given at less than optimal levels, and after administration of 7-glutamyl transpeptidase can be variously interpreted since appropriate controls were not reported. GSH present in the intestinal lumen probably arises from the diet, bile, desquamated epithelial cells, and by export from the epithelial cells of the stomach and intestines. Since these cells (and the pancreatic juice) are well equipped with 7-glutamyl transpeptidase and dipeptidase activities, it is reasonable to suppose that a substantial fraction, but perhaps not all, of intestinal lumenal GSH is split anJ absorbed as dipeptides and free amino acids. This is consistent with the finding of high cysteine levels in portal blood after giving oral GSH (Vifia et al., 1989). However, small amounts of GSH may be taken up into the plasma, as observed with other peptides (Matthews, 1987; Gardner, 1987), presumably by leakage through junctions between epithelial cells. GSH administered intraperitoneally to mice did not significantly increase the GSH levels of the jejunal or colon mucosa suggesting that there is little or no basolateral transport of GSH into these epithelial cells. Oral administration of GSH led to an increase of mucosal GSH in colon, jejunum and stomach (M~rtensson et al., 1990a).

Glutathione deficiency These cells may take up intact GSH from the lumen, but the increased cellular level of GSH after oral administration of GSH might also arise in other ways. Thus, 2:-glutamylcysteine might be formed from luminal GSH by carboxypeptidase-type cleavage of GSH or by transpeptidation between GSH and cysteine followed by transport and conversion to GSH by GSH synthetase. Another possible pathway involves formation of 2:-glutamyl GSH followed by conversion to GSH by the action of 2:-glutamylcyclotransferase (Abbott et al., 1986). ~-glutamyltraaspeptidase

2GSH

~2:-GIu-GSH + CySH-Gly y-glutamylcyclotransfer ase

7-Glu-GSH

,5-Oxoproline + GSH

It has been suggested that lumenal GSH, much of which normally arises probably from the bile, provides a protective function for the intestinal mucosa (M~trtensson et al., 1990a). Bile contains high levels of GSH (1-6mM) (Abbott and Meister, 1986) and this could lead to high local levels of GSH in the small intestine. Pancreatic juice, which contains high levels of y-glutamyl transpeptidase and dipeptidase, understandably does not have detectable levels of GSH (Abbott and Meister, 1986). Although there appears to be little uptake of intact GSH from the intestinal lumen into the blood plasma under normal conditions, it is conceivable that uptake of GSH might occur after giving larger oral doses of GSH. GSH can also be given parenteraUy, but the potential significance of this approach needs to be considered in relation to findings made with very high plasma GSH levels (M~rtensson et al., 1989a, 1990). Thus, in the mouse, plasma levels of GSH, normally in the micromolar range, can be increased to levels of 5 mM or higher by intraperitoneal injection of GSH. That such markedly increased levels of plasma GSH do not lead to appreciable increase of the tissue levels of GSH or protect against the effects of GSH deficiency (see Section 4.4), indicates that plasma GSH is not rapidly taken up by the cells. The physiological utilization of plasma GSH seems to involve extracellular degradation of GSH followed by transport of the resulting amino acids and dipeptides into cells and intracellular GSH synthesis. Possibly a small amount of intact GSH is taken up, but it would not seem to represent a quantitatively significant fraction of cellular GSH. Transport of GSH from plasma to brain across the blood-brain barrier has been considered. Early studies did not reveal evidence of such transport (Cornford et al., 1978), but recently Kannan et al. (1990) reported data interpreted to show carriermediated transport across the blood-brain barrier in the rat; uptake was saturable with apparent Km of 5.84 m~l. No value was found for uptake at physiological plasma levels (20-30 #M). If such a transport system exists it should be possible to demonstrate uptake into the brain of mice given high doses of

161

GSH which develop millimolar levels of plasma GSH. The relevant studies (Jain et al., 1991) do not provide evidence for significant transport of GSH into the cerebral cortex even after 200- to 1000-fold increase of plasma GSH. The possibility that GSH or a portion of this molecule may be transferred between cells is suggested by recent studies on spermatogenic cell GSH (Li et al., 1989; Den Boer et aL, 1989). These investigations, which show that the formation of spermatogenic cell GSH requires Sertoli cells, suggest that the process involves germ cell-somatic cell interactions. Studies in which several types of Chinese hamster V79 cells were cocultured and analyzed for GSH by flow cytometry suggest occurrence of 'metabolic sharing' in which GSH or "~-glutamylcysteine may be transferred between cells via gap junctions (Kavanagh et al., 1988). Many of the studies on GSH metabolism and transport have been carried out in rodents; although species differences have been observed, it is generally believed that the metabolism and transport of GSH are closely similar in various mammalian species. However, it may be noted that rodent plasma contains little or no y-glutamyl transpeptidase activity as compared to human plasma. Human plasma contains about 1-5 #~ total GSH as compared to 10-40 #M in the rat. Other species differences have been noted (Purucker and Wernze, 1990). Human plasma contains cystinylbisglycine (Armstrong, 1979) as well as cystine. The level of GSH is significantly different in blood plasma obtained from different blood vessels in the rat (Anderson et al., 1980) and mouse (M~trtensson et al., 1989a); that there is a significant arteriovenous difference across the lung may complicate interpretation of values on samples obtained by heart puncture.

4. G L U T A T H I O N E DEFICIENCY 4.1.

INBORN DEFECTS OF SYNTHESIS

Several inborn errors of GSH metabolism have been found in humans (Meister and Larsson, 1989). These include patients who have an inborn deficiency of GSSG reductase associated with increased tendency to hemolysis and early development of cataracts. Patients with glucose 6-phosphate dehydrogenase deficiency exhibit increased sensitivity to oxidative stress; their attacks of acute hemolytic anemia may be triggered by various drugs, infection, and foods such as lava beans. The biochemical defect leads to insufficient maintenance of GSH levels in erythrocytes, an effect that results from insufficient NADPH formation. A few patients with inherited deficiencies of GSH synthetase or y-glutamylcysteine synthetase have been described. In one type of GSH synthetase deficiency, apparently restricted to the erythrocyte, there is compensated hemolytic disease. The genetic defect apparently leads to synthesis of an

162

A. MElSTEg

unstable GSH synthetase molecule which can be replaced in most cells by synthesis of active enzyme. Such enzyme synthesis is impossible in erythrocytes which cannot synthesize proteins. Severe GSH synthetase deficiency and 7-glutamylcysteine synthetase deficiency are associated with hemolytic disease and defective central nervous system function. A most interesting aspect of severe GSH synthetase deficiency is the associated 5-oxoprolinuria and 5oxoprolinemia (Wellner et al., 1974). Since GSH feedback inhibits 7-glutamylcysteine synthetase, this enzyme does not normally catalyze dipeptide synthesis at maximal rate (Richman and Meister, 1975). In the absence of appreciable GSH synthetase activity intracellular GSH levels are very low thus releasing 7-glutamylcysteine synthetase from feedback inhibition. The formation of ~-glutamylcysteine proceeds at least several times more rapidly under these conditions leading to excessive formation of ),-glutamylcysteine. 7-Glutamylcysteine is a good substrate of 7-glutamylcyclotransferase and there is therefore a marked increase in the formation of 5-oxoproline. The formation of 5-oxoproline exceeds the capacity of 5-oxoprolinase and 5-oxoproline therefore accumulates in the blood and is excreted in the urine. The accumulation of 5-oxoproline leads to marked acidosis and individuals with this inborn error develop life threatening acidosis soon after birth (Meister and Larsson, 1989).

4.2. EXPERIMENTALPRODUCTION OF GLUTATHIONE DEFICIENCY

4.2.1. Nonspecific Agents Depletion of cellular GSH has been a frequent objective in many investigations of the function of this tripeptide. Cellular GSH levels may be substantially decreased by administration of oxidizing agents, irradiation, or by treatment with compounds that interact with the thiol group of GSH. Oxidizing agents such as diamide (Kosower and Kosower, 1969), and t-butylhydroperoxide (Plummer et al., 1981), can produce rapid decrease of cellular GSH levels. Disappearance of GSH is, however, accompanied by formation of high levels of GSSG and there is also increased formation of mixed disulfides between GSH and proteins. In many instances depletion of GSH is quite temporary because the GSSG formed is rapidly reduced by GSSG reductase. Marked alteration of the ratio of GSH to GSSG and excessive utilization of N A D P H may be expected. The oxidizing agents used thus far are not specific for GSH and therefore their application leads to oxidation of other cellular components. Compounds that react with the thiol group of GSH such as diethylmaleate and phorone (diisopropylidine acetone) have been extensively used for GSH depletion in in vitro and in vivo studies (Plummer et al., 1981; Deneke et al., 1989). Although application of

these compounds may lead to substantial decrease in cellular GSH levels, a variety of additional effects are produced. Commercially available diethylmaleate contains diethylfumarate and undoubtedly has other impurities. It is most probably converted in tissues to maleate, which is known to affect certain enzymes (see, for example Tate and Meister, 1975). Administration of maleate has been reported to have toxic effects on the kidney (Rosenberg and Segal, 1964; Harrison and Harrison, 1954). Administration of diethylmaleate leads to lipid peroxidation, but it was concluded that this is not mediated by GSH depletion but by other effects (Reiter and Wendel, 1982). Diethylmaleate has several effects on microsomal mixed function oxidations (Anders, 1978); it stimulates heme oxygenase activity (Burk and Correia, 1979) and inhibits protein synthesis (Costa and Murphy, 1986). Phorone also increases heme oxygenase activity and affects other liver enzymes (Yoshida et al., 1987). A major difficulty associated with the use of diethylmaleate and similar compounds is that the GSH deficiency produced is accompanied by removal of cysteine moieties from cells via the mercapturate pathway. Another problem is that the decrease in GSH level achieved by use of diethylmaleate and related compounds is shortlived. Since GSH feedback inhibits its own synthesis (Richman and Meister, 1975), an early effect of treatment with oxidizing agents or with thiol-reactive compounds is increased synthesis of GSH. The increased synthesis of GSH that follows treatment with diethylmaleate and related compounds can lead to higher than normal levels of GSH. This produces an anomalous result in which administration of a compound thought to be a 'GSH depletor' actually leads to an increase in GSH level (Deneke and Fanburg, 1989; Plummer et al., 1981). 4.2.2. Amino Acid Sulfoxim&es The considerations reviewed above indicate that nonspecific thiol-reactive compounds and nonspecific oxidizing agents have major limitations for studies involving GSH depletion. An alternative approach is inhibition of GSH synthesis. Inhibition of GSH synthetase seems not to be a promising approach because, as discussed above (Section 4.1), patients with severe inborn GSH synthetase deficiency develop marked acidosis due to overproduction of 5-oxoproline. Inhibition of ~,-glutamylcysteine synthetase offers a more attractive basis for production of experimental GSH deficiency. Several inhibitors of this enzyme are known (Sekura and Meister, 1977; Simondsen and Meister, 1986; Moore et al., 1987; Griffith et al., 1979; Griffith and Meister, 1979a); the sulfoximine inhibitors, especially buthionine sulfoximine, have been widely used. Earlier studies showed that glutamine synthetase is irreversibly inhibited by the convulsant compound methionine sulfoximine, which binds to the glutamate

