(-)Schisandrin B ameliorates paraquat-induced oxidative stress by suppressing glutathione depletion and enhancing glutathione recovery in differentiated PC12 cells

Exposure to paraquat (PQ; N,N'-dimethyl-4-4'-bipyridium), a potent herbicide, can lead to neuronal cell death and increased risk of Parkinson's disease because of oxidative stress. In this study, we investigated the effect of (-)schisandrin B [(-)Sch B, a potent enantiomer of schisandrin B] on PQ-induced cell injury in differentiated pheochromocytoma cells (PC12). PQ treatment caused cell injury in PC12 cells, as indicated by the significant increase in lactate dehydrogenase (LDH) leakage. Pretreatment with (-)Sch B (5 μM) protected against PQ-induced toxicity in PC12 cells, as evidenced by the significant decrease in LDH leakage. (-)Sch B induced the cytochrome P-450-mediated reactive oxygen species generation in differentiated PC12 cells. The cytoprotection afforded by (-)Sch B pretreatment was associated with an increase in cellular reduced glutathione (GSH) level as well as the enhancement of γ-glutamylcysteine ligase (GCL) and glutathione reductase (GR) activity in PQ-challenged cells. Both GCL and GR inhibitors abrogated the cytoprotective effect of (-)Sch B in PQ-challenged cells. The biochemical mechanism underlying the GSH-enhancing effect of (-)Sch B was further investigated in PC12 cells subjected to an acute peroxide challenge. Although the initial GSH depletion induced by peroxide was reduced through GR-catalyzed regeneration of GSH in (-)Sch B-pretreated cells, the later enhanced GSH recovery was mainly mediated by GCL-catalyzed GSH synthesis. The results suggest that (-)Sch B treatment may increase the resistance of dopaminergic cells against PQ-induced oxidative stress through reducing the extent of oxidant-induced GSH depletion and enhancing the subsequent GSH recovery.     

Schisandrin B stereoisomers protect against hypoxia/reoxygenation-induced apoptosis and inhibit associated changes in Ca2+-induced mitochondrial permeability transition and mitochondrial membrane potential in H9c2 cardiomyocytes

The effects of schisandrin B stereoisomers, (+/-)gamma-schisandrin [(+/-)gamma-Sch] and (-)schisandrin B [(-)Sch B], on hypoxia/reoxygenation-induced apoptosis were investigated in H9c2 cardiomyocytes. Changes in cellular reduced glutathione (GSH) levels, Ca(2+)-induced mitochondrial permeability transition (MPT), and mitochondrial membrane potential (Deltapsi(m)) values, were examined in (+/-)gamma-Sch-pretreated and (-)Sch B-pretreated cells, without or with hypoxia/reoxygenation challenge. The (+/-)gamma-Sch and (-)Sch B (2.5-5.0 microM) pretreatments protected against hypoxia/reoxygenation-induced apoptosis of H9c2 cells in a concentration-dependent manner, with (-)Sch B being more potent. The degrees of protection decreased, however, at the higher drug concentrations of 7.5 microM in both (+/-)gamma-Sch-pretreated and (-)Sch B-pretreated cells. The anti-apoptotic effects of the drugs were further evidenced by the suppression of hypoxia/reoxygenation-induced mitochondrial cytochrome c release and the subsequent cleavage of caspase 3 and poly-ADP-ribose polymerase after (-)Sch B pretreatment. Both (+/-)gamma-Sch and (-)Sch B pretreatments increased GSH levels in H9c2 cells, with (-)Sch B being more potent. Hypoxia/reoxygenation challenge caused a depletion in cellular GSH and the cytoprotection afforded by (+/-)gamma-Sch/(-)Sch B was associated with enhancement of cellular GSH in H9c2 cells, as compared to the drug-unpretreated control. Whereas hypoxia/reoxygenation challenge increased the extent of Ca(2+)-induced MPT pore opening and decreased Deltapsi(m) in H9c2 cardiomyocytes, cytoprotection against hypoxia/reoxygenation-induced apoptosis afforded by (+/-)gamma-Sch/(-)Sch B pretreatments was associated with a decreased sensitivity to Ca(2+)-induced MPT and an increased Deltapsi(m) in both unchallenged and challenged cells, as compared to the respective drug-unpretreated controls. The degrees of protection against apoptosis correlated negatively with the extents of Ca(2+)-induced MPT (r=-0.615, P<0.01) and positively with the values of Deltapsi(m) (r=0.703, P<0.01) in (+/-)gamma-Sch/(-)Sch B-pretreated and hypoxia/reoxygenation challenged cells. The results indicate that (+/-)gamma-Sch/(-)Sch B pretreatment protected against hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes and that the cytoprotection afforded by (+/-)gamma-Sch/(-)Sch B may at least in part be mediated by a decrease in cellular sensitivity to Ca(2+)-induced MPT, which may in turn result from enhancement of cellular GSH levels by drug pretreatments.     

