Glutathione Ethyl Ester Research Articles

Regulation of endothelial glutathione by ICAM-1 governs VEGF-A-mediated eNOS activity and angiogenesis

Previous studies suggest that inflammatory cell adhesion molecules may modulate endothelial cell migration and angiogenesis through unknown mechanisms. Using a combination of in vitro and in vivo approaches, herein we reveal a novel redox-sensitive mechanism by which ICAM-1 modulates endothelial GSH that controls VEGF-A-induced eNOS activity, endothelial chemotaxis, and angiogenesis. In vivo disk angiogenesis assays showed attenuated VEGF-A-mediated angiogenesis in ICAM-1 −/− mice. Moreover, VEGF-A-dependent chemotaxis, eNOS phosphorylation, and nitric oxide production were impaired in ICAM-1 −/− mouse aortic endothelial cells (MAEC) compared to WT MAEC. Decreasing intracellular GSH in ICAM-1 −/− MAEC to levels observed in WT MAEC with 150 μM buthionine sulfoximine restored VEGF-A responses. Conversely, GSH supplementation of WT MAEC with 5 mM glutathione ethyl ester mimicked defects observed in ICAM-1 −/− cells. Deficient angiogenic responses in ICAM-1 −/− cells were associated with increased expression of the lipid phosphatase PTEN, consistent with antagonism of signaling pathways leading to eNOS activation. PTEN expression was also sensitive to GSH status, decreasing or increasing in proportion to intracellular GSH concentrations. These data suggest a novel role for ICAM-1 in modulating VEGF-A-induced angiogenesis and eNOS activity through regulation of PTEN expression via modulation of intracellular GSH status.

Role of intracellular calcium and phospholipase A2 in arachidonic acid-induced toxicity in liver cells overexpressing CYP2E1

Liver cells (HepG2 and primary hepatocytes) overexpressing CYP2E1 and exposed to arachidonic acid (AA) were previously shown to lose viability together with enhanced lipid peroxidation. These events were blocked in cells pre-incubated with antioxidants (α-tocopherol, glutathione ethyl ester), or in HepG2 cells not expressing CYP2E1. The goal of the current study was to evaluate the role of calcium and calcium-activated hydrolases in these CYP2E1–AA interactions. CYP2E1-expressing HepG2 cells treated with AA showed an early increase in cytosolic calcium and partial depletion of ionomycin-sensitive calcium stores. These changes in calcium were blocked by α-tocopherol. AA activated phospholipase A2 (PLA2) in CYP2E1-expressing liver cells, and this was inhibited by PLA2 inhibitors or α-tocopherol. PLA2 inhibitors prevented the cell death caused by AA, without affecting CYP2E1 activity or lipid peroxidation. AA toxicity and PLA2 activation were inhibited in calcium-depleted cells, but not by removal of extracellular calcium alone. Removal of extracellular calcium inhibited the early increase in cytosolic calcium caused by AA. CYP2E1 overexpressing HepG2 cells exposed to AA showed a decrease in mitochondrial membrane potential, which was prevented by the PLA2 inhibitors. These results suggest that AA-induced toxicity to CYPE1-expressing cells: (i) is associated with release of Ca 2+ from intracellular stores that depends mainly on oxidative membrane damage; (ii) is associated with activation of PLA2 that depends on intracellular calcium and lipid peroxidation; (iii) does not depend on increased influx of extracellular calcium, and (iv) depends on the effect of converging events (lipid peroxidation, intracellular calcium, activation of PLA2) on mitochondria to induce bioenergetic failure and necrosis. These interactions may play a role in alcohol liver toxicity, which requires polyunsaturated fatty acids, and involves induction of CYP2E1.

