Multidrug-resistance-associated protein plays a protective role in menadione-induced oxidative stress in endothelial cells

AimsMenadione, a redox-cycling quinone known to cause oxidative stress, binds to reduced glutathione (GSH) to form glutathione S-conjugate. Glutathione S-conjugates efflux is often mediated by multidrug-resistance-associated protein (MRP). We investigated the effect of a transporter inhibitor, MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), on menadione-induced oxidative stress in bovine aortic endothelial cells (BAECs).Main methodsBAECs were treated with menadione and MK571, and cell viability was measured. Modulation of intracellular GSH levels was performed with buthionine sulfoximine and GSH ethyl ester treatments. Intracellular superoxide was estimated by dihydroethidium oxidation using fluorescence microscopy or flow cytometry. Expression of MRP was determined by flow cytometry using phycoerythrin-conjugated anti-MRP monoclonal antibody.Key findingsIntracellular GSH depletion by buthionine sulfoximine promoted the loss of viability of BAECs exposed to menadione. Exogenous GSH, which does not permeate the cell membrane, or GSH ethyl ester protected BAECs against the loss of viability induced by menadione. The results suggest that GSH binds to menadione outside the cells as well as inside. Pretreatment of BAECs with MK571 dramatically increased intracellular levels of superoxide generated from menadione, indicating that menadione may accumulate in the intracellular milieu. Finally, we found that MK571 aggravated menadione-induced toxicity in BAECs and that MRP levels were increased in menadione-treated cells.SignificanceWe conclude that MRP plays a vital role in protecting BAECs against menadione-induced oxidative stress, presumably due to its ability to transport glutathione S-conjugate.     

A role of vitamin E in protection against cell injury. Maintenance of intracellular glutathione precursors and biosynthesis

The depletion of cell calcium from isolated rat hepatocytes results in stimulated lipid peroxidation, loss of intracellular and mitochondrial GSH (reduced glutathione), and enhancement of both efflux and oxidation of GSH. These events are followed by cell injury and enhance the susceptibility of the cells to toxic chemicals. It is shown herein that an initial event in the generation of such injury is the depletion of cellular alpha-tocopherol. alpha-Tocopheryl succinate addition (25 microM) to the calcium-depleted cells markedly elevated the alpha-tocopherol content of the cells, inhibited the associated lipid peroxidation, and maintained intracellular GSH levels without affecting its efflux or redox status. This resulted in an enhanced formation of total glutathione after a 5-h incubation, which correlated with the alpha-tocopherol content of the cells, and was greater than that expected by a direct sparing action of vitamin E. Inhibition of hepatocyte glutathione biosynthesis by buthionine sulfoximine (0.5 mM) eliminated the enhancement of GSH formation by vitamin E. Analysis of endogenous and 35S-labelled precursors of glutathione biosynthesis by high-performance liquid chromatography demonstrated that the depletion of cellular alpha-tocopherol resulted in the efflux of glutathione precursors. It is concluded that cell injury associated with alpha-tocopherol depletion is partly the result of the efflux of glutathione precursors, and hence diminished biosynthesis and intracellular levels of GSH. These losses and resultant cell injury are preventable by maintenance of cellular alpha-tocopherol levels.     

Glutathione changes occurring after S-adenosylhomocysteine hydrolase inhibition

Freshly isolated rat hepatocytes, which metabolize methionine through the cystathionine pathway, and cultured L5178Y cells, which do not, were compared for their response to the inhibition of S-adenosylhomocysteine (SAH) hydrolase (EC 3.3.1.1). When cells were incubated in Fischer's medium lacking cystine but containing 0.67 mM methionine and 10% serum, the addition of periodate-oxidized adenosine (POA), an inhibitor of SAH hydrolase, increased the level of SAH approximately 4-fold in L5178Y cells (5 mM POA) and 30-fold in hepatocytes (1 mM POA). POA treatment also decreased the amount of intracellular glutathione (GSH) in hepatocytes by 6-fold, and in L5178Y cells by 3-fold. Incubation of hepatocytes with adenosine plus homocysteine, 2-chloroadenosine, or 2',3'-acyclic adenosine increased intracellular SAH and also lowered GSH levels. Neither GSH oxidation nor efflux of GSH or GSH conjugates appeared to account for the GSH loss. Intracellular GSH, covalently bound to proteins as mixed disulfides, increased when hepatocytes were incubated with POA, but the increase was insufficient to account for the total GSH loss. In hepatocytes with prelabeled [35S]GSH, POA caused the cellular GSH content to decrease while the specific activity of [35S]GSH remained constant, suggesting that inhibitor treatments that caused elevated SAH levels may have increased the degradation of GSH while GSH synthesis was inhibited.     