Beneficial effect of (-)schisandrin B against 3-nitropropionic acid-induced cell death in PC12 cells

Huntington's disease (HD) is characterized by the dysfunction of mitochondrial energy metabolism, which is associated with the functional impairment of succinate dehydrogenase (mitochondrial complex II), and pyruvate dehydrogenase (PDH). Treatment with 3-nitropropionic acid (3-NP), a potent irreversible inhibitor of succinate dehydrogenase, replicates most of the pathophysiological features of HD. In the present study, we investigated the effect of (-)schisandrin B [(-)Sch B, a potent enantiomer of schisandrin B] on 3-NP-induced cell injury in rat differentiated neuronal PC12 cells. The 3-NP caused cell necrosis, as assessed by lactate dehydrogenase (LDH) leakage, and mitochondrion-dependent cell apoptosis, as assessed by caspase-3 and caspase-9 activation, in differentiated PC12 cells. The cytotoxicity induced by 3-NP was associated with a depletion of cellular reduced glutathione (GSH) as well as the activation of redox-sensitive c-Jun N-terminal kinase (JNK) pathway and the inhibition of PDH. (-)Sch B pretreatment (5 and 15 μM) significantly reduced the extent of necrotic and apoptotic cell death in 3-NP-challenged cells. The cytoprotection afforded by (-)Sch B pretreatment was associated with the attenuation of 3-NP-induced GSH depletion as well as JNK activation and PDH inhibition. (-)Sch B pretreatment enhanced cellular glutathione redox status and ameliorated the 3-NP-induced cellular energy crisis, presumably by suppressing the activated JNK-mediated PDH inhibition, thereby protecting against necrotic and apoptotic cell death in differentiated PC12 cells.     

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.     

Species-related variations in tissue antioxidant status--II. Differences in susceptibility to oxidative challenge.

1. In order to investigate possible species-related variations in antioxidant capacity, the susceptibility of red cells from various species (e.g. rat, rabbit, pig and quail) to depletion of glutathione (GSH) and formation of malondialdehyde (MDA), an indirect measure of lipid peroxidation, following in vitro oxidative challenge with t-butylhydroperoxide (tBHP) has been examined. 2. Marked differences in sensitivity were found, although the relative order of susceptibilities varied depending on the index of oxidation used. 3. For example, pig erythrocytes showed the highest sensitivity to depletion of GSH but the greatest resistance to tBHP-induced MDA formation. 4. Red cell susceptibility to oxidative stress under the experimental conditions used was not predictable from basal levels of GSH or from the activities of antioxidant enzymes, suggesting a prominent role of non-enzymatic antioxidants. 5. Species-dependent differences in antioxidant capacity were also found to extend to myocardial tissue homogenates and some degree of parallelism was noted with tBHP-induced MDA formation in red cells of the same species. 6. Thus, the relative resistance of both tissues from pig contrasted with the high susceptibility of red cells and myocardium from rat and quail. 7. This parallelism allows the suggestion that the functional consequences of antioxidant interventions might be discernible from measurements involving red cells. 8. Our findings may have potentially important implications in the interpretation and comparison of data obtained with experimental models of disease states in which oxidative processes are implicated when differences in species are involved.     

