Glutathione depletion in rested and exercised mice: biochemical consequence and adaptation

The effect of chronic in vivo glutathione (GSH) depletion by L-buthionine-[S,R]-sulfoximine (BSO) on intracellular and interorgan GSH regulation was investigated in mice both at rest and after an acute bout of exhaustive swim exercise. BSO treatment for 12 days decreased concentrations of GSH in the liver, kidney, quadriceps muscle, and plasma to 28, 15, 7, and 35%, respectively, compared to GSH-adequate mice. In most tissues, with the exception of the kidney, this decrease was associated with a concomitant decrease of glutathione disulfide (GSSG) such that the GSH/GSSG ratio was maintained. GSH depletion caused adaptive changes in several enzymes related to GSH regulation, such as liver glutathione peroxidase (-25%), kidney gamma-glutamyltranspeptidase (+20%), glutathione disulfide reductase (+131%) and glutathione sulfur-transferase (+53%). There was an apparent down-regulation of muscle gamma-glutamyltranspeptidase (-56%) in the GSH-depleted mice, which contributed to a conservation of plasma GSH. Exhaustive exercise in the GSH-adequate state severely depleted GSH content in the liver (-55%) and kidney (-35%), whereas plasma and muscle GSH levels remained constant. However, exercise in the GSH-depleted state exacerbated GSH deficit in the liver (-57%), kidney (-33%), plasma (-65%), and muscle (-25%) in the absence of adequate reserves of liver GSH. Hepatic lipid peroxidation increased by 220 and 290%, respectively, after exhaustive exercise in the GSH-adequate and -depleted mice. We conclude that GSH homeostasis is essential for the prooxidant-antioxidant balance during prolonged physical exercise.     

Cisplatin induces N-acetyl cysteine suppressible F2-isoprostane production and injury in renal tubular epithelial cells

In the low intracellular chloride milieu, chloride ions of cisplatin may exchange for cellular SH moieties resulting in glutathione depletion, H2O2 accumulation, and lipid peroxidation. Cisplatin-induced lipid peroxidation, in addition to causing direct cellular injury, may further contribute to cisplatin-induced renal dysfunction by generating vasoconstrictive E2- and F2-isoprostanes. The aim of this study was to determine whether cisplatin-induced renal epithelial (LLC-PK1 and primary human proximal tubular) cell injury is associated with increased production of isoprostanes, and whether this can be suppressed with a thiol donor, N-acetyl cysteine. It was confirmed that incubation of renal epithelial cells with cisplatin resulted in N-acetyl cysteine-inhibitable glutathione depletion, H2O2 accumulation, lipid degradation, and lactate dehydrogenase release. In additional experiments, incubation of cells with cisplatin for 48 h was accompanied by a dose-related increase in total (free plus esterified) F2-isoprostanes. An increase in F2-isoprostanes was discernible at 16.5 microM cisplatin and doubled at 66.0 microM. N-Acetyl cysteine at 50 microM concentration effectively suppressed 66.0 microM cisplatin-induced increase in isoprostanes. Similar findings were also obtained in human cells. Thus, cisplatin-induced tubular cell injury is accompanied by increased isoprostane production through a mechanism involving thiol depletion. On the basis of this new finding, it is hypothesized that these arachidonic acid peroxidation products may be partially responsible for the cisplatin-induced renal vasoconstriction demonstrable in the in vivo models.     

