Assessment of the Role of the Glutathione and Pentose Phosphate Pathways in the Protection of Primary Cerebrocortical Cultures from Oxidative Stress

Abstract: Reactive oxygen species have been implicated in neuronal injury associated with various neuropathological disorders. However, little is known regarding the relationship between antioxidant enzyme capacity and resultant toxicity. The antioxidant pathways of primary cerebrocortical cultures were directly examined using a novel technique that measures pentose phosphate pathway (PPP) activity, which is enzymatically coupled to glutathione peroxidase (GPx) detoxification of hydrogen peroxide (H₂O₂). PPP activity was quantified from data obtained by gas chromatography/mass spectrometry analysis of released labeled lactate following metabolic degradation of [1,6-¹³C₂,6,6-²H₂]glucose by cerebrocortical cultures. The antioxidant capacity of these cultures was systematically evaluated using H₂O₂, and the resultant toxicity was quantified by lactate dehydrogenase release. Exposure of primary mixed and purified astrocytic cultures to H₂O₂ caused stimulation of PPP activity in a concentration-dependent fashion from 0.25 to 22.2% and from 6.9 to 66.7% of glucose metabolized to lactate through the PPP, respectively. In the mixed cultures, chelation of iron before H₂O₂ exposure was protective and resulted in a correlation between PPP saturation and toxicity. Conversely, addition of iron, inhibition of GPx, or depletion of glutathione decreased H₂O₂-induced PPP stimulation and increased toxicity. These results implicate the Fenton reaction, reflect the pivotal role of GPx in H₂O₂ detoxification, and contribute to our understanding of the etiological role of free radicals in neuropathological conditions.     

Role of labile iron in the toxicity of pharmacological ascorbate

Pharmacological ascorbate has been shown to induce toxicity in a wide range of cancer cell lines. Pharmacological ascorbate in animal models has shown promise for use in cancer treatment. At pharmacological concentrations the oxidation of ascorbate produces a high flux of H₂O₂ via the formation of ascorbate radical (Asc^•-). The rate of oxidation of ascorbate is principally a function of the level of catalytically active metals. Iron in cell culture media contributes significantly to the rate of H₂O₂ generation. We hypothesized that increasing intracellular iron would enhance ascorbate-induced cytotoxicity and that iron chelators could modulate the catalytic efficiency with respect to ascorbate oxidation. Treatment of cells with the iron-chelators deferoxamine (DFO) or dipyridyl (DPD) in the presence of 2 mM ascorbate decreased the flux of H₂O₂ generated by pharmacological ascorbate and reversed ascorbate-induced toxicity. Conversely, increasing the level of intracellular iron by preincubating cells with Fe-hydroxyquinoline (HQ) increased ascorbate toxicity and decreased clonogenic survival. These findings indicate that redox metal metals, e.g., Fe³⁺/Fe²⁺, have an important role in ascorbate-induced cytotoxicity. Approaches that increase catalytic iron could potentially enhance the cytotoxicity of pharmacological ascorbate in vivo.     

Iron chelator Deferoxamine protects human neuroblastoma cell line SH-SY5Y from 6-Hydroxydopamine-induced apoptosis and autophagy dysfunction

BackgroundIntracellular iron involves in Fenton’s reaction-mediated Hydroxyl radical (OH·) generation by reacting with the neurotoxic agent 6-Hydroxydopamine (6-OHDA) autoxidation derivative Hydrogen Peroxide (H₂O₂). Several studies have been conducted so far on the neuroprotective activities of the iron chelator Deferoxamine (DFO) but little or no clear evidence about the underlying cellular mechanism is available.MethodsThe present study was conducted on Human neuroblastoma cell line SH-SY5Y in the absence or presence of 6-OHDA or H₂O₂ and / or DFO. Following incubation, cell viability assay, intracellular reactive oxygen species (ROS) determination, flow cytometric quantification of apoptotic cells followed by nuclear staining, intracellular tracking of transfected fusion construct of microtubule-associated protein 1B-light chain with Green fluorescent protein - Red fluorescent protein (LC3B-GFP-RFP reporters) and immunocytochemistry of intracellular Cathepsin protein by confocal microscopy, were conducted. In addition, western blotting was carried out to detect expressions of apoptotic and autophagy related proteins.ResultsThis study confirmed the neuroprotective potential of DFO by inhibiting 6-OHDA-mediated cell death and ROS generation. Reduced percentage of apoptotic cells and appearance of altered nuclei architecture followed by a reduced expression of cleaved PARP (Poly-ADP-ribose Polymerase) and cleaved Caspase-3 were observed upon DFO treatment against 6-OHDA, and as well as against H₂O₂ in SH-SY5Y cell lines. Besides, DFO induced the intracellular autophagolysosome formation (red puncta) rather than autophagosome (yellow puncta) only. Thereafter it was observed that DFO restored the expression of intracellular lysosomal protease Cathepsin and reduced the expression of the LC3-II.ConclusionTaken together, this study clearly demonstrated that the anti-Fenton activity of DFO inhibited apoptosis and caused blockade in ALP or autophagy dysfunction in SH-SY5Y cell lines. These outcomes further suggest that DFO provides neuroprotection by inhibiting apoptosis and inducing the progression of Autophagy- lysosomal pathway (ALP).     

