Inevitable glutathione, then and now

Transcription

1 Indian Journal of Experimental Biology Vol. 40, June 2002, pp Inevitable glutathione, then and now S v S Rana, Tanu Allen & Rajul Singh Toxicology Laboratory, Department o f Zoology, Ch. Charan Singh University., Meerut , India Tel: , Fax: Glutathione a predominant tripeptide thiol compound of many prokaryotes and eukaryotes, is synthesized from its precursor amino acids ego y-glutarnate, cysteine and glycine. It is mainly involved in detoxication mechanisms through conjugation reactions. Other functions include thiol transfer, destruction of free radicals and metabolism of various exogenous and endogenous compounds. It becomes mandatory for a cell to manage high concentration of intracellular GSH to protect itself from chemical/dug abuse. Glutathione dependent enzymes viz: glutathione-s-transferases, glutathione peroxidase, glutathione reductase and y-glutamate transpeptidase facilitate protective manifestations. Liver serves as a glutathionegenerating factor which supplies the kidney and intestine with other constituents of glutathione resynthesis. The principal mechanism of hepatocyte glutathione turnover appears to be cellular efflux. Kidney too plays an important role in organismic GSH homeostasis. Role of GSH in organs like lung, intestine and brain has recently been described. GSH involvement in programmed cell death has also been indicated. Immense interest makes the then "thee glutathione" as "inevitable glutathione". This article describes the role of this vital molecule in cell physiology and detoxication mechanisms in particular. '''Lest I forget thee, glutathione" is the serene phrase coined by Kosowers in 1960's. The last couple of years have seen a remarkable surge in interest on this ubiquitous tripeptide, the glutathione. Scientific meetings held in 1973 in Tubingen, in 1975 in Santa Ynez and in 1978 in SchloB Reisensburg, Germany and others have discussed a multitude of pharmacological and toxicological aspects related to glutathione mainly centered on the events leading to liver cell damage and protective mechanisms. The discovery of glutathione dependent enzymes and their precise role in detoxication mechanisms has made "thee glutathione" as "inevitable glutathione" now. An attempt has been made in this review to summarize much of the information available till now on this tripeptide. Glutathione was first discovered by J de. Rey Pailhade over hundred years ago and its structure (L-y-glutamyl-L-cysteinyl-glycine, or y-glu-cys-gly) was deduced in 1930's. Glutathione is the predominant thiol compound in many cells, both prokaryotes and eukaryotes, where its total intracellular concentration lies between 0.5 to 12 mm. It is present in most eukaryotes except those that do not have mitochondria. It is absent in many archaebacteria, but it is replaced by y-glu-cys in holobacteria. Likewise some eubacteria do not have glutathione but it is found in cyanobacteria and purple bacteria. It is believed that glutathione arose initially in the prokaryotic ancestors of mitochondria and chloroplasts and was acquired by eukaryotes along with these organelles. Glutathione is a tripeptide and its unique structure holds the key to its diverse functional activities. Evidently, it is a combination of 3 amino acids. First, the side chain of y-carboxyl group of glutamic acid is joined by a peptide bond to the a-amino group of cysteine. The a-carboxyl group of the resulting y-glu Cyst is then joined by a normal peptide bond with the a-amino group of glycine. Naturally, glutathione is found as a mixture of the thiol form (GSH), and with a disulphide bond (GSSG) linking two glutathione moieties'. y-glu. Cys. Gly. NH H I II I 0= C - C - CH 2 - CH 2 - C - N - C - N - CH 2 - C = 0 I I I I o H CH 2 0 I SH y-glutamyl cysteinyl glycine (GSH) Glutathione synthesis Glutathione is synthesized directly from precursor amino acids. Two cytosolic enzymes are involved, both of which require ATP and Mg2+ or Mn 2 +. The first, y-glutamylcysteine synthetase which is ratelimiting and the second, glutathione synthetase adds

2 RANA et al.: INEVITABLE GLUTATHIONE, THEN & NOW 707 glycine. The product, glutathione, regulates its own synthesis by non-allosteric competitive inhibition of the y-glutamate binding site of the first enzyme 2. The Km for both, glutathione and glutamate are approximately 2 mm; the physiologic concentration of glutamate in liver is approximately 2 mm whereas the concentration of glutathione in liver is approximately 7 mm 3 Nearly 90% of hepatic glutathione is found in cytosol. Glutathione binding in cytosol is accounted mainly for the glutathione S-transferases, which have 0.1 mm Km for glutathione and account for 10% of cytosol protein 4. Only a small fraction of cytosol glutathione could be accommodated by binding to these enzymes. Therefore, it can be concluded that the bulk of cellular glutathione exists unbound. The availability of the precursor amino acid, cysteine, is of critical importance in glutathione synthesis. The physiologic concentration of cysteine is about one order of magnitude lower than the Km of y glutamylcysteine synthetase for cysteine 5. Moreover, the source of cysteine in hepatocytes appears to be principally methionine 6. Cysteine is poorly taken up by hepatocytes whereas methionine is readily taken up and metabolized to S adenosylmethionine, homocysteine, cystathione, and cysteine in sequence 7. The kidney differs in that cysteine, as cystine is readily taken up by tubular epithelium, supporting the requirements for the rapid turnover of glutathione in kidneys. The fate of oxidized glutathione Within cells, the oxidized glutathione can undergo reactions with tissue thiols to form mixed disulfides catalyzed by thiol-disulfide transferases 9. Substantial amount of glutathione seem to be "stored" in this way and the normal diurnal variation in reduced/oxidized glutathione is closely associated with this phenomenon. In addition, oxidized glutathione can be enzymatically reduced by glutathione reductase and NADPH'o. Oxidized glutathione (GSSG) usually exists in very small concentrations in the liver cells (5% of total glutathione equivalents in liver). The maintenance of low GSSG concentration is accounted for by its rapid reactions with protein thiols, its reduction by NADPH, glutathione reductase and its active transport out of the cells. In liver the latter seems to occur preferentially into bile. Under conditions of oxidative stress associated with increased formation of GSSG, the concentration of GSSG does not increase significantly in liver. Thus, the mechanisms handling GSSG ensure that it does not accumulate. Rapid efflux (export) appears to be of critical importance in ridding cells of GSSG and avoiding its potential noxious effects". Enzymatic degradation of glutathione Glutathione may be consumed by conjugation reactions (glutathione S-transferases) which mainly involve metabolism of xenobiotics 12. However, the major enzymatic degradation normally involves the action of y-glutamyltranspeptidase 13, a brush border enzyme of renal tubular cells'4, and intestinal epithelium'5 for which no significant role has been demonstrated in liverl7. The principal mechanism of hepatocyte glutathione turnover appears to be efflux '6. This contention is based on comparison of the rate of efflux of hepatic glutathione into the perfusate of isolated perfused liver compared to published estimates of hepatic glutathione half-life. However, no direct comparison of quantitative efflux versus steady-state hepatic glutathione turnover has been made's. Functions of glutathione The multiple physiological and metabolic functions of GSH include thiol transfer reactions that protect cell membranes and proteins. These include thiol disulfide reactions that are involved in protein assembly, protein degradation and catalysis. GSH participates in reactions that destroy hydrogen peroxide, organic peroxides, free radicals and certain foreign compounds. GSH participates by number of chemical mechanisms in the metabolism of various endogenous compounds. It serves catalytically in some cases and as a reactant in others. GSH functions in the transport of amino acids. GSH itself is transported across cell membranes, but the reverse process does not occur appreciably. The intracellular concentration of GSH is much greater than that of cysteine. The liver is very active in GSH biosynthesis and translocates GSH to the blood plasma and to the bile. Kidney is active in removing GSH from blood plasma. GSH is thus a major interorgan transport form of cysteine. Role of GSH in detoxication Cysteinyl residue of GSH offers a nucleophilic thiol which is important for detoxication of electrophilic metabolites and metabolically produced oxidizing agents. Its net negative charge and overall hydrophilicity greatly increase the aqueous solubility of the lipophilic moieties with which it becomes conjugated. Its molecular weight (307) ensures that its adducts are preferentially secreted via the biliary system which

3 708 INDIAN J EXP SIOL, JUNE 2002 selects molecules of molecular weight greater than 300 to 500 according to the species '8. In mammals, GSH conjugates are often further metabolized by hydrolysis and N-acetylation, either in the gut or in the kidney, to give N-acetylcysteinyl conjugates known as mercapturic acids which are excreted in the urine l9. A number of electrophiles undergo GSH conjugation by substitution or addition reactions. Many, but not all, such reactions are also catalyzed by GSH transferases. Paracetamol is oxidized at endoplasmic reticulum to N-acetyliminoquinone, which undergoes nucleophilic addition of GSH followed by rearrangement to give 3[glutathione-S-yl] paracetamol 2o. The GSH-dependent biliary secretion mechanism was observed by Ballatori and Clarkson 21. They reported that same mechanism may be applicable to other heavy metals that have a selective affinity for SH groups. Observations on the biliary secretion of zinc 22, copper 23, silver 24 and cadmium 25 provide evidence for an important role of GSH in regulating the biliary secretion of each of these metals. Munog and co-workers 26 reported that chronic ethanol administration profoundly modifies the hepatic metabolism of glutathione and may thus have important effects on the detoxication of xenobiotics by the liver. Role of glutathione in protecting endothelial cells against hydrogen peroxide oxidant injury was also studied 27. Protection against paraquat induced injury by exogenous GSH in pulmonary alveolar type II cells was reported 28. Exogenous GSH provided rat small intestinal epithelial cells with significant protection against injury induced by t-butylhydroperoxide or menadione. This protection was found to be dependent upon uptake of intact GSH 29. Exogenous glutathione offered protection against CCl4 induced liver injury in rats 30. Studies with a variety of cell types have shown that decrease in GSH increases the sensitivity to oxidative and chemical injury. The possible use of GSH as a direct therapeutic agent to protect against chemical and reperfusion injury, radiation damage, chemical toxicity or chemical induced carcinogenesis was also reported 31. Metal-GSH interaction has been studied. Many of these observations further support the pharmacological role of glutathione. In mammals as well as in fish, most of the cadmium is sequestered in kidney and hepatic tissues bound to metallothionein, some is excreted into the bile bound to GSH Cadmium causes lipid peroxidation in tissues 35 to initiate a process which is inhibited by GSH peroxidase. These studies suggested that an interaction might exist between cadmium intoxication and GSH metabolism in different species. The role of GSH in the formation of conjugates with electrophilic drug metabolites most often formed by the cytochrome P-450 linked mono-oxygenase is now well established. Thus, studies with a number of model compounds including halogenated benzenes and acetaminophen have shown that hepatotoxicity of such xenobiotics often is preceeded by GSH depletion and prevented under conditions of facilitated biosynthesis of this thio! It was further suggested 38 that cysteine and methionine protect hepatocytes from bromobenzene toxicity by facilitating GSH synthesis and not by direct interaction with the reactive metabolite. They demonstrated that glutathione participates in hepatic detoxication reactions in three different ways (Fig. 1). The cytochrome P-450 monoxygenase system in isolated hepatocytes can generate H20 2, HCHO and epoxides, which are in tum detoxified by enzyme systems which utilize GSH either as reductant, as a cofactor, or as a nucleophile in conjugation reactions. Since intracellular accumulation of any of these reactive reaction products is associated with cell damage, the maintenance of a high intracellular concentration of GSH appears essential for prevention of cytotoxicity as a consequence of drug metabolism. GSH-dependent enzymes Glutathione peroxidase (EC )-Glutathione peroxidases (GSH-Px) are selenoenzymes which catalyse the reduction of hydroperoxides at the expense of GSH In this process, hydrogen peroxide is reduced to water whereas organic hydroperoxides are reduced to alcohols. ROOH + 2GSH --->~ ROH + H GSSG or H GSH --...;>~ 2H20 + GSSG GSH peroxidase resides in the cytosol and mitochondrial matrix 41. It exhibits following properties. (i) It acts as an enzyme protecting hemoglobin from oxidative destruction by H (ii) It acts as a "contraction factor" of mitochondria i.e. as a compound preventing loss of contractability of mitochondria under special conditions. (iji) It catalyses reduction of H20 2 and organic hydroperoxides including those derived from unsaturated lipids to alcohol.