Cellular thiol status-dependent inhibition of tumor cell growth via modulation of p27kip1 translocation and retinoblastoma protein phosphorylation by 1′-acetoxychavicol acetate

1'-Acetoxychavicol acetate (ACA) has been shown to inhibit tumor cell growth, but there is limited information on its effects on cell signaling and the cell cycle control pathway. In this study, we sought to determine how ACA alters cell cycle and its related control factors in its growth inhibitory effect in Ehrlich ascites tumor cells (EATC). ACA caused an accumulation of cells in the G1 phase and an inhibition of DNA synthesis, which were reversed by supplementation with N-acetylcysteine (NAC) or glutathione ethyl ester (GEE). Furthermore, ACA decreased hyperphosphorylated Rb levels and increased hypophosphorylated Rb levels. NAC and GEE also abolished the decease in Rb phosphorylation by ACA. As Rb phosphorylation is regulated by G1 cyclin dependent kinase and CDK inhibitor p27(kip1), which is an important regulator of the mammalian cell cycle, we estimated the amount of p27(kip1) levels by western blotting. Treatment with ACA had virtually no effect on the amount of p27(kip1) levels, but caused a decrease in phosphorylated p27(kip1) and an increase in unphosphorylated p27(kip1) as well as an increase in the nuclear localization of p27(kip1). These events were abolished in the presence of NAC or GEE. These results suggest that in EATC, cell growth inhibition elicited by ACA involves decreases in Rb and p27(kip1) phosphorylation and an increase in nuclear localization of p27(kip1), and these events are dependent on the cellular thiol status.

Deferoxamine Reduces Tissue Damage During Endotoxin-Induced Mastitis in Dairy Cows

The protective effects of 3 antioxidants on polymorphonuclear neutrophil-induced damage to mammary cells were evaluated in vivo using an endotoxin-induced mastitis model. Fifteen healthy, midlactation cows with no history of clinical Escherichia coli mastitis were randomly assigned to 1 of the 3 treatment groups corresponding to each modulator to be evaluated, that is, deferoxamine, catechin, and glutathione ethyl ester. Each cow had 1 quarter infused with saline and 1 quarter infused with the selected modulator; a third quarter was infused with lipopolysaccharides (LPS), whereas the fourth quarter received a combination of LPS and the modulator. Infusion of LPS caused acute mastitis as determined by visual observations and by large increases in milk somatic cell count, BSA, and proteolytic activity. These parameters were not affected by antioxidant administration. The extent of cell damage was evaluated by measuring milk levels of lactate dehydrogenase and N-acetyl-β-D-glucosaminidase activity. Levels of these parameters were several times higher after LPS administration. Intramammary infusions of catechin or glutathione ethyl ester did not exert any protective effect, whereas infusion of deferoxamine, a chelator of iron, decreased milk lactate dehydrogenase and NA-Gase activity, suggesting a protective effect against neutrophil-induced damage. The protective effect of deferoxamine was also evidenced by a lower milk level of haptoglobin. The proteolytic activity of mastitic milk was not influenced by the presence of deferoxamine. Overall, our results suggest that local infusion of deferoxamine may be an effective tool to protect mammary tissue against neutrophil-induced oxidative stress during bovine mastitis.

Glutathione is required for efficient production of infectious picornavirus virions

Glutathione is an intracellular reducing agent that helps maintain the redox potential of the cell and is important for immune function. The drug l-buthionine sulfoximine (BSO) selectively inhibits glutathione synthesis. Glutathione has been reported to block replication of HIV, HSV-1, and influenza virus, whereas cells treated with BSO exhibit increased replication of Sendai virus. Pre-treatment of HeLa cell monolayers with BSO inhibited replication of CVB3, CVB4, and HRV14 with viral titers reduced by approximately 6, 5, and 3 log 10, respectively. The addition of glutathione ethyl ester, but not dithiothreitol or 2-mercaptoethanol, to the culture medium reversed the inhibitory effect of BSO. Viral RNA and protein synthesis were not inhibited by BSO treatment. Fractionation of lysates from CVB3-infected BSO-treated cells on cesium chloride and sucrose gradients revealed that empty capsids but not mature virions were being produced. The levels of the 5S and 14S assembly intermediates, however, were not affected by BSO treatment. These results demonstrate that glutathione is important for production of mature infectious picornavirus virions.

