The role of low molecular weight thiols in T lymphocyte proliferation and IL-2 secretion

Glutathione (GSH) is an abundant intracellular tripeptide that has been implicated as an important regulator of T cell proliferation. The effect of pharmacological regulators of GSH and other thiols on murine T cell signaling, proliferation, and intracellular thiol levels was examined. l-Buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH synthesis, markedly reduced GSH levels and blocked T cell proliferation without significant effect on cell viability. N-acetylcysteine markedly enhanced T cell proliferation without affecting GSH levels. Cotreatment of T cells with N-acetylcysteine and BSO failed to restore GSH levels, but completely restored the proliferative response. Both 2-ME and l-cysteine also reversed the BSO inhibition of T cell proliferation. Intracellular l-cysteine levels were reduced with BSO treatment and restored with cotreatment with NAC or l-cysteine. However, 2-ME completely reversed the BSO inhibition of proliferation without increasing intracellular cysteine levels. Therefore, neither GSH nor cysteine is singularly critical in limiting T cell proliferation. Reducing equivalents from free thiols were required because oxidation of the thiol moiety completely abolished the effect. Furthermore, BSO did not change the expression of surface activation markers, but effectively blocked IL-2 and IL-6 secretion. Importantly, exogenous IL-2 completely overcame BSO-induced block of T cell proliferation. These results demonstrate that T cell proliferation is regulated by thiol-sensitive pathway involving IL-2.     

Differential sensitivities of murine melanocytes and melanoma cells to buthionine sulfoximine and anticancer drugs.

High levels of intracellular glutathione (GSH) may result in resistance of tumor cells to cytotoxic drugs. Because of the innate refractory nature of melanoma cells to chemotherapy, we have used a syngeneic murine system consisting of nontumorigenic Mel-ab melanocytes, tumorigenic H-ras-transformed melanocytes (C9.1), and the highly metastatic BL6 melanoma cells to examine the GSH content, glutathione S-transferase (GST) activity, and sensitivity to buthionine sulfoximine (BSO) and other cytotoxic drugs. Compared to the nontumorigenic melanocytes, both C9.1 and BL6 melanoma cells have nearly fivefold higher GSH content, and BL6 cells have increased GST activity. C9.1 and BL6 cells are more resistant to the cytotoxic effects of BCNU and adriamycin; however, the degrees of resistance do not reflect the increased GSH content in these cells. Pretreatment of BL6 melanoma cells with 50 microM BSO depleted over 90% of their GSH content and enhanced the growth-inhibitory effects of L-dopa methylester, BCNU, bleomycin, and dacarbazine. Exposure to BSO alone was not toxic to the tumor cells for up to 24 hr, but was significantly cytotoxic in the melanocytes after 9 hr. The sensitivity of these cells to BSO appears to depend on a critical level of GSH depletion which is not related to the initial GSH content. These studies suggest that the resistance of melanoma cells to cytotoxic drugs is only partially attributed to changes in the GSH system caused during cellular transformation.     

Schisandrin B-induced increase in cellular glutathione level and protection against oxidant injury are mediated by the enhancement of glutathione synthesis and regeneration in AML12 and H9c2 cells

To define the relative role of reduced glutathione (GSH) synthesis and regeneration in schisandrin B (Sch B)-induced increase in cellular GSH level and the associated cytoprotection against oxidative challenge, the effects of L-buthionine-[S,R]-sulfoximine (BSO, a specific inhibitor of gamma-glutamate cysteine ligase (GCL)) and 1,3-bis(2-chloroethyl)-1-nitrourea (BCNU, a specific inhibitor of glutathione reductase (GR)) treatments or their combined treatment were examined in control and Sch B-treated AML12 and H9c2 cells, without and/or with menadione intoxication. Both BSO and BCNU treatments reduced cellular GSH level in AML12 and H9c2 cells, with the effect of BSO being more prominent. The GSH-enhancing effect of Sch B was also suppressed by BSO and BCNU treatments, with the effect of the combined treatment with BSO and BCNU being semi-additive. While Sch B treatment increased the GR but not GCL activity in AML12 and H9c2 cells, it increased the cellular cysteine level. BSO treatment also suppressed the Sch B-induced increase in GR activity. BSO or BCNU treatment per se did not cause any detectable cytotoxic effect, as assessed by lactate dehydrogenase leakage, but the combined treatment with BSO and BCNU was cytotoxic, particularly in H9c2 cells. The cytotoxic effect of BSO and BCNU became more apparent following the menadione challenge. The cytoprotection afforded by Sch B pretreatment was partly suppressed by BSO or BCNU treatment, or completely abrogated by the combined treatment with BSO and BCNU. In conclusion, the results indicate that the cytoprotective action of Sch B is causally related to the increase in cellular GSH level, which is likely mediated by the enhancement of GSH synthesis and regeneration.     