Glutathione deficiency binding site of this enzyme and is phosphorylated on the sulfoximine nitrogen atom by ATP (Ronzio and Meister, 1968; Ronzio et al., 1969; Rowe et al., 1969; Meister, 1968; Gass and Meister, 1970a, b, 1974a); this reaction is analogous to the formation of 7glutamyl phosphate in the normal catalytic reaction. The resulting methionine sulfoximine phosphate binds tightly to the active site thus inhibiting the enzyme irreversibly. Of the 4 diastereoisomers of methionine sulfoximine only the 2S, S,S-form is phosphorylated on the enzyme (Manning et al., 1969). Only this isomer of methionine sulfoximine produces convulsions (Rowe and Meister, 1970). Methionine sulfoximine was examined as a possible inhibitor of 3)-glutamylcysteine synthetase after studies on this enzyme provided evidence that ),glutamyl phosphate is also an intermediate in this reaction (Orlowski and Meister, 1971). It was found that ~-glutamylcysteine synthetase is irreversibly inhibited by methionine sulfoximine, indeed by the same isomer of this compound (2,S, S,S), that inhibits glutamine synthetase (Richman et al., 1973). When methionine sulfoximine was administered to experimental animals, it decreased not only the synthesis of glutamine but also that of GSH (Palekar et al., 1975). Although methionine sulfoximine is an effective inhibitor of GSH synthesis, its usefulness in experimental work and in therapy is severely limited because of its convulsant and lethal effects, which are due largely to inactivation of brain glutamine synthetase. Studies of the active site of glutamine synthetase by mapping with a large number of glutamate analogs revealed that the S-methyl group of methionine sulfoximine binds to the ammonia binding site of glutamine synthetase (Meister, 1968, 1974a, 1978a). Sulfoximines that have larger S-alkyl moieties (e.g. prothionine sulfoximine (Griffith et al., 1979), buthionine sulfoximine (Griffith and Meister, 1979~), and others (Griffith, 1982; Meister, 1989c) do not inhibit glutamine synthetase significantly because they are hindered from binding to the ammonia binding site of this enzyme. On the other hand, these compounds effectively bind to and inactivate ~-glutamylcysteine synthetase. Thus, although the mechanisms of action of glutamine synthetase and ?-glutamylcysteine synthetase are closely similar in that both enzymes involve formation of 7-glutamyl phosphate and involve similar tetrahedral intermediates, they can be selectively inhibited by use of appropriate analogs of methionine sulfoximine (Griftith et al., 1979; Griffith and Meister, 1978, 1979a; Meister, 1978a). ct-Ethylmethionine sulfoximine produces convulsions, inhibits glutamine synthetase, but it does not inhibit 7-glutamylcysteine synthetase and therefore has little or no effect on the synthesis of GSH (Fig. 4) (Griffith and Meister, 1978). On the other hand, buthionine sulfoximine does not inhibit glutamine synthetase significantly and thus decreases GSH levels without affecting

O II

CH3-S=NH I

163 O

fl CH3-S=NH 1

e,.s-&.; CO0Metblonlne Sulfoxlmlne

CO0 a *Ethyl Methlonlne Sulfoxlmlne

CH 3

ICH3

.~.2

p.2

O = S =NH ~'/H2 CH 2 I

O=S =0 I cH2 CH 2

+

CHNH 3 I cooButhlonlrm Sulfoxlrnine

I

+

CHNH 3 I CO0Buthlonlne Suifone

FIG. 4. Chemical structures. glutamine synthesis or producing convulsions. Later studies have led to synthesis of additional compounds that inactivate ~-glutamylcysteine synthetase without affecting glutamine synthetase. Thus, S-(n-pentyl) homocysteine sulfoximine is similar in action to buthionine sulfoximine; however, the corresponding S-n-hexyl and S-n-heptyl compounds are very toxic (Griffith, 1982). Other inhibitory sulfoximines include S-2-methyl-n-butylhomocysteine sulfoximine, and the corresponding 3-methyl, 3,Y-dimethyl, 2-ethyl, and cyclohexymethyl compounds (Meister, 1989c). It appears that, as first shown for methionine sulfoximine (Richman et al., 1973), only the 2,S, S,Sdiastereoisomers of these compounds inactivate 7-glutamylcysteine synthetase (Gritfith, 1982). Although several effective inhibitors of ~-glutamylcysteine synthetase are now known, the most widely used inhibitor has been L-buthionine SR-sulfoximine, which is now commercially available. (oL-Buthionine SR-sulfoximine exhibits about one-half of the effectiveness of L-buthionine SR-sulfoximine since the D-isomers are not active.) In contrast to methionine sulfoximine, buthionine sulfoximine is relatively nontoxic and doses as high as 32 mmol/kg have been given to adult rats without causing death. The toxicity of buthionine sulfoximine is not far from that found after administration of various natural amino acids. Buthionine sulfoximine is itself not highly reactive chemically. Furthermore, in contrast to methionine sulfoximine (Rao and Meister, 1972; Cooper et al., 1976) it does not appear to be metabolized to a significant extent (Griffith 1982). Its major in vivo effect is inhibition of ~-glutamylcysteine synthetase. Buthionine sulfoximine can potentially serve as an analog of ~-glutamyl amino acids (Griffith and Meister, 1979a), so the possibility exists that buthionine sulfoximine might competitively inhibit transport of certain ~,-glutamyl compounds. Results indicating inhibition of transport into kidney of ~-glutamylcysteine by buthionine sulfoximine have been obtained (Anderson and Meister, 1983; see also Bridges and Meister, 1985). Whether this type of inhibition is involved in the reported inhibition by buthionine suifoximine of uptake of cystine (as 7-glu-cystine) by L1210 cells in vitro (Brodie and Reed, 1985) is not clear. In vivo studies by Griffith (1989) in which the separate L-S and L-R diastereoisomers of buthionine

164

A. MEISTER 0 ii CH3-S=NH I

NH ii CH3-S=O I

CH 2

CH 2

CH21

CH2

H-C-NH 2

H-C-NH 2

I

I

COOH

S

I

I

COOH

R

Diastereoisomers of L-Methionlne Sulfoxlmine

FIG. 5. Chemical structures. sulfoximine were used, indicate that the L-R isomer decreased GSH levels in kidney by about 13%; this might represent a competitive effect on transport. Such inhibition might be expected to occur with the L,S-isomer of buthionine sulfoximine as well as the corresponding L,R-isomer, which does not inhibit ),-glutamylcysteine synthetase. In in vivo studies with L-buthionine R-sulfoximine it was thus concluded that the effect of buthionine sulfoximine on such transport must be relatively small. These and other studies indicate that buthionine sulfoximine is a highly specific inhibitor of GSH synthesis. Thus far, no other in vivo effects of significance have been uncovered. The L,R-diastereoisomer is useful as a control compound for possible detection of effects of buthionine sulfoximine that are unrelated to its effect on GSH synthesis; for example, it does not inhibit proliferation of normal human T-lymphocytes, whereas L-buthionine-SR-sulfoximine does (Suthanthiran et al., 1990). When adult mice are treated with buthionine sulfoximine there is a substantial decline in the level of GSH in many tissues and in the blood plasma. T h e effects of a single injection of buthionine sulfoximine into mice on the levels of GSH in kidney and liver are given in Fig. 6 (Griffith and Meister, 1979c). T h e rate of decline of the levels of GSH in these tissues leads to estimated turnover rates which are similar to those based on rates of incorporation of isotopically labeled glutamate into GSH (Sekura and Meister, 1974). As shown in Fig. 6, the levels of GSH in liver and kidney decrease rapidly to values that are about 15-20% of the controls. A much slower further decline in GSH levels occurs when additional buthionine sulfoximine is given (Griffith and Meister, 1985). The biphasic nature of the decline of the GSH level of liver and kidney appears to be largely associated with sequestration of GSH within the mitochondria (see below). Buthionine sulfoximine is also effective when given orally (Griffith and Meister, 1979a), and as discussed below it can be given repeatedly to mice and rats for at least several weeks thus providing a means for producing a chronic GSH deficiency state. Administration of buthionine sulfoximine would not be expected to decrease cellular levels of GSH if the turnover of GSH is extremely slow, or if buthionine sulfoximine is not transported into the cell. T h e o r -

etically a very high cellular level of glutamate might decrease the rate at which inhibition of ~,-glutamylcysteine synthetase occurs, because glutamate and buthionine sulfoximine bind at the same site. The rate of regeneration of cellular GSH that follows a period of buthionine sulfoximine-induced inhibition of synthesis is usually slower than the initial rate of buthionine sulfoximine-induced decline. This is due in part to a relative deficiency of cysteine but may also be limited by the amount of available active enzyme. Questions relating to the fate of the inhibited enzyme and the rate of synthesis of new enzyme require additional study. The possibility that buthionine sulfoximine phosphate may dissociate from the inhibited enzyme in vivo with return of activity (Richman et al., 1973) needs to be studied. 4.3. MITOCHONDRIALGLUTATHIONE The finding that administration of buthionine sulfoximine to rats or mice leads to rapid decline of the GSH levels of liver and kidney to an apparent limiting value of 15-20% of the total GSH indicated that a significant fraction of cellular GSH is in a compartment separate from the general cytosolic pool, and suggested that some GSH is sequestered in mitochondria. Earlier studies had shown that mitochondria contain GSH; previous investigations were interpreted to indicate that mitochondria have a separate pool of GSH, that mitochondrial GSH does not equilibrate with cytosolic GSH, that the mitochondrial membrane is impermeable to GSH, and that mitochondrial GSH is synthesized in situ (Jocelyn and Kamminga, 1974; Jocelyn, 1975; Wahll/inder, et al., 1979; Romero and Sies, 1984). For example, since treatment of isolated hepatocytes with diethylmaleate led to depletion of cytosolic GSH but not that of mitochondrial GSH, it was therefore concluded that the mitochondrial pool of GSH of the isolated hepatocyte is metabolically separate from the cytosolic GSH pool (Meredith and Reed, 1982). Although the observed biphasic decline in the GSH levels of liver and kidney might be explained by sequestration of GSH within the mitochondria, it

I00-' z

8o60-

LIVER~

o ~ 40~

20-

o

HOURS

FIG. 6. Levels of GSH in kidney and liver after intraperitoneal injection of Dg-buthionine-SR-sulfoximine (4 mmol/kg). Reprinted from Griffith and Meister, 1979c.

