Antioxidant and bioenergetic coupling between neurons and astrocytes

Oxidative and nitrosative stress underlie the pathogenesis of a broad range of human diseases, in particular neurodegenerative disorders. Within the brain, neurons are the cells most vulnerable to excess reactive oxygen and nitrogen species; their survival relies on the antioxidant protection promoted by neighbouring astrocytes. However, neurons are also intrinsically equipped with a biochemical mechanism that links glucose metabolism to antioxidant defence. Neurons actively metabolize glucose through the pentose phosphate pathway, which maintains the antioxidant glutathione in its reduced state, hence exerting neuroprotection. This process is tightly controlled by a key glycolysis-promoting enzyme and is dependent on an appropriate supply of energy substrates from astrocytes. Thus brain bioenergetic and antioxidant defence is coupled between neurons and astrocytes. A better understanding of the regulation of this intercellular coupling should be important for identifying novel targets for future therapeutic interventions.     

Glutathione pathways in the brain

The antioxidant glutathione (GSH) is essential for the cellular detoxification of reactive oxygen species in brain cells. A compromised GSH system in the brain has been connected with the oxidative stress occuring in neurological diseases. Recent data demonstrate that besides intracellular functions GSH has also important extracellular functions in brain. In this respect astrocytes appear to play a key role in the GSH metabolism of the brain, since astroglial GSH export is essential for providing GSH precursors to neurons. Of the different brain cell types studied in vitro only astrocytes release substantial amounts of GSH. In addition, during oxidative stress astrocytes efficiently export glutathione disulfide (GSSG). The multidrug resistance protein 1 participates in both the export of GSH and GSSG from astrocytes. This review focuses on recent results on the export of GSH and GSSG from brain cells as well as on the functions of extracellular GSH in the brain. In addition, implications of disturbed GSH pathways in brain for neurodegenerative diseases will be discussed.     

Oxidative and nitrosative stress in ammonia neurotoxicity

Increased ammonia accumulation in the brain due to liver dysfunction is a major contributor to the pathogenesis of hepatic encephalopathy (HE). Fatal outcome of rapidly progressing (acute) HE is mainly related to cytotoxic brain edema associated with astrocytic swelling. An increase of brain ammonia in experimental animals or treatment of cultured astrocytes with ammonia generates reactive oxygen and nitrogen species in the target tissues, leading to oxidative/nitrosative stress (ONS). In cultured astrocytes, ammonia-induced ONS is invariably associated with the increase of the astrocytic cell volume. Interrelated mechanisms underlying this response include increased nitric oxide (NO) synthesis which is partly coupled to the activation of NMDA receptors and increased generation of reactive oxygen species by NADPH oxidase. ONS and astrocytic swelling are further augmented by excessive synthesis of glutamine (Gln) which impairs mitochondrial function following its accumulation in there and degradation back to ammonia (“the Trojan horse” hypothesis). Ammonia also induces ONS in other cell types of the CNS: neurons, microglia and the brain capillary endothelial cells (BCEC). ONS in microglia contributes to the central inflammatory response, while its metabolic and pathophysiological consequences in the BCEC evolve to the vasogenic brain edema associated with HE. Ammonia-induced ONS results in the oxidation of mRNA and nitration/nitrosylation of proteins which impact intracellular metabolism and potentiate the neurotoxic effects. Simultaneously, ammonia facilitates the antioxidant response of the brain, by activating astrocytic transport and export of glutathione, in this way increasing the availability of precursors of neuronal glutathione synthesis.     

Vitamin E, Ascorbate, Glutathione, Glutathicne Disulfide, and Enzymes of Glutathione Metabolism in Cultures of Chick Astrocytes and Neurons: Evidence that Astrocytes Play an Important Role in Antioxidative Processes in the Brain

