The effects of stress and aging on glutathione metabolism

Ageing Research Reviews 4 (2005) 288–314 www.elsevier.com/locate/arr

Review

The effects of stress and aging on glutathione metabolism Pamela Maher * The Salk Institute, 10010 N. Torrey Pines Rd., La Jolla, CA 92037, USA Received 12 January 2005; accepted 22 February 2005

Abstract Glutathione plays a critical role in many biological processes both directly as a co-factor in enzymatic reactions and indirectly as the major thiol-disulfide redox buffer in mammalian cells. Glutathione also provides a critical defense system for the protection of cells from many forms of stress. However, mild stress generally increases glutathione levels, often but not exclusively through effects on glutamate cysteine ligase, the rate-limiting enzyme for glutathione biosynthesis. This upregulation in glutathione provides protection from more severe stress and may be a critical feature of preconditioning and tolerance. In contrast, during aging, glutathione levels appear to decline in a number of tissues, thereby putting cells at increased risk of succumbing to stress. The evidence for such a decline is strongest in the brain where glutathione loss is implicated in both Parkinson’s disease and in neuronal injury following stroke. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Glutathione; Oxidative stress; Reactive oxygen species; Glutamate cysteine ligase; Antioxidant response element

1. Introduction At concentrations between 0.5 and 10 mM, the tripeptide glutathione (g-L-glutamylis the most abundant, low molecular weight thiol in plant and animal cells. As such, it plays an important role in a number of critical cellular processes including the synthesis of the deoxyribonucleotide precursors of DNA, the metabolic processing of certain endogenous compounds such as estrogens, prostaglandins, and leukotrienes and the inactivation of drugs (Meister and Anderson, 1983). GSH also L-cysteinyl-glycine)

* Tel.: +1 858 453 4100x1932; fax: +1 858 535 9062. E-mail address: [email protected]. 1568-1637/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.arr.2005.02.005

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modulates protein structure through both direct and indirect effects on protein sulfhydryl groups (Cotgreave and Gerdes, 1998; Klatt and Lamas, 2000). Perhaps most importantly, GSH and GSH-associated metabolism provide the major line of defense for the protection of cells from oxidative and other forms of stress (for reviews, see Dickinson and Forman, 2002; Hayes and McLellan, 1999; Meister and Anderson, 1983). GSH can scavenge free radicals, reduce peroxides and be conjugated with electrophilic compounds. It thereby provides cells with multiple defenses against both reactive oxygen species (ROS) and their toxic by-products. GSH is particularly effective against the highly toxic hydroxyl radical for which there are no known enzymatic defenses (Bains and Shaw, 1997) as well as several other highly reactive species such as peroxynitrite (Halliwell and Gutteridge, 1993). In addition to these functions of GSH itself, the glutathione/glutathione disulfide (GSH/GSSG) redox couple acts to maintain the redox environment of the cell (Schafer and Buettner, 2001). Furthermore, since the GSH/GSSG redox couple is the most abundant in the cell, it serves as an indicator of the cellular redox environment. Intracellular GSH levels are regulated by a complex series of mechanisms (Fig. 1) that include substrate (mainly cyst(e)ine) transport and availability, rates of synthesis and regeneration, GSH utilization and GSH efflux to extracellular compartments (Meister and Anderson, 1983). GSH is synthesized in cells by the consecutive action of two enzymes, glutamate cysteine ligase and glutathione synthase. The unusual peptide g-linkage formed in the first step of the reaction is thought to protect GSH from degradation by aminopeptidases (Sies, 1999). GSH plays multiple roles in the protection of cells from reactive oxygen species, electrophiles and xenobiotics. It can react non-enzymatically with carbon-centered radicals and is also the electron donor in the enzymatic reduction of both H2O2 and organic peroxides catalyzed by the glutathione peroxidases. The product of the oxidation of GSH

Fig. 1. Outline of GSH Metabolism. GSH is synthesized from glutamate (glu), cysteine (cys), and glycine (gly) by the sequential actions of glutamate cysteine ligase (GCL) and glutathione synthase (GS). GSH is used to eliminate reactive oxygen species such as hydrogen peroxide (H2O2) in a reaction catalyzed by glutathione peroxidase (GPX). H2O2 is produced from superoxide (O2
) by superoxide dismutase (SOD) and can also be removed from cells by catalase (CAT), although not that which is produced in mitochondria. The glutathione disulfide (GSSG) produced in this reaction can be converted back to GSH through the action of glutathione reductase (GR). GR requires NADPH for its activity, which is mainly supplied from the activity of glucose-6-phosphate dehydrogenase (G6PD), the first enzyme of the pentose phosphate shunt. GSH can also be used to detoxify endogenous and exogenous electrophiles (X) through conjugation via the glutathione-S-transferases (GST). These conjugates, as well as GSH itself, can be transported outside the cell where they can be acted upon by g-glutamyl transferase (gGT) to give g-glutamyl amino acids and cys–gly.

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by glutathione peroxidase is glutathione disulfide (GSSG). GSSG can be converted back to GSH by glutathione reductase in a reaction that requires NADPH as a reductant. Free GSSG can also be eliminated from cells by either direct conjugation to proteins (glutathionylation) (Klatt and Lamas, 2000) or export by specific transporters. Intracellular GSH is also depleted when it is used by the glutathione transferases to detoxify electrophilic compounds. The GSH conjugates with these compounds are generally much more water soluble than the original compounds and are transported across cell membranes and ultimately excreted in the urine or feces. GSH itself is also transported out of cells, thereby providing the source of plasma GSH and GSSG. Both GSH and GSSG are substrates of the extracellular, membrane bound enzyme g-glutamyl transferase (GGT) which is the only enzyme which can break the gpeptide linkage (Meister and Anderson, 1983). GGT can either transfer the g-glutamyl group of GSH, GSSG or GSH-conjugates to amino acid acceptors to give g-glutamyl peptides and cysteinylglycine or directly hydrolyze GSH to glutamate and cysteinylglycine. Cysteinylglycine can then be cleaved by a dipeptidase. The free amino acids and gglutamyl amino acids produced by both of these reactions can be transported back into cells and used to regenerate GSH. Since this pathway provides a means of recycling GSH and GSSG that have been lost from cells, its upregulation can provide an additional mechanism for maintaining GSH in cells. In this review, I will first briefly describe the different enzymatic steps in GSH metabolism to provide the background information necessary for understanding the research into the effects of stress and aging on GSH metabolism. I will also provide additional information on other aspects of GSH metabolism that are important in understanding how changes in GSH levels can have a dramatic impact on cellular function. However, before I begin I need to clarify a bit of terminology. Glutathione can exist in two forms; both as a reduced monomer (GSH) and as an oxidized dimer (glutathione disulfide or GSSG). Although technically GSH denotes only the monomeric form, many authors use it to indicate total GSH, which includes both the monomeric and dimeric forms of the tripeptide. Since GSH is the predominant form of glutathione in cells, even under conditions of stress, the levels of total GSH and GSH will not differ by a large amount. However, since the ratio GSH/GSSG itself can play a role in cellular function and is also an indictor of the redox state of the cell, knowledge of both values is useful. In this review, I will use tGSH to indicate GSH + GSSG, GSH to indicate reduced GSH and GSSG to indicate oxidized GSH. 1.1. GSH and redox balance As indicated above, the ratio of GSH/GSSG plays an important role in regulating the cellular redox status since it is the most abundant thiol-disulfide redox buffer in the cell (for review, see Schafer and Buettner, 2001). Normally, GSH is present in cells at 100-fold excess over GSSG. The oxidation of only a small amount of GSH to GSSG can significantly change this ratio and thereby the redox status of the cell. For example, if the GSH/GSSG ratio were initially 100:1 and after some insult the GSSG level doubled, this would change the ratio to 49:1. This, in turn, can have a dramatic impact on cellular function. For example, a number of studies have suggested an association between a more

