In addition to its participation in a variety of other biochemical reactions, glutathione (GSH) is a major antioxidant. It is regularly generated intracellularly from its oxidized form by glutathione reductase activity that is coupled with a series of interrelated reactions. Synthesis of GSH also takes place intracellularly by a two-step reaction, the first of which is catalyzed by rate-limiting gamma-glutamylcysteine synthetase activity. Intracellular substrates for GSH are provided both by direct amino acid transport and by a gamma-glutamyl transpeptidase reaction that salvages circulating GSH by coupling the gamma-glutamyl moiety to a suitable amino acid acceptor for transport into the cell. Although the liver is a net synthesizer of circulating GSH, organs such as the kidney salvage GSH through the gamma-glutamyl transpeptidase reaction. Intracellular GSH may be consumed by GSH transferase reactions that conjugate GSH with certain xenobiotics. Elevation of cellular GSH levels in cultured cells in response to hyperoxia or electrophilic agents such as diethylmaleate is coupled with an increase in activity of the Xc- transport system for the amino acids cystine and glutamate. Strategies may be developed for protection against oxidant injury by enhancement of transport systems for precursor amino acids of GSH or by providing substrate that circumvents feedback inhibition of GSH synthesis.

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Our results suggest that the conversion of breast tumors to a tamoxifen-resistant phenotype is associated with oxidative stress and the subsequent antioxidant response and with increased phosphorylated JNK and c-Jun levels and AP-1 activity, which together could contribute to tumor growth.

Hyperbaric oxygen exposure rapidly induces lipid peroxidation and cellular damage in a variety of organs. In this study, we demonstrate that the exposure of rats to 4 atmospheres of 100% oxygen for 90 min is associated with increased levels of lipid peroxidation products [malonaldehyde (MDA) and 4-hydroxyalkenals (4-HDA)] and with changes in the activities of two antioxidative enzymes [glutathione peroxidase (GPX) and glutathione reductase (GR)], as well as in the glutathione status in the lungs and in the brain. Products of lipid peroxidation increased after hyperbaric hyperoxia, both GPX and GR activities were decreased, and levels of total glutathione (reduced+oxidized) and glutathione disulfide (oxidized glutathione) increased in both lung and brain areas (cerebral cortex, hippocampus, hypothalamus, striatum, and cerebellum) but not in liver. When animals were injected with melatonin (10 mg/kg) immediately before the 90-min hyperbaric oxygen exposure, all measurements of oxidative damage were prevented and were similar to those in untreated control animals. Melatonin's actions may be related to a variety of mechanisms, some of which remain to be identified, including its ability to directly scavenge free radicals and its induction of antioxidative enzymes via specific melatonin receptors.

N-Acetylcysteine (NAC), a cysteine derivative with chemoprotective and radioprotective effects, was found to elevate bovine pulmonary artery endothelial cell (EC) glutathione after in vitro incubation. The elevation in glutathione was associated with enhanced uptake of radioactivity of cystine from the medium. Because cystine in medium was converted rapidly to cysteine and cysteinyl-NAC in the presence of NAC and given that cysteine has a higher affinity for uptake by EC than cystine, we conclude that the enhanced uptake of radioactivity was in the form of cysteine and at least part of the stimulatory effect of NAC on EC glutathione was due to a formation of cysteine by a mixed disulfide reaction of NAC with cystine similar to that previously reported for Chinese hamster ovarian cells (R. D. Issels et al. 1988. Biochem. Pharmacol. 37:881-888). However, NAC was more effective than cysteine in elevating cellular glutathione at equimolar concentrations, and at higher concentrations of NAC an elevation of EC glutathione occurred even in the absence of cystine in the medium through a currently unknown mechanism. Thus, at least two mechanisms are operative in the elevation of endothelial cellular glutathione by NAC. NAC may be a useful compound for elevating glutathione of the pulmonary vasculature for protection against oxidant stress.

Isolated, isovolumic rat hearts, perfused by Krebs-Henseleit buffer at constant coronary flow rate, were used to explore the hypothesis that endogenous cardiac glutathione provides protection against myocardial dysfunction associated with short periods of ischemia. Experimental animals were depleted of cardiac glutathione to 35% of control levels by intraperitoneal injections of diethylmaleate (DEM). Left ventricular pressure, coronary perfusion pressure, and glutathione levels were measured in control and experimental hearts after 60 minutes of oxygenated perfusion and after 20 minutes of global, no-flow ischemia and 30 minutes of reperfusion. With each protocol, both control and glutathione-depleted hearts received either standard buffer or one supplemented with 2 mM glutathione. Recovery of systolic function after ischemia-reperfusion was impaired in DEM-treated hearts compared with controls. In addition, the rise in perfusion pressure and chamber stiffness was also greater in DEM-treated hearts compared with controls. Recovery in glutathione-depleted hearts was improved when the reperfusate was supplemented with glutathione. In addition, the supplemented reperfusate prevented the decrease in compliance and the increase in coronary perfusion pressure in the glutathione-depleted hearts. Ischemia-reperfusion alone were not associated with a significant alteration in myocardial glutathione levels. Prewashout myocardial levels of glutathione were elevated after reperfusion with glutathione-supplemented bulfer but fell to baseline levels after a short washout period. These studies demonstrate that endogenous glutathione is important in protection of myocardium from injury after ischemia-reperfusion, presumably by modifying levels of active oxygen intermediates. The smaller changes in left ventricular pressure and coronary resistance after administration of GSH probably reflects an extracellular mechanism because benefit is seen soon after reperfusion. (Circulation 1989;80:1449-1457 D uring oxidative metabolism, cells produce potentially toxic oxygen radicals for which both specific and nonspecific scavenging mechanisms are present. Recently, it has been suggested that hypoxia or ischemia, followed by reoxygenation or reperfusion, increases production of oxygen radical species.1-5Administration of exogenous quenchers of free radicals, such as superoxide dismutase (SOD), catalase or both, improve cardiac function and limit infarct size when administered after experimental global ischemia.6,7From the Cardiology Section,

Diethyl maleate (DEM, 0.025-0.10 mM) increased glutathione (GSH) levels in calf pulmonary artery endothelial cells up to fivefold in 12-24 h of incubation. Parallel increases occurred in the rates of uptake of cystine and glutamate. The DEM-mediated increases in both GSH levels and glutamate-cystine uptake were inhibited by cycloheximide and actinomycin D, indicating a dependency on protein and RNA synthesis. The stimulatory effects of DEM on amino acid uptake and GSH levels were greater than those in endothelial cells exposed to 80% O2 for 24 h. The effect of hyperoxia on cellular transport processes was also less specific than that of DEM. Although the increase in glutamate uptake produced by hyperoxia appeared to be under the regulation of protein synthesis, the relationship with elevated GSH in the presence of hyperoxia was less clear because of elevation of control cellular GSH by cycloheximide or actinomycin D alone. Inhibition of GSH synthesis by buthionine sulfoximine also stimulated cystine and glutamate uptake. We conclude that elevation of endothelial intracellular GSH by both DEM and hyperoxia is associated with and may be produced by enhanced uptake of precursor amino acids; the effect of DEM is more pronounced and more specific than that of hyperoxia.