Inhibition of glutathione disulfide reductase by glutathione

Rat-liver glutathione disulfide reductase is significantly inhibited by physiological concentrations of the product, glutathione. GSH is a noncompetitive inhibitor against GSSG and an uncompetitive inhibitor against NADPH at saturating concentrations of the fixed substrate. In both cases, the inhibition by GSH is parabolic, consistent with the requirement for 2 eq. of GSH in the reverse reaction. The inhibition of GSSG reduction by physiological levels of the product, GSH, would result in a significantly more oxidizing intracellular environment than would be realized in the absence of inhibition. Considering inhibition by the high intracellular concentration of GSH, the steady-state concentration of GSSG required to maintain a basal glutathione peroxidase flux of 300 nmol/min/g in rat liver is estimated at 8-9 microM, about 1000-fold higher than the concentration of GSSG predicted from the equilibrium constant for glutathione reductase. The kinetic properties of glutathione reductase also provide a rationale for the increased glutathione (GSSG) efflux observed when cells are exposed to oxidative stress. The resulting decrease in intracellular GSH relieves the noncompetitive inhibition of glutathione reductase and results in an increased capacity (Vmax) and decreased Km for GSSG.     

Excretion of glutathione conjugates by primary cultured rat hepatocytes

Conjugation of xenobiotics with glutathione occurs commonly within the liver, and these glutathione conjugates are then preferentially excreted into bile. We have characterized this excretory process using primary cultured hepatocytes (24 h). 1-Chloro-2,4-dinitrobenzene rapidly entered the cells and formed a glutathione conjugate, S-(dinitrophenyl)glutathione, irrespective of the temperature of incubation. In contrast, the efflux of the glutathione conjugate was essentially absent in the cold but recovered rapidly upon rewarming of the cells. Therefore, initial rates of efflux of the conjugate at 37 degrees C were measured from cells preloaded biosynthetically at 10 degrees C. Efflux was a saturable process with respect to intracellular S-(dinitrophenyl)glutathione with an apparent Km of 0.58 +/- 0.12 mM and Vmax of 0.15 +/- 0.05 nmol/min/mg of protein. The excretion of S-(dinitrophenyl)glutathione had an energy of activation of 15.3 kcal/mol. The glutathione conjugate of p-nitrobenzylchloride when formed within the hepatocytes acted as a competitive inhibitor of S-(dinitrophenyl)glutathione efflux. Cultured hepatocytes, therefore, appeared to have a specific transport process for the excretion of glutathione conjugates. The addition of S-(dinitrophenyl)glutathione, but not GSH, GSSG, or methionine, to the medium caused a decrease in the rate of efflux of radiolabeled S-(dinitrophenyl)glutathione. The hepatocytes were able, however, to excrete the glutathione conjugate against an excess of extracellular S-(dinitrophenyl)glutathione. This observation suggested that extracellular S-(dinitrophenyl)glutathione, although capable of binding to the carrier, entered the hepatocytes quite slowly relative to rates of efflux. This carrier may function in a manner that would minimize the reuptake by hepatocytes of conjugates that have been excreted into the bile.     

