Thiol-disulfide Redox State Research Articles

Glutathione, glutathione disulfide, and S-glutathionylated proteins in cell cultures

The analysis of the global thiol–disulfide redox status in tissues and cells is a challenging task since thiols and disulfides can undergo artificial oxido-reductions during sample manipulation. Because of this, the measured values, in particular for disulfides, can have a significant bias. Whereas this methodological problem has already been addressed in samples of red blood cells and solid tissues, a reliable method to measure thiols and disulfides in cell cultures has not been previously reported.Here, we demonstrate that the major artifact occurring during thiol and disulfide analysis in cultured cells is represented by glutathione disulfide (GSSG) and S-glutathionylated proteins (PSSG) overestimation, due to artificial oxidation of glutathione (GSH) during sample manipulation, and that this methodological problem can be solved by the addition of N-ethylmaleimide (NEM) immediately after culture medium removal. Basal levels of GSSG and PSSG in different lines of cultured cells were 3–5 and 10–20 folds higher, respectively, when the cells were processed without NEM. NEM pre-treatment also prevented the artificial reduction of disulfides that occurs during the pre-analytical phase when cells are exposed to an oxidant stimulus. In fact, in the absence of NEM, after medium removal, GSH, GSSG and PSSG levels restored their initial values within 15–30min, due to the activity of reductases and the lack of the oxidant. The newly developed protocol was used to measure the thiol–disulfide redox status in 16 different line cells routinely used for biomedical research both under basal conditions and after treatment with disulfiram, a thiol-specific oxidant (0–200μM concentration range).Our data indicate that, in most cell lines, treatment with disulfiram affected the levels of GSH and GSSG only at the highest concentration. On the other hand, PSSG levels increased significantly also at the lower concentrations of the drug, and the rise was remarkable (from 100 to 1000 folds at 200μM concentration) and dose-dependent for almost all the cell lines. These data support the suitability of the analysis of PSSG in cultured cells as a biomarker of oxidative stress.

Imaging disulfide dinitroxides at 250 MHz to monitor thiol redox status

Measurement of thiol–disulfide redox status is crucial for characterization of tumor physiology. The electron paramagnetic resonance (EPR) spectra of disulfide-linked dinitroxides are readily distinguished from those of the corresponding monoradicals that are formed by cleavage of the disulfide linkage by free thiols. EPR spectra can thus be used to monitor the rate of cleavage and the thiol redox status. EPR spectra of 1H,14N- and 2H,15N-disulfide dinitroxides and the corresponding monoradicals resulting from cleavage by glutathione have been characterized at 250MHz, 1.04GHz, and 9GHz and imaged by rapid-scan EPR at 250MHz.

Protein thiols undergo reversible and irreversible oxidation during chill storage of ground beef as detected by 4,4'-dithiodipyridine.

Quantification of protein thiols and disulfides in ground beef during storage under high-oxygen atmosphere at 4 °C was performed by thiol detection using 4,4'-dithiodipyridine (4-DPS) before and after disulfide reduction using sodium borohydride. Two independent storage trials were performed, and in trial 1, only reversible thiol oxidation was observed (thiol loss was 30%). In trial 2, irreversible thiol oxidation occurred during the first days of storage, while further loss of thiols was caused by reversible disulfide formation (thiol loss was 33%, of which ca. half was lost because of irreversible oxidation). The results were compared to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of cross-linked myosin heavy chain formed by disulfide bonding. Both methods confirmed increasing disulfide formation because of thiol oxidation in meat during storage, but the 4-DPS method showed higher disulfide percentages than the SDS-PAGE method (22.2 ± 0.3% and 8.5 ± 1.2%, respectively). The 4-DPS assay provides an accurate method to evaluate the thiol-disulfide redox state in meat.

