HIV-1 tat expression and sulphamethoxazole hydroxylamine mediated oxidative stress alter the disulfide proteome in Jurkat T cells.

BACKGROUND: Adverse drug reactions (ADRs) are a significant problem for HIV patients, with the risk of developing ADRs increasing as the infection progresses to AIDS. However, the pathophysiology underlying ADRs remains unknown. Sulphamethoxazole (SMX) via its active metabolite SMX-hydroxlyamine, when used prophylactically for pneumocystis pneumonia in HIV-positive individuals, is responsible for a high incidence of ADRs. We previously demonstrated that the HIV infection and, more specifically, that the HIV-1 Tat protein can exacerbate SMX-HA-mediated ADRs. In the current study, Jurkat T cell lines expressing Tat and its deletion mutants were used to determine the effect of Tat on the thiol proteome in the presence and absence of SMX-HA revealing drug-dependent changes in the disulfide proteome in HIV infected cells. Protein lysates from HIV infected Jurkat T cells and Jurkat T cells stably transfected with HIV Tat and Tat deletion mutants were subjected to quantitative slot blot analysis, western blot analysis and redox 2 dimensional (2D) gel electrophoresis to analyze the effects of SMX-HA on the thiol proteome. RESULTS: Redox 2D gel electrophoresis demonstrated that untreated, Tat-expressing cells contain a number of proteins with oxidized thiols. The most prominent of these protein thiols was identified as peroxiredoxin. The untreated, Tat-expressing cell lines had lower levels of peroxiredoxin compared to the parental Jurkat E6.1 T cell line. Conversely, incubation with SMX-HA led to a 2- to 3-fold increase in thiol protein oxidation as well as a significant reduction in the level of peroxiredoxin in all the cell lines, particularly in the Tat-expressing cell lines. CONCLUSION: SMX-HA is an oxidant capable of inducing the oxidation of reactive protein cysteine thiols, the majority of which formed intermolecular protein bonds. The HIV Tat-expressing cell lines showed greater levels of oxidative stress than the Jurkat E6.1 cell line when treated with SMX-HA. Therefore, the combination of HIV Tat and SMX-HA appears to alter the activity of cellular proteins required for redox homeostasis and thereby accentuate the cytopathic effects associated with HIV infection of T cells that sets the stage for the initiation of an ADR.

Design and synthesis of γ-butyrolactone derivatives as potential spermicidal agents.

A series of γ-butyrolactone derivatives has been designed and synthesized from commercially available 2-acetyl butyrolactone (3-acetyldihydrofuran-2(3H)-one, 1) by aminoalkylating its active methylene followed by condensation with different aldehydes. Compounds having amino group were further converted to their respective tartrate salts and were evaluated for spermicidal activity against human sperm in vitro. Compounds showing appreciable spermicidal activity at ⩽0.5% [3c, 4d (0.5%); 2c, 3d (0.1%); 2d, 4c (0.05%)] were tested for safety studies against human cervical (HeLa) cell line. These compounds were found safer than, Nonoxynol-9. One of the two most active compounds was also found to be the safest (IC50=961 µg/ml; 4c), while the second compound exhibited lower safety against HeLa (IC50=269 µg/ml; 2d). The compound 4c significantly reduced the number of free thiols on human sperm. All the compounds were inactive against Trichomonas vaginalis.

Conversion of S-phenylsulfonylcysteine residues to mixed disulfides at pH 4.0: utility in protein thiol blocking and in protein-S-nitrosothiol detection.

A three step protocol for protein S-nitrosothiol conversion to fluorescent mixed disulfides with purified proteins, referred to as the thiosulfonate switch, is explored which involves: (1) thiol blocking at pH 4.0 using S-phenylsulfonylcysteine (SPSC); (2) trapping of protein S-nitrosothiols as their S-phenylsulfonylcysteines employing sodium benzenesulfinate; and (3) tagging the protein thiosulfonate with a fluorescent rhodamine based probe bearing a reactive thiol (Rhod-SH), which forms a mixed disulfide between the probe and the formerly S-nitrosated cysteine residue. S-Nitrosated bovine serum albumin and the S-nitrosated C-terminally truncated form of AdhR-SH (alcohol dehydrogenase regulator) designated as AdhR*-SNO were selectively labelled by the thiosulfonate switch both individually and in protein mixtures containing free thiols. This protocol features the facile reaction of thiols with S-phenylsulfonylcysteines forming mixed disulfides at mild acidic pH (pH = 4.0) in both the initial blocking step as well as in the conversion of protein-S-sulfonylcysteines to form stable fluorescent disulfides. Labelling was monitored by TOF-MS and gel electrophoresis. Proteolysis and peptide analysis of the resulting digest identified the cysteine residues containing mixed disulfides bearing the fluorescent probe, Rhod-SH.