Requirement of intracellular free thiols for hydrogen peroxide-induced hypertrophy in cardiomyocytes

Reactive oxygen species (ROS) are by-products of aerobic metabolism and are implicated in the pathogenesis of several diseases. H(2)O(2) produces oxidative stress and acts as a second messenger in several cell types. We tested whether the effect of H(2)O(2) on cellular events could be altered by changes in the intracellular redox status in a cardiomyocyte cell line. Using flow cytometric measurements, we found that adding H(2)O(2) induced hypertrophy in control cells in a time-dependent manner. Pre-incubation of the cells with buthionine sulfoximine (BSO), an inhibitor of de novo GSH synthesis, induced increase in the number of cells of small sizes by the addition of H(2)O(2) as compared to non-BSO pre-incubated control cells, and exacerbated the decrease in viability. Total thiol and GSH levels in H9c2 cells pre-incubated with BSO were about 75 and 30% of control, respectively, and GSH levels fell to below the limitation of detection after the addition of H(2)O(2), although total thiol levels were not markedly decreased. In the cells pre-incubated with BSO, hypertrophy was not observed by the addition of H(2)O(2) at any level of concentration. N-acetyl-L-cysteine and cysteine not only prevented increase in the number of cells of small sizes caused by H(2)O(2) but also induced hypertrophy in cells pre-incubated with BSO. These results suggest that the intracellular free thiol levels determine whether cell death or hypertrophy occurs in cardiomyocytes in the presence of H(2)O(2). On the other hand, the hypertrophied cells did not become larger by adding H(2)O(2), but had high levels of cellular GSH, suggesting the possibility that the hypertrophied cells have tolerance to oxidative stress.     

Enhancement of intracellular glutathione promotes lymphocyte activation by mitogen

We have examined the effect of chemically modulating intracellular glutathione (GSH) levels on murine lymphocyte activation. Lymphocyte activation was determined by the induction of polyamine synthesis (ornithine decarboxylase (ODC) induction) and DNA synthesis ([3H]thymidine([3H]Tdr) incorporation). Intracellular GSH levels were enhanced using L-2-oxothiazolidine-4-carboxylate (OTC), which delivers cysteine intracellularly, and suppressed by buthionine sulfoximine (BSO), which inhibits gamma-glutamylcysteine synthetase. In addition, the thiol 2-mercaptoethanol (2-ME) was tested for its ability to augment intracellular GSH levels. Our results indicate that both OTC and 2-ME enhance GSH concentrations and [3H]Tdr incorporation in resting and mitogen (concanavalin A)-stimulated cells. The induction of ODC by concanavalin A (Con A) was augmented by the addition of OTC or 2-ME. The GSH concentration of Con A-stimulated cells was reduced when compared to resting cells; however, it was markedly enhanced by OTC or 2-ME. The stimulatory effects of 2-ME on GSH concentrations, [3H]Tdr incorporation, and ODC induction in both resting and Con A-stimulated cells were much more potent than those of OTC. In contrast, BSO suppressed intracellular GSH and [3H]Tdr incorporation in resting and Con A-stimulated cells. BSO also inhibited the promotion of intracellular GSH concentrations and [3H]Tdr uptake by OTC or 2-ME. However, BSO did not affect the induction of ODC by Con A or its enhancement by OTC or 2-ME. We conclude that enhancement of intracellular GSH concentration results in an increased lymphocyte response to mitogen stimulation.     