4 RANA el al.: INEVITABLE GLUTATHIONE, THEN & NOW GSH as a reductant: Peroxide reduction GSH GSSG P-450 NADPH + 0 "'--= --?!. 2 NADP + + H H 2 0 (Substrate) glutathione peroxidase 2. GSH as a co-factor: Aldehyde and a-ketoaldehyde oxida tion GSH GSH P-450 NAD+ NADH R-CH ) RH+HCHO NADPH glutathione formaldehyde glutathione formyldehydrogenase glutathione hydrolase 3. GSH as a nucleophile: drug conjugation ~ S-hydroxymethyl '>.. ~. formyl - ~ HCOOH Bromobenzene P-450 NADPH 2 bromobenzene epoxides GSH ~. glutathione -S-transferase(s) or non-enzymatic bromophenyl - glutathione Fig. 1-Function of GSH in detoxification (iv) It protects biomembranes from oxidative attack. (v) It prevents lipid peroxidation by scavenging H and slowing down H dependent free radical attack on the lipids. From the kinetic studies and structural investigations, it is known that the solenocysteine residues of GSH peroxidase shuttle between different redox states during catalysis i.e. the enzyme itself is present in a reduced or oxidized state, depending on the steady state concentration of the substrates. It reduces almost every hydroperoxide to the corresponding alcohol. Subsequently, a non-selenium-dependent glutathione peroxidase activity (non-se-gsh-px) has been recognized in rat liver in addition to the seleniumdependent glutathione peroxidase (Se-GSH-Px). Several groups reported that non-se-gsh-px is due to GSH S-transferase B and possibly GSH S-transferases A and C. Kinetic studies of GSH S-transferase B reveal the Km of its non-se-gsh-px activity with cumene hydroperoxide to be 0.55 mm and that with t butyl hydroperoxide to be 2.3 rnm. Non-Se-GSH-Px will not utilize 0.25 rnm H as a substrate. In contrast Se-GSH-Px utilizes all these substrates and Km are in the range of J-lm. Non-Se-GSH-Px is found in rat liver, kidney, testis, adrenal, brain and fat. It is present in liver of hamster, rat, sheep, pig, chicken, man and guinea pig. Because of its unfavourable Km when compared with Se-GSH-Px, the role of non-se-gsh-px is uncertain. However, it increases in rat liver in selenium deficiency and using a hemoglobin-free liver perfusion system, it has been shown to remove organic hydroperoxides in a selenium-deficient liver. Thus, it may function in peroxide metabolism under some conditions 42 A wealth of information on glutathione peroxidase is available now. Induction of protective systems after exposure to free radicals or ROS may be an important adaptive response to non-lethal insults by these reactive species. Activities of GSH peroxidases in various rat tissues were found markedly higher in adults than young or aged animals 43. It was suggested that higher level of enzyme in adult rats could reflect an "inducible response", lost during ageing. Interestingly, unlike the age dependent changes with the classical GSH peroxidases, the phospholipid hydroperoxide glutathione peroxidase showed little change with age which may reflect a "house keeping" role for this membrane localized enzyme. The role of glutathione peroxidase in lipid peroxidation in vivo is still unclear, because the removal of lipid hydroproxides is probably not sufficient to prevent damage. For example, endoperoxides, epoxides and other products of the peroxidation of lipids are not substrates for glutathione peroxidase 44. However,

5 710 INmAN J EXP BIOL, JUNE 2002 protective role of Se-GSH-Px has been studied against carbon tetrachloride hepatotoxicitl 5, xylene, toluene and methanol toxicity46, cadmium toxicity in fish 47 and rat 48. Glutathione-S-transJerases (EC )-GST's are a group of detoxifying enzymes that catalyze the conjugation of reduced glutathione with a variety of compounds bearing suitable electrophilic centers in them These enzymes are dimeric in nature with molecular weight between 40,000 and 50,000 Da. In rat liver alone seven distinct forms of GST's have been separated and some of these forms have been shown to consist of various pairs of four units designated as ya, yb, yb' and yc with molecular weights of 22,000,23,500,23,500, and 25,000 respectively. These enzymes are widely distributed in higher animals and perform several detoxification functions. This group of enzymes is analogous to serum albumin in the broad spectrum of compounds that serve as ligands, but differs significantly in that the transferases have an additional and specific site for GSH. They appear to catalyze all reactions that would be expected of GSH acting as a nucleophile, providing that the electrophilic substrate is bound to the enzyme. Examples of the reactions catalyzed include conjugation to compounds with a reactive electrophilic carbon to form thioethers; reactions with other electrophilic atoms including the nitrogen of organic nitrates and the sulfur of organic thiocyanates; isomerisation; disulfide interchange; glutathione peroxidase activity with organic peroxides and formation of thiolesters. These enzymes have been shown to act as storage proteins and appear to be responsible for the binding of bilirubin and its conjugates, among many other compounds, within the hepatocyte. A more speculative scavenger function has also been suggested for them. The GSH-S-transferases are apparently identical to proteins known as ligandins, first recognized as proteins that bind a variety of anionic compounds 53 Ligandins can bind certain carcinogens, steroids and azo dyes. It has also been proposed that the "ligandin GSH-S-transferase" family of proteins has detoxication functions including (a) catalysis, (b) binding of ligands that are not substrates, and (c) covalent bond formation with very reactive compounds leading to inactivation and destruction of the protein It is believed that the GSH-S-transferases function to protect against compounds that might otherwise be toxic or carcinogenic, thus conjugation with GSH usually (but not always) results in formation of less toxic products. However, the evidence that GSH and GSH-S-transferases protect against carcinogenesis is still incomplete. Notably the liver GSH-S-transferases are induced by phenobarbital, polycyclic hydrocarbons and certain other compounds Glutathione reductase (EC )-The fact that the intracellular levels of GSH are very much higher than those of disulphide bond (GSSG) is consistent with the presence of effective enzyme activity that catalyzes the reduction of GSSG. GSSG reductase is highly active in liver, kidney, and many other mammalian tissues. The function of this enzyme is to regenerate GSH, which has been converted to GSSG by oxidation and by thiol transfer reactions. GSSG reductase has been obtained in highly purified form from several sources and the enzyme has been extensively studied The enzyme is a flavoprotein containing one mole of flavin adenine dinucleotide per enzyme subunit. The prosthetic group is linked noncovalently to the enzyme. The enzyme contains a cysteine moiety that undergoes reduction and oxidation during the catalytic cycle. GSSG reductase catalyzes the following reaction: GSSG+NADPH+H GSH+NADP+ Although the specificity of GSSG reductase requires further study, it is clear that GSSG, GSH and y glutamy1cysteine, and the mixed disulfide between GSH and coenzyme-a are substrates. The mixed disulfide between GSH and cysteine is not a substrate. Other disulfides, including mixed disulfides between GSH and various proteins, have been reported to be substrates. Such findings, however, may probably be ascribed to the occurrence of non-enzymatic transhydrogenation between GSH and mixed disulfides to form GSSG. Several hematological disorders are associated with decreased levels of GSSG reductase in erythrocytes 61 In some instances, there may be an abnormal GSSG reductase whose affinity for flavin adenine dinucleotide is decreased. In other conditions, the situation is apparently more complex and not well understood. Decreased levels of GSSG reductase have been observed occasionally in apparently normal individuals; addition of flavin adenine dinucleotide to extracts of their erythrocyte increases the reductase activity. Activity may also be increased by administration of riboflavin y-glutamyl transpeptidase (EC )-y Glutamyl transpeptidase is widely distributed in