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reducing environment (higher GSH/GSSG ratio) and cell proliferation while a more oxidizing environment (lower GSH/GSSG ratio) is associated with differentiation (see Schafer and Buettner, 2001). Thus, changes in the GSH/GSSG ratio could have profound influences on the behavior of stem cell populations in adult tissues. Indeed, small changes to the redox status of oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, which give rise to the myelin forming oligodendrocytes of the central nervous system, were shown to significantly affect the fate of the cells (Smith et al., 2000). Consistent with earlier studies, conditions which led to a more reducing environment promoted O-2A cell division whereas conditions which led to a more oxidizing environment promoted differentiation. 1.2. Intracellular distribution of GSH Although all cells contain millimolar levels of GSH, it is not distributed uniformly within the cell (Schafer and Buettner, 2001). Furthermore, increasing evidence suggests that the GSH levels in specific cellular compartments may be more important than overall cellular GSH levels when looking at the effects of changes in GSH on cell function. The majority of the GSH in cells is found in the cytoplasm where it is synthesized. However, both mitochondria and nuclei have separate pools of GSH, which appear to be at least partially independent of the cytoplasmic pool in that their concentrations do not always change in concert with that of the cytoplasmic pool (Sims et al., 2004). For example, in several studies, depletion of GSH from the cytoplasm did not result in depletion from the nucleus or mitochondria (Schafer and Buettner, 2001; Sims et al., 2004). Conversely, in some types of cells, ethacrynic acid can be used to specifically deplete the mitochondrial pool of GSH with little or no effect on the cytoplasmic pool (Muyderman et al., 2004). In astrocytes, this results in a significantly enhanced sensitivity to treatment with a generator of peroxynitrite, suggesting that the mitochondrial pool of GSH plays a critical role in the protection of cells from at least some forms of oxidative stress (Muyderman et al., 2004). This conclusion is in agreement with a series of studies by Fernandez-Checa et al. (1998) on alcoholic liver disease. In this case, chronic ethanol feeding leads to the selective reduction of mitochondrial GSH due to the impairment of the mitochondrial carrier that translocates GSH from the cytoplasm to the mitochondria. This depletion of mitochondrial GSH sensitizes hepatocytes to oxidative stress. Specific decreases in the mitochondrial GSH pool may also play a role in aging. 1.3. The interaction of GSH with proteins There are two mechanisms whereby GSH can alter protein sulfhydryl groups and thereby modulate the activity of proteins. First, changes in the redox potential of the cell due to a decrease in the GSH/GSSG ratio can induce the oxidation of protein sulfhydryls yielding an activated protein thiol (Klatt and Lamas, 2000). GSH can then interact with this activated thiol to give a mixed disulfide. Alternatively, GSSG can interact directly with sulfhydryl groups in proteins also yielding mixed protein disulfides (Cotgreave and Gerdes, 1998; Klatt and Lamas, 2000; Thomas et al., 1995). A number of proteins have been found to undergo glutathionylation, including protein chaperones, cytoskeletal proteins, cell cycle regulators and enzymes of intermediary metabolism (Lind et al., 2002).

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Glutathionylation also plays a regulatory role in mRNA transcription through its effects on the activity of a variety of transcription factors. Although both activated thiol groups and mixed protein disulfides are reversible, they often occur under conditions of oxidative stress. Therefore, the regeneration of the free protein sulfhydryl is dependent upon the reversal of that stress. If oxidative stress is maintained, the complex is likely to persist, resulting in both a decrease in GSH and a potential loss in protein function. Indeed, the activity of the glutaredoxins, which reduce both protein disulfides and mixed disulfides formed between GSH and proteins, is dependent upon GSH (Fernandes and Holmgren, 2004). Thus, GSH depletion can lead to protein denaturation and aggregation subsequent to protein thiol oxidation (Freeman et al., 1997).

2. Glutathione metabolism 2.1. Substrate transport Since glutamate and glycine occur at relatively high intracellular concentrations, cysteine is limiting for GSH biosynthesis in humans as well as other species (Wu et al., 2004). Therefore, treatments which stimulate cysteine or cystine uptake by cells usually enhance GSH biosynthesis. In the extracellular environment, cysteine is readily oxidized to form cystine, so for most cell types cystine transport mechanisms are essential to provide them with the cysteine needed for GSH synthesis. An exception to this appears to be hepatocytes, which have little or no capacity for the transport of extracellular cystine (Wu et al., 2004). In vivo evidence for the importance of cysteine for GSH biosynthesis comes from studies on severely malnourished children (Reid and Jahoor, 2001) which show that additional supplementation with N-acetylcysteine significantly increases GSH biosynthesis relative to additional supplementation with alanine. Cystine uptake in cells occurs via the Na+-independent xc
cystine/glutamate antiporter (for review, see McBean, 2002). The xc
exchanger is a member of the disulfide-linked heteromeric amino acid transporter family and consists of a light chain (xCT) that confers substrate specificity and a heavy chain (4F2hc). It transports cystine into cells in a 1:1 exchange for glutamate and thus can be inhibited by high concentrations of extracellular glutamate. This can occur in a number of conditions including advanced cancer, HIV infection, and following brain or spinal cord injury thus leading to an inhibition of GSH synthesis under conditions where it is needed most (see Section 3.4). 2.2. GSH biosynthesis Increases in the biosynthesis of GSH can play a critical role in the protection of cells from oxidative and other forms of stress. Indeed, the capacity to increase GSH synthesis in response to increased demands on GSH utilization is thought to be an important determinant of cell survival. GSH is synthesized in cells by the consecutive action of two ATP-dependent enzymes (Fig. 1). Glutamate cysteine ligase (GCL), formerly called gglutamylcysteine synthetase (g-GCS), catalyzes the first and rate-limiting step in GSH biosynthesis to form the dipeptide gGluCys, which is then combined with glycine to