Cell cycle progression of glutathione-depleted human peripheral blood mononuclear cells is inhibited at S phase

Glutathione (GSH) the most abundant nonprotein thiol, is involved in the maintenance of the cellular redox state. In this capacity it may influence lymphocyte responsiveness to various stimuli. We have investigated the requirement of GSH during the activation and proliferation of PBMC. The intracellular GSH content of PBMC was altered by continuous culture or pretreatment with buthionine-S,R-sulfoximine (BSO), a specific and irreversible inhibitor of GSH synthesis. Initial experiments demonstrated that the addition of BSO at the initiation of culture, or shortly thereafter (6 hr), inhibited DNA synthesis and produced a simultaneous decrease in intracellular GSH. It was necessary that the BSO be present in the culture for at least 24 hr prior to the initiation of DNA synthesis for maximal inhibition. Cell cycle analysis revealed that BSO did not affect the entry and progression of PBMC through G1 of the cell cycle, however, entry into S-phase was inhibited in a dose-dependent fashion. These results were further substantiated by the inability of BSO to inhibit IL-2 production and expression of the IL-2R. In addition the timely expression of the transferrin receptor by BSO-treated cells indicated that the block occurred at the G1/S transition. The influence of GSH on early activation events was determined by BSO pretreatments. Lowering the intracellular GSH level of PBMC to less than 10% of the initial content prior to mitogenic stimulation did not impair the ability of these cells to produce IL-2 and express IL-2R, indicating that GSH may not be involved in the generation and response to early activation signals. Furthermore, the removal of BSO from these cultures rapidly reversed its inhibitory effects on DNA and GSH synthesis. In the course of these studies we also observed a modest (17%) albeit consistent increase during activation in the total thiol levels of GSH-depleted PBMC. These thiols may have a key role in the activation process. These data support our hypothesis that GSH is required for lymphocyte proliferation and that additional thiols are involved during the activation process.     

Role of the antioxidant system in response of Escherichia coli bacteria to cold stress

The response of aerobically grown Escherichia coli cells to the cold shock induced by the rapid lowering of growth temperature from 37 to 20 degrees C was found to be basically the same as the oxidative stress response. The enhanced sensitivity of cells deficient in two superoxide dismutases, Mn-SOD and Fe-SOD, and the increased expression of the Mn-SOD gene, sodA, in response to cold stress were interpreted as both oxidative and cold stresses are due to a rise in the intracellular level of superoxide anion. The long-term cultivation of E. coli at 20 degrees C was also accompanied by the typical oxidative stress response reactions--an enhanced expression of the Mn-SOD and catalase HPI genes and a decrease in the intracellular level of reduced glutathione (GSH) and in the GSH/GSSG ratio.     

Glutathione depletion restores the susceptibility of cisplatin-resistant chronic myelogenous leukemia cell lines to Natural Killer cell-mediated cell death via necrosis rather than apoptosis

We investigated the effect of intracellular glutathione (GSH) levels on Natural Killer-mediated apoptosis in cisplatin-resistant K562 cells. K562/B6 and K562/C9 are cisplatin-resistant K562 cells less susceptible to lysis by natural killer cells. Cisplatin-resistant K562 cells did not present the apoptotic pattern of DNA fragmentation as it was observed for their maternal counterparts. K562/B6 and K562/C9 cell lines produce 1.6- and 1.9-times more GSH than K562 cells. Treatment of both cell lines with D,L-buthionine-(S,R)-sulfoximine (BSO, a gamma-glutamyl cysteine synthetase inhibitor) decreased GSH levels and augmented cell death induced by NK cells via a necrotic rather than an apoptotic process. Proliferating cell nuclear antigen (PCNA) expression was elevated in cisplatin-resistant K562 subclones, and the reduction of GSH levels after treatment with BSO decreased the expression of PCNA. These results suggest that the GSH level affects the NK cell-mediated cell death of cisplatin-resistant K562 cells by inducing necrosis rather than apoptosis.     

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.     

Apoptosis in arsenic trioxide-treated Calu-6 lung cells is correlated with the depletion of GSH levels rather than the changes of ROS levels