Gastroprotection of suaveolol, isolated from Hyptis suaveolens, against ethanol-induced gastric lesions in Wistar rats: role of prostaglandins, nitric oxide and sulfhydryls.

Hyptis suaveolens is a medicinal plant that is, according to traditional medicine, considered useful in the treatment of gastric ulcers. Although its gastroprotective activity was reported, the active compounds have not been identified. Therefore, the aim of the present study was to identify at least one active compound potentially responsible for the gastroprotective activity of H. suaveolens by using a bioassay guided study with an ethanol-induced gastric ulcer experimental model in rats. The results show that the hexane extract had protective activity (close to 70% when using doses between 10 and 100 mg/kg), and that the compound suaveolol, isolated from this extract, was one of the active gastroprotective agents. This is the first report about the gastroprotective activity of suaveolol. Rats treated with this compound at 3, 10, 30 and 100 mg/kg showed 12.6, 21.3, 39.6 and 70.2% gastroprotection respectively. The effect elicited by suaveolol (at 100 mg/kg) was attenuated by pretreatment with either N(G)-nitro-L-arginine methyl ester (70 mg/kg, i.p.), a nitric oxide (NO) synthase inhibitor, indomethacin (10 mg/kg, s.c.), a blocker of prostaglandin synthesis, or N-ethylmaleimide (10 mg/kg, s.c.), a blocker of sulfhydryl groups. This suggests that the gastroprotective mechanism of action of this compound involves NO, prostaglandins and sulfhydryl groups.

Increase in osmiophilia of axonal membranes of crayfish as a result of electrical stimulation, asphyxia, or treatment with reducing agents.

Certain axonal membranes of crayfish abdominal nerve cord display ultrastructural changes if the axons are fixed, during electrical stimulation, by aldehydes followed by osmium. Such changes are characterized by an increase in electron opacity and thickness of the unit membranes' dense strata in the axon surface, endoplasmic reticulum, and outer mitochondrial membranes. The electron opacity completely disappears if the sections are treated with hydrogen peroxide solutions. This suggests that the changes represent an increase in the membranes' reactivity for osmium. An unmasking of SH groups could explain such increased osmiophilia, since SH groups are very reactive with osmium, while disulfide bonds are considerably less reactive. This hypothesis was tested by treating control, glutaraldehydefixed nerve cords with disulfide reducing agents. In these preparations an increase in electron opacity and thickness was observed to be localized in the same axonal membranes which reacted as a result of electrical stimulation. The phenomenon did not appear if the SH groups were blocked by maleimide or N-ethylmaleimide before treatment with osmium. These findings seem to suggest that certain axonal membranes of crayfish contain proteins rich in sulfur whose SH groups are unmasked as a result of electrical stimulation. In preliminary experiments an increase in osmiophilia localized in the same membranes with the same characteristics and distribution was observed also in axons from nerve cords asphyxiated either in vitro or in the living animal.

Human aldehyde dehydrogenase: mechanism of inhibition of disulfiram.

Disulfiram labeled with carbon-14 reacts specifically with human liver aldehyde dehydrogenase E1 with loss of catalytic activity and no incorporation of label. Carbon-14-labeled diethyldithiocarbamate is formed and the number of enzyme sulfhydryl groups decreases from 34 to 30 during this process. Activity is recovered by-mercaptoethanol but not by glutathione, the physiological reducing agent.

Calcium uptake by isolated sarcoplasmic reticulum treated with dithiothreitol.

