I. Introduction

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Glutathione (GSH)^b performs a variety of important physiological and metabolic functions in all mammalian cells, including the detoxification of free radicals, metals, and other electrophilic compounds. One important detoxification mechanism involves the binding of GSH to electrophilic chemicals and the export of the resulting GSH S-conjugates from the cell. These conjugation reactions have been extensively characterized for a multitude of foreign chemicals, but they are also critical for the metabolism of endogenous reactive intermediates and for the formation of specific biological mediators. GSH forms thioether conjugates with leukotrienes, prostaglandins, hepoxilin, nitric oxide, hydroxyalkenals, ascorbic acid, dopa, dopamine, and maleic acid, and it forms thioesters with cysteine, coenzyme A, proteins, and other cellular thiols. The glycine carboxyl group of GSH binds to the amino group of spermidine to produce GSH-amides in reactions catalyzed by glutathionylspermidine synthetase and trypanothione synthetase in bacterial systems. GSH also binds endogenous metals, such as copper, selenium, chromium, and zinc, via nonenzymatic reactions.

The binding of GSH to these endogenous compounds serves several important roles: (a) it serves to limit and regulate the reactivity of the chemicals; (b) it facilitates their membrane transport and elimination from the cell and organism; and (c) in some cases, it leads to the formation of essential biological mediators. The cysteinyl leukotrienes, for example, are involved in inflammatory and anaphylactic reactions. The GSH conjugate of 9-deoxy-⁹, ¹²-prostaglandin D₂ may modulate the antiproliferative activity of the parent compound (Atsmon et al., 1990a), whereas S-nitrosoglutathione, a relatively stable intermediate derived from the nonenzymatic reaction of nitric oxide with intracellular GSH, seems to have the same biological functions as nitric oxide itself (Ignarro, 1990).

Many proteins are activated or inhibited in vitro by the disulfide exchange between the protein and GSH. Glutathione disulfide (GSSG) can activate enzymes such as glucose-6-phosphatase, acid phosphatase, -aminolaevulinate synthetase, and fructose 16-bisphosphatase; however, it inhibits glycogen synthetase, pyruvate kinase, adenylate cyclase, phosphorylase/phosphatase, ribonucleotide reductase, phosphofructokinase, glycogen debranching enzyme, and fatty acid synthase.

The present discussion reviews the occurrence and biological functions of the endogenous GSH S-conjugates.

	II. Glutathione Homeostasis

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GSH is a tripeptide (L--glutamyl-L-cysteinyl-glycine) that serves several essential functions within the cell (Ballatori, 1994; Deleve and Kaplowitz, 1991; Meister and Tate, 1976; Meister and Anderson, 1983). It is the most abundant nonprotein thiol in almost all aerobic species, occurring at intracellular concentrations of 0.5 to 10 mM. In contrast, extracellular GSH concentrations are usually 3 to 4 orders of magnitude lower. Under physiological conditions, GSSG reductase maintains more than 98% of intracellular GSH in the reduced, thiol form (GSH). The rest is present within the cell as mixed disulfides (mainly GS-S-protein), as the disulfide (GSSG), and as thioethers.

The key functional element of the GSH molecule is the cysteinyl moiety, which provides the reactive thiol group and is responsible for the many functions of GSH. These functions include (a) the maintenance of protein structure and function by reducing the disulfide linkages of proteins, (b) the regulation of protein synthesis and degradation, (c) the maintenance of immune function, (d) protection against oxidative damage, and (e) detoxification of reactive chemicals. GSH also serves as a storage and transport form of the cysteine moiety, and it functions in (a) leukotriene and prostaglandin metabolism, (b) the reduction of ribonucleotides to deoxyribonucleotides, (c) the modulation of microtubule-related processes, and (d) bile formation (for reviews see Deleve and Kaplowitz, 1991; Gilbert, 1982; Hinchman and Ballatori, 1994; Kosower and Kosower, 1978; Meister and Tate, 1976; Meister and Anderson, 1983).

The key structural elements of GSH are the -carboxyl peptide linkage of glutamate and the presence of the C-terminal glycine, which directly determine its metabolism and function. The N-terminal glutamyl and cysteinyl moieties are linked through the -carboxyl group of glutamate instead of the more common -carboxyl peptide linkage, restricting cleavage to -glutamyl transpeptidase (GT), which occurs on the external surface of certain cell membranes. Therefore, GSH is resistant to intracellular degradation and can only be cleaved by cell types that have Gt on the cell membrane. The presence of the C-terminal glycine protects the peptide against cleavage by intracellular -glutamylcyclotransferase.