Arsenic trioxide (ATO) can regulate many biological functions such as apoptosis and differentiation in various cells. We investigated an involvement of ROS such as H(2)O(2) and O(2)(*-), and GSH in ATO-treated Calu-6 cell death. The levels of intracellular H(2)O(2) were decreased in ATO-treated Calu-6 cells at 72 h. However, the levels of O(2)(*-) were significantly increased. ATO reduced the intracellular GSH content. Many of the cells having depleted GSH contents were dead, as evidenced by the propidium iodine staining. The activity of CuZn-SOD was strongly down-regulated by ATO at 72 h while the activity of Mn-SOD was weakly up-regulated. The activity of catalase was decreased by ATO. ROS scavengers, Tiron and Trimetazidine did not reduce levels of apoptosis and intracellular O(2)(*-) in ATO-treated Calu-6 cells. Tempol showing a decrease in intracellular O(2)(*-) levels reduced the loss of mitochondrial transmembrane potential (DeltaPsi(m)). Treatment with NAC showing the recovery of GSH depletion and the decreased effect on O(2)(*-) levels in ATO-treated cells significantly inhibited apoptosis. In addition, BSO significantly increased the depletion of GSH content and apoptosis in ATO-treated cells. Treatment with SOD and catalase significantly reduced the levels of O(2)(*-) levels in ATO-treated cells, but did not inhibit apoptosis along with non-effect on the recovery of GSH depletion. Taken together, our results suggest that ATO induces apoptosis in Calu-6 cells via the depletion of the intracellular GSH contents rather than the changes of ROS levels.     

Osmotic stress induces loss of glutathione and increases the sensitivity to oxidative stress in H9c2 cardiac myocytes

It has been observed that H9c2 cardiac cells cultured in physiologic solutions exhibit delayed cell death after repeated medium replacements, of which the cause was the relatively mild osmotic challenges during the renewal of the culture medium. Interestingly, the cell damage was associated with altered intracellular GSH homeostasis. Therefore, this study attempted to elucidate the effects of osmotic stress on GSH metabolism. In cells subjected to osmotic stress by lowering the NaCl concentration of the medium, the cell swelling was rapidly counterbalanced, but the intracellular GSH content was significantly lower in 3 h. Meanwhile, the ratio of GSH-to-GSSG was not affected. As expected, osmotic stress also increased the sensitivity to H₂O₂, which was attributable to the decrease of GSH content. The decrease of GSH content was similarly evident when the synthetic pathways of GSH were blocked by BSO or acivicin. It was concluded that osmotic stress induced the decrease of intracellular GSH content by increased consumption and this loss of GSH rendered the cells susceptible to a subsequent oxidative stress.     

Differential cell death and Bcl-2 expression in the mouse retina after glutathione decrease by systemic D,L-buthionine sulphoximine administration

Glutathione (GSH) plays a critical role in cellular defense against unregulated oxidative stress in mammalian cells including neurons. We previously demonstrated that GSH decrease using [D, L]-buthionine sulphoximine (BSO) induces retinal cell death, but the underlying mechanisms of this are still unclear. Here, we demonstrated that retinal GSH level is closely related to retinal cell death as well as expression of an anti-apoptotic molecule, Bcl-2, in the retina. We induced differential expression of retinal GSH by single and multiple administrations of BSO, and examined retinal GSH levels and retinal cell death in vivo. Single BSO administration showed a transient decrease in the retinal GSH level, whereas multiple BSO administration showed a persistent decrease in the retinal GSH level. Retinal cell death also showed similar patterns: transient increases of retinal cell death were observed after single BSO administration, whereas persistent increases of retinal cell death were observed after multiple BSO administration. Changes in the retinal GSH level affected Bcl-2 expression in the retina. Immunoblot and immunohistochemical analyses showed that single and multiple administration of BSO induced differential expressions of Bcl-2 in the retina. Taken together, the results of our study suggest that the retinal GSH is important for the survival of retinal cells, and retinal GSH appears to be deeply related to Bcl-2 expression in the retina. Thus, alteration of Bcl-2 expression may provide a therapeutic tool for retinal degenerative diseases caused by retinal oxidative stress such as glaucoma or retinopathy.     

Differential roles of 3H-1,2-dithiole-3-thione-induced glutathione, glutathione S-transferase and aldose reductase in protecting against 4-hydroxy-2-nonenal toxicity in cultured cardiomyocytes