Glutathone and glutathione ethyl ester supplementation of mice alter glutathione homeostasis during exercise

The present study examined the effect of glutathione (GSH) and glutathione ethyl ester (GSH-E) supplementation on GSH homeostasis and exercise-induced oxidative stress. Male Swiss-Webster mice were randomly divided into 4 groups: starved for 24 h and injected with GSH or GSH-E (6 mmol/kg body wt, i.p.) 1 h before exercise, starved for 24 h and injected with saline (S); and having free access to food and injected with saline (C). Half of each group of mice was killed either after an acute bout of exhaustive swimming (E) or after rest (R). Plasma GSH concentration was 100-160% (P < 0.05) higher in GSH mice vs. C or S mice at rest, whereas GSH-E injection had no effect. Plasma GSH was not affected by exercise in C or S mice, but was 44 and 34% lower (P < 0.05) in E vs. R mice with GSH or GSH-E injection, respectively. S, GSH- and GSH-E-treated mice had significantly lower liver GSH concentration and the GSH:glutathione disulfide (GSSG) ratio than C mice. Hepatic and renal GSH and the GSH:GSSG ratio were significantly lower in E vs. R mice in all groups. GSH-E-treated mice had a significantly smaller exercise-induced decrease in GSH vs. C, S, and GSH-treated mice and no difference in the GSH:GSSG ratio in the kidney. Activities of gamma-glutamylcysteine synthetase and gamma-glutamyltranspeptidase in the liver and kidney were not affected by either GSH treatment or exercise. GSH concentration and the GSH:GSSG ratio in quadriceps muscle were not different among C, S and GSH-treated mice, but significantly lower in GSH-E-treated mice (P < 0.05). Hepatic malondialdehyde (MDA) content was greater in exercised mice in all but GSH-E-treated groups. GSH and GSH-E increased MDA levels in the kidney of E vs. R mice, but attenuated exercise-induced lipid peroxidation in muscle. Swim endurance time was approximately 2 h longer in GSH (351 +/- 22 min) and GSH-E (348 +/- 27) than S mice (237 +/- 17). We conclude that 1) acute GSH and GSH-E supplementation at the given doses does not increase tissue GSH content or redox status; 2) both GSH and GSH-E improve endurance performance and prevent muscle lipid peroxidation during prolonged exercise; and 3) while both compounds may impose a metabolic and oxidative stress to the kidney, this side effect is smaller with GSH-E supplementation.     

Preparation and standardization of U.S. Standard Pertussis Vaccine, Lot No. 11

Novel glutathione (GSH) analogs, previously shown to inhibit glutathione S-transferase (GST) activity at about 1 microM in vitro, were tested for their ability to potentiate the killing of cultured tumor cells by chemotherapeutic drugs. When tested at doses up to 200 microM, the analogs were neither toxic nor capable of potentiating drug toxicity unless the diethyl ester (DEE) form was used for treatment of the cells. HPLC analysis revealed rapid internalization of the DEE and intracellular conversion to a monoethyl ester form that accumulated in the cell, followed by a more gradual loss of the second ester to generate the active parent form. For the four GSH analogs tested, the ability of the DEE forms to potentiate chlorambucil (CMB) toxicity in HT-29 human colon adenocarcinoma cells strongly correlated with the in vitro ability of the parent form to inhibit recombinant human P1-1. This isozyme is the dominant form of GST present in HT-29 cells. Of the four analog DEEs tested, gamma-glutamyl-S-(benzyl)cysteinyl-R(-)-phenyl glycine (TER 117) DEE was the most effective in potentiating CMB toxicity in several cell lines: HT-29, HT4-1 (HT-29 subclone), SKOV-3 ovarian carcinoma, and SK VLB (vinblastine-resistant variant of SKOV-3) cells. gamma-Glutamyl-S-(octyl)cysteinyl-glycine (TER 143) DEE potentiated mitomycin C (MTC) toxicity in HT4-1 and SK VLB cells while TER 117 DEE did not. TER 117 DEE enhanced melphalan effects on xenografts of HT4-1 in mice to a similar extent as that achieved with the previously described nonspecific GST inhibitor, ethacrynic acid. Taken together, our results indicate that cell-permeable analogs of GSH can potentiate cytotoxicity of common chemotherapeutic drugs and this effect has a strong positive correlation with the ability of the analogs to inhibit specific GST isozymes.     