GSH is synthesized in all mammalian cells (Meister and Tate, 1976), and the liver is a major site of biosynthesis (Deleve and Kaplowitz, 1991). The synthesis of GSH from its three amino acid precursors L-glutamate, L-cysteine, and glycine takes place in the cytosol (fig. 1). It is a two-step reaction, catalyzed by -glutamylcysteine synthetase and by GSH synthetase, that requires two moles of adenosine triphosphate (ATP) per mole of GSH (fig. 1). The first step, catalyzed by -glutamylcysteine synthetase, is controlled by negative feedback from its end product, GSH (Richman and Meister, 1975). However, feedback inhibition can be partially prevented by an excess of glutamate that blocks the regulatory site on the enzyme (Meister, 1984; Meister and Anderson, 1983; Richman and Meister, 1975). When GSH is consumed and feedback inhibition is lost, the availability of cysteine as a precursor can become the limiting factor.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   GSH synthesis and degradation. In the first step of GSH synthesis, an amide linkage is formed between cysteine and glutamate catalyzed by the -glutamylcysteine synthetase. GSH synthetase then catalyzes the reaction between glycine and the cysteine carboxyl of -glutamylcysteine dipeptide to form GSH. GSH is transported out of the cell and broken down by the membrane-bound enzyme GT, which removes the -glutamyl moiety, and by dipeptidases, which remove the glycine moiety. The resulting amino acids can be reabsorbed and used for additional GSH synthesis. ADP, adenosine 5'diphosphate; Cys, cysteine; Glu, glutamate; Gly, glycine; Pi, inorganic phosphate.

Although GSH is synthesized inside the cell, its biodegradation occurs outside of cells (fig. 1). The enzymes that catalyze its breakdown are GT and dipeptidases, which are membrane-bound proteins that are located predominantly on the apical surface of epithelial tissues. GT is the only enzyme that removes the -glutamyl moiety from GSH under physiological conditions. Dipeptidases remove the glycyl moiety. The breakdown products (glutamate, glycine, and cysteine) can be reabsorbed into the cell for GSH synthesis (fig. 1).

The GSH S-conjugates are metabolized by the same degradative enzymes that metabolize GSH (fig. 2). The breakdown products of the GSH S-conjugates are glutamate, glycine, and cysteine, which can also be reabsorbed into the cell. The glutamate and glycine may then be used for GSH synthesis, whereas the cysteine S-conjugates can be acetylated on the amino group of the cysteinyl residue by intracellular N-acetyltransferases to form the corresponding mercapturic acids (N-acetylcysteine S-conjugates). Mercapturic acids are released into the circulation or bile (Hinchman et al., 1991); some are eventually excreted in urine, and some may undergo further metabolism. The addition of the N-acetylcysteine moiety generally increases a compound's polarity and water solubility, and converts neutral compounds to anions, facilitating their transport across cell membranes and their excretion from the organism (Boyland and Chasseaud, 1969).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Metabolic and transport steps in mercapturic acid biosynthesis. The GSH S-conjugate is made intracellularly and then transported out of the cell for subsequent degradation by the ectoproteins like GT and the dipeptidases. The cysteine S-conjugate that is formed is transported back into the cell and N-acetylated to form mercapturic acid. (This figure was adapted with permission from Hinchman and Ballatori, 1994, J Toxicol Environ Health 41:387-409.) Ac, acetyl; CoA, coenzyme A; Cys, cysteine; E, electrophile; Glu, glutamate; Gly, glycine; S-E, thiol-conjugated electrophile; SH, sulfhydryl group.

B. Biological Functions

1. Regulation of cellular sulfhydryl status. a. REDOX REACTIONS. All aerobic organisms are subject to physiological oxidant stress as a consequence of aerobic metabolism. The intermediates that are formed, including superoxide and hydrogen peroxide, lead to the further production of toxic oxygen radicals that can cause lipid peroxidation and disrupt metabolic processes. GSH is the predominant defense against these toxic products of oxygen, particularly in the mitochondria, a major site for the synthesis of reactive oxygen intermediates (Deleve and Kaplowitz, 1991). Mitochondrial GSH is critical in the defense against both physiologically and pathologically generated oxidant stress. Mitochondria do not have the enzymes necessary for GSH synthesis, and they import cytosolic GSH. Because catalase is compartmentalized in the peroxisome, mitochondrial GSH in the presence of GSH peroxidase is the main defense against toxic oxygen intermediates.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   The detoxification functions of GSH include the conjugation of electrophilic chemicals and reactive metals and the maintenance of the cellular thiol redox status. AH2 and A, reduced and oxidized forms, respectively, of compounds that participate in the synthesis of NADPH; GSH, reduced glutathione; GSSG, glutathione disulfide; NADPH and NADP+, reduced and oxidized forms, respectively, of nicotinamide adenine dinucleotidephosphate; RH and R, reduced and oxidized forms, respectively, of some organic molecules; ROOH, a hydroperoxide; RDH, an alcohol; RS-SG, a glutathione thioester; RS-SR', a mixed disulfide of two organic molecules.

2. Conjugation of electrophiles and metals. GSH plays a major role in detoxifying many reactive metabolites by either spontaneous conjugation or by a reaction catalyzed by the GSH S-transferases (Boyland and Chasseaud, 1969; Coles and Ketterer, 1990; Hinchman and Ballatori, 1994; Jakoby, 1978; Mannervik, 1985). GSH S-transferases have broad and overlapping substrate specificities, which allow them to participate in the detoxification of a chemically diverse group of compounds. The most common reactions involve nucleophilic attack by GSH on an electrophilic carbon: saturated carbon atoms (e.g., alkyl halides, lactones and epoxides), unsaturated carbon atoms (e.g., , -unsaturated compounds, quinones and quinonimines, and esters), or aromatic carbon atoms (e.g., aryl halides and aryl nitro compounds; Douglas, 1988). The substrates have in common a degree of hydrophobicity and possess electrophilic centers that undergo nucleophilic substitution, nucleophilic addition to , -unsaturated ketones or epoxides or, in the case of hydroperoxides, nucleophilic attack on electrophilic oxygen, resulting in reduction.