6 RANA et at. : INEVITABLE GLUTATHIONE, THEN & NOW 711 mammalian tissues 64 The enzyme is especially concentrated in certain epithelial structures that are involved in transport processes, e.g., the epithelia of the proximal renal tubule, jejunal villi, choroid plexus, bile ductules, semjnal vesicles and ciliary body. The enzyme is present in many other locations in lesser amounts, including central nervous system neurons. Histochemical studies show that the transpeptidase is localized in the brush border of the proximal renal tubule and that it is bound to the external (lurrunal) surface of the cell membrane. In the liver, y-glutamyl transpeptidase is localized in the bile duct epithelium and in the canalicular regions of the hepatocytes. Fetal liver exhibits much higher y-glutamyl transpeptidase activity than does adult liver. On the other hand, the kidneys of fetal and newborn rats exhibit low transpeptidase activity. Activity increases during development, ultimately leading to high activity levels typical of adult kjdney. The free bile acids (cholate, chenodeoxycholate, and deoxycholate) stimulate transpeptidation, whereas the corresponding glycine and taurine conjugates inhibit transpeptidation. These and related studies suggest a potential feedback role for bile ductule y glutamyl trans peptidase, in which free bile acids activate the enzyme to catalyze biliary GSH utilization and thus increase the pool of amino acid precursors required for conjugation (glycine directly, and taurine through cysteine oxidation). Conjugated bile acids would have the reverse effect by inhibiting ductule y glutamyl transpeptidase 65 The pathway of GSH transport and metabolism in the liver involves transport of GSH by hepatocytes into the bile canaliculi and metabolism by the action of y-glutamyl transpeptidase and dipeptidase located on the biliary ductular epithelium. This pathway was revealed by the finding of high levels of cysteinylglycine, y-glutamylglutathione, y-glutamy1cysteine, glutamate, glycine, and cysteine in bile, by studies in which GSH synthesis was inhibited. The canalicular transport of GSH, as estimated from the total of metabolites found is much greater than the GSH found in bile. GSH and GSH metabolites found in bile increase with age in association with an increase in hepatic GSH. In younger rats, there is apparent uptake of cysteine and glycine moieties, which may reflect uptake of cysteinylglycine at the ductular level. This intrahepatic pathway of GSH transport and metabolism may serve as a cellular mechanism in the processing of GSH conjugates and as a recovery system for cysteine moieties 66. Although y-glutamyl transpeptidase can catalyze hydrolysis of GSH and other y-glutamyl compounds in vitro, there is substantial evidence that transpeptidation is a major function of the enzyme in vivo 67. A variety of neutral amino acids and dipeptides are active as acceptors of the y-glutamyl group of GSH; the most active arruno acid acceptors include cystine 68, glutamine, and methionine 69. GSH itself can serve as an acceptor. When y-glutamyltranspeptidase is incubated with physiological levels (1-10 mm) of GSH, the initial rates of formation of y-glutamyl-gsh are greater than the rates of formation of glutamate (hydrolysis). y-glutamyl-gsh has been found to occur in bile and kidne/o. Transpeptidase activity is greatly inhibited in vivo by injecting animals with inhibitors of this enzyme leading to extensive glutathionuria 7 1 Administration of an inhibitor of GSH synthesis to mice leads to a prompt decrease in the GSH level of the liver and other tissues and a consequent decrease in plasma GSH levels72. Adrrunistration of an inhibitor of transpeptidase leads to an increase in plasma GSH levels. Transport of GSH from the liver is a significant pathway for transport of amino acid sulfur from liver to kidney and to other tissues. The kidney uses GSH that is transported from the liver into the blood plasma as well as GSH transported from renal cells into the renal tubular lumen. Animals treated with inhibitors of y-glutamyl transpeptidase and patients with y-glutamyl transpeptidase deficiency excrete, in addition to GSH and GSSG, large amounts of y-glutamy1cysteine and cysteine moieties 73. This observation indicates that the function of y-glutamyl transpeptidase is closely associated with the metabolism or transport, or both, of cysteine and y-glutamy1cysteine. Transport of y-glutamy1cysteine into cells appears to be inhibited by the high levels of GSH that accompany reduced transpeptidase activity. The finding of urinary y-glutamy1cysteine and cysteine moieties in transpeptidase deficiency or inhibition reflects accumulation of y-glutamylcyst(e)ine and is in accord with the fact that cysteine is an excellent acceptor substrate of y glutamyl transpeptidase. GSH in liver Glutathione plays a key role in the liver in detoxification reactions and in regulating the thio-sulphide status of the cell. Liver is viewed as a glutathionegenerating factor which supplies the kidney and intestine with other constituents for glutathione resynthesis. The glutathione content of rat liver is usually 7 to

7 712 INDIAN J EX? BIOL, JUNE ~M/g. The GSH content of the liver is divided into two pools with apparently different half-lives (1.7 and 28.5 hr). The "labile" pool of glutathione functions as a reservoir of cysteine. Glutathione releases cysteine for protein synthesis when other conditions are fulfilled and the amount of cysteine becomes ratelimiting. The availability of the precursor amino acid cysteine is of critical importance in glutathione synthesis. Moreover, the source of cysteine in hepatocytes appears to be principally methionine. Cysteine is poorly taken up by hepatocytes whereas methionine is readily taken up and metabolized to S-adenosylmethionine, homocysteine, cystathione and cysteine in sequence Substantial amounts of glutathione seem to be "stored" in this way and the normal diurnal variation in reduced/oxidized glutathione is closely associated with this phenomenon. Glutathione may be consumed by conjugation reactions (glutathione-s-transferases) which mainly involve metabolism of xenobiotic agents. However, the principal mechanism of hepatocyte ~lutathione turnover appears to be cellular efflux Apparently all cells efflux reduced glutathione, and those cells with significant y-glutamyltranspeptidase degrade the extracellular glutathione by oxidation and hydrolysis. The component amino acids either redistribute to the liver and other organs or are used to maintain intrarenal glutathione 79 The efflux of liver glutathione into bile and blood not only is a major control mechanism for hepatic glutathione homeostasis, but is also important for supplying other organs rich in surface membrane y-gluamyltranspeptidase (e.g. kidney and intestine) with the constituent amino acids necessary for glutathione synthesis. Thus, the regulation of GSH in the liver is based on a homeostatic feedback inhibition mechanism. The availability of cysteine is a critical factor in the regulation of synthesis. Turnover in liver is determined mainly by the efflux of glutathione in both sinusoidal blood and bile and its subsequent degradation. The