Insights into potential cellular mechanisms of cisplatin nephrotoxicity and their clinical application

Cisplatin preferentially accumulates in cells of the S3 segment of the renal proximal tubule and is toxified intracellularly by hydration. The earliest manifestation of toxicity is inhibition of protein synthesis. GSH depletion is another important mechanism causing CP toxicity. Intracellular binding to SH groups leads to GSH depletion, resulting in lipid peroxidation and eventually mitochondrial damage. New measures to prevent GSH depletion and scavenge intracellular free oxygen radicals have been tried in clinical studies. Promising results indicate that cisplatin nephrotoxicity can be further reduced in the future.     

Depletion of glutathione during bovine oocyte maturation reversibly blocks the decondensation of the male pronucleus and pronuclear apposition during fertilization

Oocyte-produced glutathione (the tripeptide gamma-glutamyl-cysteinyl-glycine; GSH) has been implicated in the reduction of disulfide bonds in the sperm nucleus during fertilization and thus in the development of the male pronucleus (PN). In this study, we show that the depletion of endogenous glutathione by 10 mM buthionine sulfoximine (BSO; specific inhibitor of GSH synthesis) during bovine oocyte maturation (24 h in vitro; represents prophase I to metaphase II transition in this species) blocks the formation of a male PN in > 85% of treated oocytes (vs. 6.8% in controls) and prevents the assembly of the sperm aster microtubules in approximately 35%. Consequently, the pronuclear migration and apposition do not occur. Ultrastructural observations suggest that the effect of BSO on pronuclear apposition might be due to incomplete disassembly of the sperm tail connecting piece, which normally leads to the release of the sperm centriole and to the reconstitution of the zygotic centrosome during fertilization. The sperm nucleus decondensation and migration blocks were reversed by the treatment of the GSH-depleted oocytes with 1-10 mM dithiothreitol (a disulfide bond-reducing agent) applied 8 h after insemination: 82% of these oocytes exhibited a normal male PN and pronuclear apposition 20 h after insemination. The pool of glutathione seems to be generated during oocyte maturation since > 80% of oocytes that were matured in the absence of BSO displayed a normal male PN, as apposed to a female PN, when inseminated and cultured in the presence of 10 mM BSO. These data suggest that the reduction of disulfide bonds in the sperm after incorporation is important for the formation of the male PN, as well as for the disassembly of the sperm tail connecting piece and pronuclear apposition. The lack of disulfide-reducing power in the GSH-depleted oocytes can be reversed by treatment with disulfide bond-reducing agents.     

The reactivity of selected acrylate esters toward glutathione and deoxyribonucleosides in vitro: structure-activity relationships

Acrylate esters are alpha,beta-unsaturated esters used as plastic monomers whose toxicity may involve reaction with tissue nucleophiles via Michael addition. Structure-activity relationships for reactivity of selected esters with glutathione (GSH) and deoxyribonucleosides were investigated in the present studies. The esters investigated were methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, butyl acrylate, butyl methacrylate, tetraethyleneglycol diacrylate, tetraethyleneglycol dimethacrylate, and ethyleneglycol dimethacrylate. To compare their reactivities toward GSH, esters were incubated for up to 1 hr at 37 degrees C and pH 7.4 with either GSH or red blood cells in phosphate-buffered saline followed by measurement of free thiol. In both systems acrylate electrophilic reactivity decreased with alpha-methyl substitution; however, the decrease in electrophilic reactivity was more evident in the cell-free system than in the red blood cell model. Increased alcohol chain length moderately affected the apparent second-order rate constant for the spontaneous reaction of acrylate esters with GSH, but did not affect potency relative to cellular GSH depletion. The apparent second-order rate constants of bifunctional esters are more than twice the rate constants of the much smaller monofunctional esters. Ethyl acrylate, a reactive acrylate ester based upon glutathione alkylation, has been designated a class 2B (suspect human) carcinogen by the International Agency for Research on Cancer. To detect possible DNA alkylation by acrylate esters in vitro, ethyl acrylate was incubated with deoxyribonucleosides for up to 24 hr at pH 6.7 or 7.4 and 37 degrees C or up to 8 hr and 50 degrees C. HPLC analysis revealed no detectable adduct formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of some sulfur-containing antioxidants on lead-exposed lenses