Thiol redox modulation of doxorubicin mediated cytotoxicity in cultured AIDS-related Kaposi's sarcoma cells

The chemotherapeutic, doxorubicin, is currently used empirically in the treatment of AIDS- related Kaposi's sarcoma (AIDS-KS). Although often employed in a chemotherapeutic cocktail (doxorubicin, bleomycin, vincristine) single-agent therapy has recently been attempted with liposome encapsulated doxorubicin. Although doxorubicin's mechanism of action against AIDS-KS is unknown, we hypothesized that doxorubicin's ability to undergo redox cycling is associated with its clinical efficacy. The current study was conducted to investigate the effects of doxorubicin on selected xenobiotic-associated biochemical responses of three cellular populations: KS lesional cells, nonlesional cells from the KS donors, and fibroblasts obtained from HIV- aged matched men. Our results show that during doxorubicin challenge, there are strong positive correlations between cellular glutathione (GSH) levels and viability (r = 0.94), NADPH levels and viability (r = 0.93), and GSH and NADPH levels (r = 0.93), and demonstrate that as a consequence of their abilities to maintain cellular thiol redox pools HIV- donor cells are significantly less susceptible to doxorubicin's cytotoxic effects relative to AIDS-KS cells. Additional studies further supported the contribution of reduced thiols in mediating doxorubicin tolerance. While pretreatment with the GSH precursor, N-acetylcysteine was cytoprotective for all cell groups during doxorubicin challenge, GSH depletion markedly enhanced doxorubicin's cytotoxic effects. Studies to investigate the effects of a hydroxyl scavenger and iron chelator during doxorubicin challenge showed moderate cytoprotection in the AIDS-KS cells but deleterious effects in the HIV control cells. Inactivation of the longer lived membrane generated ROI in the cytoprotective deficient AIDS-KS cells, as well as an impairment of endogenous defenses in the HIV- donor control cells, may account for these scavenger and chelator associated findings. In summary, our findings show that doxorubicin mediates, at least in part, its AIDS-KS cellular cytotoxic effects by a redox related mechanism, and provides a biochemical rationale for doxorubicin's clinical efficacy in AIDS-KS treatment.     

Schisandrin B elicits a glutathione antioxidant response and protects against apoptosis via the redox-sensitive ERK/Nrf2 pathway in AML12 hepatocytes

Abstract This study examined the effects of (-)schisandrin B [(-)Sch B] on MAPK and Nrf2 activation and the subsequent induction of glutathione antioxidant response and cytoprotection against apoptosis in AML12 hepatocytes. Pharmacological tools, such as cytochrome P-450 (CYP) inhibitor, antioxidant, MAPK inhibitors and Nrf2 RNAi, were used to delineate the signalling pathway. (-)Sch B caused a time-dependent activation of MAPK in AML12 cells, particularly the ERK1/2. The MAPK activation was followed by an enhancement in Nrf2 nuclear translocation and the eliciting of a glutathione antioxidant response. Reactive oxygen species arising from a CYP-catalysed reaction with (-)Sch B seemed to be causally related to the activation of MAPK and Nrf2. ERK inhibition by U0126 or Nrf2 suppression by Nrf2 RNAi transfection almost completely abrogated the cytoprotection against menadione-induced apoptosis in (-)Sch B-pre-treated cells. (-)Sch B pre-treatment potentiated the menadione-induced ERK activation, whereas both p38 and JNK activations were suppressed. Under the condition of ERK inhibition, Sch B treatment did not protect against carbon tetrachloride-hepatotoxicity in an in vivo mouse model. In conclusion, (-)Sch B triggers a redox-sensitive ERK/Nrf2 signalling, which then elicits a cellular glutathione antioxidant response and protects against oxidant-induced apoptosis in AML12 cells.     

Protective effect of S-adenosylmethionine on cellular and mitochondrial membranes of rat hepatocytes against tert-butylhydroperoxide-induced injury in primary culture

Accumulating evidence that administration of S-adenosylmethionine (SAMe) protects hepatocytes against oxidative stress-mediated injury led us to evaluate the effect of SAMe on hepatocyte injury induced in culture by oxidant substance tert-butylhydroperoxide (1.5 mM tBHP) with regard to prevent mitochondrial injury. The pretreatment of hepatocyte culture with SAMe in doses of 0.25, 0.5, 1, 2.5, 5, 10, 25 and 50 mg/l for 30 min prevented the release of LDH from cells incubated for 30 min with tBHP in a dose dependent manner. The inhibitory effect of SAMe on lipid peroxidation paralleled the effect on cell viability. SAMe also moderated the decrease of the mitochondrial membrane potential induced by tBHP. Our results indicate that the inhibition of lipid peroxidation by SAMe can contribute to the prevention of disruption of both cellular and mitochondrial membranes. While the protective effect of SAMe against tBHP-induced GSH depletion was not confirmed, probably the most potent effect of SAMe on membranes by phospholipid methylation should be verified.     