Isolated vesicles of the sarcoplasmic reticulum are known to take up calcium when provided with magnesium adenosine triphosphate as an energy source. Preparations of high activity are obtained by keeping the vesicles in 5 millimolar dithiothreitol (a reagent that reduces disulfide groups), and these preparations retain activity for a week or longer. The highly active preparations lend themselves to a spectrophotometric method for following calcium uptake, and continuous uptake kinetics are readily obtained. Calcium uptake appears to follow Michaelis-Menten kinetics (K(m) = 8x10(-6); V(mas), = 7x10(-7) mole per second per milligram of protein). These preparations are also useful for studying the effects of inhibitors of uptake, such as quinine. When extrapolated to the intact muscle, the results from these isolated vesicles should give a better estimate than has been available of the actual rates of calcium uptake and of the physiological effect of inhibitors of uptake.

Energy transduction: inhibition of cockroach feeding by naphthoquinone.

1,4-Naphthoquinones inhibit feeding of Periplaneta americana by complexing with sulfhydryl groups of receptor protein in sensory neurons, by oxidizing the sulfhydryl groups, and by being reduced.

Beneficial Effects of N-Acetyl-L-cysteine or Taurine Pre- or Post-treatments in the Heart, Spleen, Lung, and Testis of Hexavalent Chromium-Exposed Mice.

Hexavalent chromium[Cr(VI)] compounds may induce toxic effects, possibly via reactive intermediates and radicals formed during Cr(VI) reduction. In this study, we probed the possible effects of N-acetyl-L-cysteine (NAC) and taurine pre- or post-treatments on Cr(VI)-induced changes in lipid peroxidation and nonprotein thiols (NPSH) in mice heart, lung, spleen, and testis tissues. The mice were randomly assigned to six groups, consisting of control, Cr(VI)-exposed (20 mg Cr/kg, intraperitoneal ,ip), NAC (200 mg/kg, ip) as pre-treatment and post-treatment, and taurine (1 g/kg, ip) pre-treatment and post-treatment groups. Lipid peroxidation and NPSH levels were determined and the results were compared with regard to tissue- and antioxidant-specific basis. Exposure to Cr(VI) significantly increased lipid peroxidation in all tissues as compared to the control (p < 0.05); and consistent with this data, NPSH levels were significantly decreased (p < 0.05). Notably, administration of NAC and taurine, either before or after Cr(VI) exposure, was able to ameliorate the lipid peroxidation (p < 0.05) in all tissues. In the case of NPSH content, while the decline could be alleviated by both NAC and taurine pre- and post-treatments in the spleen, diverging results were obtained in other tissues. The effects of Cr(VI) on the lung thiols were abolished by pre-treatment with NAC and taurine; however, post-treatments could not exert significant effect. While thiol depletion in the heart was totally replenished by NAC and taurine administrations, NAC pre-treatment was partially more effective than post-treatment. In contrast with lipid peroxidation data, NAC treatment could not provide a statistically significant beneficial effect on NPSH content of the testis, whereas the effect in this tissue by taurine was profound. Thus, these data highlight the importance of tissue-specific factors and the critical role of administration time. Overall, our data suggest that NAC and taurine may have potential in prevention of Cr(VI)-induced toxicity in the heart, lung, spleen, and testis tissues.

Blocking the Thiol at Cysteine-322 Destabilizes Tau Protein and Prevents Its Oligomer Formation.

Abnormal accumulation of tau protein into oligomers contributes to neuronal dysfunction. Reduction of tau level is potentially able to prevent its accumulation. Here we uncover a critical role of the free thiol at Cys-322 in determining tau stability. We found that the application of thiol-blocking agents like NEM or MMTS blocks this thiol, by which it destabilizes tau protein and prevents its oligomer formation. Furthermore, we identified a tau-interacting protein, selenoprotein W, which attenuates tau accumulation by forming disulfide linkage between SelW Cys-37 and tau Cys-322. These findings provide a promising strategy to prevent tau accumulation and oligomer formation.

The thiosemicarbazone Me2NNMe2 induces paraptosis by disrupting the ER thiol redox homeostasis based on protein disulfide isomerase inhibition.