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generate GSH in a reaction catalyzed by glutathione synthase (GS). The synthesis of GSH in cells is normally regulated by feedback inhibition of GCL by GSH. GCL can also be inhibited by buthionine sulfoximine (BSO), which has been used by many investigators to look at the role of GSH biosynthesis in a wide range of cellular functions. A number and variety of different compounds have been shown to increase GSH levels in cells by increasing GCL activity (Table 1). The activity of GCL is regulated at the transcriptional, translational and post-translational levels and the complex interplay of these different levels of regulation is just beginning to be understood (for reviews, see Soltaninassab et al., 2000; Wild and Mulcahy, 2000; Dickinson and Forman, 2002). GCL is a heterodimer composed of a 73 kDa catalytic (GCLC) subunit and a 31 kDa regulatory (GCLR) subunit. GCLC has all of the catalytic activity and is the site of GSH feedback inhibition. Although it was originally thought that GCLC only functions in concert with GCLR, a recent study suggests that GCLC alone may be responsible for the constitutive synthesis of GSH (Dickinson et al., 2004). The association of GCLC with GCLR is needed in order to overcome feedback inhibition by GSH when a higher rate of synthesis is required such as during stress. In agreement with this conclusion, mice deficient in GCLC are early embyronic lethal whereas mice deficient in GCLR are viable but show a significantly enhanced sensitivity to stress (Dalton et al., 2004). The two subunits of GCL are transcriptionally regulated by a wide variety of compounds (for reviews, see Soltaninassab et al., 2000; Wild and Mulcahy, 2000; Dickinson et al., 2004). A number of cis elements are implicated in the transcriptional activation of both GCLR and GCLC mRNAs including AP-1, AP-2, NF-kB, SP-1 and ARE (aka EpRE, StRE). However, the pathways for the transcriptional up-regulation of the two subunits appear to be independent and vary with both inducing agent and cell type. Furthermore, the sequences of the rat promoters for both GCLC and GCLR (Yang et al., 2001a,b) are very different from those for the corresponding human genes (Dickinson and Forman, 2002) suggesting that studies on the transcriptional regulation of GCL in rats may not translate well to humans. Recently, the role of the ARE in regulating the transcription of the two GCL subunits as well as other phase II detoxification enzymes has received a great deal of attention. The family of phase II detoxification enzymes includes several other proteins involved in GSH metabolism, including the light subunit of the cystine/glutamate antiporter (see Section 2.1) and the glutathione transferases (see Section 2.5). The transcriptional activation of these proteins is mediated at least in part by a cis-acting enhancer termed the antioxidant response element (ARE). Transcriptional activation through the ARE is dependent upon the transcription factor NF-E2-related factor 2 (Nrf2), a member of the Cap‘n’Collar family of bZIP proteins (for reviews, see Chen and Kong, 2004; Nguyen et al., 2003). Very little Nrf2 is found in unstimulated cells and that which is present is held in the cytoplasm by the actin-bound protein Keap1, which also promotes the degradation of Nrf2 by the proteasome. Upon stimulation by agents, which activate the ARE, Nrf2 is released from Keap1 which leads to its accumulation and its translocation to the nucleus, where it can induce the expression of genes containing an ARE. Not a lot is known about the post-translational regulation of GCL activity although there is evidence that the activity of GCLC can be negatively regulated by phosphorylation (Soltaninassab et al., 2000). A number of kinases have been reported to phosphorylate GCL

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Table 1 Compounds and treatments, which can increase GCL expression Adriamycin 1-(4-Amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitrosourea Apigenin Apocynin L-Azetidine-2-carboxylic acid b-Naphthoflavone (b-NF) Butylated hydroxyanisole (BHA) Butylated hydroxytoluene (BHT) Cigarette smoke condensate Ciprofibrate Cisplatin Copper chloride Curcumin Cycloheximide Diethyl maleate (DEM) Dimethoxy-1,4-naphthoquinone (DMNQ) 15-deoxy-D(12,14)-prostaglandin J2 Diquat Erythropoietin Estradiol Ethoxyquin Heat shock Hydrocortisone Hydrogen peroxide 6-Hydroxydopamine 4-Hydroxy-2-nonenal (4-HNE) Hydrogen sulfide Hypoxia Insulin Interleukin-1b Iodoacetamide Ionizing radiation (0.05–30 Gy) Kaempferol Menadione Methyl mercury hydroxide Nitric oxide Okadaic acid Oltipraz Oxidized low density lipoproteins (ox-LDL) Phorone Prostaglandin A2 Pyrrolidine dithiocarbamate (PDTC) Quercetin Sodium aresenite tert-Butylhydroquinone (t-BHQ) Tumor necrosis factor-a (TNFa) Zinc chloride Adapted from (Wild and Mulcahy, 2000; Dickinson and Forman, 2002; Myhrstad et al., 2002; Kimura and Kimura, 2004).

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including protein kinase A (PKA), protein kinase C (PKC) and Ca2+/calmodulindependent kinase (Soltaninassab et al., 2000). Glutathione synthase (GS), the second enzyme required for GSH biosynthesis, is a 118 kDa homodimer (Dickinson and Forman, 2002). Almost nothing is known about its role, if any, in regulating GSH biosynthesis. Furthermore, since the 50 untranslated region of the GS gene has not been cloned and sequenced, potential regulatory elements which control the transcription of the gene are unknown as well. 2.3. Glutathione reductase Both enzymatic (via glutathione peroxidases) and non-enzymatic detoxification of ROS by GSH results in the production of GSSG. Since an increase in GSSG is harmful to cells, GSSG is often transported outside of cells, resulting in a depletion of tGSH. The more economical way to remove GSSG is via the activity of glutathione reductase (GR), which regenerates GSH from GSSG in a reaction that is absolutely dependent upon NADPH. Increases in GR activity can be mediated by two distinct mechanisms: (1) an increase in the level and/or activity of GR or (2) an increase in the levels of NADPH by increasing the activity of the pentose phosphate shunt, the main source of NADPH in the cell [see Schubert, this volume]. GR belongs to the family of FAD-containing pyridine nucleotide:disulfide oxidoreductases which also includes the thioredoxin reductases. The active enzyme is composed of two identical subuits of 52,400 kDa, each of which contains binding sites for FAD, NADPH and GSSG (Lopez-Barea et al., 1990). The enzyme also contains a redox-active disulfide, which plays a critical role in the flow of electrons from NADPH to GSSG. Recent evidence suggests that GR may be particularly susceptible to oxidative damage brought about by GSH depletion (Barker et al., 1996), although whether the damage is due to effects on the redox-active disulfide was not explored. 1,3-bis(2-chloroethyl)-N-nitrosurea (BCNU; aka carmustine), which carbamoylates a lysine residue in the active site of GR in an NADPH-dependent manner, can be used to inhibit GR and thereby study its roles in cellular function. However, BCNU also inhibits thioredoxin reductase so this needs to be taken into account when considering data obtained with this drug. 2.4. Glutathione peroxidases Glutathione peroxidases (GPxs) catalyse the reduction of H2O2 and alkyl hydroperoxides at the expense of GSH (for reviews, Arthur, 2000; Brigelius-Flohe, 1999). Although catalase can also remove H2O2, the relative levels of GPxs and catalase vary greatly from tissue to tissue (Halliwell and Gutteridge, 1993). In particular, the brain has very low levels of catalase activity and relatively high levels of GPx activity, while the liver has high levels of both. Furthermore, GPxs but not catalase (except in the heart) are found in the mitochondria and so play a critical role in removing H2O2 produced in the mitochondria. Mitochondria also contain GR. There are four different GPxs (GPx1–4) in mammals, all of which contain selenocysteine in the active site and therefore are dependent upon an adequate supply of dietary selenium. The best characterized of the GPxs is GPX1, which is expressed in a variety of tissues with the highest levels of expression in liver and kidney.