Investigation of bioactivation and toxicity of styrene in CYP2E1 transgenic cells

Styrene has been found to be toxic to the respiratory system, and the toxicity of styrene is metabolism-dependent. CYP2E1 is suggested to be one of the cytochrome P450 enzymes responsible for the bioactivation of styrene. Our work focused on the roles of CYP2E1 and epoxide, a metabolite of styrene epoxidation, in the cytotoxicity of styrene. Styrene was found to be more toxic to h2E1 cells than to the wild type, while there was no difference found when styrene oxide was administered. Both soluble and microsomal epoxide hydrolase inhibitors dramatically enhanced styrene toxicity. Glutathione and glutathione ethyl ester showed protection against styrene cytotoxicity. Cytotoxicity of a selection of styrene analogues, such as ethylbenzene, vinylcyclohexane, and ethylcyclohexane, was assessed to determine if unsaturation is required for styrene toxicity. Ethylbenzene and vinylcyclohexane were found to be as toxic as styrene to h2E1 cells, whereas little toxicity of ethylcyclohexane to h2E1 cells was observed. This indicates the importance of vinyl group of styrene in its cytotoxicity, but saturation of the vinyl group does not necessarily eliminate styrene toxicity. An N-acetylcysteine conjugate derived from styrene oxide was identified by LC/MS/MS in the sample obtained from the incubation of h2E1 cell lysate with styrene in the presence of N-acetylcysteine. Formation of the N-acetylcysteine conjugate was found to be NADPH-dependent. These studies provided strong evidence in support of toxic role of styrene epoxide metabolite in styrene toxicity.

Chemical and Cytotoxic Constituents from the Leaves of Cinnamomum kotoense

Three new butanolides, kotomolide A (1), isokotomolide A (2), and kotomolide B (3), and a new secobutanolide, secokotomolide A (4), along with 21 known compounds were isolated from the leaves of Cinnamomum kotoense. Their structures were determined by spectroscopic analyses. Compound 4 was found to induce significant cell death in the human HeLa cell line. Apoptotic-related DNA damage can be positively related to the dose of compound 4. The DNA damage was measured by the percentage of subG1 (24 h after the treatment of compound 4) as determined by cell cycle analysis and TUNEL assay. Treatment with 4 significantly increased intracellular H2O2 and/or peroxide, nitric oxide (NO) at 1, 3, and 24 h. Our results also showed that compound 4 induced (a) noticeable reduction of mitochondrial transmembrane potential (DeltaPsi(m)), (b) activation of caspase 3/7, and (c) up-regulation of the p53 expression. Compound 4-induced DNA damage was found to markedly decrease when the cells were pretreated with an intracellular glutathione supplement (glutathione ethyl ester). These results suggest that an increase of H2O2 and/or peroxide by compound 4 is the initial apoptotic event. The intracellular GSH depletion is a critical event in compound 4-induced apoptosis in HeLa cells.

Transcription Factor Nrf2 Protects HepG2 Cells against CYP2E1 plus Arachidonic Acid-dependent Toxicity

Nrf2 is a transcription factor that regulates important antioxidant and phase II detoxification genes. Arachidonic acid (AA) causes CYP2E1-dependent toxicity in HepG2 cells. The ability of Nrf2 to protect against CYP2E1-dependent AA toxicity and its possible mechanism were evaluated. AA activates Nrf2 in CYP2E1-expressing HepG2 cells (E47 cells), increasing Nrf2 protein and mRNA levels, Nrf2 nuclear translocation, and Nrf2-ARE binding activity. These increases in Nrf2 are associated with elevated expression of Nrf2-regulated antioxidant genes. Overexpression of Nrf2 by transient transfection of plasmid Nrf2 confers resistance of E47 cells against AA toxicity. Blocking Nrf2 with small interfering RNA (siRNA)-Nrf2 potentiates the CYP2E1-dependent AA toxicity. This enhanced toxicity is accompanied by decreases of cellular GSH levels and increases in production of reactive oxygen species and lipid peroxidation. There is also a potentiation of mitochondrial damage in the presence of siRNA-Nrf2. The protective effects of Nrf2 against CYP2E1-dependent toxicity can be blocked by l-buthionine-(S,R)-sulfoximine, a specific inhibitor of glutamate-cysteine ligase, which is a rate-limiting enzyme in the synthesis of GSH and is regulated by Nrf2. Elevation of GSH by supplementing with glutathione ethyl ester can partially reverse the enhanced AA toxicity by siRNA-Nrf2. Moreover, in contrast to AA, l-buthionine-(S,R)-sulfoximine toxicity is not prevented by plasmid Nrf2 probably because protective GSH cannot be synthesized. Together, these results suggest that Nrf2, through up-regulation of glutamate-cysteine ligase and increase of GSH levels, protects against CYP2E1-dependent AA toxicity.