Mechanism of RPE cell death in α-crystallin deficient mice: a novel and critical role for MRP1-mediated GSH efflux

Absence of α-crystallins (αA and αB) in retinal pigment epithelial (RPE) cells renders them susceptible to oxidant-induced cell death. We tested the hypothesis that the protective effect of α-crystallin is mediated by changes in cellular glutathione (GSH) and elucidated the mechanism of GSH efflux. In α-crystallin overexpressing cells resistant to cell death, cellular GSH was >2 fold higher than vector control cells and this increase was seen particularly in mitochondria. The high GSH levels associated with α-crystallin overexpression were due to increased GSH biosynthesis. On the other hand, cellular GSH was decreased by 50% in murine retina lacking αA or αB crystallin. Multiple multidrug resistance protein (MRP) family isoforms were expressed in RPE, among which MRP1 was the most abundant. MRP1 was localized to the plasma membrane and inhibition of MRP1 markedly decreased GSH efflux. MRP1-suppressed cells were resistant to cell death and contained elevated intracellular GSH and GSSG. Increased GSH in MRP1-supressed cells resulted from a higher conversion of GSSG to GSH by glutathione reductase. In contrast, GSH efflux was significantly higher in MRP1 overexpressing RPE cells which also contained lower levels of cellular GSH and GSSG. Oxidative stress further increased GSH efflux with a decrease in cellular GSH and rendered cells apoptosis-prone. In conclusion, our data reveal for the first time that 1) MRP1 mediates GSH and GSSG efflux in RPE cells; 2) MRP1 inhibition renders RPE cells resistant to oxidative stress-induced cell death while MRP1 overexpression makes them susceptible and 3) the antiapoptotic function of α-crystallin in oxidatively stressed cells is mediated in part by GSH and MRP1. Our findings suggest that MRP1 and α crystallin are potential therapeutic targets in pathological retinal degenerative disorders linked to oxidative stress.     

Protective role of intracellular glutathione against oxidized low density lipoprotein in cultured endothelial cells

We examined the role of intracellular glutathione (GSH) in the defense of endothelial cells against oxidized low density lipoprotein (OX-LDL). Incubation of cultured bovine endothelial cells with OX-LDL produced a loss of intracellular GSH, followed by lysis. A decrease in the cellular stores of GSH by treating the endothelial cells with buthionine sulfoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, increased the susceptibility of endothelial cells to lysis by OX-LDL. In contrast, an increase in cellular GSH level by treatment with L-2-oxothiazolidine-4-caboxylate, an effective intracellular cysteine delivery agent, reduced the toxicity of OX-LDL. These findings suggest that intracellular GSH plays an important role in the defense of endothelial cells against OX-LDL, and that the mechanism of OX-LDL toxicity is related to the depletion of intracellular GSH.     

H7, a protein kinase C inhibitor, increases the glutathione content of neuroblastoma cells.

It is shown that the intracellular glutathione (GSH) concentration of neuroblastoma-2a cells in culture increases with a maximum at 24 h after starting treatment with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), an inhibitor of protein kinase C (PKC). Other inhibitors of this and other protein kinases, e.g. sphingosine, staurosporine, and HA 1004, at the concentrations tested, had a less marked or negligible effect on intracellular GSH concentration. 12-O-Tetradecanoylphorbol-13-acetate (TPA) was also tested and showed no significant effect 24 h after addition.     

Dissecting the role of glutathione biosynthesis in Plasmodium falciparum

Glutathione (γ-glutamylcysteinyl-glycine, GSH) has vital functions as thiol redox buffer and cofactor of antioxidant and detoxification enzymes. Plasmodium falciparum possesses a functional GSH biosynthesis pathway and contains mM concentrations of the tripeptide. It was impossible to delete in P. falciparum the genes encoding γ-glutamylcysteine synthetase (γGCS) or glutathione synthetase (GS), the two enzymes synthesizing GSH, although both gene loci were not refractory to recombination. Our data show that the parasites cannot compensate for the loss of GSH biosynthesis via GSH uptake. This suggests an important if not essential function of GSH biosynthesis pathway for the parasites. Treatment with the irreversible inhibitor of γGCS L-buthionine sulfoximine (BSO) reduced intracellular GSH levels in P. falciparum and was lethal for their intra-erythrocytic development, corroborating the suggestion that GSH biosynthesis is important for parasite survival. Episomal expression of γgcs in P. falciparum increased tolerance to BSO attributable to increased levels of γGCS. Concomitantly expression of glutathione reductase was reduced leading to an increased GSH efflux. Together these data indicate that GSH levels are tightly regulated by a functional GSH biosynthesis and the reduction of GSSG.     