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It is found in both the cytoplasm and mitochondria and reduces mainly soluble inorganic and organic hydroperoxides. GPX1 is a tetrameric protein consisting of four identical 22– 23 kDa subunits. Depending on the mouse line, mice deficient in GPx1 are completely normal (Esposito et al., 2000), more sensitive to certain forms of oxidative stress than their wild type counterparts (de Haan et al., 1998) or have reduced body weight relative to wild type mice (Esposito et al., 2000). In the latter strain, mitochondria from the liver showed an increase in H2O2 production and lipid peroxides and a decrease in energy output, suggesting that the activity of GPx1 may play an important role in safeguarding the function of the tissues where it is highly expressed. GPx2 (gastrointestinal GPx) and GPx3 (plasma GPx) are mainly expressed in the gastrointestinal tract and kidney, respectively, and have a similar structure and substrate specificity to GPx1 except that GPx3 is an extracellular glycoprotein. In contrast, GPx4 (phospholipid hydroperoxide GPx) is a 20– 22 kDa monomer, which can also reduce hydroperoxides of complex lipids and can act on hydroperoxides embedded in membranes. Mice deficient in GPx4 are early embryonic lethal (Yant et al., 2003) suggesting that this activity is essential for development. GPx4 is expressed most highly in brain and testis (Esposito et al., 2000). In sperm it also appears to serve a structural function that is independent of its enzymatic activity. GPxs can be inhibited by mercaptosuccinate. 2.5. Glutathione transferases Glutathione transferases (GSTs) play a critical role in defending the organism against reactive chemicals formed both from the breakdown of endogenously produced compounds and the biotransformation of foreign compounds (for reviews, see Hayes and McLellan, 1999; Rinaldi et al., 2002). There are over 21 structurally diverse GSTs in humans, which includes both soluble and membrane-bound proteins. The GSTs are encoded by two separate multigene families; one comprising the soluble GSTs and the other the membrane-bound GSTs. The family of soluble GSTs contains six classes of transferases with different but overlapping substrate specificity. All of the GSTs use GSH to detoxify metabolites of xenobiotics as well as reactive a,b-unsaturated carbonyls, epoxides and hydroperoxides. As with GCL and the light subunit of the cystine/glutamate antiporter, GSTs are phase II detoxification enzymes. Thus, their transcription is mediated by an ARE, which can be activated by the same compounds which activate the transcription of these other genes involved in GSH metabolism. The coordinated upregulation of GSH biosynthesis along with the GSTs makes sense since the abundance and catalytic properties of GSTs indicate that they could empty the liver GSH pool in a few seconds when a suitable substrate is present (Rinaldi et al., 2002). Thus, their protective role is absolutely dependent upon an adequate supply of GSH. 2.6. Regulation of GSH/GSSG efflux Regulation of intracellular GSH levels can be mediated by controlling the efflux of GSH or GSSG. GSH is transported out of cells by a carrier dependent mechanism. GSH efflux has been studied primarily in hepatocytes because the liver is the source of most of the GSH in plasma. Two different transport systems, sinusoidal and canicular, have been described

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in hepatocytes and there is evidence for the presence of these transport systems in other types of cells (Ghibelli et al., 1998; Meredith et al., 1998; Pullar and Hampton, 2002). However, the rates of GSH efflux can be underestimated in cells which express g-glutamyl transferase. The GSH transporters are poorly characterized and have not yet been cloned. GSSG is also exported from cells via a distinct pathway involving the multi-drug resistance proteins (Hirrlinger et al., 2001). Normally, GSSG is rapidly reduced by GR within the cell but since it is toxic to cells, export provides an additional layer of protection. However, export of GSSG leads to a depletion of tGSH.

3. Stress and glutathione 3.1. Oxidative and oxidant-induced stress Although severe oxidative stress, which is defined as an imbalance between the production and removal of reactive oxygen species (Halliwell and Gutteridge, 1993), can cause a decrease in tGSH (see Section 3.4), a number of reports have shown that moderate stress often increases tGSH. This increase is likely to provide protection of cells from both the ongoing stress and from subsequent, more severe stress. For example, H9c2 cells pretreated with a non-toxic dose of H2O2 for 24 h showed an increase in GSH levels and an increased resistance to a toxic dose of H2O2 that was dependent on GCL activity (Seo et al., 2004). Similarly, treatment of lung-derived epithelial cells with 4-hydroxy-2-nonenal (4HNE), an a,b-unsaturated aldehyde derived from lipid peroxidation that is produced endogenously during inflammation or exposure to air pollutants leads to a dose dependent increase in tGSH levels (Dickinson and Forman, 2002). The increase in tGSH levels correlates in a time and dose dependent fashion with an increase in the levels of GCLC and GCLR. Although the authors saw similar changes in tGSH and GCL levels in both rat and human lung epithelial cells (Dickinson and Forman, 2002) in response to 4HNE, the mechanisms regulating the increase in GCL levels appear to be species specific. Oxidized LDL (oxLDL), which is implicated in the development of coronary artery disease, can also elevate tGSH levels in macrophages, monocytes and endothelial cells through a similar mechanism involving the induction of the transcription of the GCLC and GCLR genes (Bea et al., 2003; Moellering et al., 2002). Furthermore, inhibition of GSH biosynthesis in monocytes and macrophages increases the toxicity of oxLDL (Gotoh et al., 1993), suggesting that this is also a tolerance phenomenon. In addition, these data suggest that genetic variations in the ability to induce GSH biosynthesis or in the activity of the biosynthetic enzymes themselves could contribute to the development of coronary artery disease. These studies highlight a common theme with regard to many compounds that increase tGSH levels. This is that many of these compounds initially decrease tGSH levels before eventually leading to an increase well above control levels. Since GCL activity is feedback inhibited by GSH, it is not surprising that treatment with compounds that decrease tGSH can lead to a transient increase in tGSH levels, for the decrease in tGSH would be expected to increase GCL activity. Surprisingly, the increases in tGSH levels are frequently sustained and the compounds also increase the levels of GCL. Upon closer examination, it