Differential regulation of tissue thiol-disulfide redox status in a murine model of peritonitis

BackgroundGlutathione (GSH)/glutathione disulfide (GSSG) and cysteine (Cys)/cystine (CySS) are major redox pools with important roles in cytoprotection. We determined the impact of septic peritonitis on thiol-disulfide redox status in mice.MethodsFVB/N mice (6–12 week old; 8/group) underwent laparotomy with cecal ligation and puncture (CLP) or laparotomy alone (control). Sections of ileum, colon, lung and liver were obtained and GSH, GSSG, Cys and CySS concentrations determined by HPLC 24 h after laparotomy. Redox potential [Eh in millivolts (mV)] of the GSH/GSSG and Cys/CySS pools was calculated using the Nernst equation. Data were analyzed by ANOVA (mean ± SE).ResultsGSH/GSSG Eh in ileum, colon, and liver was significantly oxidized in septic mice versus control mice (ileum: septic −202±4 versus control −228±2 mV; colon: -195±8 versus −214±1 mV; and liver: -194±3 vs. -210±1 mV, all P<0.01). Lung GSH/GSSG redox was similar in each group (−191±3 versus −190±2 mV). In contrast, ileal and colonic Cys/CySS Eh was unchanged with CLP, while liver and lung Cys/CySS Eh became significantly more reducing (liver: septic = −103±3 versus control −90±2 mV; lung: -101±5 versus −81±1 mV, each P<0.05).ConclusionsSeptic peritonitis induced by CLP oxidizes ileal and colonic GSH/GSSG redox but Cys/CySS Eh remains unchanged in these intestinal tissues. In liver, CLP oxidizes the GSH/GSSG redox pool and CyS/CySS Eh becomes more reducing; in lung, CLP does not alter GSH/GSSG Eh, and Cys/CySS Eh is less oxidized. CLP-induced infection/inflammation differentially regulates major thiol-disulfide redox pools in this murine model.

Tyrosine administration decreases glutathione and stimulates lipid and protein oxidation in rat cerebral cortex

Tyrosine levels are abnormally elevated in tissues and physiological fluids of patients with inborn errors of tyrosine catabolism especially in tyrosinemia type II which is caused by deficiency of tyrosine aminotransferase (TAT) and provokes eyes, skin and central nervous system disturbances. We have recently reported that tyrosine promoted oxidative stress in vitro but the exact mechanisms of brain damage in these disorder are poorly known. In the present study, we investigated the in vivo effect of L-tyrosine (500 mg/Kg) on oxidative stress indices in cerebral cortex homogenates of 14-day-old Wistar rats. A single injection of L-tyrosine decreased glutathione (GSH) and thiol-disulfide redox state (SH/SS ratio) while thiobarbituric acid-reactive substances, protein carbonyl content and glucose-6-phosphate dehydrogenase activity were enhanced. In contrast, the treatment did not affect ascorbic acid content, and the activities of superoxide dismutase, catalase and glutathione peroxidase. These results indicate that acute administration of L-tyrosine may impair antioxidant defenses and stimulate oxidative damage to lipids and proteins in cerebral cortex of young rats in vivo. This suggests that oxidative stress may represent a pathophysiological mechanism in hypetyrosinemic patients.

Kinetic and Thermodynamic Aspects of Cellular Thiol–Disulfide Redox Regulation

Regulation of intracellular thiol-disulfide redox status is an essential part of cellular homeostasis. This involves the regulation of both oxidative and reductive pathways, production of oxidant scavengers and, importantly, the ability of cells to respond to changes in the redox environment. In the cytosol, regulatory disulfide bonds are typically formed in spite of the prevailing reducing conditions and may thereby function as redox switches. Such disulfide bonds are protected from enzymatic reduction by kinetic barriers and are thus allowed to exist long enough to elicit the signal. Factors that affect the rate of thiol-disulfide exchange and stability of disulfide bonds are discussed within the framework of the underlying chemical foundations. This includes the effect of thiol acidity (pK(a)), the local electrostatic environment, molecular strain, and entropy. Even though a thiol-disulfide exchange reaction is thermodynamically favorable, it will only take place if the activation energy to form the transition state complex can be overcome. This is accomplished by enzymes, such as the oxidoreductases, that direct reactions in thermodynamically favorable directions by decreasing the activation energy barrier.