Abstract: GSH, GSSG, vitamin E, and ascorbate were measured in 14-day cultures of chick astrocytes and neurons and compared with levels in the forebrains of chick embryos of comparable age. Activities of enzymes involved in GSH metabolism were also measured. These included -γ-glutamylcysteine synthetase, GSH synthetase, γ-glutamyl cyclotransferase, γ-glutamyltranspeptidase, glutathione transferase (GST), GSH peroxidase, and GSSG reductase. The concentration of lipid-soluble vitamin E in the cultured neurons was found to be comparable with that in the forebrain. On the other hand, the concentration of vitamin E in the astrocytes was significantly greater in the cultured astrocytes than in the neurons, suggesting that the astrocytes are able to accumulate exogenous vitamin E more extensively than neurons. The concentrations of major fatty acids were higher in the cell membranes of cultured neurons than those in the astrocytes. Ascorbate was not detected in cultured cells although the chick forebrains contained appreciable levels of this antioxidant. GSH, total glutathione (i.e., GSH and GSSG), and GST activity were much higher in cultured astrocytes than in neurons. γ-Glutamylcysteine synthetase activity was higher in the cultured astrocytes than in the cultured neurons. GSH reductase and GSH peroxidase activities were roughly comparable in cultured astrocytes and neurons. The high levels of GSH and GST in cultured astrocytes appears to reflect the situation in vivo. The data suggest that astrocytes are resistant to reactive oxygen species (and potentially toxic xenobiotics) and may play a protective role in the brain. Because enzymes of GSH metabolism are generally well represented in cultured astrocytes and neurons these cells may be ideally suited as probes for manipulating GSH levels in neural tissues in vitro. Cultured astrocytes and neurons should be amenable to the study of the effects of various metabolic insults on the GSH system. Such studies may provide insights into the design of therapeutic strategies to combat oxidative and xenobiotic stresses.     

Glial perspectives of metabolic states during cerebral hypoxia--calcium regulation and metabolic energy

Cooperation between astrocytes and neurons is a unique interaction between two highly specialized cell types of the brain. Therefore, lack of nutrient supply during ischemia requires tight coordination of metabolism between astrocytes and neurons to keep the brain functions intact. To understand the impact of energy limitation on astrocytes, the functions of astrocytes have to be considered: (i) supplementation of neuronal cells, (ii) modulation of the extracellular milieu, mainly of the glutamate level, and (iii) elimination of reactive oxygen species (ROS). In cultured astrocytes and neurons inhibition of oxidative phosphorylation, using rotenone, was tested. Interestingly, this had only a negligible effect on Ca2+ homeostasis in astrocytes, even in combination with a severe glutamate stress. In contrast, in neurons glutamate in the presence of rotenone induced Ca2+ deregulation. Ca2+ homeostasis is very critical for cell survival. A massive and prolonged Ca2+ rise will lead to deregulation of many processes in such a way that the cells affected can hardly survive. Ca2+ homeostasis depends on the energy-consuming processes, which maintain the steep gradient between intracellular and extracellular Ca2+ concentration. Deprivation of oxygen and glucose during ischemia leads to a depletion of ATP in the brain, due to inhibited glycolytic and mitochondrial activity, whereas energy-consuming processes like ion pumps drain the ATP pools. On the other hand, specific mechanisms can protect brain structures against the massive insult of ischemia. Glycogen, stored in astrocytes, can maintain both neurons and astrocytes alive during short limitation of oxygen and glucose. Moreover, astrocytes can fuel ATP generation by providing lactate for neurons.     

Uptake,Metabolic Effects and Toxicity of Arsenate and Arsenite in Astrocytes

The inorganic arsenic species arsenate and arsenite are common environmental toxins which contaminate the drinking water in many countries. Chronic intoxication with arsenicals has been connected with various diseases, but causes also neurological complications and impairs cognitive development, learning and memory. In brain, astrocytes have a pivotal role as partners of neurons in homeostatic and metabolic processes. In addition, astrocytes are the first parenchymal brain cell type which encounters substances which cross the blood–brain barrier and are considered as first line of defence against the toxic potential of xenobiotics. Therefore, astrocytes are likely to play a prominent role in the metabolism and potential detoxification of arsenicals in brain. This article summarizes the current knowledge on the uptake and toxicity of arsenate and arsenite in astrocytes and discusses the modulation of the astrocytic glucose and glutathione metabolism by arsenicals.     