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becomes apparent that many if not all of these compounds are able to directly or indirectly (i.e. after metabolism) activate the ARE (Wild and Mulcahy, 2000; Dickinson and Forman, 2002; Dickinson et al., 2004). The induction of GCL synthesis is often, although not always, dependent upon Nrf2-driven ARE mediated transcription. In agreement with this idea, tGSH levels are decreased in the liver and fibroblasts from Nrf2-deficient mice (Chan and Kwong, 2000) as are the levels of GCLC and GCLR mRNA. However, since synthesis of the light chain of the cystine/glutamate antiporter is also dependent upon Nrf2-driven ARE mediated transcription (Ishii et al., 2000), at least some of the effects of these compounds on tGSH levels in certain tissues may be mediated by their ability to increase cystine uptake and thereby the rate limiting substrate for GSH biosynthesis. 3.2. Heat shock/thermotolerance The effects of heat shock on GSH levels received a great deal of attention in the 1980s. Of particular interest was the role of GSH in the development of heat-induced tolerance or thermotolerance. Thermotolerance is the ability of a mild heat stress to induce resistance to a more severe heat stress, which would normally kill the cell. Early studies suggested that tGSH levels increased during induction of thermotolerance and that the development of thermotolerance was compromised in cells exposed to mild heat stress in the presence of either BSO to block GSH synthesis or DEM to deplete tGSH (Mitchell et al., 1983). These data are supported by more recent studies in rat (Harris et al., 1991) and mouse (Arechiga et al., 1995) embryos. However, other studies, particularly those performed with tumor cell lines, suggest that the relationship between GSH levels and thermotolerance is more complex (Freeman et al., 1985) and likely to be cell-type dependent (Anderstam and Harms-Ringdahl, 1986). Despite these ambiguities regarding the effects of heat stress on GSH levels, there is both correlative and direct evidence that the small heat shock proteins (Hsps), including human Hsp27 and its mouse homologue Hsp25, can regulate GSH levels through a mechanism dependent upon glucose-6-phosphate dehydrogenase (G6PD), the first and rate limiting enzyme of the pentose phosphate shunt. Several studies have shown that overexpression of Hsp25/27 in fibroblasts (Baek et al., 2000; Preville et al., 1999) or muscle cells (Escobedo et al., 2004) increases GSH levels and cellular resistance to various types of stress. Interestingly, differentiation of muscle cells also results in increases in the expression of Hsp25/27 and tGSH along with resistance to oxidative stress (Escobedo et al., 2004). Furthermore, mice deficient in two small Hsps related to Hsp25/27 (aBcrystallin and HspB2) show both decreased tGSH levels in the heart and an increased susceptibility to ischemia-reperfusion injury (Morrison et al., 2004). To understand the mechanism underlying this effect of Hsp25/27 expression on GSH metabolism, the activities of the enzymes involved in GSH metabolism were characterized in control and Hsp25/27-overexpressing cells (Preville et al., 1999; Baek et al., 2000). Increases in GR activity (Preville et al., 1999; Baek et al., 2000) were seen which correlated with an increase in the level of G6PD activity and protein (Preville et al., 1999). These results are consistent with both the dependence of GR on NADPH and G6PD being a major source of NADPH in cells. Although nucleated cells contain other pathways for NADPH biosynthesis, recent studies with G6PD-deficient cells indicate that under conditions of

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oxidative stress, G6PD becomes essential for the production of NADPH (Filosa et al., 2003). Direct demonstrations that G6PD can modulate GSH levels were obtained in studies where G6PD was overexpressed in fibroblasts (Preville et al., 1999) or HeLa cells (Salvemini et al., 1999) and the effects of this overexpression on tGSH levels examined. In both cases, a 2–3-fold increase in G6PD activity resulted in 2-fold increase in GSH levels as well as a very significant increase in resistance to oxidative stress (Salvemini et al., 1999). These studies highlight the importance of multiple aspects of GSH metabolism in maintaining GSH levels and indicate that studies wherein changes in GSH levels are seen need to examine all aspects of GSH metabolism in order to be understood. Indeed, G6PD activity is also increased by compounds which deplete GSH levels (Salvemini et al., 1999). In addition, G6PD is very sensitive to oxidation and loss of G6PD activity is seen in several models of aging (Beckman and Ames, 1998). Therefore, while it has not been explored, decreases in G6PD activity could play an important role in the loss of GSH seen in aging. 3.3. Hypoxia Oxygen levels are also known to alter GSH levels in cells and tissues. However, no clear picture has emerged from these studies, suggesting that the effects are dependent upon both the cell or tissue type being examined and the severity of the treatment. Both low oxygen (hypoxia) and high oxygen (hyperoxia) have been shown to either deplete or to increase GSH levels. Many of these studies are likely to reflect the repeated observation that mild stress often increases GSH levels through various pathways. A good example comes from a comparison of a study on hypoxic preconditioning in nerve cells in vitro designed to mimic ischemia-reperfusion injury in vivo (Arthur et al., 2004) versus bona fide ischemia in the brain (Anderson and Sims, 2002). Many investigators have shown that non-lethal ischemia (aka hypoxic preconditioning) can protect various tissues and cells from lethal ischemia although the mechanisms underlying this protection are still under debate. Using two different in vitro models of ischemia/reperfusion injury in nerve cells, Arthur et al. (2004) showed that hypoxic preconditioning results in significant increases in the activities of both GR and GPx as well as Mn superoxide dismutase but not other antioxidant enzymes, as well as increased cell survival. This suggests that at least part of the protection afforded by preconditioning could be mediated by increases in tGSH (although GSH levels were not measured directly). Using an in vivo model of ischemia-reperfusion injury, a loss of mitochondrial tGSH was seen during both ischemia and reperfusion, which correlated with the subsequent development of tissue damage (Anderson and Sims, 2002). Thus, conditions, which induce mild hypoxia may upregulate tGSH levels whereas more severe hypoxia leads to a possibly fatal decrease in tGSH levels. 3.4. Oxidative glutamate toxicity Since tGSH depletion is implicated in the nerve cell death seen in stroke/ischemia, Parkinson’s disease and aging, understanding how it kills cells could lead to the development of new therapeutic approaches for the treatment of these conditions. We have developed a model for studying the pathways leading from tGSH depletion to death in nerve cells. Our model utilizes glutamate to gradually deplete cells of tGSH (for primary

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references see Tan et al., 2001). Glutamate is the major excitatory neurotransmitter in the central nervous system, but it has also been implicated in nerve cell death following acute neurologic insults. Although glutamate is present in synaptic nerve terminals at millimolar concentrations, the extracellular concentrations are normally high only during the brief periods of synaptic transmission. However, certain forms of injury can result in extended periods of elevated extracellular glutamate levels. High levels of extracellular glutamate have been shown to be toxic to nerve cells in culture via two distinct processes: excitotoxicity, which occurs through the activation of ionotropic glutamate receptors, and a programmed cell death pathway called oxidative glutamate toxicity or oxytosis, which is mediated by a series of disturbances to the intracellular redox system. Increases in the endogenous levels of ROS are key elements in the cell death cascade in both of these processes. The majority of our studies on oxidative glutamate toxicity utilized the mouse hippocampal cell line, HT22, and were confirmed with primary cortical neurons. In this system, high concentrations of extracellular glutamate block the uptake of cystine, which enters the cell through the cystine/glutamate antiporter, thereby resulting in the inhibition of GSH biosynthesis (Fig. 2). Concentrations of extracellular glutamate as low as 100 mM inhibit the import of cystine. When GSH is depleted by more than 80% for a period of a few hours, cells commit to die by a form of programmed cell death. The physiological relevance of this cell death mechanism has been debated, for it has incorrectly been assumed that in pathological conditions the level of extracellular glutamate does not become sufficiently high to alter cystine transport. However, extracellular glutamate can reach levels exceeding 500 mM in ischemia and over 300 mM in cell culture excitotoxicity paradigms. Furthermore, oxidative glutamate toxicity can occur as a consequence of receptor-mediated excitotoxicity. This, in turn, leads to a delayed form of cell death that extends over many hours. The exposure of HT22 cells to glutamate leads to the depletion of tGSH over 6 h which is paralleled by a slow increase in ROS levels to about 10% of its maximal value (Fig. 3). Once tGSH levels fall below 20% of control values, ROS levels increase exponentially, eventually reaching levels 100–200 times higher than control values by

Fig. 2. Mechanism of oxidative glutamate toxicity. High concentrations of extracellular glutamate inhibit cystine uptake via the cystine/glutamte antiporter (xc
) thereby leading to tGSH depletion.