Lead (Pb) is known to negatively affect glutathione (GSH) metabolism in the lens. The present study examined the effects of Captopril, Taurine, and alpha-Lipoic acid on the Pb-induced GSH depletion and lipid peroxide increase in the lenticular system. Captopril administration returned the GSH, cysteine (CYS), and malondialdehyde (MDA) levels to near normal. Following Taurine administration the GSH, CYS and MDA levels were intermediate between the control group and the Pb group levels. Alpha-Lipoic acid administration, however, only increased the CYS levels. No significant changes in oxidized glutathione (GSSG) levels were observed in any treatment group.     

Docosahexaenoic acid accumulates in cardiolipin and enhances HT-29 cell oxidant production

The objective of this study was to investigate membrane fatty acids for their effects on mitochondrial function in live cells. Mitochondrial potential and oxidant production were measured in human colonic adenocarcinoma (HT-29) cells with membranes enhanced in either oleic acid, linoleic acid, arachidonic acid, eicosapentaenoic acid, or docosahexaenoic acid. Docosahexaenoic acid-enriched cells had increased mitochondrial potential and produced 5-fold more cellular oxidants than did cells enriched with any other fatty acid. Oxidant production in fatty acid-enriched HT-29 cells did not correlate with the degree of unsaturation for total membrane fatty acids. However, there was a strong correlation between the degree of fatty acid unsaturation of cardiolipin, a critical inner-mitochondrial membrane phospholipid, and oxidant production. Cardiolipin acyl composition is known to influence the activity of electron transport complexes, an effect that can increase mitochondrial oxidant production. Docosahexaenoic acid was enriched to 48 mol% of the fatty acids present in HT-29 cell cardiolipin. These results demonstrate the importance of membrane acyl composition to mitochondrial potential and oxidant production in live cells. Additionally, results suggest that docosahexaenoic acid increases cell oxidant production by accumulating in cardiolipin, where its presence alters electron transport efficiency.     

Cytotoxicity and apoptosis produced by cytochrome P450 2E1 in Hep G2 cells

Two Hep G2 subclones overexpressing CYP2E1 were established with the use of transfection and limited dilution screening techniques. The Hep G2-CI2E1-43 and -47 (E47) cells (transduced Hep G2 subclones that overexpress CYP2E1) grew at a slower rate than parental Hep G2 cells or control subclones that do not express CYP2E1, but remained fully viable. When GSH synthesis was inhibited by treatment with buthionine sulfoximine, GSH levels rapidly declined in E47 cells but not control cells, which is most likely a reflection of CYP2E1-catalyzed formation of reactive oxygen species. Under these conditions of GSH depletion, cytotoxicity and apoptosis were found only with the E47 cells. Low levels of lipid peroxidation were found in the E47 cells, which became more pronounced after GSH depletion. The antioxidants vitamin E, vitamin C, or trolox prevented the lipid peroxidation as well as the cytotoxicity and apoptosis, as did transfection with plasmid containing antisense CYP2E1 or overexpression of Bcl-2. Levels of ATP were lower in E47 cells because of damage to mitochondrial complex I. When GSH was depleted, oxygen uptake was markedly decreased with all substrates in the E47 extracts. Vitamin E completely prevented the decrease in oxygen uptake. Under conditions of CYP2E1 overexpression, two modes of CYP2E1-dependent toxicity can be observed in Hep G2 cells: a slower growth rate when cellular GSH levels are maintained and a loss of cellular viability when cellular GSH levels are depleted. Elevated lipid peroxidation plays an important role in the CYP2E1-dependent toxicity and apoptosis. This direct toxicity of overexpressed CYP2E1 may reflect the ability of this enzyme to generate reactive oxygen species even in the absence of added metabolic substrate.     