Hypoxia‐induced apoptosis of astrocytes is mediated by reduction of Dicer and activation of caspase‐1

Hypoxia is a condition in which the whole body or a region of the body is deprived of oxygen supply. The brain is very sensitive to the lack of oxygen and cerebral hypoxia can rapidly cause severe brain damage. Astrocytes are essential for the survival and function of neurons. Therefore, protecting astrocytes against cell death is one of the main therapeutic strategies for treating hypoxia. Hence, the mechanism of hypoxia‐induced astrocytic cell death should be fully elucidated. In this study, astrocytes were exposed to hypoxic conditions using a hypoxia work station or the hypoxia mimetic agent cobalt chloride (CoCl₂). Both the hypoxic gas mixture (1% O₂) and chemical hypoxia‐induced apoptotic cell death in T98G glioblastoma cells and mouse primary astrocytes. Reactive oxygen species were generated in response to the hypoxia‐mediated activation of caspase‐1. Active caspase‐1 induced the classical caspase‐dependent apoptosis of astrocytes. In addition, the microRNA processing enzyme Dicer was cleaved by caspase‐3 during hypoxia. Knockdown of Dicer using antisense oligonucleotides induced apoptosis of T98G cells. Taken together, these results suggest that astrocytic cell death during hypoxia is mediated by the reactive oxygen species/caspase‐1/classical caspase‐dependent apoptotic pathway. In addition, the decrease in Dicer levels by active caspase‐3 amplifies this apoptotic pathway via a positive feedback loop. These findings may provide a new target for therapeutic interventions in cerebral hypoxia.     

Antibodies to synthetic fragment 95–123 of the prion protein protect neurons and astrocytes from beta-amyloid toxicity

Molecular mechanisms of β-amyloid toxic effect on brain cells during Alzheimer’s disease is associated with oxidative stress, intracellular Ca²⁺ increase in neurons and astrocytes and activation of reactive oxygen species production. Prion protein is known to be involved in beta-amyloid toxicity. Here we investigated an effect of affinity purified antibodies to synthetic fragment 95–123 of the prion protein (PrP-(95–123)) on β-amyloid induced cell death, Ca²⁺-signal, reactive oxygen species production and caspase 3 activation. We have shown that antibodies to PrP-(95–123) are able to protect neurons and astrocytes from beta-amyloid induced cell death with no effect on the intracellular Ca²⁺ concentration altered by beta-amyloid treatment. However, the antibodies significantly reduced β-amyloid induced reactive oxygen species production in astrocytes and decreased caspase 3 activation in neurons and astrocytes. Thus, induction of antibodies to PrP-(95–123) of the prion protein provides a new approach to anti-Alzheimer’s disease vaccine design.     

Methylene Blue Protects Astrocytes against Glucose Oxygen Deprivation by Improving Cellular Respiration

Astrocytes outnumber neurons and serve many metabolic and trophic functions in the mammalian brain. Preserving astrocytes is critical for normal brain function as well as for protecting the brain against various insults. Our previous studies have indicated that methylene blue (MB) functions as an alternative electron carrier and enhances brain metabolism. In addition, MB has been shown to be protective against neurodegeneration and brain injury. In the current study, we investigated the protective role of MB in astrocytes. Cell viability assays showed that MB treatment significantly protected primary astrocytes from oxygen-glucose deprivation (OGD) & reoxygenation induced cell death. We also studied the effect of MB on cellular oxygen and glucose metabolism in primary astrocytes following OGD-reoxygenation injury. MB treatment significantly increased cellular oxygen consumption, glucose uptake and ATP production in primary astrocytes. In conclusion our study demonstrated that MB protects astrocytes against OGD-reoxygenation injury by improving astrocyte cellular respiration.     

Protective Effects of Resveratrol on Hydrogen Peroxide Induced Toxicity in Primary Cortical Astrocyte Cultures

It is well established that the brain is particularly susceptible to oxidative damage due to its high consumption of oxygen and that astrocytes are involved in a variety of important activities for the nervous system, including a protective role against damage induced by reactive oxygen species (ROS). The use of antioxidant compounds, such as polyphenol resveratrol found in red wine, to improve endogenous antioxidant defenses has been proposed for neural protection. The aim of this study is to evaluate the putative protective effect of resveratrol against acute H₂O₂-induced oxidative stress in astrocyte cultures, evaluating ROS production, glutamate uptake activity, glutathione content and S100B secretion. Our results confirm the ability of resveratrol to counteract oxidative damage caused by H₂O₂, not only by its antioxidant properties, but also through the modulation of important glial functions, particularly improving glutamate uptake activity, increasing glutathione content and stimulating S100B secretion, which all contribute to the functional recovery after brain injury.     