Glutathione deficiency might also be related to the finding that buthionine sulfoximine is not transported at a significant rate into mitochondria (Meister and Griffith, 1983). However, it was discovered that mitochondria do not have ~,-glutamylcysteine synthetase or GSH synthetase activities (Griffith and Meister, 1985), and therefore the failure of buthionine sulfoximine to enter mitochondria readily is not relevant. The absence of the synthetases from mitochondria is significant, however, because it indicates that mitochondrial GSH arises from the cytosol. When isolated rat liver mitochondria were suspended in solutions containing high (5-10 mM) levels of GSH, the total level of GSH in the mitochondrial increased significantly (Fu et al., 1989; Kurosawa et al., 1990; M~rtensson et aL, 1990b). Studies on the kinetics of GSH uptake by isolated rat liver mitochondria were carried out in which GSH transport into and out of the mitochondrial matrix was examined at low and high external GSH levels and at short time intervals (e.g. 15 see) (M,~rtensson et al., 1990b). The results show that mitochondrial GSH homeostasis is effected by a multicomponent transport system; this appears to underlie the remarkable ability of mitochondria to take up and retain GSH. At external GSH levels of less than I mM, GSH is transported into the mitochondrial matrix by a high affinity component (Kin~ 60/~M; limax ~ 0.5 nmol/ min/mg of protein), which is saturated at levels of 1-2 mM and stimulated by ATP. Another component has lower affinity (Kin ~ 5.4mM; Vm,x~ 5.9mmol/ min/mg protein) and is stimulated by ATP and ADP. These studies provide evidence that increased levels of extramitochondriai GSH promote uptake and exchange. The intermembranous space appears to function as a recovery zone that facilitates efficient cycling of matrix GSH. The results of these studies on isolated liver mitochondria are in accord with previous in vivo studies (Griffith and Meister, 1985) on the incorporation of cysteine into mitochondrial and cytosolic GSH which indicate that there is rapid exchange of GSH between the mitochondrial matrix, the intermembranous space, and the cytosol. The findings also seem to explain the observation that decreasing cytosolic GSH levels (produced by administration of buthionine sulfoximine) decreases net export of GSH from mitochondria to cytosoi. In vivo studies show (see Section 4.4) that administration of GSH monoesters to mice leads to increased mitochondrial GSH levels in various tissues, suggesting that mitochondria are highly effective in transporting GSH from the cytosol. It is conceivable that GSH monoesters are taken up by mitochondria and then de-esterified within them. The experimental conditions necessary to check this point have not yet been achieved. However, it appears more likely that the increase in mitochondrial GSH that follows administration of GSH monoesters is associated with the high affinity of mitochondria for GSH. Thus, mitochondria can take up GSH

165

z

t CYTOPLAS M

x ® GssG -

®

FIG. 7. Scheme for synthesis and transport of GSH in mitochondria and cytosol. See the text for details. Reprinted from Grit~th and Meister, 1985, effectively even when the cytosolic GSH levels are very low. Reversible conversion of GSH to GSSG occurs in both mitochondria and cytosol, but the synthesis of GSH occurs only in the cytosol (Fig. 7). GSH, rather than GSSG, is probably the major transport form since GSH is the predominant intracellular form. The observed rapid labeling of mitochondrial GSH after administration of labeled cysteine indicates that there are exchange carriers in the mitochondrial membrane that are accessible to both mitochondrial and cytosolic GSH. The finding that the net efflux of GSH from mitochondria is very slow in the presence of low levels of extramitochondrial GSH indicates that this transport mechanism functions in a manner that conserves mitochondrial GSH during periods of cytosolic GSH depletion which may arise as a consequence of nutritional factors or through phenomena associated with oxidation or conjugation in the presence of toxic compounds. As discussed below (Section 4.4.4), a small but significant fraction of the oxygen utilized by mitochondria is converted to hydrogen peroxide. Since mitochondria do not contain catalase, they depend almost entirely on GSH-dependent reactions for destruction of hydrogen peroxide. 4.4. GLUTATHIONE DEFICIENCY AND ITS REVERSAL BY ADMINISTRATION OF GSH ESTERS 4.4.1. General Findings The effectiveness of buthionine sulfoximine as an inhibitor of 3,-glutamylcysteine synthetase in vivo has led to several applications. It is a useful selective agent for turning off GSH synthesis and thus decreasing cellular GSH levels for a variety of experimental purposes (Meister, 1983a). For example, depletion of GSH by treatment with buthionine sulfoximine sensitizes cells to the toxic effects of HgC12 (Naganuma et al., 1990), CdCI 2 (Singhal et al., 1987), phenylalanine mustard (Roizin-Towle, 1985), radiation (Dethmers and Meister, 1981; Clement et al., 1981),

166

A. MEISTER

cisplatin (Anderson et al., 1990), cyclophosphamide (Ishikawa et al., 1989a, b), morphine (McCartney, 1989), compounds that produce oxidative cytolysis (Arrick et al., 1982) and other substances (Perez et al., 1990). Effects on tumors are considered below (see Section 5). Buthionine sulfoximine was initially given to mice in a single subcutaneous dose; 2 hr later, the GSH levels of liver, pancreas, kidney, skeletal muscle and plasma were found to be appreciably decreased. When buthionine sulfoximine was supplied in the drinking water (20 mu) for 15 days, there were significant decreases in the levels of GSH in these tissues as well as in brain, heart, lung, spleen, small intestinal mucosa, and colon mucosa (Griffith and Meister, 1979a). Subsequent studies on rats and mice in our laboratory and in others have provided confirmation of these results. Drew and Miners (1984) studied fed male C3H mice and found that liver and kidney GSH levels were depleted by buthionine sulfoximine in a dosedependent manner; maximal effects were observed 2~4hr after buthionine sulfoximine administration. Control levels of 7-glutamylcysteine synthetase activity and GSH content were restored about 16 hr after administration of buthionine sulfoximine. Buthionine sulfoximine had no effect on hepatic microsomal cytochrome P-450 levels, a range of cytochrome P-450 dependent enzyme activities or p-nitrophenol glucuronyl transferase activity. Buthionine sulfoximine had no effect on phenol sulphotransferase and on two GSH S-transferase activities, nor did it affect duration of hexobarbitone induced narcosis in mice. Pretreatment of mice with buthionine sulfoximine decreased the proportion of a 50 mg/kg dose of paracetamol excreted in the urine as GSH-derived conjugates without affecting clearance of paracetamol through the glucuronidation or sulfation pathways. Minchinton et al. (1984) gave single and repeated doses of buthionine sulfoximine to mice and studied the pattern of GSH depletion in liver, kidney, skeletal muscle, and 3 types of murine tumors. Liver and kidney showed rapid depletion of GSH to levels about 20% of controls after a single dose of 1-5 mmol/kg of buthionine sulfoximine. Depletion of muscle GSH followed a similar pattern but the rate of decline was slower. The tumors examined required repeated administration of buthionine sulfoximine over several days to achieve a degree of GSH depletion similar to that found in the normal tissues. Sun et al. (1985) gave buthionine sulfoximine in the drinking water to mice for various periods up to 28 days. With 30 mM buthionine sulfoximine in the drinking water, GSH levels were significantly decreased in lungs, lung lavage fluid, liver, kidney, and blood. The GSH levels returned to control levels 7 days after buthionine sulfoximine was withdrawn. The levels of alkaline phosphatase, lactate dehydrogenase, glucose 6-phosphate dehydrogenase, GSH

peroxidase, and GSSG reductase in lungs and lung lavage fluid, and total and differential cell counts from lung lavage fluid were not different between control and buthionine sulfoximine-treated mice. Serum aspartyl transferase and 7-glutamyl transpeptidase activities were also unaffected by treatment with buthionine sulfoximine. Lung and liver cytochrome P-450 concentrations were similarly unaffected by treatment with buthionine sulfoximine. Lee et al. (1987) studied the effects of buthionine sulfoximine on the GSH levels of normal tissues and 3 experimental tumors in mice. Attempts to preferentially deplete tumor GSH by exploiting the differences in recovery rates between normal tissues and tumors were only partially successful. Dose-depletion relationship studies showed that, with the exception of the lungs, GSH depletion could be achieved in tumors with doses of buthionine sulfoximine that were lower than those required for normal tissues. Smith et al. (1989) examined the pharmacokinetics of buthionine sulfoximine in mice. Intravenous doses of buthionine sulfoximine were rapidly eliminated from mouse plasma. The initial phase had a half time of 4.9 min whereas the half time of the terminal phase was 36.7 min. Plasma clearance of buthionine sulfoximine was 28.1 ml/min/kg and the steady state volume of distribution was 280ml/kg. Although oral bioavailability of buthionine sulfoximine based on plasma levels was low, comparable GSH depletion was found after intravenous and oral doses of buthionine sulfoximine suggesting rapid tissue uptake. These authors noted that plasma buthionine sulfoximine levels do not correlate directly with GSH depletion in the tissues. They found little apparent toxicity after intravenous administration of multiple doses of buthionine sulfoximine at doses of 400-1600 mg/kg/dose. Many studies in our laboratory and in others failed to reveal significant toxic effects after administration of buthionine sulfoximine to adult rats or mice; however, the tissue levels of GSH obtained in these experiments were not markedly decreased nor were they maintained at low level for long periods. These observations, however, were encouraging in that they did not immediately exclude the possibility of treatment of humans with buthionine sulfoximine as an adjuvant to cancer chemotherapy or to radiation therapy (see Section 5). However, they did not exclude the likely possibility that combination of treatment with buthionine sulfoximine and certain other drugs might lead to toxicity; indeed subsequent work has demonstrated such effects. Nevertheless, the early experience with buthionine sulfoximine has led to the impression that this compound is essentially 'nontoxic'. Presumably most tissues have a large excess of GSH and therefore a substantial decrease in GSH level might not inevitably be accompanied by toxicity. However, it seemed of interest to administer buthionine sulfoximine for longer periods to determine the effects of prolonged GSH deficiency. Since