Due to their high biological activity, thiosemicarbazones have been developed for treatment of diverse diseases, including cancer, resulting in multiple clinical trials especially of the lead compound Triapine. During the last years, a novel subclass of anticancer thiosemicarbazones has attracted substantial interest based on their enhanced cytotoxic activity. Increasing evidence suggests that the double-dimethylated Triapine derivative Me2NNMe2 differs from Triapine not only in its efficacy but also in its mode of action. Here we show that Me2NNMe2- (but not Triapine)-treated cancer cells exhibit all hallmarks of paraptotic cell death including, besides the appearance of endoplasmic reticulum (ER)-derived vesicles, also mitochondrial swelling and caspase-independent cell death via the MAPK signaling pathway. Subsequently, we uncover that the copper complex of Me2NNMe2 (a supposed intracellular metabolite) inhibits the ER-resident protein disulfide isomerase, resulting in a specific form of ER stress based on disruption of the Ca2+ and ER thiol redox homeostasis. Our findings indicate that compounds like Me2NNMe2 are of interest especially for the treatment of apoptosis-resistant cancer and provide new insights into mechanisms underlying drug-induced paraptosis.

Glutathione synthesis in human erythrocytes. II. Purification and properties of the enzymes of glutathione biosynthesis.

The two enzymes required to synthesize glutathione de novo have been purified from human erythrocytes. Glutamylcysteine synthetase was purified 4300-fold and was approximately 80% pure based on polyacrylamide gel electrophoresis. The purified enzyme catalyzes the formation of 30.5 mumoles of gamma-glutamyl-cysteine per mg of protein per hr and is inhibited by sulfhydryl inhibitors. Glutathione synthetase was purified 6000-fold from erythrocytes to homogeneity as determined by polyacrylamide gel electrophoresis. The erythrocyte enzyme has a molecular weight of 150,000 and catalyzes the formation of 35.9 mumoles of glutathione per mg of protein per hr. Comparison of the amino acid composition and some kinetic parameters of yeast glutathione synthetase and the erythrocyte enzyme demonstrate similarities between these enzymes.

Isolation and characterization of soluble beta-galactoside-binding lectins from mammalian liver.

Soluble beta-galactoside-binding lectins were isolated by chromatography on asialofetuin-Sepharose-4B column in 10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl, 5 mM CaCl2 and 1 mM 2-mercaptoethanol. The three lectins moved essentially as single polypeptide bands, of 18, 22 and 24 kDa, respectively, for sheep, goat and buffalo hepatic lectins. Sheep and goat lectins each contained 4 mol of hexose, whereas the hexose content of the buffalo lectin was 7 mol. The number of sulfhydryl groups in sheep, goat and buffalo lectins were determined to be 3.2, 4.3 and 4.8, respectively. The optical properties of the three lectins were similar to those of tryptophan-containing proteins. Lectin-mediated hemagglutination of trypsinized rabbit erythrocytes was most effectively inhibited by lactose, followed by o-nitrophenyl beta-galactopyranoside and galactose, but remained unaffected by glucose, mannose, fucose and fructose. Calcium ions substantially enhanced their hemagglutinating activity. Goat and buffalo lectins, but not sheep lectin, were also stimulated by Mg2+, Mn2+, Sr2+ and Ni2+ ions. The lectins lost activity after treatment with para-hydroxy-mercuribenzoate and N-ethylmaleimide. However, iodoacetamide treatment had no effect on the activity. The results show that the three lectins are different from the soluble beta-galactoside-binding lectins studied thus far.

Molecular characteristics of small intestinal and renal brush border thiamin transporters in rats.

The molecular characteristics of thiamin (T) transport were studied in the small intestinal and renal brush border membrane vesicles of rats, using [(3)H]T at high specific activity. The effects of various chemical modifiers (amino acid blockers) on T uptake were examined and their specificity assessed. Treatment with the carboxylic specific blockers 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate, (1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride and N-ethyl-5-phenylisoaxolium-3'-sulfonate (Woodward's Reagent K) and with the sulfhydryl specific blocker p-chloromercuribenzene sulfonate inhibited T transport in both types of vesicles. Phenylglyoxal, but not ninhydrin, both reagents for arginine residues, and diethylpyrocarbonate, a reagent for histidine residues, specifically decreased T transport only in renal and small intestinal vesicles respectively. Similarly 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reacted, but not N-acetylimidazole, both of which are reagents for tyrosine residues. However, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole inhibition was aspecific. Acetylsalicylic acid, a reagent for lysine and serine residues, decreased T transport, but the lysine effect was aspecific. Acetylsalicylic acid serine blockage also eliminated T/H(+) exchange in small intestinal vesicles. Taken together, these results suggest that for T transport carboxylic and sulfhydryl groups and serine residues are essential in both renal and small intestinal brush border membrane vesicles. In addition, arginine and histidine residues are also essential respectively for renal and small intestinal transporters. Serine was essential for the T/H(+) antiport mechanism.