Protection of HepG2 cells against acrolein toxicity by 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide via glutathione-mediated mechanism

Acrolein is an environmental toxicant, mainly found in smoke released from incomplete combustion of organic matter. Several studies showed that exposure to acrolein can lead to liver damage. The mechanisms involved in acrolein-induced hepatocellular toxicity, however, are not completely understood. This study examined the cytotoxic mechanisms of acrolein on HepG2 cells. Acrolein at pathophysiological concentrations was shown to cause apoptotic cell death and an increase in levels of protein carbonyl and thiobarbituric acid reactive acid substances. Acrolein also rapidly depleted intracellular glutathione (GSH), GSH-linked glutathione-S-transferases, and aldose reductase, three critical cellular defenses that detoxify reactive aldehydes. Results further showed that depletion of cellular GSH by acrolein preceded the loss of cell viability. To further determine the role of cellular GSH in acrolein-mediated cytotoxicity, buthionine sulfoximine (BSO) was used to inhibit cellular GSH biosynthesis. It was observed that depletion of cellular GSH by BSO led to a marked potentiation of acrolein-mediated cytotoxicity in HepG2 cells. To further assess the contribution of these events to acrolein-induced cytotoxicity, triterpenoid compound 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) was used for induction of GSH. Induction of GSH by CDDO-Im afforded cytoprotection against acrolein toxicity in HepG2 cells. Furthermore, BSO significantly inhibited CDDO-Im-mediated induction in cellular GSH levels and also reversed cytoprotective effects of CDDO-Im in HepG2 cells. These results suggest that GSH is a predominant mechanism underlying acrolein-induced cytotoxicity as well as CDDO-Im-mediated cytoprotection. This study may provide understanding on the molecular action of acrolein which may be important to develop novel strategies for the prevention of acrolein-mediated toxicity.     

Glutathione depletion in a liver microsomal assay as an in vitro biomarker for reactive metabolite formation

Glutathione (GSH) plays a major role in cytoprotection, acting as a nucleophile trap for reactive species derived from xenobiotics. This has led to the development of an assay for the detection of reactive species generated by liver microsomal metabolism of xenobiotics. This assay has been used extensively to study reactive metabolites which initiate toxicity through a direct (non-immunological) mechanism, but there are few data on its ability to detect reactive metabolites that initiate toxicity through neo-antigen formation, or to detect xenobiotics that cause GSH loss by oxidation mediated by a redox cycling process. Accordingly, the ability of rat and human liver microsomes to metabolize xenobiotics to GSH-depleting metabolites has been investigated further. Of the five neo-antigen-forming xenobiotics tested, four (amodiaquine, phenobarbitone, procainamide, and sulphanilamide) displayed GSH reactivity that was either dependent or independent (amodiaquine) on metabolism. The other neo-antigen-forming xenobiotic (carbamazepine) was inactive in all microsomal samples tested. Four quinones believed to exert toxcity through arylation (1,4-benzoquinone) and/or redox cycling (duroquinone, menadione, mitomycin c) displayed GSH reactivity, as did nitrofurantoin and diquat, two other redox cycling xenobiotics. Induction of the mixed function oxidase system with Aroclor afforded little advantage when using rat liver microsomes, whilst there was considerable inter-individual variation in the ability of human liver microsomes to mediate metabolism-dependent GSH depletion. It is concluded that the liver microsome GSH depletion assay may be of general utility as a screen for a number of xenobiotic-derived reactive species.     