Glutathione deficiency

167

respectively, about 3% and 8% of the controls. The levels of buthionine sulfoximine were about 300nmol/g for heart and 100nmol/g for skeletal muscle. After discontinuation of buthionine sulfoxi120 mine treatment, the return of GSH was rapid in heart ioo 100 and led to values higher than the initial ones; control levels were found after 5 days, but at this time only about 40% of the control level of GSH was found in § 8C ~ , skeletal muscle. Mice depleted of GSH by treatment with buthionine sulfoximine were treated with r, 6C~ "1cysteine, glutamate, ct-ketoglutarate, glutamine, or (/~ 119 o i00 20o 300 400 ~00 glycine and a mixture containing cysteine, glutamate, (.9 40 ~ou,, ¢/~ and glycine. The findings indicated that synthesis of tissue GSH under these conditions is mainly limited 20 by availability of cysteine. -~-6. ~, The most remarkable gross effect of treatment with I I ~ 4o ',: buthionine sulfoximine was that the skeletal muscles of mice treated for 2-3 weeks showed white patches, mainly on the anterior and lateral sides of the legs, o 200I ~ buttocks, and back and on the lateral side of the CO Of ~ ~ I , I , i trunk. Light microscopic studies showed myofiber I00 200 500 400 500 600 700 necrosis and electron microscopy showed evidence Hours of mitochondrial swelling and vacuolization with FIG. 8. Effect of treatment with L-buthionine-SR-sulfox- rupture of cristae and mitochondrial membrane imine (BSO) on heart (solid symbols) and skeletal muscle (open symbols). Mice were injected intraperitoneally with disintegration. Such effects were not found in the buthionine sulfoximine twice daily and were given buthion- heart. The skeletal muscle damage seems to be reversine sulfoximine in the drinking water. Control (untreated ible since after discontinuation of buthionine sulfoximice) values of GSH were 1.15+0.071 (heart) and mine treatment for several weeks muscle damage was 0.77 + 0.053 (skeletal muscle) #mol/g. After 6 hr, 1 day, 7 not found. Mitochondria isolated from buthionine days, 14 days, and 21 days of treatment, the GSH values (Mean __+SD; n = 3-5) for heart were, respectively suifoximine treated muscle exhibited decreased citrate 0.65 ___0.051, 0.32 + 0.025, 0.11 + 0.015, 0.092 ___0.017, and synthase activity. The mitochondrial level of GSH 0.084 + 0.005. The corresponding values for skeletal muscle was decreased to about 21% of the control in skeletal were 0.51 __+0.06, 0.32 + 0.027, 0.069 __+0.015, 0.054 -I-0.004, muscle and to about 45% of the control in the heart and 0.023 + 0.003 (inset). Logarithmic plot of the data. (Table 1). When the animals were treated with GSH Reprinted from MS.rtensson and Meister, 1989. monoisopropyl ester together with buthionine sulfoximine, the levels of GSH found in the mitobuthionine sulfoximine is rapidly cleared, frequent chondria were the same as the controls in skeletal doses are necessary. muscle and somewhat higher than controls in heart mitochondria. Most interestingly, the muscles of mice treated with GSH monoester and buthionine sulfoxi4.4.2. Glutathione Deficiency in Mice mine did not show mitochondrial destruction. This approach was first examined in adult mice, Skeletal muscle degeneration associated with adminwhich were injected intraperitoneally with buthionine istration of buthionine sulfoximine was not prevented sulfoximine twice daily (dose, 2mmol/kg), and by simultaneous administration of GSH plus isobuthionine sulfoximine (20 mM) was added to the propanol. drinking water (M~rtensson and Meister, 1989). This These studies show that in the absence of applied treatment led to biphasic decline of GSH levels in the stress very marked depletion of GSH is required skeletal muscle and heart (Fig. 8). After 500 hr, before skeletal muscle mitochondrial damage occurs, skeletal muscle and heart had GSH levels that were, and that as little as 8% of the control level of total i

i

i

i

140

°I

TABLE1. Effect of Buthionine Sulfoximine (BSO) and GSH Monoester on GSH Levels of Skeletal Muscle and Heart * Skeletal Muscle Treatment None (Controls) BSO BSO + GSH + isopropanol BSO + GSH-isopropyl ester

Heart

Total ~mol/g)

Mitochondria (nmol/mg of protein)

Total (umol/g)

Mitochondria (nmol/mg of protein)

0.77 +__0.053 0.064 __+0.01 0.08 ___0.01 0.15 ___0.02

5.7 + 0.3 1.2 + 0.2 1.5 + 0.2 5.2 _.+0.4

I. 15 + 0.07 0.10 _+0.01 0.12 + 0.01 0.30 + 0.20

11.5 ___0.4 5.0 _ 0.5 7.0 ___0.6 17.4 + 0.9

*The mice were treated with BSO, GSH monoisopropyl ester, GSH, and isopropanol for 9 days as indicated. Reprinted from M~rtensson and Meister (1989).

168

A. MEISTER

TABLE2. Effect o f Administration o f Buthionine Sulfoximine (BSO), GSH Monoester and o f GSH on GSH Levels GSH levels Liver

Exp. 1 2 3 4

Lung

Treatment*

Total (pmol/g)

Mitochondrial (nmol/mg of protein)

Total ~mol/g)

None BSO BSO + GSH monoester BSO + GSH

8.26 0.98 8.29 1.10

6.4 3.8 10.8 3.9

1.69 0.36 0.71 0.42

Mitochondrial Lymphocyte (nmol/mg of total (nmol Plasma total protein) per l06 cells) (,UM) 4.7 0.86 I0.0 0.87

1.39 0.20 0.45 0.24

58.3 0.90 43.0 5080t

*Mice were treated with saline (Exp. 1) and with BSO (Exps 2~4) for 9 days. BSO was given intraperitoneally (4 mmol/kg) at 10 a.m. and 6 p.m. In Exp. 3, GSH monoisopropyl ester (5 mmol/kg; 1 1.3 ml) was injected intraperitoneally as an isosmolar solution (pH 6.5-6.8) twice daily (8 a.m. and 4 p.m.) for 9 days. In Exp. 4, mice were injected with GSH, isopropanol, and Na2SO4 in amounts equimolar to the ester given in Exp. 3. Mice (4-5 per experiment) were sacrificed at 11 a.m. on day 9. Data are given as means; SDs were +0.0443.08 for tissues and 0.2~480 for plasma. tThe GSH levels in plasma (from right ventricle) were 25,100 and 12,080/~M at 50 min and 120 min, respectively, after GSH administration. Reprinted from M~rtensson et al., 1989a. tissue GSH is apparently sufficient to protect heart mitochondria. The substantial increase of the GSH levels of mitochondria isolated from skeletal muscle and heart observed after administration of GSH monoester suggest that this compound may be useful for protection of heart and skeletal muscle against toxicity. Buthionine sulfoximine was administered to mice in a similar protocol in studies in which the levels of GSH in the lung, lymphocytes, liver, and plasma were determined (M~rtensson et al., 1989a, b). The initial t~ 2 values for the rate of GSH decline were about 25 and 45 min for lymphocytes and lung, respectively, as compared to 5.5 hr and 16 hr for heart and skeletal muscle, respectively. The decline in the plasma GSH level was parallel to that of the liver (t~/2 ~ 65 min). Treatment of mice with buthionine sulfoximine for 9 days led to substantial decline in the GSH levels of liver, lung, lymphocytes, and plasma (Table 2). The mitochondrial GSH level of the liver decreased to about 60% of the controls while that of the lung was decreased to about 18% of the controls. The level of GSH in the lymphocytes was about 14% of the controls. When mice were treated with GSH monoester together with buthionine sulfoximine, the total tissue levels of GSH were substantially higher than found after giving buthionine sulfoximine alone (Table 2). Notably, the GSH levels of liver mitochondria were significantly higher than the controls, and in the lung the mitochondrial GSH level was more than twice that of the controls. Electron microscopic studies of the liver and kidney after 3 weeks of treatment with buthionine sulfoximine showed no abnormalities. However, in type 2 cells there was a significant decrease in the number of mitochondria and marked mitochondrial swelling and degeneration. The lamellar bodies became larger and their characteristic lattice structure was disrupted (Fig. 9). There was a decrease in the number of microvilli on the alveolar surface of the type 2 cells, and swelling of mitochondria of lung capillary endothelial cells. Blunting of the microvilli of the lymphocytes was seen. These morphological changes were not found in

mice treated with buthionine sulfoximine and with GSH monoester. They were found in mice treated with buthionine sulfoximine and GSH. Coursin and Cihla (1988), who gave lower doses of buthionine sulfoximine, found substantial depletion of GSH in the lung without apparent structural change or increased susceptibility to lethal hyperoxia. It is notable that in these experiments administration of GSH intraperitoneally led to an enormous increase in the plasma level of GSH (Table 2). Thus, the plasma level of GSH after giving buthionine sulfoximine was 0.9#M (as compared to control values of 58.3 pM). After treatment with buthionine sulfoximine and GSH (5 mmol/kg twice daily), the level of plasma GSH arose to 25,100 pM, 12,080/tM, and 5,080 #M after 50, 120, and 180 min, respectively. Even with these very high levels of plasma GSH, there were no significant changes in the GSH levels of the mitochondria or the total tissue values of GSH. Such high plasma levels of GSH did not prevent the structural damage described above. In contrast, administration of the same dose of GSH monoester led to essentially normal plasma levels of GSH, to increased levels of GSH in the mitochondria and cytosol and prevented morphological damage completely. Mice given buthionine sulfoximine under the conditions indicated above did not gain weight; the controls gained an average of about 3 g during a 9-day experimental period (M~rtensson et al., 1989a, 1990a). When GSH monoester was given together with buthionine sulfoximine the animals gained about 80% of the weight gained by controls. Administration of GSH together with buthionine sulfoximine had a smaller effect on weight gain. That the mice developed diarrhea suggested study of the levels of the GSH in the gastrointestinal tract, which is known to metabolize GSH actively (Cornell and Meister, 1976). Administration of buthionine sulfoximine led to rapid decline of the GSH levels of several tissues of the digestive tract. The estimated tl/2 values for the initial rates of decline of the GSH levels were 35, 40, 40, and 30min, respectively, for jejunal

Glutathione deficiency

169

FIG. 9. Representative electron micrographs of the lungs of control mice (left) and mice treated with buthionine sulfoximine for 21 days (right); magnification 17,800. Reprinted from M~trtenssonet al., 1989.

mucosa, colon mucosa, gastric mucosa and pancreas. Depletion of GSH was significantly slower in gastric mucosa than in the other tissues examined and the rate of increase of GSH levels after discontinuation of buthionine sulfoximine treatment was greatest in the gastric mucosa. After treatment with buthionine sulfoximine for 7 days, electron microscopy showed substantial loss of the height of the epithelial cell layers in the jejunal mucosa and colon mucosa. There was marked mucosal damage with microvillus degeneration, mito.WI" 51/2--B

chondrial swelling, mitochondrial degeneration and vacuolization. Such changes were not found when the mice were treated with both buthionine sulfoximine and GSH monoester. Protection against structural damage was found when GSH monoester was given intraperitoneally and also when it was given orally. (GSH monoester is taken up intact after oral administration (Anderson et al., 1985).) In contrast, treatment with buthionine sulfoximine plus GSH intraperitoneally led to electron microscopic findings that were essentially the same as found after giving

170

A. MEISTER

buthionine sulfoximine alone. Treatment with buthionine sulfoximine plus oral GSH led to findings similar to those observed after treatment with buthionine sulfoximine and glutathione monoester, i.e. almost complete absence of the morphological changes. Administration of GSH monoester increased the GSH levels of jejunal, colon and gastric mucosa when given orally or intraperitoneally. Administration of GSH intraperitoneally increased gastric mucosa GSH levels to some extent but had no such effect on the other tissue levels of GSH. Oral administration of GSH was followed by increased GSH levels in jejunum, colon, and stomach mucosa, but less than that found after administration of GSH ester.