Tumor necrosis factor-alpha mediates osteopenia caused by depletion of antioxidants.

We recently found that estrogen deficiency leads to a lowering of thiol antioxidant defenses in rodent bone. Moreover, administration of agents that increase the concentration in bone of glutathione, the main intracellular antioxidant, prevented estrogen-deficiency bone loss, whereas depletion of glutathione by buthionine sulfoximine (BSO) administration provoked substantial bone loss. It has been shown that the estrogen-deficiency bone loss is dependent on TNFalpha signaling. Therefore, a model in which estrogen deficiency causes bone loss by lowering antioxidant defenses predicts that the osteopenia caused by lowering antioxidant defenses should similarly depend on TNFalpha signaling. We found that the loss of bone caused by either BSO administration or ovariectomy was inhibited by administration of soluble TNFalpha receptors and abrogated in mice deleted for TNFalpha gene expression. In both circumstances, lack of TNFalpha signaling prevented the increase in bone resorption and the deficit in bone formation that otherwise occurred. Thus, depletion of thiol antioxidants by BSO, like ovariectomy, causes bone loss through TNFalpha signaling. Furthermore, in ovariectomized mice treated with soluble TNFalpha receptors, thiol antioxidant defenses in bone remained low, despite inhibition of bone loss. This suggests that the low levels of antioxidants in bone seen after ovariectomy are the cause, rather than the effect, of the increased resorption. These experiments are consistent with a model for estrogen-deficiency bone loss in which estrogen deficiency lowers thiol antioxidant defenses in bone cells, thereby increasing reactive oxygen species levels, which in turn induce expression of TNFalpha, which causes loss of bone.

Chemical modification of membranes. II. Permeation paths for sulfhydryl agents.

The amino-reactive reagent, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS),(1) considerably reduces the uptake of the sulfhydryl agent, parachloromercuriphenylsulfonic acid (PCMBS), but does not reduce its effects on cation permeability and on cation transport. These data indicate that PCMBS enters the membrane by at least two channels, one sensitive and the other insensitive to SITS, with only the latter leading to the cation-controlling sulfhydryl groups. Substitution of phosphate or sulfate for chloride results in an inhibition of PCMBS uptake via the SITS-insensitive pathway. These and other data lead to the conclusion that the SITS-sensitive pathway is the predominant one for anion permeation, and the insensitive one for cation permeation. Parachloromercuribenzoate (PCMB), an agent that is more lipid-soluble than PCMBS, penetrates faster but has a smaller effect on cation permeability. Its uptake is less sensitive to SITS. These and other observations suggest that the cation permeation path involves an aqueous channel in the membrane.

Chemical modification of membranes. I. Effects of sulfhydryl and amino reactive reagents on anion and cation permeability of the human red blood cell.

Four different amino-reactive reagents, 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (SITS),(1) 1-fluoro-2,4-dinitrobenzene (FDNB), 2,4,6-trinitrobenzene sulfonic acid (TNBS), and 2-methoxy-5-nitrotropone (MNT) decrease the anion permeability of the human red blood cell, as measured by sulfate fluxes, whereas the sulfhydryl agent, parachloromercuriphenyl sulfonic acid (PCMBS), does not. In contrast, PCMBS increases the cation permeability as measured by K(+) leakage, whereas SITS does not. Of the other agents, FDNB increases the cation permeability to the same extent as PCMBS but MNT and TNBS produce smaller increases. PCMBS does not protect against FDNB as it does against other sulfhydryl agents (X-irradiation) and the FDNB effect on cations is attributed to amino groups. Studies of the binding of SITS indicate that it does not penetrate into the membrane and its failure to influence cation permeability is attributed to its inability to reach an internal population of amino groups. It is concluded that two ion permeability barriers, both involving proteins, are present in the red blood cell. The more superficial barrier contains amino groups and controls anion flow; the more internal barrier contains sulfhydryl and amino groups and controls cation flow. The amino groups contribute to the control of permeability by virtue of their positive charges, but the role of sulfhydryl groups is not clear. Only a small fraction of the membrane protein amino and sulfhydryl is involved in the barriers.