constituents of exported glutathione are conserved by hydrolysis and cellular uptake mainly in kidney and intestine as governed by brush border y-glutamyltrans peptidase. It is generally agreed that the intrahepatic glutathione is able to afford protection against liver dysfunction by at least two ways, firstly as a substrate of glutathione peroxidase, GSH serves to reduce a large variety of hydroperoxides before they attack unsaturated lipids or convert already formed lipid hydroperoxides to the corresponding hydroxy compounds. Secondly as a substrate of glutathione-s-transferases, it enables the liver to detoxify many foreign compounds or their metabolites and to excrete the product, preferably into the bile 8o. GSH in kidney The kidney is a heterogenous tissue that contains numerous cell populations, each possessing distinct morphological, physiological and biochemical properties 8 1 Glutathione, is found in higher concentration in the kidney and has been directly or indirectly implicated in the maintenance of normal kidney function 82. A lowering of the GSH level in the kidney cortex leads to the reversible inhibition of protein kinases, Na-K-dependent ATPase, glucose-6-phosphatase, amino acid and a-methyl-d-glucoside uptake and gluconeogenesis. GSH is believed to play an important role for expression of full activity of the transport systems and they get disturbed in case of its unavailability. The glutathione uptake by the kidney occurs not only by glomerular filtration and subsequent degradation by the y-glutamyl transpeptidase of the tubular brush border membrane, but also by an additional uptake mechanism. It was also recognized that the capacity for enzymatic hydrolysis of glutathione is localized mainly in the kidney. Studies on interorgan relationship in the tumover of glutathione, have established that the kidney is a major site of glutathione degradation to the constituent amino acids, whereas other organs e.g. the liver, synthesize their own glutathione pool and contribute to the plasma pool. The steady state levels of GSH in the kidney result from a balance between the rate of glutathione synthesis and the rate of its utilization by transpeptidase. The regulation of cellular GSH status in the kidneys as well as in several other tissues is compartmentalized involving processes that occur at the plasma membrane, cytosol, endoplasmic reticulum, nucleus and mitochondria 83. At the plasma membrane, GSH is transported into renal proximal carrier 84 and can function to provide exogenous GSH to proximal tubular cells to protect them from oxidative injurl 5. 86, GSH is transported out of renal proximal tubule cells 87 and functions in the turnover of cellular GSH by delivering the tripeptide to the active site of y-glutamyltranspeptidase 79. The proximal and distal tubular cells, show cell type dependent differences in activities of GSH-dependent enzymes in sub-

8 RANA et al.: INEVITABLE GLUTATHIONE, THEN & NOW 713 cellular fractions and also a markedly different capacity to transport GSH into mitochondria. An electrogenic GSH transport system has also been described in the renal brush-border membrane vesicles. This transporter has characteristics compatible with a role in GSH efflux from the cell into the tubular lumen, and recent studies have indicated that kidneys secrete GSH into the lumen. Thus, current evidence indicates that both uptake and efflux can occur in the kidney. This transport may be important both in regulation of renal and organismic GSH homeostasis. GSH in other organs Oxidative injury in the lung occurs from inhaled air pollutants, oxygen toxicity, antitumour agents and redox cycling agents. When GSH supply becomes limiting, cell injury caused by these compounds is increased. Berggreu et al. 88 found that isolated perfused lung can synthesize GSH from the precursor amino acids and that the exogenous GSH may be as useful as a direct therapeutic agent to protect lung against certain types of chemkal and oxidative injury. High GSH concentrations are therefore observed in lung cells 89,9o. Lungs are unique in that the extracellular milieu at the alveolar epithelial surface is rich in GSH and is about 140 times of the plasma level 9 1 Drug-metabolizing and oxidation-reduction systems in the epithelium of the small intestine represent a first line of defence against ingested xenobiotics and toxins. Epithelial cells in the intestine have a characteristically rapid turnover rate that appears to be essential to protect against injury from exogenous physical, chemical and infectious agents. Isolated intestinal epithelial cells are known to be as susceptible as chemical injury and these cells are known to be susceptible to hepatocytes in their sensitivity to chemical inj ury92,93. In absence of extracellular GSH or amino acid precursors, the intracellular GSH concentration decreased up to 60%, indicating that intestinal epithelial cells require a continuous supply of GSH, in the form of either the intact tripeptide or the precursors, to maintain intracellular GSH levels. The brain is an extremely heterogenous organ with a large number of different neuronal and non-neuronal cell types, and extensive morphological differentiation and biochemical compartmentalization within a given ce1l 94. The neurons also show variability in chemical composition at the soma, dendrites, axons and terminals. GSH contributes to the overall redox state of cells in brain. It has been implicated in brain neuropathology such as that seen in brain ischemia 95 or Parkinson's disease 96. GSH appears to be present at relatively low concentrations in neuronal cell bodies in rodent and monkey brain, as well as in apical dendrites of monkey cortex and cerebellum. Also studies indicate that it is localized in the non-neuronal elements, as well as fibrous and terminal regions of neurons. The major interest in brain GSH relates to aspects of oxidative stress and ageing. GSH and apoptosis Aerobic organisms faced with the threat of oxidation from molecular oxygen (0 2 ) have evolved antioxidant defences to cope with this potential problem. However, cellular antioxidants can become overwhelmed by oxidative damage including supraphysiologic concentration of O 2. Oxidative cellular injury involves modifications of cellular macromolecules by reactive oxygen intermediates (ROI) often leading to cell death97- loo. The apoptotic processes in these cells are often associated with decreased levels of GSH due to increased efflux of this antioxidant from the cells. Because GSH is essential for maintaining the reducing capacity of cells, the loss of this capacity may cause the cell to die. This mechanism of cell death is mediated by mitochondrial dysfunction caused due to the absence of a minimum concentration of GSH required by mitochondria which also affects the ATP production in cells 97. Conclusion In summary, glutathione is a vital substance in detoxification and cell physiology. Its regulation in liver is based on a homeostatic feedback inhibition mechanism. The availability of cysteine is a critical factor in the regulation of synthesis. Turnover in liver is determined mainly by the efflux of glutathione in both sinusoidal blood and bile and its subsequent degradation. The constituents of exported glutathione are conserved by hydrolysis and cellular uptake mainly in the kidney and intestine as governed by brush-border y glutamyltranspeptidase. Thus, one can view the liver as a glutathione generating factor which supplies the kidney and intestines with the constituents for glutathione resynthesis. References 1 Creighton T E, Glutathione in Encyclopedia of molecular biology (John Wiley & Sons Inc, New York) Vol. II (1999) 1023.