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4.4.3. Glutathione Deficiency in Newborn Rats and Mice Calvin et al. (1986), who gave buthionine sulfoximine to mice (9-12 days of age) as part of an investigation of GSH metabolism in testes, observed that they developed cataracts in association with markedly decreased levels of GSH in the lens. Many earlier studies suggested that there is a relationship between decreased levels of lens GSH and formation of cataracts (see, e.g. Bellows and Shoch, 1950; Kinsey and Merriam, 1950). Although it is now evident that the effect of buthionine sulfoximine is mediated through GSH deficiency, it was initially conceivable that another mechanism might be involved. When newborn mice and rats were given buthionine sulfoximine soon after birth, they were found to have cataracts when they opened their eyes about 12-16 days later (M~rtensson et al., 1989b). No cataracts were found after treating mice older than 14 days. This seems to be related to the development of a blood-lens barrier. When pregnant mice were given buthionine sulfoximine at different periods during gestation cataracts were found in the offspring only after buthionine sulfoximine was given on days 15-19 of gestation, the period that corresponds to one in which there is fetal eye development. No cataracts occurred in the treated mothers. In the studies described in Fig. 10, newborn rats were injected with buthionine sulfoximine on the second and third days of life, and the levels of lens GSH were determined. (Rats injected with buthionine sulfoximine on the first day of life exhibit about 50% mortality; see below.) In the controls, the level of GSH decreased on the first day of life and then increased reaching a maximum at about 4 days; there was then a decrease to levels similar to those found in adults. Treatment with buthionine sulfoximine led to a marked decrease in the level of GSH. This low level persisted for several days and then increased. Administration of GSH together with buthionine sulfoximine did not affect the levels of GSH in the lens significantly whereas treatment with buthionine sulfoximine plus GSH monoester led to 3-fold higher

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GSH monoester on lens GSH levels. Newborn rats (6g) were injected intraperitoneally with saline (controls) or with BSO (3 mmol/kg) 26 and 50 hr after birth (arrows) and, as indicated, twice daily (10a.m. and 10p.m.) with GSH (isosmolar, adjusted to pH 6.8 with NaOH; 5 mmol/kg) plus isopropanol at 5 mmol/kg and Na2SO4 at 2.5 mmol/kg, or with GSH monoisopropyl ester.I/2 (H2SO4) (isosmolar, adjusted to pH 6.8 with NaOH; 5 mmol/kg). Rats were sacrificed at the times indicated and their lenses were analyzed for GSH. The lenses of controls weighed 3.5, 6.0, 8.6, and 14rag, at days 0, 4, 8, and 14 respectively. The corresponding weights were 3.5, 4.5, 5.0-5.2 and 8.5-9.5 mg for the BSO (and BSO plus GSH) -treated rats, and 3.5, 5.8, 8.3, and 14 mg for the BSO plus GSH monoester-treated rats. Data are given as means + SD (error bars) (n = 3-5). The number of cataracts found when rats treated in the same way spontaneously opened their eyes is recorded in Table 3. The controls and rats treated with BSO and GSH monoester opened their eyes on day 14; the other rats opened their eyes on day 15. Reprinted from M~rtensson et al., 1989b. levels of GSH on days 3~-6. The lens GSH level reached the control level by day 14 when the mice opened their eyes. All of the mice treated with buthionine sulfoximine, as well as those treated with buthionine sulfoximine and GSH, developed cataracts, whereas none of the control rats or those treated with buthionine sulfoximine plus GSH monoester developed cataracts (Table 3). It is of interest that when the rats were given only a single dose of buthionine sulfoximine, 50-70% of them developed cataracts. When the dose of GSH monoester was reduced by 50%, about half of the rats developed cataracts. Thus, the dose of buthionine sulfoximine given in these experiments is close to that minimally required to produce cataracts, and the dose of GSH ester required to protect against cataracts is also close to that minimally required. Lens mitochondrial GSH decreased after administration of buthionine sulfoximine to about 21% of

Glutathione deficiency

171

TABLE3. Prevention of Buthionine Sulf oximine (BSO )-induced Cataracts by G S H Monoester Exp. 1 2 3 4

Treatment

No. of cataracts/ no. of rats

Total lens GSH* (~mol/g)

Controls BSO BSO + GSH BSO + GSH monoester

0/31 20/20, 1l/l 1,, 11/11t 11/11 0/20

9.50 _ 0.50 (100) 0.30 + 0.03 (3) 0.33 +__0.04 (3) 0.70 _ 0.05 (7)

Mitochondrial GSH* (nmol per mg of protein) 11.0 + 0.7 (100) 2.3 + 0.3 (21) 2.4 + 0.3 (22) 5.1 _ 0.3 (46)

Newborn rats were injected intraperitoneally with two daily doses of saline (n = 20) or Na2SO4 (2.5 #mol/kg) (n = 11) (controls; Exp. l), or with BSO (Exps 2-4) as described in Fig. 10. The rats were also injected twice daily with GSH (Exp. 3) or with GSH monoisopropyl ester (Exp. 4) as described in Fig. I0. The number of rats with cataracts (in all cases, bilateral) is given. *Values are given as means _% . _SD (n = 3). Values in parentheses are percent control value. tAlso treated with two daily doses of Na2SO4 (2.5 mmol/kg) and with isopropanol or ethanol (5 mmol/kg). Reprinted from M~.rtensson et al., 1989a.

the controls, and a similar value was found after treatment with buthionine sulfoximine and GSH. Treatment with buthionine sulfoximine and GSH monoester led to a lens mitochondrial GSH level that was about 46% of the controls. Electron microscopy of the lenses of newborn rats and mice after treatment with buthionine sulfoximine showed dramatic changes in the epithelial cells including formation of many vacuoles with apparent loss of cytoplasm and mitochondrial swelling and degeneration. There was also increased density of the chromatin pattern of the nuclei with indentation and shrinkage. These changes were not found after treatment with buthionine sulfoximine and GSH monoester, but they were found after treatment with buthionine sulfoximine and GSH (M~rtensson et al., 1989a). Fig. 11 shows electron microscopy of the lens epithelial zone of a newborn mouse whose mother was treated with buthionine sulfoximine. Administration of buthionine sulfoximine to adult mice or rats produces only a relatively small decrease in the GSH level of brain; no damage to brain was detected by electron microscopy. Presumably the brain is protected by the blood-brain barrier and indeed analyses of brain show that buthionine sulfoximine is not effectively transported into the brain of adult animals. However, administration of buthionine sulfoximine leads to a substantial decrease in the GSH levels of the brains of newborn mice, an effect consistent with an undeveloped blood-brain barrier (Slivka et al., 1988). The turnover of brain GSH has generally been thought to be relatively slow; thus, a t-,2 value of 70 hr was estimated for rat brain (Douglas and Mortensen, 1956). Because buthionine sulfoximine is poorly transported into the brain, this approach is less useful for estimating GSH turnover rates than it is for other tissues. However, buthionine sulfoximine ethyl ester is readily transported into the brain and studies with this compound provided evidence for a fraction of cerebral cortex GSH that turns over rapidly in adult mice (Steinherz et al., 1990). Buthionine sulfoximine may also be given to adult rats in 15% dimethylsulfoxide which increases buthionine sulfoximine uptake by brain (Steinherz et al., 1990). Another procedure, pioneered by Pileblad and Magnusson (Pileblad and Magnusson, 1988;

Pileblad et al., 1989), involves administration of buthionine sulfoximine directly to the brain by means of an indwelling catheter implanted in the lateral ventricles. These approaches have led to substantial decrease of brain GSH levels in adult animals. Treatment of newborn rats with buthionine sulfoximine leads to rapid initial decline in the cerebral cortex GSH level followed by slower rates of decrease; t½values of 8 min, 60 hr and 160 days were estimated (Fig. 12; Jain et al., 1991). The level of cortex GSH increases with age in newborn rats. The effects of giving buthionine sulfoximine to newborn rats on the GSH levels of cerebral cortex and on the levels of GSH in the mitochondrial fraction were studied. After 3 days, the GSH level of the cerebral cortex decreased to about 19% of that of untreated controls and the mitochondrial GSH decreased to about 40% of controls (Table 4). When GSH was administered with buthionine sulfoximine there was no significant difference between the levels of cortex GSH or of the mitochondrial fraction as compared with the values obtained on rats given buthionine sulfoximine alone, However, when GSH monoester was given together with buthionine sulfoximine, the levels of GSH in the cortex and in the mitochondrial fraction were substantially higher than found after giving buthionine sulfoximine alone. When treatment with buthionine sulfoximine was continued for 9 days, the cortex GSH level declined to 13% of controls and the mitochondrial fraction GSH was 16% of controls. These values were within experimental error the same when GSH was given together with buthionine sulfoximine. However, when GSH monoester was given together with buthionine sulfoximine, the GSH level of the cortex was much higher and the level of GSH in the mitochondrial fraction was about 6 times higher than that found after treatment with buthionine sulfoximine alone; this value was about the same as that of untreated controls. These changes in GSH levels were accompanied by striking electron microscopic findings (Fig. 13). Sections of cerebral cortex from buthionine sulfoximine-treated rats showed relatively few mitochondria; those present were strikingly enlarged as compared with control mitochondria. The remarkable mitochondrial swelling and degeneration seen

A. MEISTER

172

FIG. 11. Representative electron micrographs of the epithelial zone of the lens of a control mouse 6 hr after birth (left) and an age-matched mouse from a mother treated with buthionine sulfoximine (right); Note marked mitochondrial swelling and degeneration with vacuole formation. Reprinted from MArtensson et al.. 1989a.

after treatment with buthionine sulfoximine were also found in rats treated with both buthionine sulfoximine and GSH. However, sections of cerebral cortex obtained from newborn rats treated with buthionine sulfoximine plus GSH monoester showed mitochondria that were indistinguishable from controls. 4.4.4.