The antioxidant defense system of isolated guinea pig Leydig cells

Utilization of highly enriched preparations of steroidogenic Leydig cells have proven invaluable for studying the direct effects of various hormones and agents on Leydig cell functionin vitro. However, recent work indicates that isolated Leydig cells are often subjected to oxygen (O₂) toxicity when cultured at ambient (19%) oxygen concentrations. Because intracellular antioxidants play an important role in protecting cells against oxygen toxicity, we have investigated the intracellular antioxidant defense system of isolated Leydig cells. The cellular levels of several antioxidants including catalase, glucose-6-phosphate dehydrogenase (G-6-PDH), superoxide dismutase (SOD) of the Cu/Zn & Mn variety, glutathione peroxidase, glutathione reductase and total glutathione were quantitated using enriched populations of Leydig cells isolated from adult male guinea pig testes. Compared to whole testicular homogenates, Leydig cells contained significantly (P<0.01) less G-6-PDH, total SOD, glutathione reductase and total glutathione, but significantly (P<0.001) more glutathione peroxidase. Compared to hepatic values previously reported in the guinea pig, Leydig cells contain nearly 400 times less catalase, about 14 times less glutathione peroxidase and almost 11 times less glutathione reductase. Since G-6-PDH and glutathione reductase are both necessary to regenerate reduced gluthathione (GSH) which couples with glutathione peroxidase to breakdown hydrogen peroxide (H₂O₂) under normal conditions, it is plausible that the oxygen toxicity observed in isolated Leydig cells is due to the intracellular accumulation of H₂O₂. Using the dichlorofluorescin diacetate (DCF-DA) assay, we found that Leydig cells incubated in the presence of 19% O₂ produced significantly (P<0.001) higher levels of H₂O₂ with time in culture compared to Leydig cells maintained at 3% O₂. These results support the hypothesis that the increased susceptibility of isolated Leydig cells to oxygen toxicity may be due, in part, to decreased amounts of certain antioxidant defenses and an increased production of the reactive oxygen species H₂O₂.     

Desferrioxamine as an Electron Donor. Inhibition of Membranal Lipid Peroxidation Initiated by H202 –Activated Metmyoglobin and Other Peroxidizing Systems

Desferoxamine (DFO) involvement in several peroxidative systems was studied. These sytems included: a) membranal lipid peroxidation initiated by H₂O₂-activated metmyoglobin (or methemoglobin); b) phenol-red oxidation by activated metmyoglobin or horseradish peroxidase (HRP): c) β-carotene-linoleate couple oxidation stimulated by lipoxygenase or hemin. Desferrioxamine was found to inhibit all these systems but not ferrioxamine (FO). Phenol-red oxidation by H₂0₂-horseradish peroxidase was inhibited competitively with DFO. Kinetic studies using the spectra changes in the Soret region of metmyoglobin suggest a mechanism by which H₂0₂ reacts with the iron-heme to form an intermediate of oxy-ferryl myoglobin that subsequently reacts with DFO to return the activated compound to the resting state. These activities of DFO resemble the reaction of other electron donors.     

Extinction of cells of cyanobacterium Anabaena circinalis in the presence of humic acid under illumination

Laboratory experiments targeting the effect of humic acid (HA) on the cell lysis of cyanobacterium Anabaena circinalis have been performed. Light irradiation was found to be an important factor for the cell lysis phenomenon, whereas intracellular hydrogen peroxide (H₂O₂) might be a chemical factor for the process. An exogenous H₂O₂ concentration of 1.0 mg l⁻¹ was determined as the threshold for cell survival. Our results indicated that HA or its possible product(s) of photochemical reaction can induce damage to intracellular catalase under artificial illumination, which leads intracellular H₂O₂ to be accumulated to an abnormally high concentration, eventually resulting in cell death. Moreover, H₂O₂ released into the culture from dead cells can damage other cells, which in turn brings about the population extinction.     