Effect of a triclosan/PVM/MA copolymer/fluoride dentifrice on volatile sulfur compounds in vitro.

OBJECTIVE: The objective of the investigation was to document the in vitro efficacy of a triclosan/PVM/MA copolymer/fluoride (TCF) dentifrice against the formation of volatile sulfur compounds (VSC) as well as the growth of H2S-producing bacteria. Clinical studies using organoleptic judges, gas chromatography, or a portable sulfide monitor have generally been employed in the assessment of treatments for the control of oral malodor. However, these studies are not appropriate for screening purposes because of the expense and time required. METHODS: An in vitro method was developed for the purpose of screening new compounds, agents or formulations for their ability to control VSC formation and for determining bio-equivalence of efficacy when implementing changes in existing formulations. The method combines basic microbiological methods, dynamic flow cell techniques and head space analysis. The in vitro VSC method was validated by comparing the efficacy of two dentifrices containing TCF with a control fluoride dentifrice as the TCF products have been clinically proven to control oral malodor. RESULTS: In the validation studies, the TCF-containing dentifrices were significantly better (P < 0.05) than the control dentifrice in inhibiting VSC formation and reducing H(2)S-producing bacteria. For example, when compared with baseline, the TCF dentifrices reduced VSC formation between 42 and 49% compared with the control dentifrice which reduced VSC formation 3%. There was no significant difference (P > 0.05) between the two TCF dentifrice formulations. CONCLUSION: Using an in vitro breath VSC model, it has been demonstrated that two variants of a dentifrice containing triclosan, PVM/MA copolymer and fluoride have efficacy that is significantly better than a control fluoridated dentifrice and that there is no significant difference between the triclosan/PVM/MA copolymer/fluoride dentifrice variants.

Ascorbate-mediated electron transfer in protein thiol oxidation in the endoplasmic reticulum.

Addition of, or gulonolactone oxidase-dependent in situ generation of, ascorbate provoked the oxidation of protein thiols, which was accompanied by ascorbate consumption in liver microsomal vesicles. The maximal rate of protein thiol oxidation was similar upon gulonolactone, ascorbate or dehydroascorbate addition. Cytochrome P450 inhibitors (econazole, proadifen, quercetin) decreased ascorbate consumption and the gulonolactone or ascorbate-stimulated thiol oxidation. The results demonstrate that the ascorbate/dehydroascorbate redox couple plays an important role in electron transfer from protein thiols to oxygen in the hepatic endoplasmic reticulum, even in gulonolactone oxidase deficient species.

Toxicity by pyruvate in HepG2 cells depleted of glutathione: role of mitochondria.

Several studies have shown that pyruvate can scavenge H(2)O(2) and protect from H(2)O(2)-mediated cell injury. Mitochondria are critical participants in the control of apoptotic and necrotic cell death. Mitochondrial GSH plays an important role in the maintenance of cell functions and viability by metabolism of oxygen free radicals generated by the respiratory chain. Since loss of GSH, especially mitochondrial GSH, is associated with increased production of reactive oxygen species and cell toxicity, the ability of pyruvate to protect against these actions was evaluated. Adding pyruvate to HepG2 cells depleted of GSH by treatment with l-buthionine sulfoximine (BSO) surprisingly caused loss of viability after 24 and 48 h of incubation. Anoxia, treatment with antioxidants, and infection with cytosolic catalase, and interestingly, catalase expressed in the mitochondrial compartment were able to rescue the HepG2 cells from this pyruvate plus BSO injury, suggesting a key role for H(2)O(2), and lipid peroxides as mediators in the cytotoxicity. This toxicity and cell death observed was linked to damage to the mitochondria as evidenced by the increased lipid peroxidation in total homogenate and mitochondrial fraction, loss of mitochondrial membrane potential, and a decrease in protein-sulfhydryl groups. The type of cell death observed under these conditions was a mixture of apoptosis and necrosis. These results suggest that the protective ability of pyruvate against oxidant damage requires a functional GSH pool, especially in the mitochondrial compartment, and that in the absence of GSH, pyruvate increases cell injury by damaging the mitochondria, presumably as a consequence of enhanced electron flow and reactive oxygen production by the respiratory chain.