Indian Journal of Experimental Biology Vol. 40, June 2002, pp. 706-716

Inevitable glutathione, then and now

S v S Rana, Tanu Allen & Rajul Singh

Toxicology Laboratory, Department o f Zoology, Ch. Charan Singh University., Meerut 250004, India

Tel: 0121-761190, Fax: 0121-760577

E-mail: sureshvs_rana @yahoo.com

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. Rey­Pailhade 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-Glu­Cyst 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.

NH3+ 0 H

I II I 0= C - C - CH2 - CH2 - C - N - C - N - CH2 - C = 0

I I I I o H CH2 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 Mn2+. The

first, y-glutamylcysteine synthetase which is rate­limiting and the second, glutathione synthetase adds

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 enzyme2

. 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 protein4. 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 y­glutamylcysteine synthetase for cysteine5.

Moreover, the source of cysteine in hepatocytes appears to be principally methionine6

. Cysteine is poorly taken up by hepatocytes whereas methionine is readily taken up and metabolized to S­adenosylmethionine, homocysteine, cystathione, and cysteine in sequence7

. 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 transferases9

. 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 (ex-

port) 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 reac­

tions (glutathione S-transferases) which mainly in­volve metabolism of xenobiotics 12

. However, the ma­jor enzymatic degradation normally involves the ac­tion of y-glutamyltranspeptidase13

, a brush border en­zyme of renal tubular cells'4, and intestinal epithe­lium'5 for which no significant role has been demon­strated in liver l7. 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

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] paracetamol2o.

The GSH-dependent biliary secretion mechanism was observed by Ballatori and Clarkson21 . 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 zinc22, copper23, silver24 and cadmium25 provide evi­dence for an important role of GSH in regulating the biliary secretion of each of these metals. Munog and co-workers26 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 studied27. Protection against paraquat induced injury by exogenous GSH in pulmonary alveolar type II cells was reported28. 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 GSH29. Exogenous glu­tathione offered protection against CCl4 induced liver injury in rats30. 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 reported31 .

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 GSH32.34. Cadmium causes lipid peroxidation in tissues35 to initiate a proc­ess which is inhibited by GSH peroxidase. These studies suggested that an interaction might exist be-

tween 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!36.37. It was further suggested38 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 1.11.1.9)-Gluta­

thione peroxidases (GSH-Px) are selenoenzymes which catalyse the reduction of hydroperoxides at the expense of GSH39.40. In this process, hydrogen perox­ide is reduced to water whereas organic hydroperox­ides are reduced to alcohols.

ROOH + 2GSH --->~ ROH + H20 + GSSG

or

H20 2 + 2GSH --......;>~ 2H20 + GSSG

GSH peroxidase resides in the cytosol and mitochon­drial matrix41 . It exhibits following properties.

(i) It acts as an enzyme protecting hemoglobin from oxidative destruction by H20 2.

(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.

RANA el al.: INEVITABLE GLUTATHIONE, THEN & NOW 709

1. GSH as a reductant: Peroxide reduction

GSH GSSG P-450

NADPH + 0 2 "'--= --?!. NADP + + H20 2 H20

(Substrate) glutathione peroxidase

2. GSH as a co-factor: Aldehyde and a-ketoaldehyde oxida tion

GSH GSH NAD+ NADH

~ S-hydroxymethyl '>.. ~. formyl -~ HCOOH P-450

R-CH ) • RH+HCHO NADPH glutathione formaldehyde glutathione formyl-

dehydrogenase glutathione hydrolase

3. GSH as a nucleophile: drug conjugation

GSH P-450

Bromobenzene NADPH

°2 bromobenzene epoxides

~. 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

H20 2 and slowing down H20 2 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 t­butyl hydroperoxide to be 2.3 rnM. Non-Se-GSH-Px will not utilize 0.25 rnM H20 2 as a substrate. In con­trast Se-GSH-Px utilizes all these substrates and Km are in the range of 10-50 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 unfa-

vourable 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 conditions42

•

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 animals43

. 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 peroxidase44

. However,

710 INmAN J EXP BIOL, JUNE 2002

protective role of Se-GSH-Px has been studied against carbon tetrachloride hepatotoxicitl5

, xylene, toluene and methanol toxicity46, cadmium toxicity in fish47

and rat48.

Glutathione-S-transJerases (EC 2.5.1.18)-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 them49

-52

. 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 compounds53

•

Ligandins can bind certain carcinogens, steroids and azo dyes. It has also been proposed that the "ligandin­GSH-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 protein54

.55

•

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 compounds56

-58

.

Glutathione reductase (EC 1.6.4.2)-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 studied59

.60

. 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+ ----7 2GSH+NADP+

Although the specificity of GSSG reductase re­quires further study, it is clear that GSSG, GSH and y­glutamy1cysteine, 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 erythrocytes61

•

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 ribo­flavin62

-63

.

y-Glutamyl transpeptidase (EC 2.3.2.2)-y­Glutamyl transpeptidase is widely distributed in

RANA et at. : INEVITABLE GLUTATHIONE, THEN & NOW 711

mammalian tissues64• The enzyme is especially con­

centrated 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 y­glutamyl 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 y­glutamyl transpeptidase65

•

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 moieties66

.

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 vivo67

. 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 cystine68

,

glutamine, and methionine69. 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 glutathionuria7 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 moieties73

. 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 y­glutamyl transpeptidase.

GSH in liver Glutathione plays a key role in the liver in detoxifi­

cation 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

712 INDIAN J EX? BIOL, JUNE 2002

8 ~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 sequence75

•76

. 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 efflux77

•7

. 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 glutathione79

• 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 hydroper-

oxides 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 bile8o.

GSH in kidney The kidney is a heterogenous tissue that contains

numerous cell populations, each possessing distinct morphological, physiological and biochemical proper­ties8 1

• Glutathione, is found in higher concentration in the kidney and has been directly or indirectly impli­cated in the maintenance of normal kidney function82

.

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 unavail­abi lity.

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 mitochondria83

. At the plasma membrane, GSH is transported into renal proximal carrier84 and can function to provide exogenous GSH to proximal tubular cells to protect them from oxida­tive injurl5

.86

, GSH is transported out of renal proxi­mal tubule cells87 and functions in the turnover of cel­lular GSH by delivering the tripeptide to the active site of y-glutamyltranspeptidase79

. The proximal and distal tubular cells, show cell type dependent differ­ences in activities of GSH-dependent enzymes in sub-

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 cells89,9o. 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 level91

•

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 ce1l94

. 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 ischemia95

or Parkinson's disease96. 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 oxida­

tion from molecular oxygen (02) have evolved anti­oxidant defences to cope with this potential problem. However, cellular antioxidants can become over­whelmed by oxidative damage including supraphysi­ologic concentration of O2. 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 cells97.

Conclusion In summary, glutathione is a vital substance in de­

toxification 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 y­glutamyltranspeptidase. Thus, one can view the liver as a glutathione generating factor which supplies the kidney and intestines with the constituents for glu­tathione resynthesis.

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