Regulation of tyrosine kinase B activity by the Cyp46/cholesterol loss pathway in mature hippocampal neurons: relevance for neuronal survival under stress and in aging

It is well established that memory formation and retention involve the coordinated flow of information from the post-synaptic site of particular neuronal populations to the nucleus, where short and long-lasting modifications of gene expression occur. With age, mnemonic, motor and sensorial alterations occur, and it is believed that extra failures in the mechanisms used for memory formation and storage are the cause of neurodegenerative pathologies like Alzheimer's disease. A prime candidate responsible for damage and loss of function during aging is the accumulation of reactive oxygen species, derived from normal oxidative metabolism. However, dysfunction in the aged brain is not paralleled by an increase in neuronal death, indicative that the brain is better suited to fight against the death signals generated from reactive oxygen species than against loss-of-function stimuli. A main aim of this laboratory is to understand how neurons perform and survive in the constitutive stress background represented by aging. In this report, we summarize our recent findings in relation to survival.     

Oxidative stress decreases antioxidant enzyme activities in reaggregation cultures of rat brain cells

The brain has been suggested to be especially sensitive to damage by reactive oxygen species. In this study, we examined the effects of hyperoxic conditions on the activities and mRNA levels of antioxidant enzymes in reaggregation cultures of rat forebrain cells. Cultures were exposed to 80% oxygen for 12–60 h starting on Days 17 and 33 in culture. Superoxide dismutase activities and mRNA levels were not affected by hyperoxia, whereas catalase activity was slightly decreased after 24 h in 80% oxygen at Day 17. Glutathione peroxidase activity was markedly decreased already after 12 h of hyperoxia, and decreased activities of glutathione reductase and glucose-6-phosphate dehydrogenase were also noted. The glutathione peroxidase mRNA levels were increased in hyperoxic cultures at Day 17 but not at Day 33. These results suggest that the enzymatic defense mechanisms against reactive oxygen species in the brain are rather weak and deteriorate during oxidative stress but that a potential for compensatory upregulation exists at least during the first postnatal weeks.     

Effects of glutathione depletion on the viability of human NT2-derived neuronal and astroglial cultures

The level of glutathione (GSH) is often reduced in brains that are affected by neurodegeneration. It is not known, however,whether this is a cause or a consequence of the disease. Here we have examined the effects of GSH depletion on the viability of human neurons cultured in either the presence or the absence of astrocytes, both derived from NT2/D1 cells. We established that the endogenous concentration of GSH is 10 times lower in neurons than in astrocytes (1.42 versus 18.9 pmol microg protein(-1)) and that pure neuronal cultures begin to die by apoptosis within 24 h of GSH depletion. By contrast, neurons that are co-cultured with astrocytes remain viable for several days, even with a profoundly decreased GSH content. However, they die rapidly when challenged additionally with nitrative stress. In addition, astrocytes survive for prolonged periods of time (>12 days) under severely reduced GSH concentrations. Our study shows clear differences in the content and sensitivity to depletion of GSH in neurons and astrocytes and establishes the significance of neuronal-glial interactions for the maintenance of neuronal viability under reduced GSH content. However, with chronic GSH depletion, these interactions might not be sufficient to protect neurons from other injurious factors (i.e. reactive oxygen and nitrogen species), which indicates that defective GSH metabolism might facilitate the progression of neurodegeneration.     

Involvement of selenoprotein P in protection of human astrocytes from oxidative damage

Selenoprotein P (SeP) is a highly glycosylated, selenium-rich plasma protein. Aside from its role as selenium carrier protein, an antioxidative function of SeP has been suggested. Astrocytes, which detoxify reactive oxygen species in the brain, were described as potential target cells of SeP. We investigated the expression of SeP in human astrocytes and its involvement in the protection of these cells against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage. We show that primary human astrocytes and the human astrocytoma cell line MOG-G-CCM express SeP as an unglycosylated protein, which is not secreted. SeP expression in astrocytes is constitutive. Preincubation of astrocytes with hepatocyte-derived SeP mimicks the protective effect of low-molecular-weight selenocompounds such as sodium selenite or selenomethionine against oxidative damage, shielding astrocytes from t-BHP-induced cytotoxicity. Selenium supplementation of astrocytes counteracts oxidative stress via an increase in expression and activity of the selenoenzyme cytosolic glutathione peroxidase (cGPx). Furthermore, specific downregulation of SeP expression by small interfering RNA decreases cell viability of human astrocytes and makes them more susceptible to t-BHP-induced cytotoxicity. Our results implicate an antioxidant activity of constitutively expressed SeP in selenium-deficient astrocytes, while during adequate selenium supply the enhanced protection against oxidative stress is exerted by cGPx.     