Tyrosine promotes oxidative stress in cerebral cortex of young rats

Tyrosine accumulates in inborn errors of tyrosine catabolism, especially in tyrosinemia type II, where tyrosine levels are highly elevated in tissues and physiological fluids of affected patients. In tyrosinemia type II, high levels of tyrosine are correlated with eyes, skin and central nervous system disturbances. Considering that the mechanisms of brain damage in these disorders are poorly known, in the present study, we investigated whether oxidative stress is elicited by l-tyrosine in cerebral cortex homogenates of 14-day-old Wistar rats. The in vitro effect of 0.1–4.0mM l-tyrosine was studied on the following oxidative stress parameters: total radical-trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR), ascorbic acid content, reduced glutathione (GSH) content, spontaneous chemiluminescence, thiobarbituric acid-reactive substances (TBA-RS), thiol-disulfide redox state (SH/SS ratio), protein carbonyl content, formation of DNA-protein cross-links, and the activities of the enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glucose-6-phosphate dehydrogenase (G6PDH). TRAP, TAR, ascorbic acid content, SH/SS ratio and CAT activity were significantly diminished, while formation of DNA-protein cross-link was significantly enhanced by l-tyrosine in vitro. In contrast, l-tyrosine did not affect the other parameters of oxidative stress evaluated. These results indicate that l-tyrosine decreases enzymatic and non-enzymatic antioxidant defenses, changes the redox state and stimulates DNA damage in cerebral cortex of young rats in vitro. This suggests that oxidative stress may represent a pathophysiological mechanism in tyrosinemic patients, in which this amino acid accumulates.

15-deoxy-Δ12,14-prostaglandin J2 inhibits the expression of microsomal prostaglandin E synthase type 2 in colon cancer cells

Prostaglandin (PG) E(2) (PGE(2)) plays a predominant role in promoting colorectal carcinogenesis. The biosynthesis of PGE(2) is accomplished by conversion of the cyclooxygenase (COX) product PGH(2) by several terminal prostaglandin E synthases (PGES). Among the known PGES isoforms, microsomal PGES type 1 (mPGES-1) and type 2 (mPGES-2) were found to be overexpressed in colorectal cancer (CRC); however, the role and regulation of these enzymes in this malignancy are not yet fully understood. Here, we report that the cyclopentenone prostaglandins (CyPGs) 15-deoxy-Delta(12,14)-PGJ(2) and PGA(2) downregulate mPGES-2 expression in the colorectal carcinoma cell lines Caco-2 and HCT 116 without affecting the expression of any other PGES or COX. Inhibition of mPGES-2 was subsequently followed by decreased microsomal PGES activity. These effects were mediated via modulation of the cellular thiol-disulfide redox status but did not involve activation of the peroxisome proliferator-activated receptor gamma or PGD(2) receptors. CyPGs had antiproliferative properties in vitro; however, this biological activity could not be directly attributed to decreased PGES activity because it could not be reversed by adding PGE(2). Our data suggest that there is a feedback mechanism between PGE(2) and CyPGs that implicates mPGES-2 as a new potential target for pharmacological intervention in CRC.

Thiol antioxidant and thiol-reducing agents attenuate 15-deoxy-Δ 12,14-prostaglandin J 2-induced heme oxygenase-1 expression

Heme oxygenase-1 (HO-1) is induced as a beneficial and adaptive response in cells and tissues exposed to oxidative stress. Herein we examined how various eicosanoids affect the induction of HO-1, and the possible mechanism underlying 15-deoxy-Δ 12,14- prostaglandin J 2 (15d-PGJ 2)-induced HO-1 expression. PGH 2, PGD 2 and its metabolites of the PGJ 2 series, and PGA 1 markedly induced the protein expression of HO-1. Arachidonic acid (AA), docosahexaenoic acid (DHA), PGE 2, PGF 2α, and thromboxane B 2 (TXB 2) were shown to have no effect on the induction of HO-1. 15d-PGJ 2 was the most potent activator achieving significance at 5 μM. Although 15d-PGJ 2 significantly activated the MAPKs of JNK and ERK, the activation of JNK and ERK did not contribute to the induction of HO-1 as determined using transfection of dominant-negative plasmids and MAPKs inhibitors. Additional experiment indicated that 15d-PGJ 2 induced HO-1 expression through peroxisome proliferator-activated receptor (PPAR)-independent pathway. 15d-PGJ 2 significantly decreased the intracellular level of reduced glutathione; and the thiol antioxidant, N-acetyl-L-cysteine (NAC), and the thiol-reducing agent, dithiothreitol (DTT), inhibited the induction of HO-1 by 15d-PGJ 2. Finally, NAC and DTT exhibited significant inhibition of HO-1 mRNA and HO-1 promoter reporter activity induced by 15d-PGJ 2. These results suggest that thiol antioxidant and reducing agents attenuate the expression of HO-1 induced by 15d-PGJ 2, and that the cellular thiol-disulfide redox status may be linked to HO-1 activation.