	III. Endogenous Glutathione Thioethers

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In addition to the many exogenous electrophilic chemicals, a multitude of endogenous compounds also react with GSH to form adducts (tables 1 and 2). Some of the endogenous electrophiles are produced as byproducts of lipid peroxidation, which may be caused by free radicals or high-energy irradiation (Slater, 1984). Many of these compounds are cytotoxic or genotoxic. Without detoxification, the result is a cascade of reactions leading to further radical damage, membrane decomposition, and free-radical attack on other cellular targets, such as DNA.

A. Leukotriene C₄ (LTC₄)

1. Synthesis and biological actions. Leukotrienes are potent biological mediators that are formed in response to a variety of immunological and inflammatory stimuli (Denzlinger et al., 1985; Hammarstrom, 1983; Lewis and Austen, 1984; Piper, 1984; Samuelsson, 1983). They are synthesized by a variety of white blood cells, including macrophages, monocytes, neutrophils, eosinophils, and mast cells (connective tissue cells derived from the blood-forming tissues that secrete substances that mediate inflammatory and allergic reactions), as well as cells in the lung, spleen, brain, and heart (Samuelsson, 1983; Lewis and Austen, 1984; Hammarstrom, 1983; Piper, 1984). In addition, Kupffer cells release LTC₄(D₄)-like material upon stimulation (Decker, 1985). After production, leukotrienes are released by the cells and can be detected in various body fluids.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   The formation of LTA4 from arachidonic acid via the 5-lipoxygenase pathway and the conversion of LTA4 to LTC4 and LTB4. 5-HPETE, 5-hydroperoxyeicosatetraenoic acid.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Structures of LTC4 and its degradation products. The sequential cleavage of glutamate and glycine from LTC4 by the ectoenzymes GT and dipeptidase yield LTD4 and LTE4, respectively. The acetylation of LTE4 yields N-acetyl-LTE4. Intracellular -oxidation of LTE4 results in the formation of -hydroxy-LTE4 and -carboxy-LTE4. Subsequent -oxidation from the -end leads to -carboxy-dinor-LTE4 and -carboxy-tetranor-dihydro-LTE4.

2. Leukotriene metabolism. The rapid removal of the cysteinyl leukotrienes in inflamed tissues is an important mechanism for their inactivation. The cysteinyl leukotrienes are inactivated in primates by (a) intravascular degradation, (b) hepatic and renal uptake from the blood circulation, (c) intracellular metabolism, and (d) biliary and renal excretion of LTC₄ degradation products (Huber et al., 1990)

Almost all mammalian cells produce prostaglandins (Eling et al., 1990; Smith, 1992). They were first identified in human semen in the early 1930s via their ability to stimulate uterine contractions and lower blood pressure. The physiological effects of prostaglandins have now been well described; they are similar to those produced by other eicosanoids, such as leukotrienes. They are involved in the inflammatory response, the production of pain and fever, the regulation of blood pressure, the induction of blood clotting, the control of several reproductive functions (such as the induction of labor), and the regulation of the sleep/wake cycle.

Prostaglandins are synthesized from the same precursor as leukotrienes, arachidonic acid, in a reaction catalyzed by the enzyme prostaglandin endoperoxide synthase (Miyamoto et al., 1976; fig. 6). This enzyme has two catalytic activities: a cyclooxygenase activity and a hydroperoxidase activity. The former catalyzes the addition of two molecules of oxygen to arachidonic acid, forming prostaglandin G₂. The latter mediates a GSH-dependent reaction that converts the hydroperoxy function of prostaglandin G₂ to a hydroxyl group and forms prostaglandin H₂ (PGH₂). PGH₂ is the immediate precursor of all of the major biologically active products of this pathway, including those that form prostaglandin D₂ (PGD₂), prostaglandin E₂ (PGE₂), and prostaglandin F_2. (fig. 6). Christ-Hazelhof et al. (1976) found that GSH S-transferase isozymes had PGH₂-converting activity; this was further confirmed by Ujihara et al. (1988). They demonstrated that PGD₂, PGE₂, and prostaglandin F₂ were formed from PGH₂ by various rat GSH S-transferase isozymes.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Biosynthesis of PGE2, PGF2, and PGD2 from arachidonic acid.