9 7]4 INDIAN J EXP BIOL, JUNE Dav is J S, Balinsky J B & Harington J S, Assay, purification, properties and mechani sm o f action of y g lutamylcysteine synthetase from the li ver of rat and Xenopus laevis, Biochem J, 133 (1 973) Wahllander A, Soboll S & Si es H, Hepatic mitochondrial and cytosolic glutathio ne content and the subcellular d istribution of GSH-S-transferases, FEBS Leu. 75 (1 979) Kaplowitz N, T he physiologic signifi cance o f the g lutathione S-transferases, Am J Physiol. 239 (1 980) Ri chman P G & Meister A, Regul ati on of y-glutamylcysteine synthetase by nonallosteric feedback inhibition by glu tathione, J Bioi Chem. 250 ( 1975) Thor H, Moldeus P, Hermanson R. Hogberg J. Reed OJ & Orrenius S, Metabolic acti vati on and hepatotoxicity : Toxicit y o f bromobenzene in hepatocytes isoiated from phenobarbital and diethy l- maleate treated rats, Arch Biochem Biophys. 188 (1978) Beatty P W & Reed 0 J, In volvemcnt o f the cystath ionine pathway in the bi osynthesis of g lutathione by isolated rat hepatocytes, Arch Biochem and Biophys. 204 (1 980) Ormstad K. Joncs 0 P & Orreni us S. Characteri st ics of glutathi one biosynthesis by Freshl y isolated rat kid ney cclls, J Bioi Chem. 255 ( 1980) Issacs J & Binkley F. Glutathione dependent cont rol o f protein disulfide-sulfhydryl content by subcellular fractions of hepatic ti ssue, Biochem Biophys Acta. 497 ( 1977) Kosower N S & Kosower E M, T he glutathione status o f cells, Review of Cytology. 54 ( 1978) 109. II Kaplowitz N, The importance and regul ation of hepatic glutathione, The Yale Joumal of Biology alld Medicin e. 54 ( 198 1) Tate S & Meister A, Interaction of y-g lutamyltranspeptidase wi th amino acids, dipeptides and dcri vati vcs and analogs of glutathione, J Bioi Chem. 249 (1 974) Thompson G A & Meister A, Interaction o f y-glutamyltranspeptidases, evidence for separate substrate sites and for high affinity o f a.-cystinc, iliochem Biophys Res Commun. 71 ( 1976) Horiuchi S, Inoue M & Morino Y, y - Gluta myltranspeptidase: Sidcdness of its acti ve site on renal brushborder membrane, Eur J Biochem. 87 ( 1978) Cornell J S & Meister A, Glutathione and y-glutamyl cycle enzymes in crypt and villus tip cells of rat jejunal mucosa, Proc Natl Acad Sci. 73 ( 1976) Sics H, Bartoli G M, Burk R F & Waydhas C. Glutathione e fou x from perfused rat liver aftcr phenobarbital treatment, during drug oxidati ons and in selenium deficiency, Eu r J Biochem. 89 ( 1978) Kaplowitz N, The import ance and regul ation of hepatic glutathione, The Yale Journal of Biology and Medicill e. 54 (1 98 1) Milburn P, Excretion of xenobiotic compounds in bi le. in The hepato - biliary system, edited by W Taylor (Plenum Press, New York ) 1976, Chasseaud L F, Conjugation with glutathione and mercaptu ric acid excretion, in "Glutathione Metabo li sm and Function", edited by L MArias & W B Jakoby, (Raven Press New York) 1976, Hi nson J A, Monks T J, Horg M K, Hyatt R J & Pohl L R, 3(Glutathione-S-yl) acetaminophen a biliary me tabolite of acetaminophen, Drug Metab Disp. 10 (1 982) Ballatori N & Clarkson T W. Developmental changes in th e excreti on of methyl mercury and g lutathione, Science (1 982) Alexander J. Aaseth J & Refsvik T, Excretion of zinc in rat bile- a role of glutathione, Acta Pharmacol Toxicol. 49 (1981) Alexander J & Aaseth J, Biliary excretion of copper and zinc in the rat as influenced by diethylmaleate, selenitc and di ethyldithiocarbamate, Biochelll Pharmacol. 29 ( 1980) Alexander J & Aaseth J, Hepatobili ary transport and organ distribution of silver in the rat as inll uenced by selenitc, Toxicology. 21 ( 198 1) Cheri an M G & Vostal J J, Biliary excreti on o f cadmium in rat. I. Dose- dependent biliary excretion and the form of cadmi um in the bile, J Toxicol Environ Healrh. 2 ( 1977) Munog M E, Martin M I, Fermoso I. Gonjalez I & Estel ler A, Effec t of chronic ethanol feeding on glutathi one and glutathione re latcd enzyme acti vitics in the liver, Drug alld Alcohol Dependence. 20 ( 1987) Andreoli S P, Mall et S P & Bergstein J M, Role o f glutathione in protecting endothe li al cells against hydrogen peroxide oxidant injury. J Lab Clill Med. 108 (1986) Hagen T M. Brown L A & Jones 0 P, Protecti on against paraquat induced inj ury by exogenous GSH in pu lmonary alveolar type II cell s. Biochem Pharlnacol, 35 (1986) Lash L H, Hagen T M & Jones 0 P, Exogenous glutathi one protects intestinal epit heli al cells fro m ox idative injury, Proc Narl Acad Sci USA. 83 (1986) Rana S V S ' & Tayal M K, Influence o f zinc, vit- B J2 and g lutathione on the li ver of rats exposed to carbon tetrachlori de, Industrial Health (Japan). 19 ( 198 1) Hagen T M, Aw T Y & Jones 0 P, Glutathione uptakc and protection against oxidati ve injury in isolated ki dney ccll s, Kidney Illtem ational. 34 ( 1988) Graf P & Sies H, Hepatic uptakc o f cadmium and its bil iary release as affected by dith ioerythritol and glutathi one, Biochem Pharmacol. 33 ( 1984) Noel-Lambot F, Gerda)' C H & Di steche A, Distribution of Cd, Zn and Cu in liver and gills o f the eel Anguilla anguilla with special reference to metal lothi oneins, Comp Biochem Physiol. 6 1C ( 1978) Stacey N H, Cantilena L R & Kl aassen C 0, Cadmium toxici ty and lipid peroxidation in isolated rat hepatocytes, Toxicol Appl Pharlnacol. 53 ( 1980) Rana S V S & Kumar A, Si gnifi cance of lipid peroxidation in liver injury after heavy metal poisoning in rats, Curr S.:i. 53 ( 1984) Mitchell J R, Jo llow 0 J, Potter W Z, Gill ett e J R & Brodie B B, Acetaminophen induced hepati c necrosis. IV. Protective role o f glutathione, J Pharmacal Exp Ther. 187 ( 1973) Jollow 0 J, Mitchell J R, Zampaglione N & Gillete J R, Bromobenzene induced liver necro is. Protective role of g lutathione and e vidence for 3,4-bromobenzene oxide as the hepatotoxic metabolite, Pharmacology. II (1974) Nemoto N, Gelboin H V, Habig W H, Ketley J N & Jakoby W B. K-region Benzo(a) pyre ne-4,5-oxide is conj ugated by homogenous g lutathione S- transferases. Nature. 255 ( 1975) 5 12.