Glutathione depletion in a liver microsomal assay as an in vitro biomarker for reactive metabolite formation

Glutathione (GSH) plays a major role in cytoprotection, acting as a nucleophile trap for reactive species derived from xenobiotics. This has led to the development of an assay for the detection of reactive species generated by liver microsomal metabolism of xenobiotics. This assay has been used extensively to study reactive metabolites which initiate toxicity through a direct (non-immunological) mechanism, but there are few data on its ability to detect reactive metabolites that initiate toxicity through neo-antigen formation, or to detect xenobiotics that cause GSH loss by oxidation mediated by a redox cycling process. Accordingly, the ability of rat and human liver microsomes to metabolize xenobiotics to GSH-depleting metabolites has been investigated further. Of the five neo-antigen-forming xenobiotics tested, four (amodiaquine, phenobarbitone, procainamide, and sulphanilamide) displayed GSH reactivity that was either dependent or independent (amodiaquine) on metabolism. The other neo-antigen-forming xenobiotic (carbamazepine) was inactive in all microsomal samples tested. Four quinones believed to exert toxcity through arylation (1,4-benzoquinone) and/or redox cycling (duroquinone, menadione, mitomycin c) displayed GSH reactivity, as did nitrofurantoin and diquat, two other redox cycling xenobiotics. Induction of the mixed function oxidase system with Aroclor afforded little advantage when using rat liver microsomes, whilst there was considerable inter-individual variation in the ability of human liver microsomes to mediate metabolism-dependent GSH depletion. It is concluded that the liver microsome GSH depletion assay may be of general utility as a screen for a number of xenobiotic-derived reactive species.     

Alterations in susceptibility to carbon tetrachloride toxicity and hepatic antioxidant/detoxification system in streptozetocin-induced short-term diabetic rats: Effects of insulin and Schisandrin B treatment

The streptozotocin-induced short-term (2 week) diabetic rats showed an increase in susceptibility to carbon tetrachloride (CCl4)-induced hepatocellular damage. This diabetes-induced change was associated with a marked impairment in the hepatic glutathione antioxidant/detoxification response to CCl₄ challenge, as indicated by the abrogation of the increases in hepatic reduced glutathione (GSH) level, glucose-6-phosphate dehydrogenase and microsomal glutathione S-transferases (GST) activities upon challenge with increasing doses of CCl₄. While the hepatic GSH level was increased in diabetic rats, the hepatic mitochondrial GSH level and Se-glutathione peroxidase activity were significantly reduced. Insulin treatment could reverse most of the biochemical alterations induced by diabetes. Both insulin and schisandrin B (Sch B) pretreatments protected against the CCl₄ hepatotoxicity in diabetic rats. The hepatoprotection was associated with improvement in hepatic glutathione redox status in both cytosolic and mitochondrial compartments, as well as the increases in hepatic ascorbic acid level and microsomal GST activity. The ensemble of results suggests that the diabetes-induced impairment in hepatic mitochondrial glutathione redox status may at least in part be attributed to the enhanced susceptibility to CCl4 hepatotoxicity. Sch B may be a useful hepatoprotective agent against xenobiotics-induced toxicity under the diabetic conditions. (Mol Cell Biochem 175: 225–232, 1997)     

Hepatoprotective action of schisandrin B against carbon tetrachloride toxicity was mediated by both enhancement of mitochondrial glutathione status and induction of heat shock proteins in mice

In the present study, we investigated the differential role of the mitochondrial glutathione status and induction of heat shock proteins (HSPs) 25/70 in protecting against carbon tetrachloride (CCl_4) hepatotoxicity in schisandrin B (Sch B)-pretreated mice. The time-course of Sch B-induced changes in these hepatic parameters were examined. Dimethyl diphenyl bicarboxylate (DDB), a non-hepatoprotective analog of Sch B, was studied for comparison. Sch B treatment (2 mmol/kg) produced maximal enhancement in hepatic mitochondrial glutathione status as well as increases in hepatic HSP 25/70 levels at 24 h post-dosing. The stimulatory effect of Sch B then gradually subsided, but the activities of hepatic mitochondrial glutathione reductase (GR) and glutathione S-transferases (GST) as well as the level of HSP 25 remained relatively high even at 72 h post-dosing. CCl_4 challenge caused significant impairment in mitochondrial glutathione status and a decrease in HSP 70 level, but the HSP 25 level was significantly elevated. While the extent of hepatoprotection afforded by Sch B pretreatment against CCl_4 was found to inversely correlate with the time elapsed after the dosing, the protective effect was associated with the ability of Sch B to maintain the mitochondrial glutathione status and/or induce further production of HSP 25 in CCl_4-intoxicated condition. On the other hand, DDB treatment (2 mmol/kg), which did not increase mitochondrial GSH level and GST activity or induce further production of HSP 25 after CCl_4 challenge, could not protect against CCl_4 toxicity. The results suggest that the enhancement of mitochondrial glutathione status and induction of HSP 25/70 may contribute independently to the hepatoprotection afforded by Sch B pretreatment.     