PGE₂ can be transformed to the dehydration product prostaglandin A₂ (PGA₂), and PGE₁ to PGA₁ (fig. 7; Santoro et al., 1986; Turner et al., 1982; Suzuki et al., 1988; Ikai et al., 1987). Both PGA₁ and PGA₂ are potentially toxic, and GSH can conjugate with both to form S-(PGA)-GSH derivatives (Bogaards et al., 1997; Cagen et al., 1975; Ham et al., 1975; fig. 7). The inhibitory effect of PGA on the proliferation of tumor cells was markedly diminished when GSH was added to cell culture medium containing PGA (Honn and Marnett, 1985). Conversely, GSH-depleted cells are insensitive to the cytotoxicity of PGA₂, suggesting that PGA₂-GSH conjugates may be involved in the cytotoxicity of PGA₂ (Parker and Ankel, 1992). Parker and Ankel (1992) demonstrated that PGA₂ is initially conjugated to GSH and then reduced at the 9-keto position to form S-(9-hydroxy-PGA₂)-GSH. This conjugate is then secreted from the cells and is apparently degraded to form the CysGly and Cys derivatives.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Conversion of PGE1 to PGA1, and PGE2 to PGA2, conjugation of PGA1 and PGA2 with GSH, and reduction at the keto group to form the respective (9-hydroxyl-PGA)-GSH conjugates.

PGD₂-derived compounds are members of a large group of eicosanoids that display cytotoxic activity. A common feature in all these compounds is the presence of a reactive , -unsaturated ketone in the cyclopentenone ring. Prostaglandins that lack an , -unsaturated ketone are not cytotoxic, and it has been assumed that the , -unsaturated ketone is essential for exerting cytotoxic activity (Kato et al., 1986; Bregman et al., 1986; Honn and Marnett, 1985). As illustrated in figure 8, PGD₂ can be transformed to the dehydration product 9-deoxy- ⁹-PGD₂ (also called prostaglandin J₂), 9-deoxy-¹²-PGD₂, and 9-deoxy- ⁹, ¹²-PGD₂ (also called ¹²-prostaglandin J₂; Kikawa et al., 1984; Fitzpatrick and Wynalda, 1983). These compounds exert much greater cytotoxic activity than PGD₂ and most likely are responsible for the cytotoxicity originally ascribed to PGD₂ (Atsmon et al., 1990a; Fukushima et al., 1982; Tanaka et al., 1985; Narumiya and Fukushima, 1985; Kikawa et al., 1984).

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Conversion of PGD2 to 9-deoxy-9-PGD2 (also called prostaglandin J2), 9-deoxy-12-PGD2, and 9-deoxy-9, 12-PGD2, and the conjugation of these metabolites with GSH.

, -Unsaturated ketones are very susceptible to nucleophilic addition reactions with thiols such as GSH. Atsmon et al. (1990b) found that 9-deoxy- ⁹, ¹²-PGD₂, a potent inhibitor of cell proliferation, and 9-deoxy-¹²-PGD₂ are conjugated in vitro with GSH to form S-(9-deoxy- ⁹, ¹²-PGD₂)-GSH conjugate and S-(9-deoxy-¹²-PGD₂)-GSH conjugate, respectively (fig. 8). After conjugation, further metabolism leads to reduction by the enzyme prostaglandin 11-ketoreductase at the 11-keto and (12-13) moieties to form S-(11-hydroxy-9deoxy- ⁹, ¹²-PGD₂)-GSH and S-(11-hydroxy-9-deoxy-¹²-PGD₂)-GSH (fig. 8). Prior depletion of intracellular GSH decreases the amount of intracellular conjugated prostaglandin and significantly enhances the antiproliferative effect of 9-deoxy- ⁹, ¹²-PGD₂ on cell growth (Atsmon et al., 1990a). Intracellular GSH also suppresses the binding of 9-deoxy- ⁹, ¹²-PGD₂ to nuclei, and inhibits 9-deoxy- ⁹, ¹²-PGD₂-induced synthesis of heme oxygenase and P67 (a 67-kDa heat shock protein) in porcine aortic endothelial cells (Koizumi et al., 1992). Thus, intracellular GSH may modulate the antiproliferative activity of 9-deoxy- ⁹, ¹²-PGD₂ and, possibly, of other cytotoxic prostaglandins.

Hepoxilins are biologically active epoxy alcohols formed from arachidonic acid by initial 12-lipoxygenation and subsequent intramolecular rearrangement of (12S)-hydroperoxy-eicosatetraenoic acid (Pace-Asciak et al., 1983; Pace-Asciak, 1984; fig. 9). There are two position-isomeric hepoxilins isolated, hepoxilin A₃ and hepoxilin B₃. Hepoxilin A₃ is capable of modulating synaptic neurotransmission and neuronal excitability, suggesting a role in neurotransmission (Carlen et al., 1989).

View larger version (12K): [in this window] [in a new window]   Fig. 9.   Formation of hepoxilin A3 from arachidonic acid, and the GSH conjugation of hepoxilin A3 to form hepoxilin A3-C.

The epoxide moiety of hepoxilins is subject to reaction with epoxide hydrolase or with GSH. As illustrated in figure 9, GSH S-transferase catalyzes the conjugation reaction in which GSH is attached to the 11-carbon position of hepoxilin A₃ to form hepoxilin A₃-C (Carlen et al., 1994; Pace-Asciak et al., 1989, 1990). The biological significance of this GSH conjugation is unknown, although Pace-Asciak et al. (1990) and Carlen et al. (1994) demonstrated that the GSH conjugate of hepoxilin A₃ causes a hyperpolarization of hippocampal CA1 neurons, once again suggesting a role in neurotransmission.