10 RANA et ai.: INEVITABLE GLUTATHIONE, THEN & NOW Flohe L, The selenoprotein glutathione peroxidase, in Glutathione: Ch emical Biochemical and MedicaL aspects. Part A eds. D. Dolphin D, Avramovic and 0, Poulson R, (John Wiley & Sons New York) (1989) Ursini F, Maiorino M & Brigelius- Flohe R, The diversity of glutathione peroxidase, Methods in Enzymol, 252 ( 1995) Mills G C, Glutathione peroxidase and the destruction of hydrogen peroxide in animal tissues, Arch Biochem Biophys, 86 (1960) I. 42 Burk R F, Trumble M J & Lawrence R A. Rat hepatic cytosolic glutathione dependent enzyme protection against lipid peroxidation in the NADPH microsomal lipid peroxidation system, Biochem Biophys Acta, 618 (1980) Zhang L, Maiorino M, Roveri A & Ursini F, Phospholipid hydroperoxide glutathione peroxidase: Specific activity in tissues of rats of different age and comparison with other glutathione peroxidases, Biochem Biophys Acta, 1006 (1989) Flohe L, in Free Radicals in Biology edited by W A Pryor (Academic Press, New York and London) Vol V, 1982, Rana S V S & Rastogi S, Antioxidative enzymes in the liver of rat treated with carbon tetrachloride after parathyoridectomy, Physiol Chem Phys and Med NMR, 25 (1993) Rana S V S & Kumar S, Antioxidative enzymes in the liver of rat after indi vidual and combined exposure to xylene, toluene and meth yl alcohol, Physol Ch em Phys & Med NMR, 27 (1995) Rana S V S & Singh R, Species difference in glutathione dependent enzymes in the liver and kidney of two fresh water fishes and their implications in cadmium toxicity, Ichthyological Res, 43 (1996) Rana S V S & Rastogi N, Antioxidant enzymes in the liver and kidney of diabetic rats and their implications in cadmium toxicity, Physiol Chem Phy & Med NMR, 32 (2000) Boyland E & Chasseaud L F, Enzyme-catalysed conjugations of glutathione with unsaturated compounds, Biochem J, 104 (1967) Habig W H, Pabst M J & Jakoby W B, Glutathione-Stransferase. The first enzymatic step in mercapturic acid formation, J Bioi Chem, 249 (1974) Wood J L, Metabolic conjugation and metabolic hydrolysis, in Biochemistry of mercapturic acid formation edited by Fishman W H (Academic Press, New York) Vol 2 ( 1970) Jakoby W B, The glutathione-s-transferase: A group of multifunctional detoxification proteins, Adv Ellzymol, 46 ( 1978) Arias I M & Jakoby W B, eds. Glutathione metabolism alld function, (Raven Press, New York) Habig W H, Pabst M J & Jakoby W B, Glutathione-Stransferases A. A novel kinetic mechanism in which the major reaction pathway depends on substrate concentration, J Biochem Chem, 249 (1971) Ketley J N, Habig W H & Jakoby W B, Binding of nonsubstrate ligands to the glutathione-s-transferases, J Bioi Chem, 250 (1975) Chasseaud L F, The role of glutathione and glutathione-stransferases in the metabolism of chemical carcinogens and other electrophi lic agents, Adv Cancer Res, 29 (1979) Benson A M, Chan Y N, Bneding E, Heine H S & Talalay P, Elevation of extrahepatic glutathione-s-transferases and epoxide hydratase actlvlttes by 2(3)- fert-butyl-4- hydroxyanilole, Cancer Res, 39 (1979) Rana S V S & Gupta S, Phenobarbital alters liver and kidney function in toluene and trichloroethylene-treated rats, Toxic Substance Mechanisms, 18 (1999) I. 59 Mapson L W & Goddard D R, The reduction of glutathione by plant tissues, Biochem J, 49 (1951) 60 I. 60 Racker E, Glutathione reductase from baker's yeast and beef liver, J Bioi Chel1l, 217 (1955) Flohe L, Benohr H C, Sies H, Waller H D & Wendel A, eds. Symposium on glutathione, Thielne stuttgart, (1973). 62 Beutler E, Glutathione reductase: Stimulation in normal subjects by ribooavin supplementation, Science, 165 (1969) Paniker N V, Srivastava S K & Beutler E, Glutathione metabolism of red cells effect of glutathione reductase deficiency on the stimulation of hexose monophosphate shunt under oxidative stress, Biochem Biophys Acta, 215 (1970) Meister A, Tate S S & Ross L L, Membrane-bound y glutamyl transpeptidase. in The Enzymes of Biological Membranes, edited by A. Martinosi (Plenum Press, New York) Vol 3 (1976) Abbott W A & Meister A, Modulation of y-glutamyl transpeptidase activity by bile acids, J Bioi Chem, 258 (1983) Abbott D & Meister A, Intrahepatic transport and utilization of biliary glutathione and its metabolites, Proc Natl Acad Sci USA, 83 (1986) Allison D & Meister A, Evidence that transpeptidation is a significant function of y-glutamyltranspeptidase, J Bioi Chem, 256 (1981) Thompson G A & Meister A, Utilization of L-cystine by the y-glutamyl transpeptidase- y-glutamyl cyclotransferase pathway, Proc Natl Acad Sci USA, 72 (1975) Tate S S & Meister A, Interaction of y-glutamyl transpeptidase with amino acids, dipeptides, and derivatives and analogs of glutathione, J Bioi Chem, 249 (1974) Abbott W A, Griffith 0 W & Meister A, y-glutamylglutathione; natural occurrence and enzymology, J Bioi Chem, 261 (1986) Griffith 0 W & Meister A, Translocation of intracellular glutathione to membrane-bound y-glutamyl trans peptidase as a discrete step in the y-glutamyl cycle: Glutathionuria after inhibition of transpeptidase, Proc NatL Acad Sci USA, 76 (1979) Griffith 0 W, Bridges R J & Meister A, Evidencc that thc y-glutamyl cycle functions in vivo using intracellular glutathione: Effects of amino acids and selective inhibition of enzymes, Proc Natl Acad Sci USA, 75 (1978) Griffith 0 W & Meister A, Excretion of cysteine and y glutamylcysteine moieties in human and experimental animal y-glutamyl transpeptidase deficiency, Proc Natl Acad Sci USA, 77 (1980) Tateishi N & Higashi T, Turnover of glutathione in rat liver, in Functions of glutathione in liver and kidney, edited by Sies H, Wendel A, (Springer-Verlag Berlin) (1978) 3.