Protective effect of glutathione on the cytotoxicity caused by a combination of aluminum and iron in suspension-cultured tobacco cells

The role of endogenous glutathione (GSH) in the protection of suspension-cultured tobacco cells from aluminum (Al) toxicity was examined. Cells at the logarithmic phase of growth were treated with or without Al in nutrient medium prepared without P_i and EDTA. In the absence of Al, total GSH content (including oxidized glutathione [GSSG]) increased gradually. In the presence of Al, the increase of GSH was repressed. This effect was observed before the loss of plasma membrane integrity and the loss of cell viability. In contrast, GSSG content in cells increased in the presence of Al. GSH-deprived cells were prepared by culturing cells with buthionine sulfoximine (an inhibitor of Γ -glutamylcysteine synthetase) for 24 h. Total GSH content in GSH-deprived cells was 6% of that in normal cells. The GSH-deprived cells exhibited a higher degree of lipid peroxidation, increased accumulation of Al, and greater loss of viability than normal cells. These results suggest that GSH protects cells from the oxidative membrane damage caused by a combination of Al and Fe(II) possibly by both direct consumption of GSH and oxidation of GSH.     

Suppression of Human T Cell Proliferation Mediated by the Cathepsin B Inhibitor,z-FA-FMK Is Due to Oxidative Stress

The cathepsin B inhibitor, benzyloxycarbonyl-phenylalanine-alanine-fluoromethyl ketone (z-FA-FMK) readily inhibits anti-CD3-induced human T cell proliferation, whereas the analogue benzyloxycarbonyl-phenylalanine-alanine-diazomethyl ketone (z-FA-DMK) had no effect. In contrast, benzyloxycarbonyl-phenylalanine-alanine-chloromethyl ketone (z-FA-CMK) was toxic. The inhibition of T cell proliferation mediated by z-FA-FMK requires not only the FMK moiety, but also the benzyloxycarbonyl group at the N-terminal, suggesting some degree of specificity in z-FA-FMK-induced inhibition of primary T cell proliferation. We showed that z-FA-FMK treatment leads to a decrease in intracellular glutathione (GSH) with a concomitant increase in reactive oxygen species (ROS) levels in activated T cells. The inhibition of anti-CD3-induced T cell proliferation mediated by z-FA-FMK was abolished by the presence of low molecular weight thiols such as GSH, N-acetylcysteine (NAC) and L-cysteine, whereas D-cysteine which cannot be metabolised to GSH has no effect. The inhibition of anti-CD3-induced up-regulation of CD25 and CD69 expression mediated by z-FA-FMK was also attenuated in the presence of exogenous GSH. Similar to cell proliferation, GSH, NAC and L-cysteine but not D-cysteine, completely restored the processing of caspase-8 and caspase-3 to their respective subunits in z-FA-FMK-treated activated T cells. Our collective results demonstrated that the inhibition of T cell activation and proliferation mediated by z-FA-FMK is due to oxidative stress via the depletion of GSH.     

Increased efflux rather than oxidation is the mechanism of glutathione depletion by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Incubation of isolated hepatocytes in the presence of either the parkinsonian-inducing compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or its putative toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) led to a depletion of intracellular reduced glutathione (GSH), which was mostly recovered as glutathione disulfide (GSSG). However, both MPTP- and MPP+-induced glutathione perturbances were relatively unaffected by the prior inhibition of glutathione reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), suggesting that intracellular oxidation was not the major mechanism involved in the GSH loss. Inclusion of cystine in the incubation mixtures revealed a time-dependent formation of cysteinyl glutathione (CySSG), indicating that an increased efflux was mostly responsible for the MPTP- and MPP+-induced GSH depletion. Therefore, the measurement of GSSG, which is apparently formed extracellularly, was not associated with oxidative stress.     