Inhibition of glutathione depletion by retinoic acid and tocopherol protects cultured neurons from staurosporine-induced oxidative stress and apoptosis

Cellular redox status is an important factor during neuronal apoptosis. In primary cultures of chick embryonic neurons, serum deprivation and treatment with staurosporine (200 nM) for 24 h increased the percentage of apoptotic neurons from 13% in controls to 28%, and 68%, respectively. Both exposure to staurosporine and serum deprivation resulted in a four-fold increase in the mitochondrial reactive oxygen species production 4 h after the onset of the injury. Whereas the intracellular glutathione content remained unchanged by serum deprivation, it was markedly reduced by staurosporine suggesting that an increased reactive oxygen species production was more deleterious at a low intracellular glutathione content. Treatment with L-buthionine-(S,R)-sulfoximine, an inhibitor of the glutathione synthesis, decreased the intracellular glutathione content, but did not significantly alter the percentage of apoptotic neurons. Tocopherol (10 microM) and retinoic acid (0.1 microM) inhibited staurosporine-induced glutathione depletion as well as the increase in the percentage of apoptotic neurons. We conclude that under conditions of an increased reactive oxygen species production a high intracellular glutathione content could protect neurons from apoptotic injury and that drugs inhibiting the glutathione depletion could prevent neurons from oxidative damage.     

Reconstruction and flux analysis of coupling between metabolic pathways of astrocytes and neurons: application to cerebral hypoxia

Background

It is a daunting task to identify all the metabolic pathways of brain energy metabolism and develop a dynamic simulation environment that will cover a time scale ranging from seconds to hours. To simplify this task and make it more practicable, we undertook stoichiometric modeling of brain energy metabolism with the major aim of including the main interacting pathways in and between astrocytes and neurons.

Model

The constructed model includes central metabolism (glycolysis, pentose phosphate pathway, TCA cycle), lipid metabolism, reactive oxygen species (ROS) detoxification, amino acid metabolism (synthesis and catabolism), the well-known glutamate-glutamine cycle, other coupling reactions between astrocytes and neurons, and neurotransmitter metabolism. This is, to our knowledge, the most comprehensive attempt at stoichiometric modeling of brain metabolism to date in terms of its coverage of a wide range of metabolic pathways. We then attempted to model the basal physiological behaviour and hypoxic behaviour of the brain cells where astrocytes and neurons are tightly coupled.

Results

The reconstructed stoichiometric reaction model included 217 reactions (184 internal, 33 exchange) and 216 metabolites (183 internal, 33 external) distributed in and between astrocytes and neurons. Flux balance analysis (FBA) techniques were applied to the reconstructed model to elucidate the underlying cellular principles of neuron-astrocyte coupling. Simulation of resting conditions under the constraints of maximization of glutamate/glutamine/GABA cycle fluxes between the two cell types with subsequent minimization of Euclidean norm of fluxes resulted in a flux distribution in accordance with literature-based findings. As a further validation of our model, the effect of oxygen deprivation (hypoxia) on fluxes was simulated using an FBA-derivative approach, known as minimization of metabolic adjustment (MOMA). The results show the power of the constructed model to simulate disease behaviour on the flux level, and its potential to analyze cellular metabolic behaviour in silico.

Conclusion

The predictive power of the constructed model for the key flux distributions, especially central carbon metabolism and glutamate-glutamine cycle fluxes, and its application to hypoxia is promising. The resultant acceptable predictions strengthen the power of such stoichiometric models in the analysis of mammalian cell metabolism.     

Inhibition of astrocyte glutamate uptake by reactive oxygen species: role of antioxidant enzymes.