Protective effects of glutathione isopropyl ester on the sensitivity of cultured cells to UVB irradiation

The effect of glutathione (GSH) isopropyl ester on cellular sensitivity to UVB irradiation was investigated in HeLaS3 cells. Pretreatment with 0.1-0.5 mM GSH isopropyl ester for 4 h significantly inhibited the decrease of thymidine (TdR) incorporation caused by UVB irradiation at a dose of 500 J/m2, whereas pretreatment with a high dose (1 mM) had no effect. The colony formation ability of the pretreated cells (0.3 mM) was significantly better than that of cells that received irradiation only. When the cells were treated with GSH isopropyl ester, their intracellular GSH level increased dose-dependently over a 4 h period, suggesting that GSH isopropyl ester was transported into the cells and there converted to GSH. Within 2 min of exposure, the intracellular GSH level depleted rapidly to about 75% of that in non-irradiated normal cells. In contrast, the GSH level in cells pretreated with 0.3 mM GSH isopropyl ester was maintained at the same level as that in normal cells, indicating that the maintenance of intracellular GSH level is due to converted GSH from GSH isopropyl ester. These results clearly show that intracellular GSH is involved in cell protection against photodamage, and that GSH isopropyl ester is a useful antioxidant for protection against photooxidative injury.     

2-Methyl-thiazolidine-2,4-dicarboxylic acid protects against paracetamol induced toxicity in human liver derived HepG2 cells

The effects of 2-methyl-thiazolidine-2,4-dicarboxylic acid (CP) on paracetamol-induced toxicity were investigated and evaluated in a human liver derived HepG2 cell line. Incubation of the cells with CP (2 mM and 10 mM) drastically attenuated the GSH and cysteine depletion caused by toxic concentrations of paracetamol (1 mM and 5 mM). When CP (10 mM) was introduced alone into the medium, the level of malondialdehyde and the reactive oxygen species were maintained at the control levels with a simultaneous increase of non-protein sulfhydryl in the cells. Thus, the results of our work prove that CP is a non-toxic precursor of cysteine and GSH, and successfully prevents paracetamol toxicity in HepG2 cells.     

Modulation of intracellular glutathione and cysteine metabolism in bovine oviduct epithelial cells cultured In vitro

The objective of this study was to determine how alterations in intracellular thiol levels of oviduct epithelium occur in response to chemical or environmental conditions that could result in oxidative stress. Bovine oviducts were classified as follicular (F) or luteal (L) according to the reproductive stage of the ovary. Epithelial cells were harvested from the ampulla (AMP) and isthmus (ISTH) region of each oviduct, suspended in culture medium, and then plated into collagen-coated culture plates and grown to confluency. Basal levels of intracellular cysteine (Cys) and glutathione (GSH) were determined in oviduct epithelial cells and found to range from 0.36 to 0.46 pmol/ microg protein for Cys and from 5.3 to 6.4 pmol/ microg protein for GSH. Oxidized Cys values ranged from 21% to 39% of total Cys, whereas the oxidized GSH levels observed were from 21% to 28% of total GSH except in luteal ISTH, where they were significantly lower (6%). Confluent cells were exposed to GSH-depleting agents, L-buthionine-S,R-sulfoximine (BSO) or diethyl maleate (DEM), at doses ranging from 10 to 5000 microM. Both compounds depleted GSH in a dose-dependent manner, and 500 microM concentrations were chosen for subsequent studies with each compound. Cys levels in BSO (500 microM)-treated oviduct epithelial cells were transiently elevated over control values during the initial 5-h incubation; there was then a decrease in Cys levels by AMP but not ISTH oviduct epithelial cells. BSO-treated oviduct epithelial cells displayed a continued depletion of GSH over the incubation period and by 24 h were depleted by 38% to 61%. These results demonstrate a difference in GSH turnover in oviduct epithelial cells associated with reproductive stage. Exposure to DEM (500 microM) caused a decline in both Cys and GSH levels, which were partially restored after DEM removal. In general, L-staged oviduct epithelial cells were observed to be more competent at replenishing thiol stores than F-staged oviduct epithelial cells. Results from this study suggest that reproductive stage and region influence intracellular oviduct epithelium thiol status and therefore may affect how this tissue responds to chemicals or environmental conditions leading to oxidative stress.     