4-hydroxy-2-nonenal (HNE) plays an important role in the pathogenesis of cardiac disorders. While conjugation with glutathione (GSH) catalyzed by GSH S-transferase (GST) has been suggested to be a major detoxification mechanism for HNE in target cells, whether chemically upregulated cellular GSH and GST afford protection against HNE toxicity in cardiac cells has not been investigated. In addition, the differential roles of chemically induced GSH and GST as well as other cellular factors in detoxifying HNE in cardiomyocytes are unclear. In this study, we have characterized the induction of GSH and GST by 3H-1,2-dithiole-3-thione (D3T) and the protective effects of the D3T-elevated cellular defenses on HNE-mediated toxicity in rat H9C2 cardiomyocytes. Treatment of cardiomyocytes with D3T resulted in a significant induction of both GSH and GST as well as the mRNA expression of gamma-glutamylcysteine ligase catalytic subunit and GSTA. Both GSH and GST remained elevated for at least 72 h after removal of D3T from the culture media. Treatment of cells with HNE led to a significant decrease in cell viability and an increased formation of HNE-protein adducts. Pretreatment of cells with D3T dramatically protected against HNE-mediated cytotoxicity and protein-adduct formation. HNE treatment caused a significant decrease in cellular GSH level, which preceded the loss of cell viability. Either depletion of cellular GSH by buthionine sulfoximine (BSO) or inhibition of GST by sulfasalazine markedly sensitized the cells to HNE toxicity. Co-treatment of cardiomyocytes with BSO was found to completely block the D3T-mediated GSH elevation, which however failed to reverse the cytoprotective effects of D3T, suggesting that other cellular factor(s) might be involved in D3T cytotprotection. In this regard, D3T was shown to induce cellular aldose reductase (AR). Surprisingly, inhibition of AR by sorbinil failed to potentiate HNE toxicity in cardiomyocytes. In contrast, sorbinil dramatically augmented HNE cytotoxicity in cells with GSH depletion induced by BSO. Similarly, in BSO-treated cells, D3T cytoprotection was also largely reversed by sorbinil, indicating that AR played a significant role in detoxifying HNE only under the condition of GSH depletion in cardiomyocytes. Taken together, this study demonstrates that D3T can induce GSH, GST, and AR in cardiomyocytes, and that the above cellular factors appear to play differential roles in detoxification of HNE in cardiomyocytes.     

The Role of Antioxidant Systems in the Cold Stress Response of Escherichia coli

The response of aerobically grown Escherichia coli cells to the cold shock induced by the rapid lowering of growth temperature from 37 to 20°C was found to be basically the same as the oxidative stress response. The enhanced sensitivity of cells deficient in two superoxide dismutases, Mn-SOD and Fe-SOD, and the increased expression of the Mn-SOD gene, sodA, in response to cold stress were interpreted as both oxidative and cold stresses are due to a rise in the intracellular level of superoxide anion. The long-term cultivation of E. coli at 20°C was also accompanied by the typical oxidative stress response reactions—an enhanced expression of the Mn-SOD and catalase HPI genes and a decrease in the intracellular level of reduced glutathione (GSH) and in the GSH/GSSG ratio.     

Infection of H69AR cells with retroviral particles harboring interfering RNAi significantly reduced the multidrug resistance of these small cell lung cancer cells

Incubation of the drug-sensitive H69, a small cell lung cancer cell line, with increased concentrations of adriamycin yielded multidrug resistant (MDR) H69AR cells that over-express multidrug resistance-associated protein (MRP1). MRP1 co-transports its substrate with glutathione (GSH), leading to lower intracellular GSH. In this report we tested whether depleting intracellular GSH in MRP1-expressing cells could hyper-sensitize them to anticancer drugs or not. We have found that the GSH contents in MRP1-expressing cells are significantly lower than their corresponding control cells. The treatment with MRP1 substrate verapamil or the GSH synthetase inhibitor buthionine sulfoxi-mine significantly reduced the intracellular GSH contents in MRP1-expressing cells. Interestingly, depleting intracellular GSH contents can hyper-sensitize the MRP1-cDNA transfected BHK cells to daunomycin, but not the adriamycin-selected H69AR cells. Further analyses indicated that anti-apoptotic factor Bcl2 might be a factor responsible for the fact that depleting intracellular GSH could not hyper-sensitize H69AR cells to daunomycin. We hypothesized that knocking down the expression of Bcl2 could hyper-sensitize H69AR cells to daunomycin. Interestingly, infection of H69AR cells with retroviral particles harboring Bcl2 interfering RNAi not only reduced the expression of Bcl2, but also many factors that contribute to MDR, such as Bcl-xl, MRP1 and ABCC3, etc., leading to the MDR H69AR cells more sensitive to daunomycin than the parental H69 cell. Thus, although the mechanisms of the down-regulation of the genes contributing to MDR remain to be elucidated, retroviral particles harboring Bcl2 interfering RNAi could be used as an alternative way to sensitize the MDR cancer cells to anticancer drugs.     