Conclusions

These studies show that a major effect of GSH deficiency produced by inhibition of its synthesis is mitochondrial damage. The findings indicate that there is a relationship between the extent of mito-

chondrial GSH depletion and cellular damage as evaluated by citrate synthase determinations and electron microscopy. Degeneration of skeletal muscle was associated with mitochondrial GSH levels of about 20% of the controls. Mitochondrial and lamellar body damage in lung type 2 cells was observed when mitochondrial GSH levels were about 21% of the controls. Jejunal mucosal damage was associated with mitochondrial GSH levels of about 13% of the controls. On the other hand, prolonged treatment with buthionine sulfoximine did not lead to cellular damage in adult mice in the liver and heart which were found to have mitochondrial GSH levels that were greater than about 40% of the controls.

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FzG. 12. Effect of administration of buthionine sulfoximine (BSO) on cerebral cortex GSH levels. Newborn rats (7.5-8.0 g) were injected with BSO (2 mmol/kg) i.p. twice daily at 9 a.m, and 9 p.m. Preweaning rats (34--36 g) were given two daily doses of BSO (3 mmol/kg). Adult rats (400-500 g) were given two daily doses of BSO of 4 mmol/kg in 15% dimethyl sulfoxide. GSH levels were determined in the cerebral cortex initially and after BSO injection, Inset: Values after a single dose of BSO (2-4 mmol/kg); adult rats were injected with BSO in 15% dimethylsulfoxide. Control values were 1.36 _ 0.08, 1.27 _.+0.07, and 1.22 +_0.5/LM for newborn, preweaning, and adult rats, respectively. All determinations were done in duplicate on samples from three to six animals. Values are means _+SD. Reprinted from Jain et al., 1991.

173

The kidneys of adult mice were also essentially unaffected by this degree of G S H deficiency. The formation of cataracts in newborn rats occurred with lens mitochondrial G S H levels that were about 20% of the controls; treatment with G S H ester, which prevented cataract formation, led to lens mitochondrial G S H levels of 46%. On the basis of the data that have thus far been collected it would seem that mitochondrial G S H levels can be decreased to approximately half of the control levels without serious effect, but that decrease to much lower values of G S H is associated with mitochondrial damage. Mitochondrial damage would be expected to decrease the available cellular energy for many other functions including synthesis of macromolecules, for example. It is of interest that G S H deficiency leads to a significant decrease in lens mass in newborn rats (Fig. 10, legend). Although it appears that mitochondrial damage is an important consequence of G S H deficiency induced by administration of buthionine sulfoximine, other types of cellular damage were seen by electron microscopy including nuclear effects and, in the lungs, an effect on lamellar bodies. It is emphasized that these effects occurred without application of external stress such as increased oxygen, drugs, or radiation. Furthermore, they were completely prevented by administration of G S H monoesters. It is now known (see Section 4.3) that mitochondria obtain G S H by transport from the cytosol. N o t all of the oxygen utilized by mitochondria is reduced to water; a significant fraction of it (perhaps 2 - 5 % ) is converted, apparently through superoxide, to hydrogen peroxide (Loschen and Floh6, 1971; Boveris et al., 1972; Boveris and Chance, 1973; F o r m a n and

TABLE4. Effects of Buthionine Sulfoximine (BSO), GSH, and GSH Ester on GSH Levels of Cerebral Cortex of Newborn Rats GSH

Treatment 3 days None (controls) BSO BSO + GSH BSO + GSH ester 9 days None (controls) BSO BSO + GSH BSO + GSH ester

Experiment

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Mitochondria, nmol/mg of protein

1 2 3 4

1.01 __+0.05 0.19+0.01 0.22 + 0.01 0.31 +__0.01

12.2 _ 0.75 4.92+0,22 4.83 -I- 0.08 8.78 -t-0,41

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1.12+__0.11 0.13-t-0.01 0.14+__0.01 0.38 + 0.02

8.16+0.83 1.29+0.11 1.22+0.09 7.55 + 0.68

Reprinted from Jain et al., 1991. GSH levels are given as means_ SD (n = 3-4). Newborn rats (36-48 hr of age) were injected i.p. with saline or BSO (2 mmol/kg) twice daily for 3 or 9 days at 9 a.m. and 9 p.m. In Exps 4 and 8, the rats were also given (twice daily at 11 a.m. and 11 p.m.) GSH monoethyl ester (2.5 mmol/kg). In Exps 3 and 7, the rats were also given an isosmolar (pH 6.5~.8) solution containing GSH (2.5mmol/kg), ethanol, and Na2SO 4 (in amounts equivalent to those of GSH ester given in Exps 4 and 8). Equal volumes of saline were given in Exps 2 and 6 to match those given in Exps 3, 4, 7, and 8.

174

A. ME1STER

FIG. 13. Mitochondrial damage in cerebral cortex of newborn rats after administration of buthionine sulfoximine (BSO). The rats were given saline (a), BSO (b), BSO + G S H (c), or BSO + G S H ester (d) from 48 hr to 11 days o f age. In b and c there is marked mitochondrial swelling and degeneration (see the text) that is not seen in controls (a) or G S H ester-treated animals (d). In a~l, two representative mitochondria are indicated by arrows. ( x 6600; bar in lower left-hand corner = 0.37 pm.) Reprinted from Jain et al.~ 1991.

Glutathione deficiency Boveris, 1982). The accumulation of hydrogen peroxide, or of closely related species, produces extensive mitochondrial damage when GSH levels are greatly decreased. Mitochondria continually produce reactive oxygen species but contain no catalase; they are therefore largely, if not entirely, dependent upon GSH and GSH peroxidases. It is possible that other antioxidants are involved in the protection of mitochondria, but it would appear that under normal physiological conditions GSH is the principal functional antioxidant. It not only reacts with reactive oxygen species but also functions in the maintenance in the reduced state of other protective antioxidants such as ascorbate and ct-tocopherol. Recent studies have provided additional evidence that the newborn rat is highly sensitive to the effects of GSH deficiency. In contrast to the results observed in adult mice, treatment of newborn rats with buthionine sulfoximine leads to severe kidney and liver damage (MS.rtensson and Meister, 1991). Other tissues are also affected and the animals exhibit a high mortality rate. Mortality is decreased when GSH monoester is given together with buthionine sulfoximine. It is notable that administration of ascorbate in high doses (2 mmol/kg) also decreases mortality (as well as formation of cataracts) in newborn rats. The protective effect of ascorbate observed in the presence of marked GSH deficiency is consistent with the conclusion that ascorbate and GSH share common actions in the destruction of reactive oxygen intermediates. Thus, both ascorbate and GSH react effectively with hydrogen peroxide. The possibility that buthionine sulfoximine has a toxic effect on cells separate from, and in addition to, its effect on ),-glutamylcysteine synthetase needs to be considered. The experimental results indicate that such a hypothetical effect would apparently be prevented by GSH. Reports describing 'toxicity' of buthionine sulfoximine have appeared (see, for example, Dethlefsen et al., 1986), and in earlier work (Dethmers and Meister, 1981) decreased viability of cells was observed in cells that were markedly depleted of GSH by treatment with buthionine sulfoximine. Such 'toxic' effects may probably be ascribed to the effects of GSH depletion. Nevertheless, the possibility that buthionine sulfoximine has an additional toxic effect needs to be borne in mind. It is relevant to note that a closely related structure, buthionine sulfone, which does not inactivate ),-glutamylcysteine synthetase, did not produce cataracts or other structural damage in mice (M~rtensson and Meister, 1989; M~rtensson et al., 1989a, 1990a, b). Several studies have been carried out with L-buthionine-R-sulfoximine, which does not inhibit GSH synthesis (see Section 4.2.2); this isomer does not exhibit the effects described above which are found after giving L-buthionine SR-sulfoximine. The data (Tables 1-4) are in accord with the earlier findings which indicate that there is no appreciable transport of intact GSH into cells (Section 3). Thus,

175

the GSH values found after giving buthionine sulfoximine and GSH are, within experimental error, not different from those found after giving buthionine sulfoximine alone. The very slight differences found do not appear to be significant, and may be ascribed to technical phenomena such as incomplete removal of extraceUular GSH during preparation of the tissues. The findings suggest that if transport of intact GSH occurs under these conditions, its extent is well within the experimental error of the determinations.

5. G L U T A T H I O N E DEPLETION A N D T U M O R THERAPY

5.1, GENERAL(ANDBRIEFHISTORICAL) CONSIDERATIONS The general concept that GSH protects cells against heavy metal ions, radiation, oxidation, and various toxic compounds began with the early studies of Hopkins (Hopkins, 1921; Hopkins and Morgan, 1936, 1938), and was further developed by GuzmanBarron (1951) and later workers. Lipke and Kearns (1959) found that houseflies that had become resistant to DDT (dichlorodiphenyltrichloroethane) develop a GSH-dependent mechanism for the dehydrochlorination of this insecticide. In this way DDT is converted to a relatively harmless hydroxy derivative; as discussed below, analogous phenomena have turned out to be of significance in relation to certain drug resistant tumors. Hirono (1961) found that tumor cells that were resistant to alkylating agents had increased levels of nonprotein thiols as compared to cells that were sensitive to alkylating agents. Increased levels of nonprotein thiols, later recognized to be GSH, were also found in other resistant tumor cells (Calcutt and Connors, 1963; Ball et al., 1966). In one study, the increase in GSH content of the resistant tumor was not considered to be highly significant, but it was the only difference that could be demonstrated (Ball et al., 1966). In another study, the question was raised as to how the increased concentration of thiol might account for the large degree of resistance; at the time, GSH was not considered to be particularly reactive toward alkylating agents. Therapy based on application of thiol protection was considered, but it was felt that this approach would not lead to a gain in selectivity (Connors, 1966). Later studies on the effects of phenylalanine mustard on resistant and sensitive leukemia cells led to the finding that the doses of phenylalanine mustard required to kill the cells varied considerably and that this was related to the cellular GSH levels (Vistica, 1979). Growth of the tumors in media containing decreased levels of cystine led to lower cellular GSH levels and to increased sensitivity to phenylalanine mustard (Suzukake et al., 1982). After development of the sulfoximine inhibitors of ~-glutamylcysteine synthetase (Griflith et al., 1979; Meister, 1978a), it was suggested that treatment with

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FIG. 14. Effect of inhibition of GSH synthesis by buthionine sulfoximine (BSO) on the intracellular GSH concentration. Cell suspensions (106 per ml) of the CEM (closed circles), 8226 (open squares), and HSB (open circles) lines were grown in standard medium containing 1 mM buthionine sulfoximine. Intracellular GSH was determined in samples removed at various intervals. The ordinate is logarithmic. The data given in the Inset were obtained with CEM cells. Reprinted from Dethmers and Meister, 1981.