Induction of reactive oxygen species in neurons by haloperidol

Haloperidol (HP) is widely prescribed for schizophrenia and other affective disorders but has severe side effects such as tardive dyskinesia. Because oxidative stress has been implicated in the clinical side effects of HP, rat primary cortical neurons and the mouse hippocampal cell line HT-22 were used to characterize the generation of reactive oxygen species (ROS) and other cellular alterations caused by HP. Primary neurons and HT-22 cells are equally sensitive to HP with an IC50 of 35 microM in the primary neurons and 45 microM in HT-22. HP induces a sixfold increase in levels of ROS, which are generated from mitochondria but not from the metabolism of catecholamines by monoamine oxidases. Glutathione (GSH) is an important antioxidant for the protection of cells against HP toxicity because (1) the intracellular GSH decreases as the ROS production increases, (2) the exogenous addition of antioxidants, such as beta-estradiol and vitamin E, lowers the level of ROS and protects HT-22 cells from HP, and (3) treatments that result in the reduction of the intracellular GSH potentiate HP toxicity. The GSH decrease is followed by the increase in the intracellular level of Ca2+, which immediately precedes cell death. Therefore, HP causes a sequence of cellular alterations that lead to cell death and the production of ROS is the integral part of this cascade.     

Intestinal and peritoneal bleeding: detection with an intravascular contrast agent and fast three-dimensional MR imaging--preliminary experience from an experimental study

We investigated transcobalamin II (TC) isoelectrofocusing (IEF) phenotype and codon 259 polymorphism, in Caco-2 and HT-29 cells and in blood drawn from 39 healthy Caucasians. Caco-2 cells expressed a single TC variant (259-Arg), while HT-29 cells expressed TC with either Arg or Pro at codon 259 and exhibited two isoproteins in IEF with urea, but only one in IEF without urea. Among the Caucasians, 7 subjects expressed the TC 259-Arg variant, 10 the 259-Pro variant, and 22 were heterozygous. The TC 259-Pro isoprotein issued from HT-29 cells and heterozygous caucasian sera, was, respectively, 2. 4-fold and 1.6-fold higher than the TC 259-Arg isoprotein. Apo-TC and vitamin B12 serum concentrations in 259-Pro homozygotes were, respectively, 1.7 and 1.4-fold higher than those in 259-Arg homozygotes (p<0.005 and p=0.05). In conclusion, the 259-Arg/Pro polymorphism yields two TC variants only titratable in denaturing conditions and affects the blood level of both Apo-TC and vitamin B12.     