Correlation of oxygen utilization and hydrogen peroxide accumulation with oxygen induced enzymes in Lactobacillus plantarum cultures

Two strains of Lactobacillus plantarum accumulated H₂O₂ when grown aerobically in a complex glucose based medium. The H₂O₂ accumulation did not occur immediately on exposure of the culture to O₂ but was delayed for a time which, in the case of one strain, was dependent on the amount of inoculum used to seed the culture. The accumulation was always preceded by an increase in the rate of O₂ utilization by the cultures. The latter coincided approximately with an increase in specific activity of NADH oxidase, pyruvate oxidase and NADH peroxidase. H₂O₂ was not a product of NADH oxidase in vitro but was formed in substantial quantities from O₂ during oxidation of pyruvate. The three enzymes were induced by O₂ and H₂O₂; the induction of NADH oxidase responded to lower levels of O₂ (but not of H₂O₂) than the pyruvate oxidase or the NADH peroxidase.Abbreviations MRSG Mann, Rogosa and Sharpe medium (1960) with glucose as fermentation source - TPP thiamin pyrophosphate     

Antioxidant activity of vasoactive intestinal peptide in HK2 human renal cells

Oxidative stress is a major mediator of tissue and cell injuries. The injury in chronic nephrotic syndrome, acute renal failure, myeloma kidney injury and other kidney diseases is initiated by oxidative stress. We have previously demonstrated that vasoactive intestinal peptide (VIP) acts as an antiproliferative agent in renal cancer cells. This study was designed to evaluate the renoprotective activity of VIP against H₂O₂-induced oxidative damage in a proximal tubule kidney cell line (human, non-tumor, HK2 cells) in order to investigate the potential usefulness of this peptide in the treatment of oxidative-stress related kidney diseases. HK2 cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Propidium iodide was used to identify cells undergoing apoptosis. Western blotting was performed with anti-Bcl-2, anti-Bax and anti-formyl peptide receptor (low-affinity variant FPRL-1) monoclonal antibodies whereas 2,7-dichlorofluorescein diacetate was used for measurement of levels of intracellular reactive oxygen species (ROS). HK2 cells were injured with H₂O₂ in order to induce apoptosis: the effect was time- and dose-dependent. VIP increased the levels of the antiapoptotic protein Bcl-2 and decreased those of the proapoptotic protein Bax. VIP decreased the intracellular ROS levels reached by H₂O₂-induced oxidative stress. VIP effect on ROS levels involved FPLR-1 but not VPAC_1,2 receptors as evidenced by the use of the respective antagonists WRW4 and JV-1-53. Thus, VIP protects HK2 cells from apoptosis by increasing Bcl-2 levels and this effect is initiated through FPLR1 receptor. In conclusion, VIP might exert a renoprotective effect by the suppression of oxidative stress.     

Effect of N-acetylcysteine and deferoxamine on endogenous antioxidant defense system gene expression in a rat hepatocyte model of cocaine cytotoxicity

In the present study we investigated on cultures of hepatocytes from phenobarbital-pretreated rats, the effect of the antioxidants, 0.5 mM N-acetylcysteine (NAC) or 1.5 mM deferoxamine (DFO), previously incubated for 24 h and coincubated with cocaine (0-1000 microM) for another 24 h. Cocaine cytotoxicity was monitored by either the lysis of the cell membranes or apoptosis. Lysis of the cell membranes was evidenced by lactate dehydrogenase leakage, apoptosis was observed by detecting a hypodiploid peak (<2C) in DNA histograms obtained by flow cytometry, peroxide production was quantified with 2', 7'-dichlorodihydrofluorescein diacetate and gene expression of the antioxidant enzymes: Mn- and Cu,Zn-superoxide dismutases, catalase and glutathione peroxidase were measured by Northern blot analysis. NAC and DFO significantly decreased the extent of lysis of cell membranes and apoptosis, and the antiapoptotic effect was parallel to peroxide generation. By the effect of NAC and DFO, significant increases were detected in the levels of mRNA of catalase, manganese superoxide dismutase and glutathione peroxidase. From these results we conclude that NAC or DFO, when incubated in the presence of cocaine, exerted a protective effect against cocaine toxicity at the level of both lysis of the membranes and apoptosis. This protective effect, in the case of NAC, was directed towards an increase in antioxidant enzyme expression, and in the case of DFO against reactive oxygen species generation.     