Nitric oxide (NO) is an endogenous reactive intermediate, a free radical, generated by the enzyme NO synthase (Katsuki et al., 1977). NO synthase catalyzes the conversion of L-arginine to L-citrulline and NO, with a 1:1 stoichiometry, and is competitively inhibited by analogues of L-arginine, such as N^G-monomethyl-L-arginine (Palmer et al., 1988; Palmer and Moncada, 1989). The reaction needs NADPH as a reducing cofactor, and an amino acid radical is a likely intermediate (Marletta, 1989). It is now evident that cells other than those in the vascular endothelium, including fibroblasts, macrophages, neutrophils, and neurons, synthesize NO.

NO was identified as the endothelium-derived relaxing factor (EDRF), an endogenous vasodilator (Ignarro, 1990; Palmer et al., 1987). EDRF/NO (a) activates a soluble heme-containing guanylate cyclase (Waldman and Murad, 1987); (b) stimulates guanosine 3', 5'-cyclic monophosphate (cGMP) formation; and (c) causes vascular smooth muscle relaxation, platelet aggregation inhibition, neurotransmission modulation, and macrophage cytotoxicity (Ignarro, 1990). NO also inhibits neutrophil functions, which include LTB₄ synthesis, superoxide anion (O₂) release, and neutrophil adhesion to the vascular endothelium (Ney et al., 1990; Kubes et al., 1991).

In view of the fact that the plasma and cellular milieux contain reactive species that can rapidly inactivate NO, it has been postulated that NO is stabilized by a carrier molecule that preserves its biological activity. Reduced thiol species are candidates for this role, as they readily react in the presence of NO to yield biologically active S-nitrosothiols that are more stable, and possibly more potent, than NO itself (Ignarro et al., 1981; Mendelsohn et al., 1990). Sulfhydryl groups in proteins, and free cysteine and GSH represent an abundant source of reduced thiol in biological systems. There is increasing evidence that at least part of the activity of NO is attributable to S-nitrosothiols derived from the reaction of NO with intracellular thiol compounds like cysteine or GSH (Ignarro, 1990; Myers et al., 1990). S-nitrosothiols may play the same role in the mechanism of action of EDRF as NO; the potent and long-lasting effects of vasodilation and platelet inhibition that they cause are mediated by guanylate cyclase activation (Ignarro et al., 1981; Mellion et al., 1983). These observations suggest that S-nitrosothiol groups in proteins may serve as intermediates in the cellular metabolism or bioactivity of NO and that their formation may represent an important cellular regulatory mechanism (Stamler et al., 1992). S-nitrosothiols have also been proposed as biologically active intermediates in the metabolism of organic nitrates (Ignarro et al., 1981; Mellion et al.; 1983; Loscalzo, 1985).

S-nitrosoglutathione (GS-NO) has been found endogenously in neutrophils and human airways at micromolar concentrations (Gaston et al., 1993; Clancy et al., 1994). Because GS-NO can yield NO after a one-electron reduction, several investigators have suggested that the physiological role of NO may be mediated by GS-NO and other S-nitrosothiols (Mendelsohn et al., 1990; Armstrong and Stave, 1973). Clancy and Abramson (1992) indicate that neutrophils have the potential to degrade GS-NO and to lower tissue levels of GS-NO, which may limit its biological activity. The degradation of GS-NO by activated neutrophils may contribute to tissue injury at sites of inflammation. Recently Clancy et al. (1994) found that NO reacts with intracellular GSH and activates the hexose monophosphate shunt in human neutrophils, providing additional evidence that GS-NO is a bioactive intermediary that may regulate cellular functions. GS-NO also inhibits the sarcoplasmic reticulum-bound creatine kinase (Wolosker et al., 1996), an enzyme that plays a crucial role in the process of energy transduction

NO synthesis is impaired in GSH-depleted human umbilical vein endothelial cells (Ghigo et al., 1993), suggesting that NO synthesis requires GSH and that GSH could play different roles in NO synthesis and its effects, such as (a) acting as a reducing cofactor for NO production, in concert with NADPH; (b) preventing early inactivation of NO synthase by radical intermediates or NO itself; or (c) favoring conversion of NO to GS-NO, which is relatively more stable than NO itself.

The nitrovasodilators amyl and n-butyl nitrite have been used as recreational drugs for years, and Meloche and O'Brien (1993) discovered that the NO formed when hepatocytes are incubated with n-butyl nitrite mediates many of the cytotoxic effects of this drug, including ATP depletion, lipid peroxidation, and membrane disruption. The formation of GS-NO from alkyl nitrite and GSH may be mediated by the GSH S-transferases (Meyer et al., 1994).