Direct evidence for glutathione as mediator of apoptosis in neuronal cells

Recent evidence has focused attention on the role of oxidative stress in various acute and chronic neurodegenerative diseases. Particularly, a decrease in the level of the powerful antioxidant glutathione (GSH) and death of dopaminergic neurons in substantia nigra are prominent features in Parkinson's disease. The mode of neuronal death is uncertain; however, apoptosis has been hypothesized to be mediated through the induction of free radicals via oxidative pathways. An approach to determine the role of GSH depletion in neurodegeneration and apoptosis was to create a selective modulation of this antioxidant by metabolic manipulations in a clonal cell line of neuronal origin (mouse neuroblastoma NS20Y). Intracellular GSH levels was lowered by inhibiting its biosynthesis with L-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase. This treatment led to a GSH depletion of 50% after 1 h and 98% after 24 h. A direct cause/effect relationship between GSH depletion and apoptosis was evidenced in this neuronal cell type. GSH depletion induced the death of NS20Y and promoted nuclear alterations of apoptosis as demonstrated by the in situ staining of DNA fragmentation after 5 days of BSO treatment (by terminal-deoxynucleotide transferase-mediated dUTP-nick end labeling), and the appearance of DNA laddering on agarose gel. These results suggested that redox desequilibrium induced by GSH depletion may serve as a general trigger for apoptosis in neuronal cells, and are consistent with the hypothesis that GSH depletion contribute to neuronal death in Parkinson's disease.     

Loss of GSH, oxidative stress, and decrease of intracellular pH as sequential steps in viral infection

Madin-Darby canine kidney cells infected with Sendai virus rapidly lose GSH without increase in the oxidized products. The reduced tripeptide was quantitatively recovered in the culture medium of the cells. Since the GSH loss in infected cells was not blocked by methionine, a known inhibitor of hepatocyte GSH transport, a nonspecific leakage through the plasma membrane is proposed. UV-irradiated Sendai virus gave the same results, confirming that the major loss of GSH was due to membrane perturbation upon virus fusion. Consequent to the loss of the tripeptide, an intracellular pH decrease occurred, which was due to a reversible impairment of the Na+/H+ antiporter, the main system responsible for maintaining unaltered pHi in those cells. At the end of the infection period, a rise in both pHi value and GSH content was observed, with a complete recovery in the activity of the antiporter. However, a secondary set up of oxidative stress was observed after 24 h from infection, which is the time necessary for virus budding from cells. In this case, the GSH decrease was partly due to preferential incorporation of the cysteine residue in the viral proteins and partly engaged in mixed disulfides with intracellular proteins. In conclusion, under our conditions of viral infection, oxidative stress is imposed by GSH depletion, occurring in two steps and following direct virus challenge of the cell membrane without the intervention of reactive oxygen species. These results provide a rationale for the reported, and often contradictory, mutual effects of GSH and viral infection.     

Glutathione regulation of neutral sphingomyelinase in tumor necrosis factor-alpha-induced cell death

Tumor necrosis factor-alpha (TNFalpha)-induced cell death involves a diverse array of mediators and regulators including proteases, reactive oxygen species, the sphingolipid ceramide, and Bcl-2. It is not known, however, if and how these components are connected. We have previously reported that GSH inhibits, in vitro, the neutral magnesium-dependent sphingomyelinase (N-SMase) from Molt-4 leukemia cells. In this study, GSH was found to reversibly inhibit the N-SMase from human mammary carcinoma MCF7 cells. Treatment of MCF7 cells with TNFalpha induced a marked decrease in the level of cellular GSH, which was accompanied by hydrolysis of sphingomyelin and generation of ceramide. Pretreatment of cells with GSH, GSH-methylester, or N-acetylcysteine, a precursor of GSH biosynthesis, inhibited the TNFalpha-induced sphingomyelin hydrolysis and ceramide generation as well as cell death. Furthermore, no significant changes in GSH levels were observed in MCF7 cells treated with either bacterial SMase or ceramide, and GSH did not protect cells from death induced by ceramide. Taken together, these results show that GSH depletion occurs upstream of activation of N-SMase in the TNFalpha signaling pathway. TNFalpha has been shown to activate at least two groups of caspases involved in the initiation and "execution" phases of apoptosis. Therefore, additional studies were conducted to determine the relationship of GSH and the death proteases. Evidence is provided to demonstrate that depletion of GSH is dependent on activity of interleukin-1beta-converting enzyme-like proteases but is upstream of the site of action of Bcl-2 and of the execution phase caspases. Taken together, these studies demonstrate a critical role for GSH in TNFalpha action and in connecting major components in the pathways leading to cell death.