Gene knockdown of gamma-glutamylcysteine synthetase by RNAi in the parasitic protozoa Trypanosoma brucei demonstrates that it is an essential enzyme

The parasitic protozoa Trypanosoma brucei utilizes a novel cofactor (trypanothione, T(SH)2), which is a conjugate of GSH and spermidine, to maintain cellular redox balance. gamma-Glutamylcysteine synthetase (gamma-GCS) catalyzes the first step in the biosynthesis of GSH. To evaluate the importance of thiol metabolism to the parasite, RNAi methods were used to knock down gene expression of gamma-GCS in procyclic T. brucei cells. Induction of gamma-GCS RNAi with tetracycline led to cell death within 4-6 days post-induction. Cell death was preceded by the depletion of the gamma-GCS protein and RNA and by the loss of the cellular pools of GSH and T(SH)2. The addition of GSH (80 microM) to cell cultures rescued the RNAi cell death phenotype and restored the intracellular thiol pools to wild-type levels. Treatment of cells with buthionine sulfoximine (BSO), an enzyme-activated inhibitor of gamma-GCS, also resulted in cell death. However, the toxicity of the inhibitor was not reversed by GSH, suggesting that BSO has more than one cellular target. BSO depletes intracellular thiols to a similar extent as gamma-GCS RNAi; however, addition of GSH did not restore the pools of GSH and T(SH)2. These data suggest that BSO also acts to inhibit the transport of GSH or its peptide metabolites into the cell. The ability of BSO to inhibit both synthesis and transport of GSH likely makes it a more effective cytotoxic agent than an inhibitor with a single mode of action. Finally the potential for the T(SH)2 biosynthetic enzymes to be regulated in response to reduced thiol levels was studied. The expression levels of ornithine decarboxylase and of S-adenosylmethionine decarboxylase, two essential enzymes in spermidine biosynthesis, remained constant in induced gamma-GCS RNAi cell lines.     

Vitamin C and vitamin E restore the resistance of GSH-depleted lens cells to H2O2

A decline in reduced glutathione (GSH) levels is associated with aging and many age-related diseases. The objective of this study was to determine whether other antioxidants can compensate for GSH depletion in protection against oxidative insults. Rabbit lens epithelial cells were depleted of > 75% of intracellular GSH by 25-200 microM buthionine sulfoximine (BSO). Depletion of GSH by BSO alone had little direct effect on cell viability, but resulted in an approximately 30-fold increase in susceptibility to H(2)O(2)-induced cell death. Experimentally enhanced levels of nonprotein sulfhydryls other than GSH (i.e., N-acetylcysteine) did not protect GSH-depleted cells from H(2)O(2)-induced cell death. In contrast, pretreatment of cells with vitamin C (25-50 microM) or vitamin E (5-40 microM), restored the resistance of GSH-depleted cells to H(2)O(2). However, concentrations of vitamin C > 400 microM and vitamin E > 80 microM enhanced the toxic effect of H(2)O(2). Although levels of GSH actually decreased by 10-20% in cells supplemented with vitamin C or vitamin E, the protective effects of vitamin C and vitamin E on BSO-treated cells were associated with significant ( approximately 70%) decreases in oxidized glutathione (GSSG) and concomitant restoration of the cellular redox status (as indicated by GSH:GSSG ratio) to levels detected in cells not treated with BSO. These results demonstrate a role for vitamin C and vitamin E in maintaining glutathione in its reduced form. The ability of vitamin C and vitamin E in compensations for GSH depletion to protect against H(2)O(2)-induced cell death suggests that GSH, vitamin C, and vitamin E have common targets in their actions against oxidative damage, and supports the preventive or therapeutic use of vitamin C and E to combat age- and pathology-associated declines in GSH. Moreover, levels of these nutrients must be optimized to achieve the maximal benefit.     