Alteration of glutathione level in human melanoma cells: effect of N-acetyl-L-cysteine and its analogues

The effects of N-acetyl-L-cysteine (L-NAC), N,N-diacetyl-L-cystine (oxidized form of L-NAC) and N-acetyl-D-cysteine on the intracellular glutathione (GSH) level and their toxicity were investigated in the human melanoma cell culture IGR1. L-NAC applied in 3 mM concentration for 24 hr decreased; when applied for 48 hr it did not alter the intracellular GSH level. Treatment with 1 mM L-NAC for 24 hr had no effect on cellular glutathione, whereas the same concentration applied for 48 hr resulted in an increase in the level of GSH. Both concentrations also induced cell injury as determined by protein assay and trypan blue staining. N,N-diacetyl-L-cystine (0.5 and 1.5 mM, 24 hr) induced a decrease in cellular glutathione content without any apparent cell toxicity. D-NAC (1 and 3 mM, 24 hr) did not influence the GSH level of the melanoma cells; however, it had toxic effects resulting in cell loss.     

Modulation of endothelial GSH concentrations: effect of exogenous GSH and GSH monoethyl ester

We studied the effects of exogenous glutathione (GSH) and GSH monoethyl ester (GSH-MEE) on the enhancement of endothelial GSH concentrations. The preparation of GSH-MEE used contained 91% GSH-MEE, approximately 9% GSH diethyl ester (GSH-DEE) and a trace amount of GSH. Both GSH and GSH-MEE markedly stimulated the intracellular concentrations of GSH in endothelial cells. GSH-MEE was more potent than GSH. The enhancement of endothelial GSH concentration by exogenous GSH was completely inhibited by buthionine sulfoximine (BSO), a potent inhibitor of gamma-glutamylcysteine synthase, or acivicin (AT-125), an inhibitor of gamma-glutamyl transpeptidase, suggesting that it was due to the extracellular breakdown and subsequent intracellular resynthesis of GSH. In contrast, the effect of GSH-MEE was largely resistant to BSO and acivicin, suggesting that it was primarily due to transport of GSH-MEE followed by intracellular hydrolysis. The GSH-MEE preparation, which contained 9% GSH-DEE, at concentrations of 2 mM or higher caused vacuolization of endothelial cells. The enhancement of GSH concentrations by exogenous GSH, but not by GSH-MEE, protected endothelial cells against H2O2-induced injury.     

Increased efflux of oxidized glutathione (GSSG) causes glutathione depletion and potentially diminishes antioxidant defense in sickle erythrocytes

Erythrocytes are both an important source and target of reactive oxygen species in sickle cell disease. Levels of glutathione, a major antioxidant, have been shown to be decreased in sickle erythrocytes and the mechanism leading to this deficiency is not known yet. Detoxification of reactive oxygen species involves the oxidation of reduced glutathione (GSH) into glutathione-disulfide (GSSG) which is actively transported out of erythrocyte. We questioned whether under oxidative conditions, GSSG efflux is increased in sickle erythrocytes. Erythrocytes of 18 homozygous sickle cell patients and 9 race-matched healthy controls were treated with 2,3-dimethoxy-l,4-naphthoquinone, which induces intracellular reactive oxygen species generation, to stimulate GSSG production. Intra- and extracellular concentrations of GSH and GSSG were measured at baseline and during 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation. While comparable at baseline, intracellular and extracellular GSSG concentrations were significantly higher in sickle erythrocytes than in healthy erythrocyte after 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation (69.9 ± 3.7 μmol/l vs. 40.6 ± 6.9 μmol/l and 25.8 ± 2.7 μmol/l vs. 13.6 ± 1.7 μmol/l respectively, P<0.002). In contrast to control erythrocytes, where GSH concentrations remained unchanged (176 ± 8.4 μmol/l vs. 163 ± 13.6 μmol/l, NS), GSH in sickle erythrocytes decreased significantly (from 167 ± 8.8 μmol/l to 111 ± 11.8 μmol/l, P<0.01) after 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation. Adding multidrug resistance-associated protein-1 inhibitor (MK571) to erythrocytes blocked GSSG efflux in both sickle and normal erythrocytes. GSSG efflux, mediated by multidrug resistance-associated protein-1, is increased in sickle erythrocytes, resulting in net loss of intracellular glutathione and possibly higher susceptibility to oxidative stress.     