these agents might make tumor cells more susceptible to certain anticancer agents and to radiation (Meister and Griffith, 1979). Administration of such an inhibitor might well be expected to decrease the GSH levels of normal tissue as well as those of tumors, and it might reasonably be questioned whether such generalized depletion of tissue GSH would be useful. Nevertheless, depletion of GSH might be therapeutically effective if the normal and tumor cells exhibit markedly different requirements for GSH. A tumor cell that has an increased requirement for GSH (possibly indicated by a higher level of cellular GSH), or a drug-resistant tumor cell which has substantially increased GSH levels might be expected to be more sensitive to GSH depletion than normal cells, which generally have a large excess of GSH. Thus, depletion of GSH by treatment of a tumor-bearing host with buthionine sulfoximine would decrease GSH synthesis in the tumor as well as in many normal cells of the host, but this might not have a significant effect on the functions of the normal cells. On the other hand, GSH depletion would place those cells with increased GSH requirements (tumor cells) at a disadvantage since they would be sensitized to a chemotherapeutic agent or to radiation. The potential usefulness of buthionine sulfoximine in the sensitization of cells to radiation was first demonstrated in studies on three human lymphoid cell lines (Dethmers and Meister, 1981). The cells were incubated in media containing buthionine sulfoximine and the GSH levels were found to decrease progressively over a period of 50hr (Fig. 14). Although cells with a level of 0.09 mM GSH (4% of the controls) were 85% viable, further decrease in

GSH level was associated with marked loss of viability. Cells that had 4-5% of the control levels of GSH were found to be much more sensitive, than were control cells, to the effects of v-irradiation. Studies by Clement et al. in 1981 (see Griffith, 1985; Meister, 1986) showed that treatment of mice bearing B16 melanomas with buthionine sulfoximine sensitizes this relatively radio-resistant tumor to radiation. In these studies, tumors were implanted in the footpads of mice and allowed to grow to a size of 250 nm 3. The mice were then treated with buthionine sulfoximine (DL-form; 8 mmol/kg) intraperitoneally. The sulfoximine was given every 4 hr for 3 injections and the tumors were irradiated 4 hr after the last dose of buthionine sulfoximine. At the time of radiation, the GSH content of the tumors was about 20% of that of untreated controls. There was a significant decrease in the size of the tumor and a significant increase in the longevity of the mice bearing the tumor. In the tumor-bearing mice that were given no therapy, or radiation therapy alone, much smaller effects were observed. These results are of interest because the tumor cells are resistant initially and the findings indicate that buthionine sulfoximine produced a significant sensitization to radiation.

5.2.

SENSITIZATION

AND

RADIATION

OF BY

TUMORS

TREATMENT

TO

CHEMOTHERAPY

WITH

BUTHIONINE

SULFOXIM1NE

In the mustard leukemia the most

studies on the toxicity of phenylalanine toward resistant and sensitive mouse cells, the dose of mustard required to kill resistant cells was much higher than that

Glutathione deficiency required to kill the most sensitive tumor cells (Vistica, 1979; Vistica and Ahmad, 1989). Resistance was not related to differences in uptake or efflux of the drug, but appeared to be correlated with the cellular level of GSH. Resistant cells were found to convert phenylalanine mustard to a nontoxic derivative in a GSH-dependent dehydrochiorination reaction. Of considerable importance, it was found that treatment of the resistant cells with buthionine sulfoximine led to resensitization of the tumor cells to phenylalanine mustard. With mice bearing resistant tumors, sensitization was achieved by continuous intraperitoneal infusion of buthionine sulfoximine. This led to an increase in the life span of the treated animals (Kramer et al., 1987; Ahmad et al., 1986, 1987; Suzukake et al., 1982, 1983; Vistica, 1983; Vistica and Ahmad, 1989). Ozols and collaborators (Hamilton et al., 1984, 1985; Ozols et al., 1986, 1987; Green et al., 1984) examined the relationship between GSH levels and the expression of both primary drug resistance and cross-resistance in human ovarian cancer cell lines as well as in an intraperitoneal model of human ovarian cancer in nude mice. These studies have been of particular interest and importance because they have led to a clinical trial of buthonine sulfoximine, which is now in progress. In the model investigations it was demonstrated that drug resistance can be reversed by depletion of GSH by use of buthionine sulfoximine. Of further interest it was found that resistance of these tumors to phenylalanine mustard is accompanied by resistance to other drugs such as adriamycin. In addition, the phenylalanine mustard resistant cells are also resistant to radiation. Depletion of GSH significantly increases the sensitivity of the resistant cells to both drugs and radiation; thus, the common denominator that underlies resistance to both modalities is increased cellular GSH. In the protocol used for one of the several current clinical trials, the patients are given 6 doses of buthionine sulfoximine (6.8 mmol/m 2) intravenously over a 3 day period and L-phenylalanine mustard (15 mg/m 2) is given 1 hr after the 5th dose (Hamilton et al., 1990). No toxicity was reported after giving buthionine sulfoximine alone, but some toxicity and neutropenia were seen after giving both drugs or the mustard alone. A significant clinical finding is that GSH levels in peripheral mononuclear cells were decreased to less than 20% of the initial value in all patients by the 5th dose of buthionine sulfoximine. It is thus apparently established that treatment with buthionine sulfoximine can decrease GSH levels, at least in these cells of the patients, with acceptable toxicity. In some instances it has been possible to sensitize tumors to chemotherapy by selectively depleting GSH in the tumor. For example, although normal brain is relatively resistant to the effects of buthionine sulfoximine, disruption of the blood-brain barrier by tumor growth leads to increased turnover of GSH in

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the tumor; as compared to the normal brain tissue, buthionine sulfoximine may enter the tumor and facilitate a selective effect which sensitizes it to the action of anticancer agents (Skapek et al., 1988a, b; Fekete et al., 1990; Lippitz et al., 1990; Friedman et al., 1989). The accumulated findings indicate that increased GSH synthesis is of significance in certain resistant tumor cells (Ozols et al., 1988; Meister, 1988a). However, there are also other cellular mechanisms that can lead to drug resistance. Earlier studies showed that resistance may be associated with decreased net transport of the drug. A mechanism that has attracted considerable attention in recent years links multidrug resistance with overexpression of a novel membrane glycoprotein (Woolley and Tew, 1988). The multidrug resistance phenotype functions to produce a net decrease in accumulation of a number of structurally unrelated drugs. At this time it is not certain as to whether this multidrug resistance pathway is completely separate from that discussed above which is based upon increased tumor cell GSH synthesis. Further studies are needed. It is of interest, however, that radiation resistance is associated with multidrug resistance that depends upon increased synthesis of GSH, whereas the multidrug P-glycoprotein resistance system is apparently not associated with radiation resistance. Relationships between the radiation response of various tumor cells and GSH levels have been extensively examined after treatment with buthionine suifoximine, treatment with diethylmaleate, administration of 2-oxothiazolidine-4-carboxylate, and after giving various radiosensitizing drugs (Nathan et al., 1980; Biaglow et al., 1983; Biaglow and Varnes, 1983; Russo and Mitchell, 1984; Shrieve et al., 1985; Russo et al., 1985; Biaglow et al., 1986; Clark, 1986; Mitchell and Russo, 1987; Mitchell et aL, 1988; Phillips et al., 1989; Kramer et al., 1989; Lehnhert et al., 1990). There is now a very large literature on this subject and also on studies relating to the sensitization of tumor cells to chemotherapy (Russo and Mitchell, 1985; Russo et al., 1986a, b; Crook et al., 1986; Tsutsui et al., 1986; Dusre et al., 1989; Lutzky et al., 1989; Karg et al., 1989; Chresta et al., 1990). Several excellent reviews have appeared (Biaglow et al., 1983; Perez et al., 1990; Doroshow et al., 1990; Bump and Brown, 1990; Mistry and Harrap, 1991). In general, there is much evidence that depletion of cellular GSH leads to sensitization of tumors to chemotherapy and to radiation, and increase of cellular GSH levels often increases resistance to these modalities. Unfortunately, in some studies diethylmaleate and other nonspecific compounds have been used either alone or together with buthionine sulfoximine and this tends to confuse interpretations of the data. Not all of the findings are as clear cut as found in the studies cited above, and factors other than GSH levels must be studied. It is likely that other

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enzymes including those involved in GSH metabolism are of significance. Although a general pattern can be seen in the various published experiments on the effects of GSH modulation on tumor sensitivity and resistance in which increased GSH levels are usually protective and decreased GSH levels usually lead to sensitization, a number of subplots within the general scheme have emerged. It seems probable that GSSG reductase and the GSH peroxidases play a significant role in some of the responses observed and that such effects may vary depending upon the type of therapy applied. For example, there is good evidence that GSH peroxidase is of major significance in the resistance of tumor cells to various anticancer quinones such as doxorubicin and donorubicin (Doroshow et al., 1990). It seems likely that the activities of various GSH S-transferases are involved in resistance to certain anticancer agents (Tew and Clapper, 1988). Another aspect of GSH metabolism that needs further consideration in relation to cancer chemotherapy relates to the GSH thioltransferases (transhydrogenases). These enzymes reversibly reduce the disulfide bonds of proteins, and convert ribonucleotides to deoxyribonucleotides. GSH plays a key role in the generation of intracellular protein thiol groups, and it is of major importance in the reduction of ribonucleotides, which bears a crucial relationship to the synthesis and repair of DNA. GSH also functions to reduce dehydroascorbate to ascorbate (SzentGyorgy, 1928; Hopkins and Morgan, 1936; Borsook et al., 1937; M~rtensson and Meister, 1991; Wells et al., 1990), and it is also involved in the maintenance of ~-tocopherol in the reduced state (Wefers and Sies, 1988; Packer et al., 1979; Niki et al., 1982; Reddy et al., 1982; Doba et al., 1985; Ursini et al., 1982; Leedle and Aust, 1990). It seems of importance in the continuing study of the relationship between GSH and tumor resistance and sensitivity to chemotherapy and radiation that these well known functions of GSH be taken into account in the design of experimental protocols. 5.3. CELLULAR LEVELS OF GSH VERSUS CELLULAR CAPACITY FOR GSH SYNTHESIS AS DETERMINANTS OF RESISTANCE AND SENSITIVITY TO RADIATION AND DRUGS