Relationship between cellular glutathione and hyperthermic toxicity in mammary carcinoma in mice

This investigation evaluates in an in vivo system the possible correlation between the intracellular content of GSH and cysteine and thermal sensitivity and thermotolerance. The studies were performed on C3H mammary carcinomas, located on the hind paw of CBA mice. Intracellular thiols were measured by the HPLC technique and the degree of thermotolerance induction was determined from tumour growth rate studies. It was found that the intracellular GSH levels did not change significantly during thermotolerance induction, and that subtoxic hyperthermia induced a pronounced transient decrease in GSH down to 30 per cent of the control level. When the intracellular GSH level was decreased to the same extent, by pretreatment with D,L-buthionine-S-R-sulphoximine (BSO), thermotolerance was still inducible. Thus, the induction of heat-induced thermal resistance did not seem to be dependent on the intracellular GSH level. When hyperthermia and BSO were combined, the GSH levels were further reduced. Treatment with BSO slightly increased the toxicity of both thermotolerance-inducing and subtoxic hyperthermia. The cysteine concentrations increased several fold after BSO and heat treatments and contributed, under these conditions, to more than 25 per cent of the intracellular free reduced thiols. In general, there was no direct correlation between GSH and cysteine levels. It is concluded that thermotolerance induction does not depend on or cause changes in intracellular GSH levels and that subtoxic heat treatments induce a pronounced transient decrease in GSH concentration.     

Ghrelin protects against cobalt chloride-induced hypoxic injury in cardiac H9c2 cells by inhibiting oxidative stress and inducing autophagy

Ghrelin is a multifunctional peptide that actively protects against cardiovascular ischemic diseases, but the underlying mechanisms are unclear. We used CoCl₂ to mimic hypoxic conditions in cardiac H9c2 cells in order to study the mechanism by which ghrelin protects cardiac myocytes against hypoxic injury by regulating the content of intracellular ROS and autophagy levels. Cell apoptosis and necrosis were evaluated by the flow cytometry assay, Hoechst staining, and LDH activity. Cell viability was detected by the WST-1 assay; ROS levels were assessed using DCFH2-DA; and Nox1, catalase and Mn-SOD were assayed by real-time PCR and activity assays. LC3II was measured by Western blot analysis. We observed that CoCl₂ induced apoptosis and death of H9c2 cells in a dose- and time-dependent manner. This was characterized by an increase in cell apoptosis, LDH activity, ROS content, Nox1 expression, and autophagy levels and a decrease in cell viability, catalase, and Mn-SOD activities. Ghrelin treatment significantly attenuated CoCl₂-induced hypoxic injury by decreasing cell apoptosis, LDH activity, ROS content, and Nox1 expression and increasing cell viability, autophagy levels, catalase, and Mn-SOD mRNA levels and activities. Further experiments revealed that inhibiting autophagy using 3-MA or AMPK pathway with compound C almost abrogated the induction of ghrelin in autophagy. This was associated with a decrease in cell viability and an increase in LDH activity. Our results indicate that ghrelin protected cardiac myocytes against CoCl₂-induced hypoxic injury by decreasing Nox1 expression, increasing the expression and activity of endogenous antioxidant enzymes, and inducing protective autophagy in an AMPK-dependent manner.     

Selenoprotein W as molecular target of methylmercury in human neuronal cells is down-regulated by GSH depletion

Methylmercury (MeHg) is well known as a neurotoxic chemical. However, little is mentioned about its neurotoxic mechanism or molecular target in human neuronal cells in particular. We show in this study that exposure of human neuronal cell line, SH-SY5Y, to MeHg dose- and time-dependently impairs viability and mRNA expression of selenoprotein W (SeW) with a significant difference, unlike other selenoenzymes such as, SeP, GPX4, 5DI, and 5'DI. Using real-time RT PCR, the influence of selenium (Se) and glutathione (GSH) on SeW expression was also investigated. While Se depletion caused a weakly reduced SeW mRNA levels, additional Se caused an increase of SeW mRNA levels. Although 2 mM GSH had induced a weak shift on SeW level, the expression of SeW mRNA was down-regulated in SH-SY5Y cells treated with 25 microM BSO, an inhibitor of GSH synthesis. To understand the relationship between a decrease of SeW expression and intracellular GSH and ROS, we measured the concentration of intracellular GSH and ROS in cells treated to 1.4 microM MeHg using fluorescence based assays. A positive correlation was found between SeW mRNA level and intracellular GSH but no significant correlation was observed between intracellular ROS and SeW mRNA level or intracellular GSH contents. Therefore, we suggest that SeW is the novel molecular target of MeHg in human neuronal cells and down-regulation of this selenoenzyme by MeHg is dependent not on generation of ROS but on depletion of GSH.