Effect of 1-methyl-4-phenylpyridinium on glutathione in rat pheochromocytoma PC 12 cells

We investigated the effect of the selective dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) on glutathione redox status and the generation of reactive oxygen intermediates (ROI) in rat pheochromocytoma PC 12 cells in vitro. Treatment with MPP+ (250 microM) led to a 63% increase of reduced glutathione (GSH) after 24 h, while a 10-fold higher concentration of MPP+ (2.5 mM) depleted cellular GSH to 12.5% of control levels within that time. Similarly, the complex I-inhibitor rotenone induced a time-dependent loss of GSH at 1 and 10 microM, whereas treatment with lower concentrations of rotenone (0.1, 0.01 microM) increased cellular GSH. Both MPP+ and rotenone increased cellular levels of oxidised glutathione (GSSG) and the higher concentrations of both compounds led to an elevated ratio of oxidised glutathione (GSSG) vs total glutathione (GSH + GSSG) indicating a shift in cellular redox balance. MPP+ or rotenone did not induce the generation of ROI or significant elevation of intracellular levels of thiobabituric acid reactive substances (TBARS) for up to 48 h. Our data suggest that MPP+ has differential effects on glutathione homeostasis depending on the degree of complex I-inhibition and that inhibition of complex I is not sufficient to generate ROI in this paradigm.     

Glutathione depletion and disruption of intracellular ionic homeostasis regulate lymphoid cell apoptosis

Intracellular glutathione (GSH) depletion is an important hallmark of apoptosis. We have recently shown that GSH depletion by its extrusion regulates apoptosis independently of excessive reactive oxygen species accumulation. However, the mechanisms by which GSH depletion regulates apoptosis are still unclear. Because disruption of intracellular ionic homeostasis, associated with apoptotic volume decrease (AVD), is necessary for the progression of apoptotic cell death, we sought to evaluate the relationship between GSH transport and ionic homeostasis during Fas ligand (FasL)-induced apoptosis in Jurkat cells. GSH depletion in response to FasL was paralleled by distinct degrees of AVD identified by differences in cellular forward scatter and electronic impedance analysis. Inhibition of GSH efflux prevented AVD, K+ loss, and the activation of two distinct ionic conductances, mediated by Kv1.3 and outward rectifying Cl- channels. Reciprocally, stimulation of GSH loss accelerated the loss of K+, AVD, and consequently the progression of the execution phase of apoptosis. Although high extracellular K+ inhibited FasL-induced apoptosis, GSH depletion was largely independent of K+ loss. These results suggest that deregulation of GSH and ionic homeostasis converge in the regulation of apoptosis in lymphoid cells.     

Intracellular thiol depletion causes mitochondrial permeability transition in ebselen-induced apoptosis