ICAM‐1 Governs Angiogenesis via Redox Regulation of PTEN Expression and eNOS activity

eNOS derived NO is critical in the angiogenic process, thus understanding the mechanisms regulating eNOS activity during angiogenesis are crucial. Here we present data showing that ICAM-1 is a novel effector of VEGF164 induced eNOS function, endothelial cell chemotaxis, and angiogenesis. VEGF164 mediated angiogenesis in WT and ICAM-1−/− mice was assessed in vivo using a Millipore disk assay. VEGF164 chemotaxis of mouse aortic endothelial cells (MAEC) was measured by transwell assay. eNOS activity was assessed using western blot analysis of phosphorylation at ser1177 and DAF-FM fluorescence. PTEN expression was assessed by western blot. VEGF164 mediated angiogenesis in vivo was significantly decreased in ICAM-1−/− mice compared to WT mice. ICAM-1−/− MAEC showed impaired VEGF164 mediated chemotaxis, eNOS phosphorylation, and NO production vs. WT MAEC. PTEN expression was significantly elevated in ICAM-1−/− MAEC which was controlled by intracellular glutathione levels, as 150 μM BSO treatment returned PTEN expression to WT levels and restored VEGF164 chemotaxis and eNOS phosphorylation. Likewise, 5 mM glutathione ethyl ester (GEE) supplementation of WT cells recapitulated ICAM-1−/− chemotactic defects, increased PTEN expression, and blocked eNOS phosphorylation. These data suggest a novel role for ICAM-1 in modulating VEGF164 induced angiogenesis and eNOS activity via regulation of intracellular glutathione levels which regulate PTEN expression.

Measurement of intracellular reactive oxygen species in mature single skeletal muscle fibres by dichlorofluorescein‐based fluorescence microscopy

Skeletal muscle fibres continually produce reactive oxygen species (ROS), which modulate several functions in muscle cells. The present study describes the validation of a novel technique to measure intracellular ROS in isolated mature mouse skeletal muscle fibres. Single muscle fibres were isolated from the Flexor Digitorum Brevis muscle of mice, cultured on collagen coated plates, loaded with 17.5 μM 2′, 7′-dichlorodihydrofluorescein-diacetate and measurements in gray scale were taken by fluorescence microscopy every 15 minutes over 45 minutes. Experimental conditions: (i) Addition of 1 μM H2O2 to the extracellular medium induced a 48% increase in intracellular ROS activity. However, this effect was not observed when fibres had been previously treated with 5 mM glutathione ethyl ester (GSHEE). (ii) Contractile activity of isolated fibres, induced by electrical stimulation, caused a moderate increase of 14% in intracellular ROS activity. (iii) Fibres that had been incubated with 5 mM GSHEE for 4 hours did not show any significant increase of intracellular ROS activity after contractile activity. These data confirm that dichlorofluorescein-based fluorescence microscopy is able to detect small changes of intracellular ROS activity. In addition, contractile activity in muscle fibres generated a net increase of intracellular ROS activity, which was abolished when the intracellular antioxidants were enhanced by glutathione loading. This work was supported by The Wellcome Trust. UK.

Glutathione-Mediated Delivery and Release Using Monolayer Protected Nanoparticle Carriers

We demonstrate here the effective delivery of a dye payload into cells using 2-nm core gold nanoparticles, with release occurring via place exchange of glutathione onto the particle surface. In vitro experiments demonstrate effective release of drug analogues upon addition of glutathione. Cell culture experiments show rapid uptake of nanoparticle and effective release of payload. The role of glutathione in the release process was demonstrated through improved payload release upon transient increase in glutathione levels achieved via introduction of glutathione ethyl ester into the cell.