Lipid peroxidation often occurs in response to oxidative stress, and many kinds of aldehydes are formed when lipid hydroperoxides break down in biological systems. The main process leading to aldehydes is likely to be the -cleavage of lipid hydroperoxides (Esterbauer et al., 1990; Grosch, 1987). The most intensively studied aldehydes so far are the 4-hydroxyalkenals, in particular, 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE). HNE is an , -unsaturated aldehyde that is produced during the oxidation of membrane lipid polyunsaturated fatty acids, such as arachidonic acid and linoleic acid. HHE is formed through both lipid peroxidation (by the degradation of  3 polyunsaturated fatty acids) and through nonperoxidative mechanisms, such as the metabolism of the alkaloid senecionine (Segall et al., 1985).

Unlike reactive free radicals, aldehydes are rather long lived and, therefore, can diffuse from their site of origin (i.e., membranes) to reach and attack other targets intracellularly or extracellularly. Numerous biological effects are associated with 4-hydroxyalkenals, including the inhibition of DNA, ribonucleic acid, and protein synthesis; cell proliferation; and the production of various genotoxic effects (Esterbauer et al., 1991).

GSH reacts with 4-hydroxyalkenals to form the structure illustrated in figure 10 (Esterbauer et al., 1975). The initial product is a saturated aldehyde with the GSH residue bound by a thioether linkage at the carbon atom, which then undergoes an intramolecular rearrangement to the five-membered cyclic hemiacetal (fig. 10). One of the primary metabolites of HNE is the GSH S-conjugate (Esterbauer et al., 1991). Grune et al. (1994) found that the main products of HNE, as identified in Ehrlich ascites cells, were GSH S-conjugate, hydroxynonenoic acid, and 14-dihydroxynonene, which is consistent with previous findings. GSH S-transferases are the main enzymes involved in the metabolism of these lipid-derived intermediates (Danielson et al., 1987). The enzyme-catalyzed reaction proceeds approximately 300 to 600 times faster than the nonenzymatic reaction. Danielson et al. (1987) have proposed that some of the GSH S-transferases may have evolved specifically for the detoxification of HNE and similar aldehydes resulting from lipid peroxidation. The HNE-GSH adduct exhibits a feedback inhibition on the GSH S-transferases. HNE is now used frequently as a substrate to characterize the various types of GSH S-transferases (Tsuchida and Sato, 1990). In rats, the liver was found to have the highest capacity to metabolize HNE (Esterbauer et al., 1985).

View larger version (16K): [in this window] [in a new window]   Fig. 10.   Conjugation of 4-hydroxyalkenals with GSH.

HHE also conjugates with GSH (fig. 10). Experiments with injecting tritium-labeled HHE into the portal veins of rats indicate that a part of the radioactivity is excreted in urine as the C-3 mercapturic acid conjugate (Winter et al., 1987). Winter et al. (1987) demonstrated that in the whole animal, the HHE-GSH conjugate is exported from the liver and further metabolized to a mercapturic acid. The degradation of HNE to its mercapturic acid can occur in the rat kidney, as well as in other tissues (Petras et al., 1995).

Ascorbic acid and GSH are among the most active reducing substances in living tissues (Meister, 1994; Winkler et al., 1994). Both of these chemicals undergo redox cycling in vivo, and there seems to be a significant interrelationship in this cycling. For example, the toxic effects of GSH deficiency can be prevented by administering ascorbate, indicating that ascorbate has a "sparing" effect on GSH (Meister, 1994). Conversely, GSH seems to be required for the regeneration of ascorbic acid from its oxidized form, dehydroascorbate (Meister, 1994; Winkler et al., 1994). The mechanism for the latter conversion most likely involves a two-electron reduction of dehydroascorbate by GSH, with the intermediate formation of a GSH-ascorbic acid conjugate (fig. 11). The initial step is the nucleophilic addition of GSH to the central carbonyl of dehydroascorbate, followed by reduction, by another GSH molecule, to yield ascorbate and GSSG (fig. 11). Winkler et al. (1994) indicate that this nonenzymatic reaction between GSH and dehydroascorbate is the major mechanism for the reduction of dehydroascorbate in mammalian tissues.

View larger version (10K): [in this window] [in a new window]   Fig. 11.   A proposed mechanism for the two-electron reduction of dehydroascorbic acid by GSH.

As illustrated in figure 12, GSH can conjugate with dopaquinone, dopaminequinone, and methyldopaquinone, which are derived from dopa, dopamine, and methyldopa, respectively; however, the physiological significance of this conjugation is not clear. Glutathionedopa was initially found in malignant melanoma (Agrup et al., 1977) and was thought to be an intermediate in the formation of 5-S-cysteinyldopa. Agrup et al. (1977) demonstrated that 5-S-cysteinyldopa, an amino acid that plays a key role in pigment cell metabolism, was formed by conjugation of GSH to dopaquinone (fig. 12), with the subsequent hydrolysis of the peptide by GT and a dipeptidase. Similar findings by Fornstedt et al. (1986) and Palumbo et al. (1995) demonstrated the occurrence of 5-S-cysteinyldopa, 5-S-cysteinyldopac, and 5-S-cysteinyldopamine in the brains of all eight mammalian species investigated (i.e., human, rhesus monkey, marmoset monkey, horse, sheep, dog, cat, and rat), indicating that the autooxidation of catechols followed by the coupling to GSH and the attack by peptidases is a normal metabolic pathway in the mammalian brain.