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Fig. 3. Schematic diagram showing the time course of the major events in the cell death pathway activated in oxidative glutamate toxicity. The lines indicate the relative increase or decrease in the molecules indicated and the colored rectangles indicate the times following the addtion of exogenous glutamate when the indicated process occur (e.g. protein synthesis). Adapted from (Tan et al., 2001).

10 h after the addition of glutamate. This late rise in ROS levels comes from mitochondria and is most likely dependent upon mitochondrial complex I. It is blocked by RNA and protein synthesis inhibitors as well as by certain caspase inhibitors but only when these agents are added within 2–4 h after the initiation of glutamate treatment. While the form of cell death seen in this model of oxidative stress has many of the characteristics of cell death seen in a variety of other systems, morphologically the cell death is more reminiscent of that seen in early neuronal development rather than the classical form (apoptosis) seen in lymphoid cells. The activation of 12-lipoxygenase (12LOX) is required for maximal late phase ROS production from mitochondria and for cell death. In the presence of glutamate, low tGSH levels cause 12-LOX activation, leading to the generation of one or more unidentified products, which is required for the generation of the maximal levels of ROS. The 12-LOX substrate, arachidonic acid, is generated at least in part by the action of phospholipase A2. A product of 12-LOX activity also activates soluble guanylyl cyclase (sGC), resulting in the production of cGMP, which, in turn, activates a cobalt-sensitive Ca2+ channel thereby mediating the large increase in intracellular Ca2+. The relationship between ROS production and intracellular Ca2+ elevation is complex. Although the increase in cytosolic Ca2+ follows the late rise in ROS production and can be blocked by agents which prevent late-phase ROS generation, inhibition of Ca2+ influx significantly reduces the late increase in ROS levels. Thus, the influx of Ca2+ and mitochondrial ROS production appear to be tightly coupled. Taken together all of the studies on stress and GSH indicate that the consequences of tGSH depletion to the cell depend upon the level of depletion and probably the intracellular site(s) of depletion. Thus, while partial depletion of tGSH, particularly if it occurs predominantly or exclusively in the cytoplasm, can lead to

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enhanced protection from oxidative insults, higher levels of tGSH loss or significant loss of tGSH from mitochondria activate a form of programmed cell death.

4. GSH in aging and age-related neurodegenerative diseases 4.1. Aging Over the years, a number of theories have been put forth to explain the mechanisms underlying the process of aging. One of the theories that has received the most attention and research support in recent years is the free radical theory of aging (for review, see Beckman and Ames, 1998). This theory proposes that there is an accumulation of oxidative damage with aging, which is the primary cause of the age-related declines in cellular function. Since GSH plays a central role in maintaining the oxidative balance of cells, a number of studies have looked at GSH levels during aging (Table 2). These studies have examined variously tGSH, GSH, GSSG and the GSH/GSSG redox couple in whole tissues and in mitochondria. Age-dependent changes in mitochondrial GSH levels are of particular interest since the mitochondria are thought to play a key role in the aging process since they are both a major source of intracellular oxidants and a target for the damaging effects of these oxidants. They are also the major source of energy in the form of ATP and compromises in their structure is thought to trigger apoptosis. In one of the first studies to look at age-dependent changes in GSH levels (Hazelton and Lang, 1980), GSH and GSSG levels were examined in various tissues from C57BL/6J mice, a well-studied model of aging. Importantly, animals of known biological ages (i.e. young, mature, old, etc.) were used. Based on the normal survival curve for this strain of mice, mature mice were defined as being between 10 and 25 months of age, old mice between 25 and 30 months and very old mice over 30 months. It is important to keep these ages in mind when reviewing the data in Table 2 since very few other studies with mice or rats used animals of known biological ages as defined by their normal survival curves. Moreover, a number of studies actually compared growing animals with mature animals or old animals rather than mature animals with old and/or very old animals when drawing their conclusions. This is a critical difference because some studies demonstrated a high level of GSH or tGSH levels in a variety of tissues during the growth phase which decreased to a plateau at maturation. Thus, a comparison of GSH levels between growing animals and mature or old animals is not necessarily a reflection of changes that are due specifically to the aging process. Nevertheless, a fairly large number of studies have shown age-dependent decreases in tGSH and/or GSH levels in a variety of tissues (Table 2). However, these changes are very tissue dependent and also appear to be somewhat species dependent. For example, the majority of studies indicate that GSH levels decline in mouse brain as the mouse passes into old age. However, in the rat the data is not as clear, perhaps because several different rat strains were used that may have different survival curves. Similarly, GSH levels appear to decrease in mouse liver at about the time the mice go from mature to old while again the data are not as clear cut with rats. However, four out of six studies with Wistar rats do show a clear decrease in liver GSH by 24 months of age. In the lung, a decrease in GSH was seen in those studies which used mice but no decreases were seen in

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Table 2 Species

Age comparison

GSH change

Reference

Brain Mouse (C57BL/6; male) CX, HC, BS

6 months versus 31 months

GSH NC GSSG

Chen et al. (1989)

Mouse (C57BL/6N; male) CX, HC, CB, BS

12 months versus 24 months

NC tGSH

Hussain et al. (1995)

Mouse (DBF; male) CX, HC, CB, ST

12 months versus 30 months

GSH

Sasaki et al. (2001)

Mouse (C57BL/6N-Nia; male)

10 months versus 26 months

GSH GSSG

Rebrin et al. (2003)

Mouse (C57BL/6; male and female) CX, CB, BS

12 months versus 24 months

GSH CX, BS male only

Wang et al. (2003)

Rat (Sprague–Dawley; male)

12 months versus 36 months 15 months versus 26 months

NC tGSH

Farooqui et al. (1987) Rikans and Moore (1988)

Rat (Sprague–Dawley; female) CX, HC, CB, ST, BS

15–18 months versus 29–30 months

tGSH all except BS

Ravindranath et al. (1989)

Rat (Wistar; male) CX

9 months versus 28 months

NC GSH NC GSSG

Barja de Quiroga et al. (1990)

Rat (Wistar; male) CX

14 months versus 27 months

GSH GSSG

Rat (Fisher; male) CX, HC, CB

12 months versus 24 months

Rat (Wistar; male) CX, CB, BS

12 months versus 24 months

tGSH

Sandhu and Kaur (2002)

17 months versus 31 months

tGSH NC GSSG

Hazelton and Lang (1980)

Mouse (C57BL/6N-Nia; male)

10 months versus 26 months

NC GSH GSSG

Rebrin et al. (2003)

Mouse (C57BL/6; male and female) Rat (Sprague–Dawley; male)

12 months versus 24 months 12 months versus 36 months 15 months versus 26 months

NC GSH

Wang et al. (2003)

Rat (Fisher; male)

Heart Mouse (C57BL/6J; mixed)

Rat (Fisher; male) Rat (Wistar; male)

14 mo vs. 27 mo

NC tGSH

GSH (HC) NC GSH (CX, CB)

tGSH NC tGSH NC GSH NC GSSG

Iantomasi et al. (1993) Liu (2002)

Farooqui et al. (1987) Rikans and Moore (1988) Stio et al. (1994)

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Table 2 (Continued ) Species

Age comparison

GSH change

Reference

Rat (Fisher; male)