Cadmium‐induced astroglial death proceeds via glutathione depletion

Cadmium is a heavy metal that accumulates in the body, and its accumulation in the brain damages both neurons and glial cells. In the current study, we explored the mechanism underlying cadmium toxicity in primary cortical astroglia cultures. Chronic treatment with 10 microM cadmium was sufficient to cause 90% cell death in 18 hr. However, unlike that observed in neurons, cadmium-induced astroglial toxicity was not attenuated by the antioxidants trolox (100 microM), caffeic acid (1 mM), and vitamin C (1 mM). In contrast, extracellular 100 microM glutathione (GSH; gamma-Glu-Cys-Gly) or 100 microM cysteine almost completely blocked cadmium-induced astroglial death, whereas 300 microM oxidized GSH (GSSG) or 300 microM cystine, which do not have the free thiol group, were ineffective. In addition, cadmium toxicity was noticeably inhibited or enhanced when intracellular GSH was, respectively, increased by using the cell-permeable glutathione ethyl ester (GSH-EE) or depleted by using buthionine sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase. In agreement with these data, intracellular GSH levels were found to be depressed in cadmium-treated astrocytes. These results suggest that the toxic effect of cadmium on primary astroglial cells involves GSH depletion and, furthermore, that GSH administration can potentially be used to counteract cadmium-induced astroglial cell death therapeutically.

Glutathione regulates susceptibility to oxidant-induced mitochondrial DNA damage in human lymphocytes

Oxidative damage to mitochondrial DNA (mtDNA) interferes with the expression of mitochondrial-encoded subunits of the electron transport complexes of oxidative phosphorylation. MtDNA is protected by several mitochondrial antioxidant systems, but the specific importance of glutathione is unknown. We hypothesized that glutathione protects mtDNA from oxidative damage in human blood lymphocytes and that glutathione depletion increases susceptibility to mtDNA depletion, which increases vulnerability to apoptosis. MtDNA damage was measured in human blood lymphocytes exposed to tert-butyl-hydroperoxide (t-BOOH) or t-BOOH plus the glutathione analog, glutathione ethyl ester (GEE). Mitochondrial oxidative stress, mtDNA damage, and susceptibility to apoptosis were analyzed after glutathione depletion with buthionine sulfoximine (BSO). The data show selective damage to lymphocyte mtDNA at low concentrations of tBOOH that is attenuated by glutathione supplementation. Moreover, inhibition of glutathione synthesis led to lymphocyte ROS generation and mtDNA damage, and increased susceptibility to receptor-mediated apoptosis. These findings implicate the glutathione system in maintaining mtDNA integrity and resistance to apoptosis in lymphocytes and suggest that assessment of mtDNA damage in blood lymphocytes may be a useful marker of oxidative stress in humans.

Antioxidants to Prevent Bovine Neutrophil-Induced Mammary Epithelial Cell Damage

Activated neutrophils are able to produce a large quantity of bactericidal molecules such as reactive oxygen species that have been associated with tissue damage in several inflammation models. The protective effects of antioxidants in a context of neutrophil-induced damage to mammary epithelial cells were first evaluated in vitro using a coculture model of activated bovine neutrophils and a bovine mammary epithelial cell line (MAC-T cells). Cell damage was determined by quantifying the release of lactate dehydrogenase by MAC-T cells in culture medium. Morphological observation of cells stained with acridine orange was used to visualize the extent of cell damage. When incubated with neutrophils activated by lipopolysaccharides and phorbol 12-myristate 13-acetate, MAC-T cells released large amounts of lactate dehydrogenase indicating significant cell damage. The addition of dimethylthiourea or bathocuproine disulfonic acid did not reduce the damage whereas catechin, deferoxamine or glutathione ethyl ester significantly reduced neutrophil-induced cytotoxicity in a dose-dependent manner. The effect of deferoxamine, an iron chelator, on the growth of Escherichia coli and the ability of bovine neutrophils to phagocytose these bacteria were then assessed in vitro. Our data showed that deferoxamine did not interfere with the phagocytic activity of neutrophils but inhibited growth of the bacteria. Overall, our results suggest that antioxidants may be effective tools for protecting mammary tissue against neutrophil-induced oxidative stress during bovine mastitis.