Schisandrin B elicits a glutathione antioxidant response and protects against apoptosis via the redox-sensitive ERK/Nrf2 pathway in AML12 hepatocytes

Abstract

This study examined the effects of (?)schisandrin B [(?)Sch B] on MAPK and Nrf2 activation and the subsequent induction of glutathione antioxidant response and cytoprotection against apoptosis in AML12 hepatocytes. Pharmacological tools, such as cytochrome P-450 (CYP) inhibitor, antioxidant, MAPK inhibitors and Nrf2 RNAi, were used to delineate the signalling pathway. (?)Sch B caused a time-dependent activation of MAPK in AML12 cells, particularly the ERK1/2. The MAPK activation was followed by an enhancement in Nrf2 nuclear translocation and the eliciting of a glutathione antioxidant response. Reactive oxygen species arising from a CYP-catalysed reaction with (?)Sch B seemed to be causally related to the activation of MAPK and Nrf2. ERK inhibition by U0126 or Nrf2 suppression by Nrf2 RNAi transfection almost completely abrogated the cytoprotection against menadione-induced apoptosis in (?)Sch B-pre-treated cells. (?)Sch B pre-treatment potentiated the menadione-induced ERK activation, whereas both p38 and JNK activations were suppressed. Under the condition of ERK inhibition, Sch B treatment did not protect against carbon tetrachloride-hepatotoxicity in an in vivo mouse model. In conclusion, (?)Sch B triggers a redox-sensitive ERK/Nrf2 signalling, which then elicits a cellular glutathione antioxidant response and protects against oxidant-induced apoptosis in AML12 cells.     

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.     

Decreased susceptibility of differentiated PC12 cells to oxidative challenge: relationship to cellular redox and expression of apoptotic protease activator factor-1

We previously showed that tert-butyl hydroperoxide (TBH) induced apoptosis in na?ve rat pheochromocytoma (nPC12) cells that correlated with cellular redox imbalance and mitochondrial apoptotic signaling. In this study, we tested the hypothesis that differentiation of nPC12 cells results in altered susceptibility to TBH utilizing a model of differentiated PC12 (dPC12) cells induced by nerve growth factor. TBH (100 microM) induced dPC12 apoptosis (12% at 24 h) at levels lower than na?ve cells (35%). This resistance was associated with elevated GSH, NADPH (reduced nicotinamide adenine dinucleotide phosphate), TBH metabolism, redox enzyme activities, reduced cellular GSH/GSSG (glutathione disulfide) status and preservation of mitochondrial membrane potential. Altering cellular GSH with ethacrynic acid or N-acetylcysteine, respectively, exacerbated or protected against dPC12 apoptosis. dPC12 apoptosis was mediated by caspase-9 and -3 activation and apoptosis protease activator protein-1 (Apaf-1) expression. These results show that nPC12 transition to dPC12 cells afforded protection against oxidative challenge due to maintenance of reduced GSH/GSSG and decreased Apaf-1 expression.     

Cytochrome P450-catalysed reactive oxygen species production mediates the (-)schisandrin B-induced glutathione and heat shock responses in AML12 hepatocytes