Ebselen, a selenoorganic compound, has recently been shown to display a novel property of inducing apoptosis through rapid depletion of intracellular thiols in human hepatoma cells, HepG(2). The present study was thus designed to explore the mechanism of how ebselen triggers apoptosis upon depletion of intracellular thiols. The results demonstrated that ebselen treatment triggered mitochondrial permeability transition rather rapidly as revealed by redistribution of calcein green fluorescence from cytosol into mitochondria. Ebselen treatment also caused a dose- and time-dependent loss of mitochondrial membrane potential (MMP) and release of cytochrome c. Pretreatment with N-acetylcysteine, a precursor of intracellular reduced glutathione (GSH) synthesis, significantly attenuated the ebselen-induced MMP disruption and subsequently inhibited the apoptosis. In contrast, pretreatment with buthionine sulfoximine, a specific inhibitor of intracellular GSH synthesis, significantly augmented the ebselen-induced MMP alteration, and enhanced the apoptosis. Although ebselen treatment significantly increased the intracellular superoxide radical and calcium concentrations, superoxide dismutase, and BAPTA (a calcium chelator), however, failed to prevent ebselen-induced MMP loss and apoptosis. Neither caspase-9 nor caspase-3 activation was detected in ebselen-treated cells. Z-VAD-FMK, a general caspase inhibitor, also had no effect on ebselen-induced MMP decrease and apoptosis. The overall findings thus suggest that mitochondrial permeability transition resulted from intracellular thiol depletion is a critical event in ebselen-induced apoptosis.     

Neurotoxicity from glutathione depletion is mediated by Cu-dependent p53 activation

Loss of intracellular neuronal glutathione (GSH) is an important feature of neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The consequences of GSH depletion include increased oxidative damage to proteins, lipids, and DNA and subsequent cytotoxic effects. GSH is also an important modulator of cellular copper (Cu) homeostasis and altered Cu metabolism is central to the pathology of several neurodegenerative diseases. The cytotoxic effects of Cu in cells depleted of GSH are not well understood. We have previously reported that depletion of neuronal GSH levels results in cell death from trace levels of extracellular Cu due to elevated Cu(I)-mediated free radical production. In this study we further examined the molecular pathway of trace Cu toxicity in neurons and fibroblasts depleted of GSH. Treatment of primary cortical neurons or 3T3 fibroblasts with the glutathione synthetase inhibitor buthionine sulfoximine resulted in substantial loss of intracellular GSH and increased cytotoxicity. We found that both neurons and fibroblasts revealed increased expression and activation of p53 after depletion of GSH. The increased p53 activity was induced by extracellular trace Cu. Furthermore, we showed that in GSH-depleted cells, Cu induced an increase in oxidative stress resulting in DNA damage and activation of p53-dependent cell death. These findings may have important implications for neurodegenerative disorders that involve GSH depletion and aberrant Cu metabolism.     

Cysteine but not glutathione modulates the radiosensitivity of human melanoma cells by affecting both survival and DNA damage

Glutathione (GSH) and its precursor cysteine (Cys) are both known to react within any cells with oxidative species and thus play an important role in cellular defense mechanisms against oxidative stress. In melanocytes, these are also important precursors of melanogenesis by reacting non-enzymatically with l-dopaquinone to form the sulfur-containing pheomelanin. Our aim was to assess pigment role in the cellular radioprotection mechanism using a human melanoma cell model of mixed-type melanin under GSH depletion to obtain a radiosensitizing effect. The latter has been achieved either by Cys deprivation or GSH specific depletion. We first compared cell survival of Cys-deprived and GSH-depleted cells vs. control cells. Cys deprivation was achieved by decreasing Cys concentration in the culture medium for 24 h. In this condition, no toxicity was observed, Cys and GSH levels decreased, melanogenesis switched to a higher eumelanin synthesis and cells were significantly more resistant to 10-Gy dose of ionizing radiations than untreated cells. Glutathione depletion was achieved with the gamma-glutamylcysteine synthetase inhibitor buthionine-S-sulfoximine (BSO) for 24 h at 50 microM, a concentration yielding no toxicity. In this condition, intracellular GSH level decreased but no change in pigmentation was observed and cells were slightly but significantly more sensitive to radiation than the control. We then compared DNA radio-induced damages by Comet assay in control cells, cells treated as above and cells with stimulated pigmentation by increasing Tyr concentration in the medium. Our results showed that, when intracellular eumelanin content increased, DNA damage decreased. By contrast, DNA damage increased in cells treated with BSO alone. It is concluded that increasing the intracellular eumelanin content by the melanin precursor Tyr or by favoring the Pheo- to Eumelanin switch, compensates for the loss of the two intracellular radioprotectors that are GSH and Cys.