View larger version (17K): [in this window] [in a new window]   Fig. 12.   Conjugation of GSH with dopamine, dopa, and -methyldopa.

The covalent interaction of quinones with essential protein sulfhydryls may result in enzyme inhibition (Monks and Lau, 1992). Dopamine and its analogs were shown to inhibit melanoma growth by the inhibition of DNA polymerase , probably by interacting with a sulfhydryl group (Wick, 1980). Ploemen et al. (1994) demonstrated that the human GSH S-transferases were inhibited by dopamine, -methyldopa, and their 5-S-glutathionyl conjugates.

-Methyldopamine (-MeDA) is a metabolite of the serotonergic neurotoxicants 34-(±)-(methylenedioxy)amphetamine (MDA) and 34-(±)-(methylenedioxy)methamphetamine (MDMA; Miller et al., 1995). -MeDA readily oxidizes, and in the presence of GSH, it forms 5-(S-glutathionyl)--methyldopamine (MeDA-SG; Miller et al., 1995). MeDA-SG is metabolized by the brain to 5-(S-cysteinyl)--MeDA and 5-(N-acetyl-L-cysteinyl)--MeDA, demonstrating that the brain possesses a functional mercapturic acid pathway (Miller et al., 1995). Because all the thiol conjugates of -MeDA remain susceptible to oxidation, the presence and persistence of these metabolites in brain tissue may contribute to the neurotoxicity of MDA and 34-(±)- (methylenedioxy)methamphetamine. Evidence for this was provided by Miller et al. (1996), who showed that intracerebroventricular administration of MeDA-SG to male Sprague-Dawley rats causes behavioral changes identical to those observed after the subcutaneous administration of MDA.

More recently, a novel antibacterial substance from immunized adult Sarcophaga (flesh fly) was purified; its molecular structure was found to be N--alanyl-5-S-glutathionyl-34-dihydroxyphenylalanine, which was synthesized enzymatically from N--alanyl-34-dihydroxyphenylalanine (-Ala-Dopa) and GSH (Leem et al., 1996).

S-(12-Dicarboxyethyl)GSH (DCE-SG; fig. 13) was isolated from calf lenses in 1963 (Calam and Waley, 1963). It was found in concentrations of 119 nmol per gram of tissues in the rat lens and 72 nmol per gram of tissues in the rat liver (Tsuboi et al., 1990a). In addition, it was found in the rat heart, but not in the rat spleen, kidney, cerebrum, or cerebellum (Tsuboi et al., 1990a).

View larger version (6K): [in this window] [in a new window]   Fig. 13.   S-(12-dicarboxyethyl)-GSH synthesis from L-malate and GSH. The enzyme that catalyzes this reaction may not belong to the GSH S-transferase family but may be a novel type of GSH-conjugating enzyme (Tsuboi et al., 1990a).

DCE-SG is synthesized in cells from GSH and L-malate (fig. 13), but the enzyme catalyzing this reaction does not belong to the GSH S-transferase family and is thought to be a novel type of GSH-conjugating enzyme (Tsuboi et al., 1990a).

DCE-SG shows strong inhibitory effects on blood coagulation and platelet aggregation (Tsuboi et al., 1990b), but the mechanism and its significance in vivo is undefined. A possible mechanism for the inhibition of platelet aggregation by DCE-SG may be related to the enhancement of cyclic AMP level and adenylate cyclase activity in platelets produced by this GSH S-conjugate (Tsuboi et al., 1993).

GSH can function as a coenzyme in several enzyme-catalyzed reactions, including the glyoxalase reaction (fig. 14). In this reaction, GSH serves as a coenzyme that converts methylglyoxal to D-lactate (Behrens, 1941; Racker, 1951; Meister and Anderson, 1983). First, methylglyoxal interacts with GSH nonenzymatically to form the hemimercaptal, the substrate for the reaction catalyzed by glyoxalase I to form S-lactyl-GSH. S-lactyl-GSH is then converted by glyoxalase II into GSH and D-lactate (fig. 14).

View larger version (11K): [in this window] [in a new window]   Fig. 14.   Glyoxalase reaction: GSH serves as a coenzyme in the conversion of methyglyoxal to D-lactate.

The biological significance of this reaction has long been a mystery. Some have suggested that ketoaldehydes play a significant role in the regulation of cell division (Szent-Gyorgyi, 1965; Egyud and Szent-Gyorgyi, 1966). According to this idea, methylglyoxal retards cell growth and glyoxalase promotes cell growth by destroying methylglyoxal, but this has not been definitively demonstrated.

	IV. Glutathione Amides: Glutathionylspermidine and Trypanothione

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The polyamine spermidine, derived from arginine and methionine, is found in all bacteria and most animal cells. It is a growth factor for some microorganisms and serves to stabilize the membrane structures of bacteria, as well as the structure of ribosomes, some viruses, and the DNA of many organisms.