Arabidopsis thaliana Plants Lacking the PSI-D Subunit of Photosystem I Suffer Severe Photoinhibition, Have Unstable Photosystem I Complexes, and Altered Redox Homeostasis in the Chloroplast Stroma

The PSI-D subunit of photosystem I is a hydrophilic subunit of about 18 kDa, which is exposed to the stroma and has an important function in the docking of ferredoxin to photosystem I. We have used an antisense approach to obtain Arabidopsis thaliana plants with only 5-60% of PSI-D. No plants were recovered completely lacking PSI-D, suggesting that PSI-D is essential for a functional PSI in plants. Plants with reduced amounts of PSI-D showed a similar decrease in all other subunits of PSI including the light harvesting complex, suggesting that in the absence of PSI-D, PSI cannot be properly assembled and becomes degraded. Plants with reduced amounts of PSI-D became light-stressed even in low light although they exhibited high non-photochemical quenching (NPQ). The high NPQ was generated by upregulating the level of violaxanthin de-epoxidase and PsbS, which are both essential components of NPQ. Interestingly, the lack of PSI-D affected the redox state of thioredoxin. During the normal light cycle thioredoxin became increasingly oxidized, which was observed as decreasing malate dehydrogenase activity over a 4-h light period. This result shows that photosynthesis was close to normal the first 15 min, but after 2-4 h photoinhibition dominated as the stroma progressively became less reduced. The change in the thiol disulfide redox state might be fatal for the PSI-D-less plants, because reduction of thioredoxin is one of the main switches for the initiation of CO2 assimilation and photoprotection upon light exposure.

Mechanisms for sensitization to TNF-induced apoptosis by acute glutathione depletion in murine hepatocytes

We previously reported that depletion of glutathione in murine hepatocytes by diethylmaleate (DEM) or acetaminophen (APAP) leads to oxidative stress—dependent necrosis and sensitizes to tumor necrosis factor (TNF)-induced apoptosis in an oxidative stress—independent fashion, which could not be explained by interference with nuclear factor κB (NF-κB) nuclear translocation. The present report explores the mechanisms of these effects. We observed that DEM led to necrosis when both mitochondrial and cytosol glutathione were depleted profoundly but sensitized to TNF-induced apoptosis when cytosol glutathione was depleted selectively. DEM and APAP lead to a significant decrease in reduced glutathione (GSH)/glutathione disulfide (GSSG) ratio. Glutathione depletion by DEM or APAP was associated with inhibition of TNF-induced NF-κB transactivation of anti-apoptotic genes, including inducible nitric oxide synthase (i-NOS). Provision of exogenous NO partially abrogated the sensitization to TNF in response to glutathione depletion. Glutathione depletion alone led to sustained increase in phospho-jun levels and c-Jun-N-terminal kinase (JNK) activity. JNK inhibitor partially blocked the sensitization to TNF-induced apoptosis accompanying glutathione depletion. In conclusion, these findings suggest that extramitochondrial glutathione depletion alters the thiol-disulfide redox state, leading to inhibition of NF-κB transactivation of survival genes and to sustained activation of JNK, both of which contribute to the sensitization to TNF-induced apoptosis. (H epatology 2003;37:1425-1434.)

Oxidation of the Glutathione/Glutathione Disulfide Redox State Is Induced by Cysteine Deficiency in Human Colon Carcinoma HT29 Cells

Glutathione (GSH) has a central role in the maintenance of the thiol-disulfide redox state in mammalian cells. GSH synthesis can be physiologically limited by the availability of cysteine (Cys), and Cys and its precursors are variable in the human diet. The purpose of this study was to determine the effect of severe Cys deficiency and readdition of Cys on the redox state of the GSH/glutathione disulfide (GSSG) pool in human colon carcinoma HT29 cells. Cells were cultured in Cys- (and cystine-)limiting medium for 48 h followed by culture in medium containing either Cys or cystine for 24 h. GSH and GSSG were measured by HPLC. Cys limitation decreased cellular GSH and GSSG concentrations with an associated >80 mV oxidation of the GSH/GSSG redox state. Upon addition of either Cys or its disulfide cystine (CySS), redox of GSH/GSSG recovered in 4 h, whereas GSH concentration continued to increase over 12 h. Maximal GSH concentrations attained were 200% of control cell values. These results show that severe Cys deficiency can have marked effects on cellular redox state but that redox recovers rapidly upon resupply. The magnitude of oxidation during Cys limitation in this cell model is sufficient to result in a >100-fold change in the reduced/oxidized ratio of redox-sensitive dithiol/disulfide motifs in proteins. If redox changes occur in vivo in association with variations in dietary Cys and its precursors, these changes could have important physiologic effects through altered redox signaling and control of cell proliferation and apoptosis.