11 716 INDIAN J EXP BIOl, JUNE Reed OJ & Fariss MW, Glutathione depletion and susceptibility, Pharmacol Rev. 36 (1984) Ormstad K. Jones D P & Orrenius S. Characteristics of glutathione biosynthesis by freshly isolated rat kidney cells. J Bioi Chem. 255 (1980) Sies H. Bartoli G M. Burk R F. & Waydhas C. Glutathione efnux from perfused rat li ver after phenobarbital treatment, during drug oxidations and in selenium deficiency, Eur J Biochem. 89 (1978) Horiuchi S, Inoue M & Morino Y. y-glutamyltranspeptidase: Sidedness of its active site on re nal brushborder membrane. Ellr J Biochelll, 87 (1978) Griffith 0 W & Meister A, Glutathione: Interorgan translocation turnover and metabolism. Proc Nati Acad Sci, 76 (1979) Wendel A, Frenkenstein S. Konz K H & Drug, Induced lipid peroxidation in mouse liver, in Functions of glutathione in liver and kidney, edited by H Sies and A Wendel, (Springer-Verlag, Berlin) ( 1978) l ash l H, Visarius T M, Sail M J, Qian W & Tokarz J J, Cellular and subcellular heterogeneity of glutathione metaboli sm and transport in rat kidney cells, Toxicology. 130 (1998) I. 82 Colowick S, l azarow A, Rucker E, Schwartz D R, Stad tman E M & Waelsh H (eds.) Glutathione. Academic Press New York (1954). 83 Smith C V, Jones D P, Guenther T M, lash l H & lauterburg B H, Compartmentation of g lutathione: Implications for the study of toxicity and disease, Toxicol Appi Pharmacal. 140 ( 1996) I. 84 Lash L H & Jones D P, Renal glutathione transport: Characteristics of the sodium- dependent system in the basallateral me mbrane, J Bioi Chelll. 259 (1984) Hagen T M, Aw T Y & Jones D P, Glutathione uptake and protection against oxidative injury in isolated kidney cells, Kidney InternaTional. Vol. 34 ( 1998) Vi sarius T M, Putt D A, Schare J M, Pegouske D M & Lash L H, Pathways of glutathione metabolism and transport in isolated proximal tubular cells from rat kidney, Biochem Pharmacol. 52 (1996) Inoue M & Morino Y, Direct evidence for the role of the membrane potential in glutathione transport by renal brush-border membranes, J Bioi Chem. 260 (1985) Berggren M, Dawson J, Moldeus P, Hagen M T & Brown L A, Protection against paraquat-induced injury by exogenous GSH in pulmonary alveolar type " cells, Biochern Phannacol. 35 (1986) Cantin A M & Begin R, Glutathione and inoammatory disorders of the lung, Lung. 169 (1991) Meister A, New aspects of glutathione biochemistry and transport: Selective alteration of glutathione metabolism, Fed Proc. 43 ( 1984) Griffith 0 W, Determination of glutathione and glutathione disulphide using glutathione reductase and 2- vinylpyridine, Anal Biochem. 106 ( 1980) Thor H, Smith M T & Hartzell P, In Exogenous glutathione protects intestinal epithelial cell s from oxidative injury. Lash H, Hagen T M et ai. Proc NaTi Acad Sci USA. 83 (1986) Orrenius S, Ormstad K, Thor H & Jewell S, Turnover and functions of glutathione studied with isolated hepatic and re nal cells, Proc Fed Am Soc Exp Bioi. 42 (1983) Asghar K, Reddy B G & Kri shna G, Histochemical localization of glutathione in ti ssues, J HislOch elll CyTochem. 23 (1975) Mills G C, Glutathione peroxidase and the destruction of hydrogen peroxide in animal ti ssue, Arch Biochelll Biophys. 86 (1960) Cohen G, The pathobiology of Parkinson's disease: Biochemical aspects of dopamine neuron senescence, J Neural TrallSm (Suppl) 19 ( 1983) Madesh M, Bernard 0 & Balasubramanian K A, Apoptotic processes in the monkey small intestine epithelium.2. Possible role of oxidative stress, Free Rad Bioi & Med. 26 (1999) Richter C, Gogradze V & Laffranchi R. Oxidants in mitochondria, from physiology to diseases, Biochell1 Biophys Acta (1995) Janssen Y M, Van H B, Born P J & Y10ssman B T, Cell and tissue responses to oxidative damage, Lab In vest. 69 (1993) Ueda N & Shah S V, Endonuclease-induced DNA damage and cell death in oxidant injury to renal tubular epithelial cells, J Ciin In vest. 90 ( 1992) 2593.