BACKGROUND: The recent literature suggests that free radicals and reactive oxygen species may account for many pathologies, including those of the nervous system. MATERIALS AND METHODS: The influence of various reactive oxygen species on the rate of glutamate uptake by astrocytes was investigated on monolayers of primary cultures of mouse cortical astrocytes. RESULTS: Hydrogen peroxide and peroxynitrite inhibited glutamate uptake in a concentration-dependent manner. Addition of copper ions and ascorbate increased the potency and the efficacy of the hydrogen peroxide effect, supporting the potential neurotoxicity of the hydroxyl radical. The free radical scavenger dimethylthiourea effectively eliminated the inhibitory potential of a mixture containing hydrogen peroxide, copper sulphate, and ascorbate on the rate of glutamate transport into astrocytes. The inhibitory effect of hydrogen peroxide on glutamate uptake was not altered by the inhibition of glutathione peroxidase, whereas the inhibition of catalase by sodium azide clearly potentiated this effect. Superoxide and nitric oxide had no effect by themselves on the rate of glutamate uptake by astrocytes. The absence of an effect of nitric oxide is not due to an inability of astrocytes to respond to this substance, since the same cultures did respond to nitric oxide with a sustained increase in cytoplasmic free calcium. CONCLUSION: These results confirm that reactive oxygen species have a potential neurotoxicity by means of impairing glutamate transport into astrocytes, and they suggest that preventing the accumulation of hydrogen peroxide in the extracellular space of the brain, especially during conditions that favor hydroxyl radical formation, could be therapeutic.     

The Pivotal Role of Astrocytes in the Metabolism of Iron in the Brain

Iron is essential for the normal functioning of cells but since it is also capable of generating toxic reactive oxygen species, the metabolism of iron is tightly regulated. The present article advances the view that astrocytes are largely responsible for distributing iron in the brain. Capillary endothelial cells are separated from the neuropil by the endfeet of astrocytes, so astrocytes are ideally positioned to regulate the transport of iron to other brain cells and to protect them if iron breaches the blood-brain barrier. Astrocytes do not appear to have a high metabolic requirement for iron yet they possess transporters for transferrin, haemin and non-transferrin-bound iron. They store iron efficiently in ferritin and can export iron by a mechanism that involves ferroportin and ceruloplasmin. Since astrocytes are a common site of abnormal iron accumulation in ageing and neurodegenerative disorders, they may represent a new therapeutic target for the treatment of iron-mediated oxidative stress.     

Handling of Copper and Copper Oxide Nanoparticles by Astrocytes

Copper is an essential trace element for many important cellular functions. However, excess of copper can impair cellular functions by copper-induced oxidative stress. In brain, astrocytes are considered to play a prominent role in the copper homeostasis. In this short review we summarise the current knowledge on the molecular mechanisms which are involved in the handling of copper by astrocytes. Cultured astrocytes efficiently take up copper ions predominantly by the copper transporter Ctr1 and the divalent metal transporter DMT1. In addition, copper oxide nanoparticles are rapidly accumulated by astrocytes via endocytosis. Cultured astrocytes tolerate moderate increases in intracellular copper contents very well. However, if a given threshold of cellular copper content is exceeded after exposure to copper, accelerated production of reactive oxygen species and compromised cell viability are observed. Upon exposure to sub-toxic concentrations of copper ions or copper oxide nanoparticles, astrocytes increase their copper storage capacity by upregulating the cellular contents of glutathione and metallothioneins. In addition, cultured astrocytes have the capacity to export copper ions which is likely to involve the copper ATPase 7A. The ability of astrocytes to efficiently accumulate, store and export copper ions suggests that astrocytes have a key role in the distribution of copper in brain. Impairment of this astrocytic function may be involved in diseases which are connected with disturbances in brain copper metabolism.     

Synthesis and role of glutathione in protection against oxidative stress in yeast

Summary

Glutathione (GSH) is an abundant and ubiquitous low-molecular-mass thiol with proposed roles in many cellular processes including amino acid transport, synthesis of proteins and nucleic acids, modulation of enzyme activity and metabolism of xenobiotics, carcinogens and reactive oxygen species. This review describes recent findings in the lower eukaryote Saccharomyces cerevisiae that are leading to a better understanding of the role of this peptide in eukaryotic cell metabolism. In particular, two gene products involved in maintaining the levels of reduced GSH have been studied; namely, GSH1 encoding γ-glutamylcysteine synthetase, the first step in the biosynthesis of GSH, and glutathione reductase, which recycles glutathione to its reduced form. These studies indicate that GSH is an essential metabolite in yeast, and that it is required for protection against oxidative stress produced by mitochondrial metabolism and exogenous reactive oxygen species. These findings are discussed in the light of analogous observations made in higher eukaryotes.