12 months versus 24 months

NC GSH NC GSSG

Liu and Dickinson (2003)

Rabbit

6 months versus >5 years

NC GSH

Cusack et al. (1991)

tGSH

Hazelton and Lang (1980) Richie et al. (1992)

Kidney Mouse (C57BL/6J; mixed) Mouse (C57BL/6-Nia; male)

17 months versus 28–31 months 12 months versus 30 months

GSH

Mouse (C57BL/6N-Nia; male)

10 months versus 26 months

NC GSH GSSG

Rebrin et al. (2003)

Mouse (C57BL/6; male and female) Rat (Sprague–Dawley; male)

12 months versus 24 months 12 months versus 36 months 15 months versus 26 months

NC GSH

Wang et al. (2003) Farooqui et al. (1987) Rikans and Moore (1988)

Rat (Fisher; male) Rat (Fisher; male)

Liver Mouse (C57BL/6J; mixed) Mouse (C57BL/6 CrSlc; mixed)

12 months versus 24 months

12 months versus 28–31 months 12 months versus 28 months

tGSH NC tGSH NC GSH NC GSSG

tGSH tGSH

Liu and Dickinson (2003)

Hazelton and Lang (1980) Teramoto et al. (1994)

Mouse (CD-1; male)

13 months versus 24 months

NC GSH NC GSSG

Nakata et al. (1996)

Mouse (C57BL/6; male and female)

12 months versus 24 months

NC GSH, male GSH, female

Wang et al. (2003)

Mouse (C57BL/6N-Nia; male)

10 months versus 26 months

NC GSH GSSG

Rebrin et al. (2003)

Rat (Sprague–Dawley; male)

12 months versus 36 months 15 months versus 26 months 15–18 months versus 29–30 months

Rat (Fisher; male) Rat (Sprague–Dawley; female)

tGSH NC tGSH NC tGSH

Farooqui et al. (1987) Rikans and Moore (1988) Ravindranath et al. (1989)

Rat (Wisar; male)

9 months versus 28 months

NC GSH NC GSSG

Barja de Quiroga et al. (1990)

Rat (Wistar; male)

12 months versus 24 months

NC tGSH

De and Darad (1991)

Rat (Wistar; male)

14 months versus 27 months

GSH GSSG

Stio et al. (1994)

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305

Table 2 (Continued ) Species

Age comparison

GSH change

Reference

Rat (Wistar; male)

6 months versus 24 months

GSH GSSG

Christon et al. (1995)

Rat (Fisher; mixed)

18 months versus 30 months

NC GSH NC GSSG

Nakata et al. (1996)

Rat (Wistar; male)

4 months versus 24 months

GSH

Palomero et al. (2001)

GSSG GSH NC GSSG

Liu and Dickinson (2003)

Rat (Fisher; male)

12 months versus 24 months

Rat (Wistar; male)

12 months versus 28 months

GSH

Mosoni et al. (2004)

15 months versus 26 months

tGSH

Rikans and Moore (1988)

20 years versus 80–92 years

GSH GSSG

Rathbun and Murray (1991)

18 months versus 28 months 12 months versus 24 months 12 months versus 36 months 15 months versus 26 months

tGSH

Teramoto et al. (1994) Wang et al. (2003) Farooqui et al. (1987) Rikans and Moore (1988)

Lens Rat (Fisher; male) Human (mixed)

Lung Mouse (C57BL/6 CrSlc; mixed) Mouse (C57BL/6; male and female) Rat (Sprague–Dawley; male) Rat (Fisher; male)

GSH NC tGSH NC tGSH

Rat (Wistar; male)

9 months versus 28 months

NC GSH NC GSSG

Perez et al. (1991)

Rat (Fisher; male)

12 months versus 24 months

NC GSH NC GSSG

Liu and Dickinson (2003)

21–30 years versus 61+ years

GSH NC GSSG

Yang et al. (1995)

Human (mixed)

<60 years versus >60 years

GSH GSSG

Samiec et al. (1998)

Human (mixed)

20–40 yrs vs. 60+ yrs

GSH GSSG

Jones et al. (2002)

Human (mixed)

12–24 years versus 41–69 years

GSH GSSG

Erden-Inal et al. (2002)

12 months versus 28 months

tGSH

Mosoni et al. (2004)

Plasma Human (male and female)

Skeletal muscle Rat (Wistar; male)

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Table 2 (Continued ) Species

Age comparison

Testis Mouse (C57BL/6N-Nia; male)

10 months versus 26 months

GSH change GSH

Reference Rebrin et al. (2003)

GSSG Rat (Fisher; male)

15 months versus 26 months

NC tGSH

Rikans and Moore (1988)

Abbreviations: NC, no change; CX, cortex; HC, hippocampus; BS, brainstem; ST, striatum; CB, cerebellum.

the four studies using rats. For both the kidney and heart, decreases in GSH were seen only in the very oldest mice and rats. The tissue that showed the most significant decreases in GSH with age is the lens of the eye. Surprisingly similar results were obtained with both rat and human lenses. Interestingly, GSH depletion using BSO, the inhibitor of GSH biosynthesis, leads to cataract development when administered to newborn rats (Martensson et al., 1989). The cataract formation could be prevented if the rats were also given GSH monoester. Taken together, these and other data suggest that one of the causes of cataract formation in older adults could be the decrease in lens GSH. Studies with human plasma have also shown a consistent decrease in GSH levels with aging as well as an increase in GSSG. However, in most of the tissues other than plasma, there is a good deal of disagreement with respect to whether or not GSSG increases with age, thereby resulting in significant changes in the GSH/GSSG ratio and redox potential of the tissue. A major reason for this discrepancy is likely to be due to difficulties in accurately measuring GSSG since the levels of GSSG are very low relative to GSH. Furthermore, GSH can be oxidized during tissue preparation, which could easily obscure true increases in the level of GSSG. However, in studies where careful attention was paid to the preparation of tissue samples, age dependent increases in GSSG were seen in a number of tissues in mice (Rebrin et al., 2003) and rats (Palomero et al., 2001) including liver, kidney, heart, brain, eye and testis. This, in turn, leads to an age dependent decrease in the GSH/GSSG ratio, suggesting a significant alteration in the redox environment of these tissues with age. A recent study, which looked at the GSH/GSSG ratio in human plasma also found a decrease in the ratio with age (Jones et al., 2002). Interestingly, this ratio remained constant until 45 years. and then declined linearly thereafter, suggesting that GSH metabolism fails to keep up with oxidizing events beginning in late middle age. The age related decreases seen in many studies in tGSH could be due to increased GSH consumption, decreased GSH production or some combination of the two. Increased consumption would be consistent with an increase in ROS production by the mitochondria with age. However, recent studies suggest that decreased production also plays an important role in the decline of tGSH with tissue age. These studies have all shown a good correlation between decreases in the level of GCL activity and decreases in GSH levels (Rathbun and Murray, 1991; Liu and Choi, 2000; Liu, 2002; Sandhu and Kaur, 2002; Mosoni et al., 2004). In addition, the decreases in GCL activity were correlated with decreases in the levels of GCL protein and/or mRNA of at least one of the two subunits (Liu