Effect of N-acetyl-cysteine on the hypoxic ventilatory response and erythropoietin production: linkage between plasma thiol redox state and O2 chemosensitivity

Oxygen-sensing chemoreceptors contribute significantly to the regulation of the respiratory drive and arterial PO(2) levels. The hypoxic ventilatory response (HVR) decreases strongly with age and is modulated by prolonged hypoxia and physical exercise. Several earlier studies indicated that the regulation of the ventilatory response and erythropoietin (EPO) production by the respective oxygen sensors involves redox-sensitive signaling pathways, which are triggered by the O(2)-dependent production of reactive oxygen species. The hypothesis that HVR and EPO production are modulated by thiol compounds or changes in the plasma thiol-disulfide redox state (REDST) was investigated. It was demonstrated that both responses are enhanced by oral treatment with N-acetyl-cysteine (NAC) and that HVR is correlated with plasma thiol level and REDST. Results suggest the possibility that age-related changes in plasma REDST may account for the age-related changes in HVR.

Rat Jejunum Controls Luminal Thiol-Disulfide Redox

The control of luminal thiol-disulfide redox state may be important for several intestinal functions, including absorption of iron or selenium and maintenance of mucus fluidity. Disulfides are present in the diet, and although luminal thiols are supplied in bile, little is known about the ability of the small intestine to reduce disulfides to maintain the luminal thiol-disulfide redox state. The objective of the current study was to determine whether the isolated, vascularly perfused jejunum, free from biliary thiols, could reduce intraluminal glutathione disulfide (GSSG) to glutathione (GSH). GSSG was introduced in a deoxygenated solution to inhibit the reoxidation of any GSH formed, and preparations were pretreated with acivicin to inhibit the degradation of GSH by gamma-glutamyltransferase. GSSG (250 micromol/L) was reduced to GSH, with the luminal redox potential (E(h)) for GSSG/2GSH changing from >0 to -111, -132 and -143 mV at 10, 20 and 30 min, respectively. The E(h) for luminal cystine/2cysteine was approximately 20 mV more reducing than that for GSSG/2GSH at each time point, suggesting that cysteine could function in the reduction of GSSG in the lumen. Measurements in specific regions showed that GSSG reduction was more rapid in the duodenum and proximal jejunum than in the distal jejunum. Preparations without acivicin treatment showed that E(h) values were unaffected by inhibition of gamma-glutamyltransferase despite differences in GSH and cysteine pool sizes. Rat intestine has a mechanism to adjust the luminal thiol-disulfide redox. In principle, dysfunction of this mechanism could contribute to malabsorption or other nutritional disorders.

Separation of cytochrome c-dependent caspase activation from thiol-disulfide redox change in cells lacking mitochondrial DNA

Release of mitochondrial cytochrome c (cyt c) is an early and common event during apoptosis. Previous studies showed that the loss of cyt c triggered superoxide production by mitochondria and contributed to the oxidation of cellular thiol-disulfide redox state. In this study, we tested whether loss of the functional electron transport chain due to depleting mitochondrial DNA (mtDNA) would affect this redox-signaling mechanism during apoptosis. Results showed that cyt c release and caspase activation in response to staurosporine treatment were preserved in cells lacking mitochondrial DNA (ρ 0 cells). However, unlike the case with ρ +cells, in which a dramatic oxidation of intracellular glutathione (GSH) occurred after mitochondrial cyt c release, the thiol-disulfide redox state in apoptotic ρ 0 cells remained largely unchanged. Thus, mitochondrial signaling of caspase activation can be separated from the bioenergetic function, and mitochondrial respiratory chain is the principal source of ROS generation in staurosporine-induced apoptosis.

Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol-disulfide status.