Knockdown of Glutamate-Cysteine Ligase by Small Hairpin RNA Reveals That Both Catalytic and Modulatory Subunits Are Essential for the Survival of Primary Neurons

Glutathione deficiency is an early biochemical feature that occurs during apoptotic neuronal death associated with certain neurological disorders such as Parkinson disease. However, whether specific targeting of glutathione biosynthesis in neurons is sufficient to trigger neurodegeneration remains undetermined. To address this issue, we used a vector-based small hairpin RNA (shRNA) strategy to knock down each subunit of glutamate-cysteine ligase (GCL; gamma-glutamylcysteine synthetase), the heterodimeric enzyme that catalyzes the rate-limiting step of glutathione biosynthesis. Independent targeting of the catalytic and modulatory subunits by shRNA caused disruption of GCL as assessed by Northern and Western blotting, enzyme activity, and glutathione concentrations. Silencing each subunit in primary cortical neurons spontaneously elicited time-dependent apoptotic death, an effect that was synergistic with glutamate or nitric oxide treatment. Moreover, neuronal apoptosis by GCL knockdown was rescued by expressing the corresponding subunit full-length cDNA carrying silent mutations within the shRNA target cDNA sequence and by incubating neurons with gamma-glutamylcysteine or glutathione ethyl ester. In contrast, supplying glutathione precursors to neurons from co-cultured astrocytes did not prevent the apoptotic death triggered by GCL knockdown. Finally, overexpressing the catalytic (but not modulatory) GCL subunit full-length cDNA increased enzyme activity and glutathione concentrations, yielding neurons more resistant to glutamate- or nitric oxide-mediated apoptosis. Thus, specific and independent disruption of each subunit of GCL in neurons can be said to cause a primary decrease in glutathione that is sufficient to promote neurodegeneration.

Cisplatin-Induced Hepatotoxicity Is Enhanced by Elevated Expression of Cytochrome P450 2E1

In this study, the possible potentiation of cisplatin-induced hepatotoxicity by cytochrome P450 2E1 (CYP2E1) was examined both in vitro and in vivo. Transfected HepG2 cells expressing CYP2E1 (E47 cells) and not expressing CYP2E1 (C34 cells) were used as an in vitro model, and mice drinking 2% acetone for 7 days to induce CYP2E1 were used as an in vivo model. Exposure of E47 cells to cisplatin caused a much greater loss of cell viability, more striking depletion of reduced glutathione (GSH), and higher reactive oxygen species (ROS) production as compared with C34 cells. The prooxidant L-buthionine-[R,S]-sulfoximine (BSO), which depletes GSH, enhanced cisplatin-induced loss of cell viability, whereas the antioxidant glutathione ethyl ester, or the iron chelator deferoxamine mesylate (DFO) protected against the cisplatin-induced loss of E47 cell viability. Diallyl sulfide (DAS), an inhibitor of CYP2E1, also protected against the cisplatin toxicity in the E47 cells. After being injected with cisplatin (ip, 45 mg/kg), mice drinking 2% acetone with increased CYP2E1 levels exhibited elevated levels of serum ALT and AST, liver caspase-3 activity and positive staining of TUNEL increased, and histopathology indicated the presence of necrotic foci in livers of acetone plus cisplatin-treated mice. Lipid peroxidation and protein oxidation as indicated by carbonyl formation, staining of 3-nitrotyrosine (3-NT) and iron were higher in the cisplatin plus acetone group, compared with cisplatin alone group. Both in vitro and in vivo results indicate that elevated CYP2E1 enhances cisplatin-induced hepatotoxicity, and the mechanism may involve increased production of ROS and oxidative stress.

Antioxidant Treatment Inhibited Glutamate-Evoked NF-κB Activation in Primary Astroglial Cell Cultures

In glial cells, glutamate exposure causes alterations in cell redox status, mainly mediated by glutathione depletion and reactive oxygen species generation. These effects finally lead to astrocyte dysfunction which contributes to the pathogenesis of several neurological disorders. This study was aimed to investigate the involvement of the NF-κB pathway in oxidative stress induced by glutamate exposure in primary cultures of astrocytes. Further, we evaluated the power of the antioxidants genistein (0.1–10 μM) and IRFI 016 (20–80 μM), a synthetic tocopherol analogue, compared with glutathione ethyl ester (10–50 μM) and cysteamine-HCl (100–500 μM), to antagonize the effects elicited by glutamate (500 μM). Alterations of cell redox status were reduced, in a dose-dependent way, by antioxidants; in particular, 80 μM IRFI 016 and 10 μM genistein almost completely restored glutathione basal levels and significantly diminished ROS production, as well as 100 μM glutathione ethyl ester. These antioxidant effects were stronger than those caused by 500 μM cysteamine-HCl. Further, glutamate promoted the up-regulation of p50 and p65 NF-κB subunits and their nuclear translocation, as revealed by Western blot analysis and electrophoretic mobility shift assay of both subunits. The activation of p50 and p65 NF-κB subunits induced by glutamate exposure was significantly reduced by IRFI 016, acting in a dose-dependent manner. Altogether, these data confirm that the NF-κB pathway is involved in cell response to oxidative stress induced by glutamate injury in primary astrocyte cultures, and suggest that the use of antioxidants, such as IRFI 016, may be a helpful pharmacological strategy for neuroprotection.