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307

and Choi, 2000; Liu, 2002). A recent paper showed that the decrease in GCL levels, at least in liver, is due to a decrease in the level of Nrf2, the transcription factor involved in the induction of the genes encoding both chains of GCL (Suh et al., 2004). Moreover, the low levels of Nrf2 in the livers of 24–28 month old rats could be restored by treatment with lipoic acid resulting in a restoration of both GCL activity and GSH levels. This suggests that the basic transcriptional response mechanism is still present in the old rats but that for some reason the basal set point is turned down during aging. Further research is clearly needed to determine if decreases in Nrf2 levels are also seen in other tissues which show an age-related decrease in GSH levels and, if so, to understand what causes the turn down in the basal set point. Only a single study showed changes in GS activity with aging (Liu and Dickinson, 2003). Whether or not there are age-dependent changes in the activity of other enzymes involved in GSH metabolism is less clear and may be tissue dependent. One of the more interesting observations to come out of the studies on GSH levels and aging is that there appear to be gender-specific differences in the age-associated changes in GSH levels. Almost all of the studies cited above used male mice or rats so a gender difference cannot account for the variations in the results of the studies shown in Table 2. However, when investigators compared changes in GSH levels between male and female mice as a function of age, significant differences in some tissues were seen. For example, in the lung and brainstem, GSH levels showed a much more dramatic decline between 12 and 24 months of age in male mice than in their female counterparts (Wang et al., 2003). However, in other tissues, such as the cerebral cortex and the cerebellum, GSH levels declined more in the males than the females between 3 and 12 months of age. Similar decreases were seen in the two sexes between 12 and 24 months of age although the GSH levels in the females at 24 months were still significantly higher than in the males. In contrast to the brain and lung, female livers showed a significantly greater decrease in GSH levels between 12 and 24 months than male livers. Although the reason for these genderdependent differences in the maintenance of GSH levels during maturation and aging have not been established, several studies suggest that they may be related to estrogen levels. Estradiol increases GSH levels in human hepatocytes both in vivo and in vitro in a process dependent upon the induction of GCL (Dabrosin and Ollinger, 2004). The liver is a major site of GSH synthesis in humans and circulating GSH can affect GSH levels in tissues (Dabrosin and Ollinger, 2004). In studies using rats, mitochondria isolated from the livers of 4–5-month-old female rats were found to have significantly higher levels of GSH than mitochondria isolated from the livers of their male counterparts (Borras et al., 2003). This difference in GSH levels correlated with a lower level of peroxide generation by female mitochondria. The gender-dependent differences in both mitochondrial GSH levels and peroxide production were eliminated in ovariectomized rats but were restored by treatment of the female rats with 17 b-estradiol. Taken together, these data suggest that estrogen may play an important role in maintaining GSH levels, at least in some tissues. Further research is needed to determine the validity of this conclusion. 4.2. Glutathione and Parkinson’s disease The major risk factor for many neurodegenerative diseases is increasing age. Thus, agedependent changes in brain levels of GSH might be expected to play a role in the

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development of these diseases. Of all the neurodegenerative diseases, evidence for a dysfunction in GSH metabolism is strongest in Parkinson’s disease (PD) (for reviews, see Anderson, 2001; Bains and Shaw, 1997; Schulz et al., 2000). PD is a neurodegenerative disease which is characterized by the loss of dopaminergic neurons in the pars compacta of the substantia nigra (SNpc). This leads to a severe depletion of dopamine in the striatum and deficits in motor function, which are the well-known hallmarks of the disease. A number of studies have found that tGSH levels are specifically decreased in the SNpc of PD patients and that there is a positive correlation between the severity of the disease and the extent of tGSH loss. tGSH levels are not reduced in other brain areas in PD patients or in patients with other neurodegenerative diseases that also affect dopaminergic neurons. In addition, tGSH levels are decreased to almost the same degree in patients with incidental Lewy body disease, a preclinical and still asymptomatic form of PD. Thus, the decrease in tGSH precedes other PD-associated changes in the SNpc such as decreases in mitochondrial complex I activity and dopamine levels. Although these data suggest a critical role for tGSH depletion in the development of PD, the depletion itself is unlikely to be the actual cause of the disease. Several in vitro and in vivo studies have shown that tGSH depletion alone does not result in the death of dopaminergic neurons (Nakamura et al., 1997; Schulz et al., 2000). However, a number of studies have shown that tGSH depletion sensitizes dopaminergic neurons to a variety of oxidative stresses including N-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) which produces irreversible clinical, biochemical and neuropathological changes that closely mimic those observed in idiopathic PD (Schulz et al., 2000). tGSH depletion was also shown to sensitize dopaminergic neurons to nitric oxide (NO) (Canals et al., 2001). In the presence of GSH, NO has protective, neurotrophic effects on dopaminergic neurons. However, following tGSH depletion, NO becomes toxic to the neurons. Taken together, these data indicate that tGSH depletion plays a critical role in the development of PD and suggest that the identification of pathways which can prevent the loss of tGSH in cells exposed to oxidative stress could have important consequences for the treatment of this disease, as well as in the treatment of other neurodegenerative diseases in which oxidative stress is thought to play a role (for review, see Halliwell, 2001). A small number of cases of PD are caused by genetic mutations, and the specific genes involved in many of these have been identified (for review, see Mouradian, 2002). Studies on the protein products of these genes suggest that a malfunction in the ubiquitin-proteasome protein degradation pathway may contribute to the loss of dopaminergic neurons in both the genetic forms of PD and in the idiopathic forms of the disease as well (Chung et al., 2001; McNaught and Jenner, 2001). Recent studies have also suggested an important role for GSH in ubiquitin-mediated protein degradation. For example, treatment of nerve cells with doses of cadmium which deplete at least 80% of the tGSH leads to an inhibition of protein ubiquitination (Figueiredo-Pereira et al., 1998), and similar reductions in tGSH levels were shown to lead to the reversible inhibition of both the ubiquitin activating enzyme (E1) (Jahngen-Hodge et al., 1997; Jha et al., 2002) and the ubiquitin-conjugating enzymes (E2) (Jahngen-Hodge et al., 1997). Clearly more work is required to understand the relationship between tGSH, redox status and the various protein degradation pathways during aging and age-associated neurodegenerative diseases.

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4.3. Summary Glutathione plays a critical role in many biological processes both directly as a co-factor in a number of enzymatic reactions but also indirectly as the major thiol-disulfide redox buffer in mammalian cells. Perhaps most importantly, glutathione provides a critical defense system for the protection of cells from many forms of stress. However, in many cases, mild stress actually increases GSH levels, thereby providing increased protection against more severe forms of stress. Thus, in line with the old adage, what doesn’t kill a cell makes it stronger. However, there is good evidence that at least in some tissues GSH levels decline with age. This decrease may contribute to many of the age-related declines in cellular function as well as the increased susceptibility to various insults. The evidence for this is strongest in the brain where GSH loss is implicated in both PD and in neuronal injury following stroke. Thus, therapies that aim to maintain GSH levels have a significant potential for reducing the functional loss associated with both severe stress and aging.

Acknowledgements Since the number of references that could be included was limited, I have used reviews in many cases instead of the original articles. I apologize to any authors whose work was not directly cited. The author’s research was supported by National Institutes of Health grant NS28212 and funding from the Mericos-TSRI Neurobiology and Vision Science Program.

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