The studies in which drug resistance was reversed by buthionine sulfoximine-induced GSH depletion can be interpreted to indicate that the degree of usefulness of GSH depletion would depend on whether differential levels of GSH can be achieved in the tumor as compared to normal tissues; thus, it might be desirable to carry out measurements of GSH on particular tumors (Mitchell et al., 1989). However, since many normal tissues have a large excess of GSH, and since certain tumors, especially those that are resistant to radiation and drugs, have GSH levels that are close to those required for

survival, even an equivalent effect of buthionine sulfoximine on the GSH levels of normal and tumor tissues would be expected to favor survival of the normal cell. The cellular level of GSH may determine the degree of drug resistance or radiation resistance of a particular tumor, and therefore analyses of tumors for GSH would provide useful information. However, the capacity of a tumor cell to synthesize GSH may be an important factor in radio-resistance and drugresistance. Thus, the ability of a cell to synthesize GSH relatively rapidly in response to a stress may be as important or even more important than the initial cellular level of GSH. The possible role of such GSH synthesizing capacity in drug and radiation resistance has generally not been given much attention. In some instances cells with high capacity for GSH synthesis may also have high levels of GSH, but this may not always be the case. This may explain some of the data in the literature which do not seem to show a close correlation between cellular GSH levels and resistance or cellular protection. An interesting bacterial model system has led to results that seem relevant to the idea expressed above. Thus, studies on a strain of Escherichia coli, enriched in its content of 7-glutamylcysteine synthetase and GSH synthetase activities by recombinant DNA techniques have suggested a different model for cell protection (Moore et al., 1989). Since mutants of E. coli that are deficient in the activities of either of the two synthetases do not exhibit increased sensitivity to radiation as compared to the wild strain, it may be concluded that the wild strain does not synthesize sufficient GSH to protect it against radiation. The gene-enriched organism was found to be much more resistant to the lethal effects of 7-irradiation that was the corresponding wild strain of origin. Although the gene-enriched strain has higher GSH levels than the wild strain, the observed radio-resistance was found to be associated with the capacity of the gene-enriched strain to synthesize GSH when irradiated rather than to the cellular levels of GSH per se. Thus, resistance was abolished in the gene enriched strain by application of buthionine sulfoximine, which does not act directly to lower cellular GSH levels, but which prevents the ability of the cells to synthesize GSH in response to a challenge. The findings seem relevant to mammalian cell phenomena. Cellular levels of GSH are typically increased (often 1.5 to 2-fold) in drug- and radiationresistant tumor cells. Such increases may reflect enhanced capacity for GSH synthesis, but the capacity itself may be a major determinant of resistance. The relative importance in cellular protection of the levels of GSH as compared to the capacity of GSH synthesis may depend upon the intensity and type of challenge. These considerations suggest the importance, in studies on cellular GSH protection, of determining not only the level of GSH but also the cellular

Glutathione deficiency capacity for GSH synthesis. Such studies seem applicable to therapy of drug- and radiation-resistant tumors and might therefore supplement other approaches, such as direct analytical studies of GSH levels. Cellular capacity of GSH synthesis may be estimated from determinations of the activity of v-glutamylcysteine synthetase, which is usually the rate-limiting step in GSH synthesis; other procedures involve the use of antibodies and determinations of the corresponding mRNA by hybridization with cDNA probes of the enzyme. Lee et al. (1989) have recently reported findings that seem to be consistent with the ideas expressed above. These investigators studied the role of GSH in drug transport in relation to sensitivity to adriamycin in a series of human ovarian cell lines obtained from biopsy samples. They found 3-fold variation in sensitivity to adriamycin which was unrelated to drug transport in terms of both influx and efflux. Furthermore, these cell lines exhibited a wide range of GSH content and there was a poor correlation between drug sensitivity and GSH content. However, when the cells were exposed to adriamycin, the GSH content of cell lines that were sensitive to the drug decreased whereas the GSH content of cell lines that were resistant increased. They estimated the rate of GSH synthesis using [35S]cysteine and [35S]methionine and found that the resistant lines had a higher steady-state rate of GSH synthesis than the sensitive lines. It was concluded that changes in GSH level during treatment may be an important indicator of tumor cell response to adriamycin. Thus their findings indicate that resistant cell lines maintained a high GSH level dunng exposure to adriamycin whereas the sensitive cell lines did not. The basis of resistance would thus seem to be related to increased capacity of the resistant cells for GSH synthesis.

6. THERAPY BASED ON INCREASE OF CELLULAR G L U T A T H I O N E 6.1. GENERALCONSIDERATIONS GSH is not required in the diets of animals. It is synthesized in many types of cells from glutamate, cysteine, and glycine, which are formed as products of cell metabolism and are also obtained from the diet. It has long been known that fasting or restriction of dietary protein decreases tissue GSH levels and that rodents exhibit a diurnal variation in tissue GSH levels that is closely associated with feeding times (Edwards and Westerfield, 1952; Beck et al., 1958; Jaeger et al., 1973; Tateishi et al., 1974, 1977). Cysteine may be obtained from dietary protein and GSH, or from dietary methionine by the transsulfuration pathway (du Vigneaud, 1952), which occurs mainly, but not exclusively, in the liver. The availability of cysteine is often the limiting factor for intracellular GSH synthesis. Therefore, to the extent that the supply of cellular cysteine is less than opti-

179

mal, supplementation with cysteine moieties would be expected to increase GSH levels. The supply of cysteine moieties for GSH synthesis is affected by a number of factors that relate to its transport, extracellular and intracellular breakdown, and its use for protein synthesis. When there is decreased transsulfuration, as may occur due to an inborn error (Mudd et al., 1989), liver disease (Horowitz et al., 1981) or prematurity (Sturman, 1980), cysteine becomes a dietary essential amino acid. Cysteine is atypical among the amino acids in exhibiting a high degree of toxicity. Thus, addition of cysteine to a basal essential amino acid diet for mice led to loss of weight and death (Birnbaum et al., 1957). Oral intake of cysteine was reported to produce necrosis of neurons in infant mouse retina and hypothalamus (Olney et al., 1971). A single injection of cysteine (1.2 mg/g body weight) into 4-day-old rats led to brain atrophy (Karlsen et al., 1981). Addition of cysteine to culture media has been reported to be toxic to cells, and toxicity was reduced by storage of the media or by addition of pyruvate (Nishiuch et al., 1976). Although several plausible mechanisms may explain the toxicity of cysteine (Cooper et al., 1982), the findings suggest that at least some of the toxicity is exerted extracellularly. Thus, administration of 2-oxothiazolidine-4-carboxylate in similar doses is nontoxic; this compound, which contains a masked thiol, is converted to cysteine intracellularly (see below). The level of cysteine within cells is regulated at low levels, for example, 10-100 #M (Anderson and Meister, 1987). Enzymes that are important for the utilization of cysteine exhibit relatively low apparent Km values; thus the Km value for cysteine for human cysteine-tRNA synthetase is equal to or less than 3 #M, and the Km value for cysteine for v-glutamylcysteine synthetase is about 0.35 mM. Fasting or deprivation of protein leads to much lower (by 20-50%) levels of tissue GSH than found in fed animals. It seems plausible that the level of cellular GSH fluctuates between a relatively high, perhaps maximal, value and a somewhat lower one. Possibly, appropriate therapy might lead to cellular levels of GSH that are constantly maximal. It is not yet known what the highest level of cellular GSH can be in vivo. Normally, it is expected that feedback inhibition of 7-glutamylcysteine synthetase will control the upper level of cellular GSH. If this is not limiting, for example, because there is an alternative delivery of GSH to the cell, one might achieve even higher cellular levels. However, such levels might not persist if they are limited by export. In situations in which GSH is consumed by intracellular reactions, for example in detoxication reactions, therapeutic delivery of GSH to the cell might be sufficiently rapid to make up for the constant utilization of GSH. Definite conclusions about 'normal' and 'maximal' cellular GSH levels cannot yet be drawn, and there must be concern about the possibility that very high levels would lead to toxic effects. For example, high

180

A. MEISTER

thiol levels in cell culture media may lead to toxicity due to peroxide formation. There are presently few published data on the toxicity of orally or parenterally administered GSH. It may not be assumed, without appropriate studies, that administration of GSH would not be accompanied by toxicity. This needs to be carefully studied before treatment with substantial doses of GSH is attempted. The situation is complicated by various sparing reactions involving other thiols, ascorbate, and other cellular components. The findings that have thus far been made relate to the effects on cellular GSH levels of administration of cellular cysteine delivery compounds and cellular GSH delivery compounds (Meister, 1985b; Meister et al., 1986). In the effort to increase tissue levels of GSH it is natural to consider the idea of giving GSH orally or parenterally. As mentioned above, there is not convincing evidence that intact GSH is transported into cells (although this has occasionally been claimed); the few exceptions, or possible exceptions, to this statement have been discussed above (Section 3). The fate of intravenously administered GSH was studied in a human (Wendel and Cikryt, 1980): a dose of 100mg of GSH was rapidly eliminated from the plasma (t½ ~ 1.6 rain). Rapid elimination of GSH was also found in studies on the pharmacokinetics of intravenously given GSH in the rat (Ammon et al., 1986). It was concluded that GSH is rapidly oxidized in plasma; in these studies bolus injections of 50-300/~mol/kg were given. As discussed below, GSH, administered orally or parenterally can serve as a source of cysteine moieties and may therefore lead to increases of the GSH levels of tissues by providing more substrate for GSH synthesis.

acid N-acylase activity (Birnbaum et al., 1952). The proposed mucolytic effect of N-acetylcysteine, which involves reductive cleavage of disulfides between glycopeptides leading to decreased viscosity of bronchial secretions (Sheffner et al., 1964), apparently needs further study (Cotgreave et al., 1987b), as does the conclusion that it is clinically effective (MacFarlane and Prescott, 1985). Following administration of N-acetyl[]4C]cysteine into the ileum, small quantities of labeled N-acetylcysteine and its disulfide were found in hepatic portal vein plasma (Cotgreave et al., 1987a). The major metabolites were found to be cysteine and sulfite which were present in the portal vein plasma at levels several times higher than that of N-acetylcysteine, 30 min after injection; GSH was a minor metabolite. There is convincing evidence that administration of N-acetylcysteine leads to increased liver GSH levels, especially after administration of toxic compounds such as acetaminophen (see for example Thor et al., 1979; Williamson et al., 1982; Miners et al., 1984; Wong et al., 1986; see Fig. 15). Although N-acetylcysteine is a standard remedy for acetaminophen intoxication, more effective compounds exist (see Figs 15 and 16). Thiazolidines have been considered as possible delivery compounds for cysteine. L-Thiazolidine-4carboxylic acid, formed by reaction of formaldehyde with L-cysteine (Schubert, 1936; Ratner and Clarke,

'r

200([) .-I ,