Augmented IL-10 production and redox-dependent signaling pathways in glucose-6-phosphate dehydrogenase-deficient mouse peritoneal macrophages

Glucose-6-phosphate dehydrogenase (G6PD) supports cellular antioxidant pathways. G6PD deficiency is associated with malaria protection but was shown to worsen the clinical course to injury. This study tested whether G6PD deficiency manifests in altered cytokine responses using peritoneal macrophages from a G6PD-deficient mouse model with a degree of defect similar to the common type A(-) human G6PD deficiency. Lipopolysaccharide (LPS)-induced interleukin (IL)-10 and IL-12 production was doubled in G6PD-deficient macrophages compared with wild-type (WT). Protein kinase C (PKC) activation by phorbol-ester prior to LPS resulted in a fivefold greater IL-10 production in G6PD-deficient macrophages compared with WT. Interferon-gamma treatment prior to LPS augmented IL-12 production in G6PD-deficient and WT macrophages and partially inhibited IL-10 production by G6PD-deficient macrophages. The antioxidants (N-acetyl-L-cysteine and glutathione ethyl-ester) blunted IL-10 and IL-12 production, indicating a role for oxidative stress in the observed response differences between deficient and WT macrophages. LPS-induced activation of nuclear factor-kappaB, cyclic adenosine monophosphate response element-binding protein, and specificity protein 3 was augmented in G6PD-deficient cells compared with WT. The PKCdelta inhibitor Rottlerin inhibited IL-10 and IL-12 production at different 50% effective-dose concentrations between deficient and WT macrophages, indicating elevated PKCdelta activity in deficient cells. This study reveals that activated G6PD-deficient macrophages display an augmented production of cytokines with a prominent impact on IL-10 production. The altered cytokine responses are associated with augmented activation of redox-dependent transcription factors and PKCdelta. Alterations in signaling pathways and associated changes in cytokine production may play a role in modulating the inflammatory responses following bacterial or malarial infections in G6PD deficiency.

W09-P-007 Glutathione ethyl ester improves antioxidant status in an experimental model of metabolic syndrome

W09-P-007 Glutathione ethyl ester improves antioxidant status in an experimental model of metabolic syndrome

Regulation of apoptosis by glutathione redox state in PC12 cells exposed simultaneously to iron and ascorbic acid

We previously reported that the levels of non-protein-bound iron (NPBI) and ascorbic acid (AA) are markedly increased in the cerebrospinal fluid of infants with perinatal asphyxia. The present study showed that FeSO 4 and AA synergistically induced apoptosis of PC12 cells, which was prevented by α-tocopherol and glutathione (GSH) ethyl ester. Markers of free radical damage, such as ortho-tyrosine, meta-tyrosine, and F 2α-isoprostane, showed a gradual increase. AA and ferrous NPBI disappeared rapidly from the culture medium, but exposure for only a few hours was sufficient to trigger apoptosis. Intracellular GSH decreased progressively along with a concomitant increase of glutathione disulfide (GSSG). The baseline half-cell reduction potential ( E hc) for GSSG, 2H +/2GSH couple was −246 mV and an E hc of −200 mV was the critical level to switch on apoptosis, although some cells escaped this fate by transient increase of intracellular GSH. Once E hc reached around −165 mV (81 mV oxidation from the baseline), all cells lost the ability to maintain an adequate intracellular GSH level and subsequently underwent apoptosis. These findings at least partly explain the mechanism of Fe–AA cytotoxicity, in that ferrous iron catalyzes hydroxyl radical generation and induces lipid peroxidation, after which subsequent depletion of GSH raises E hc to the critical level for triggering or potentiating the apoptotic cascade.