A GSH-spermidine conjugate, N¹-monoglutathionylspermidine (GspdSH), was initially detected in Escherichia coli (E. coli)under stationary or anaerobic growth conditions (Tabor and Tabor, 1975; fig. 15). Subsequently, GspdSH and the N¹, N⁸-bis(glutathionyl)spermidine conjugate, termed trypanothione, were identified in the pathogenic protozoa of genera Trypanosoma and Leishmania (Fairlamb et al., 1985; Fairlamb et al., 1986), but not in E. coli. These GSH-spermidine conjugates seem to be physiologically important to these organisms. Smith et al. (1995) showed that under anaerobic and stationary-phase conditions, 80% of the total GSH in E. coli is in the form of GspdSH, suggesting that GspdSH may be a better DNA-protectant against radical- or oxidant-induced damage than GSH. Some think that parasites maintain redox balance and defend against oxidant stress by synthesizing trypanothione, because the parasites lack typical catalase and GSH peroxidase-GSSG reductase enzyme couples (Bollinger et al., 1995). Therefore, trypanothione metabolism has been considered a possible target for new antiparasitic drugs (Fairlamb et al., 1985).

View larger version (11K): [in this window] [in a new window]   Fig. 15.   Biosynthesis of trypanothione in bacterial systems. Conjugation of the glycine carboxyl of GSH and the first amino group of spermidine is catalyzed by glutathionylspermidine synthetase. Trypanothione synthetase then catalyzes the addition of a second molecule of GSH to glutathionylspermidine to form trypanothione.

Glutathionylspermidine synthetase was initially partially purified from E. coli (Tabor and Tabor, 1966). Recently, Bollinger et al. (1995) purified E. coli glutathionylspermidine synthetase to near homogeneity, and the gene encoding it was isolated and sequenced. As illustrated in figure 15, glutathionylspermidine synthetase couples ATP hydrolysis with the formation of an amide bond between spermidine and the glycine carboxylate of GSH. This enzyme was also found to have a second catalytic activity, glutathionylspermidine amide bond hydrolysis. Thus, the bifunctional glutathionylspermidine synthetase/amidase catalyzes opposing amide bond-forming and cleaving reactions, with a net hydrolysis of ATP. Trypanothione synthase then catalyzes the addition of the second GSH molecule to the free primary amine of glutathionylspermidine to form trypanothione. The lack of the trypanothione in E. coli seems to be because of the absence of trypanothione synthase (Smith et al., 1995).

	V. Glutathione Thioesters

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It has been discovered that many enzymes are activated or inhibited in vitro by the disulfide exchange between the protein and small-molecule disulfides. The fundamental equation for a thiol/disulfide exchange is: where R and R' are either cysteine, cysteine-containing peptides, or other sulfhydryl-containing compounds.

The thiol/disulfide ratio seems to be of great importance in the regulation of cellular metabolism, and GSH plays the key role in regulating the thiol/disulfide exchange (Gilbert, 1982).

Many enzymes are activated by GSSG, including glucose-6-phosphatase, acid phosphatase, -aminolaevulinate synthetase, and fructose 16-bisphosphatase (Gilbert, 1982; Ondarza, 1989). Enzymes that are inhibited by GSSG include glycogen synthetase D, pyruvate kinase, adenylate cyclase, phosphorylase phosphatase, ribonucleotide reductase, phosphofructokinase, glycogen debranching enzyme, fatty acid synthase, and 15 OH-prostaglandin dehydrogenase. It has been reported that GSSG possesses the ability to stimulate microsomal GSH S-transferase activity (Nishino and Ito, 1989) and to cause the sensitization of calcium release to inositol 14,5-trisphosphate in permeabilized hepatocytes (Renard et al., 1992).

Similar processes are involved for coenzyme A and the GSH-coenzyme A mixed disulfide (CoASSG). CoASSG was initially identified in the rat (Ondarza and Aubanel, 1960; Ondarza, 1965) and bovine liver (Wilken and Hansen, 1961; Chang and Wilken, 1965). Numerous reports indicate that these sulfur compounds are normal components of both eukaryotic cells and many eubacteria, and that they play an important role in enzyme regulation. For instance, CoASSG was found to inhibit GSSG reductase (Ondarza, 1966), phosphofructokinase (Gilbert, 1982), and fatty acid synthase (Walters and Gilbert, 1986). In contrast, fructose 16-bisphosphatase is activated by CoASSG (Nakashima et al., 1969).

Intracellular GSSG levels rise during hydroperoxide metabolism, and these perturbations of GSH status are accompanied by significant metabolic changes that are mediated by the formation of mixed disulfides between GSSG and thiol groups present in proteins (enzymes) or low molecular weight compounds (Akerboom et al., 1982, 1984; Kosower and Kosower, 1978; Crane et al., 1982, 1983). For example, Crane et al. (1982) found that there is a rise of CoASSG during hydroperoxide metabolism in perfused rat liver but that coenzyme A disulfide levels are virtually unchanged. Thus, as a result of the metabolism of hydroperoxides, a sulfhydryl exchange occurs between the elevated levels of GSSG and cellular coenzyme A, with