The Escherichia coli transcription factor OxyR is activated by the formation of an intramolecular disulfide bond and subsequently is deactivated by enzymatic reduction of the disulfide bond. Here we show that OxyR can be activated by two possible pathways. In mutants defective in the cellular disulfide-reducing systems, OxyR is constitutively activated by a change in the thiol-disulfide redox status in the absence of added oxidants. In wild-type cells, OxyR is activated by hydrogen peroxide. By monitoring the presence of the OxyR disulfide bond after exposure to hydrogen peroxide in vivo and in vitro, we also show that the kinetics of OxyR oxidation by low concentrations of hydrogen peroxide is significantly faster than the kinetics of OxyR reduction, allowing for transient activation in an overall reducing environment. We propose that the activity of OxyR in vivo is determined by the balance between hydrogen peroxide levels and the cellular redox environment.

Thylakoid protein phosphorylation and the thiol redox state.

Illumination of thylakoid membranes leads to the phosphorylation of a number of photosystem II-related proteins, including the reaction center proteins D1 and D2 as well as the light-harvesting complex (LHCII). Regulation of light-activated thylakoid protein phosphorylation has mainly been ascribed to the redox state of the electron carrier plastoquinone. In this work, we show that this phosphorylation in vitro is also strongly influenced by the thiol disulfide redox state. Phosphorylation of the light-harvesting complex of photosystem II was found to be favored by thiol-oxidizing conditions and strongly downregulated at moderately thiol-reducing conditions. In contrast, phosphorylation of the photosystem II reaction center proteins D1 and D2 as well as that of other photosystem II subunits was found to be stimulated up to 2-fold by moderately thiol-reducing conditions and kept at a high level also at highly reducing conditions. These responses of the level of thylakoid protein phosphorylation to changes in the thiol disulfide redox state are reminiscent of those observed in vivo in response to changes in the light intensity and point to the possibility of a second loop of redox regulation of thylakoid protein phosphorylation via the ferredoxin-thioredoxin system.

Activation of Heat Shock Factor by Alkylating Agents Is Triggered by Glutathione Depletion and Oxidation of Protein Thiols

Transcriptional activation of heat shock protein genes is a common response to proteotoxic stress. Many drugs and chemicals that form reactive electrophiles modify protein structure by binding covalently to nucleophilic functional groups. Although many of these agents also activate transcription of the inducible member of the hsp70 gene family, it is not clear if covalent modification of cellular proteins per se is sufficient. Iodoacetamide (IDAM) is a prototypical alkylating toxicant that induces hsp70 transcription. However, IDAM-induced cell death is indirectly linked to protein alkylation through depletion of glutathione, induction of oxidative stress, and increased lipid peroxidation. Therefore, we determined if any of these secondary cytotoxic events might lead to activation of hsp70 transcription. IDAM treatment increased hsp70 transcription by activating heat shock transcription factor-1 (HSF1). The addition of antioxidants and iron or calcium chelators prevented cell death but did not prevent hsp70 transcription or HSF1 activation. However, the protein synthesis inhibitor cycloheximide blocked activation of hsp70 by low concentrations of IDAM. Furthermore, the addition of dithiothreitol (DTT) after IDAM removal blocked hsp70 transcription and HSF1 activation without altering IDAM binding. DTT had no effect on activation of HSF1 by hyperthermia. After IDAM treatment, cellular nonprotein and protein thiols had decreased to less than 20 and 70%, respectively, of the value in control cells. DTT treatment in situ prevented the loss of cellular protein thiols and blocked the formation of high molecular weight protein aggregates. Thus, alkylation of proteins is insufficient to activate hsp70 transcription and DNA binding of HSF1. However, cellular thiol-disulfide redox status and formation of disulfide linked aggregates of cellular proteins are linked to HSF1 activation and hsp70 transcriptional activation.

Defense mechanisms of normal and tumor cells

This paper reviews the protective systems of normal and tumor cells against chemical and radiation injury. The glutathione redox cycle is an important cell defense system that can be compromised by various chemical modifiers. Acute cell injury can involve the glutathione pools of both the cytosol and the mitochondria. Intracellular calcium may have a role in cell death following acute cell injury but extracellular calcium does not seem to initiate the events leading to cell death. Changes in the glutathione redox status affects the distribution of intracellular calcium and the protein thiol-disulfide redox status. Formation of glutathione protein-mixed disulfides is discussed in terms of a possible protective mechanism against oxidative injury.