Sch B (schisandrin B), the most abundant dibenzocyclooctadiene lignan in Fructus schisandrae, can induce glutathione antioxidant and heat shock responses, as well as protect against oxidant-induced injury in various tissues, including the liver in rodents and AML12 (alpha mouse liver 12) hepatocytes. (-)Sch B is the most potent stereoisomer of Sch B in its cytoprotective action on AML12 hepatocytes. To define the role of ROS (reactive oxygen species) arising from CYP (cytochrome P450)-catalysed metabolism of (-)Sch B in triggering glutathione antioxidant and heat shock responses, the effects of a CYP inhibitor [ABT (aminobenzotriazole)] and antioxidants [DMTU (dimethylthiouracil) and TRX (trolox)] on (-)Sch B-induced ROS production and associated increases in cellular GSH level, as well as Hsp25/70 (heat-shock protein 25/70) production, were investigated in AML12 hepatocytes. The results indicated that (-)Sch B causes a dose dependent and sustained increase in ROS production over 6 h in AML12 hepatocytes, which was completely suppressed by pre-/co-treatment with ABT or DTMU/TRX. Incubation with (-)Sch B for 6 h caused optimal and dose-dependent increases in cellular GSH level and Hsp25/70 production at 16 h post-drug exposure in AML12 hepatocytes. These cellular responses were associated with protection against menadione-induced apoptosis. Pre-/co-treatment with ABT or antioxidants completely abrogated the (-)Sch B-induced glutathione antioxidant and heat shock responses, as well as protection against menadione-induced apoptosis. Experimental evidence obtained thus far supports the causal role of ROS arising from the CYP-catalysed metabolism of (-)Sch B in eliciting glutathione antioxidant and heat shock responses in AML12 hepatocytes.     

Schisandrin B elicits a glutathione antioxidant response and protects against apoptosis via the redox-sensitive ERK/Nrf2 pathway in H9c2 cells

This study investigated the signal transduction pathway involved in the cytoprotective action of (-)schisandrin B [(-)Sch B, a stereoisomer of Sch B]. Using H9c2 cells, the authors examined the effects of (-)Sch B on MAPK and Nrf2 activation, as well as the subsequent eliciting of glutathione response and protection against apoptosis. Pharmacological tools, such as cytochrome P-450 (CYP) inhibitor, antioxidant, MAPK inhibitor, and Nrf2 RNAi, were used to delineate the signaling pathway. (-)Sch B caused a time-dependent activation of MAPK in H9c2 cells, with the degree of ERK activation being much larger than that of p38 or JNK. The MAPK activation was followed by an increase in the level of nuclear Nrf2, an indirect measure of Nrf2 activation, and the eliciting of a glutathione antioxidant response. The activation of MAPK and Nrf2 seemed to involve oxidants generated from a CYP-catalyzed reaction with (-)Sch B. Both ERK inhibition by U0126 and Nrf2 suppression by Nrf2 RNAi transfection largely abolished the cytoprotection against hypoxia/reoxygenation-induced apoptosis in (-)Sch B-pretreated cells. (-)Sch B pretreatment potentiated the reoxygenation-induced ERK activation, whereas both p38 and JNK activations were suppressed. Under the condition of ERK inhibition, Sch B treatment did not protect against ischemia/reperfusion injury in an ex vivo rat heart model. The results indicate that (-)Sch B triggers a redox-sensitive ERK/Nrf2 signaling, which then elicits a cellular glutathione antioxidant response and protects against hypoxia/reoxygenation-induced apoptosis in H9c2 cells. The ERK-mediated signaling is also likely involved in the cardioprotection afforded by Sch B in vivo.     

Vitamin E protection against chemical-induced cell injury. I. Maintenance of cellular protein thiols as a cytoprotective mechanism

Vitamin E protection against chemical-induced toxicity to isolated hepatocytes was examined during an imbalance in the thiol redox system. Intracellular reduced glutathione (GSH) was depleted by two chemicals of distinct mechanisms of action: adriamycin, a cancer chemotherapeutic agent that undergoes redox cycling, producing reactive oxygen species that consume GSH, and ethacrynic acid, a direct depleter of GSH. The experimental system used both nonstressed vitamin E-adequate isolated rat hepatocytes and compromised hepatocytes subjected to physiologically induced stress, generated by incubation in calcium-free medium. At doses whereby intracellular GSH was near total depletion, cell injury induced by either chemical was found to follow the depletion of cellular alpha-tocopherol, regardless of the status of the GSH redox system. Changes in protein thiol contents of the cells closely paralleled the changes in alpha-tocopherol contents throughout the incubation period. Supplementation of the calcium-depleted hepatocytes with alpha-tocopheryl succinate (25 microM) markedly elevated their alpha-tocopherol content and prevented the toxicities of both drugs. The prevention of cell injury and the elevation in alpha-tocopherol contents were both associated with a prevention of the loss in cellular protein thiols in the near total absence of intracellular GSH. The mechanism of protection by vitamin E against chemical-induced toxicity to hepatocytes may therefore be an alpha-tocopherol-dependent maintenance of cellular protein thiols.