AMPK-mediated increase of glycolysis as an adaptive response to oxidative stress in human cells: Implication of the cell survival in mitochondrial diseases

We report that the energy metabolism shifts to anaerobic glycolysis as an adaptive response to oxidative stress in the primary cultures of skin fibroblasts from patients with MERRF syndrome. In order to unravel the molecular mechanism involved in the alteration of energy metabolism under oxidative stress, we treated normal human skin fibroblasts (CCD-966SK cells) with sub-lethal doses of H₂O₂. The results showed that several glycolytic enzymes including hexokinase type II (HK II), lactate dehydrogenase (LDH) and glucose transporter 1 (GLUT1) were up-regulated in H₂O₂-treated normal skin fibroblasts. In addition, the glycolytic flux of skin fibroblasts was increased by H₂O₂ in a dose-dependent manner through the activation of AMP-activated protein kinase (AMPK) and phosphorylation of its downstream target, phosphofructokinase 2 (PFK2). Moreover, we found that the AMPK-mediated increase of glycolytic flux by H₂O₂ was accompanied by an increase of intracellular NADPH content. By treatment of the cells with glycolysis inhibitors, an AMPK inhibitor or genetic knockdown of AMPK, respectively, the H₂O₂-induced increase of NADPH was abrogated leading to the overproduction of intracellular ROS and cell death. Significantly, we showed that phosphorylation levels of AMPK and glycolysis were up-regulated to confer an advantage of survival for MERRF skin fibroblasts. Taken together, our findings suggest that the increased production of NADPH by AMPK-mediated increase of the glycolytic flux contributes to the adaptation of MERRF skin fibroblasts and H₂O₂-treated normal skin fibroblasts to oxidative stress.     

Oxidative Stress and Antioxidant Cell Protection Systems in the Microaerophilic Bacterium Spirillum winogradskii

The influence of oxygen availability during cultivation on the biosynthetic processes and enzymatic activities in the microaerophilic bacterium Spirillum winogradskii D-427 was studied, and the roles played by different systems of the defense against oxidation stress were determined. The metabolic adjustments caused by transition from microaerobic (2% O₂) aerobic conditions (21% O₂ of the gas phase) were found to slow down constructive metabolism and increase synthesis of exopolysaccharides as a means of external protection of cells from excess oxygen. This resulted in a twofold decline of the growth yield coefficient. Even though the low activity of catalase is compensated for by a multifold increase in the activities of other cytoplasmic enzymes that defend against toxic forms of O₂—peroxidase and enzymes of the redox system of glutathione (glutathione peroxidase and glutathione reductase)—massive lysis of cells starts in the midexponential phase and leads to culture death in the stationary phase because of H₂O₂ accumulation in the periplasm (up to 10 g/mg protein). The absence in cells of cytochrome-c-peroxidase, a periplasmic enzyme eliminating H₂O₂, was shown. It follows that the major cause of oxidative stress in cells is that active antioxidant defenses are located in the cytoplasm, whereas H₂O₂ accumulates in the periplasm due to the lack of cytochrome-c-peroxidase. The addition to the medium of thiosulfate promotes elimination of H₂O₂, stops cell lysis under aerobic conditions, lends stability to cultures, and results in a threefold increase in the growth yield.     

Replacement of media in cell culture alters oxygen toxicity: Possible role of lipid aldehydes and glutathione transferases in oxygen toxicity

Replacement of media in cell cultures during exposure to hyperoxia was found to alter oxygen toxicity. Following 100 hr of exposure to 95% or 80% O₂, the surviving fraction (SF) of Chinese hamster fibroblasts, as assayed by clonogenicity, was less than 1 × 10^?3 when the culture media was replaced only at the onset of the O₂ exposure. Media replacement every 24 hr throughout the hyperoxic exposure resulted in SFs of 1.7 × 10^?1 (95% O₂) and 1.9 × 10^?1 (80% O₂) at 95 hr. Cellular resistance to and metabolism of 4-hydroxy-2-nonenal (4HNE), a cytotoxic byproduct of lipid peroxidation, was examined in cells 24 hr following exposure to 80% O₂ for 144 hr with media replacement. These O₂-exposed cells were resistant to 4HNE, requiring 2.6 times as long in 80 μM 4HNE to reach 30% survival as compared to density-matched normoxia control. Furthermore, during 40 and 60 min of exposure to 4HNE, the O₂-preexposed cells metabolized greater quantities of 4HNE (fmole/cell) relative to control. The activity of glutathione S-transferase (GST), an enzyme believed to be involved with the detoxification of 4HNE, was significantly increased in the O₂-preexposed cells compared with controls. Catalase activity was significantly increased, but no change was found in total glutathione content, glutathione peroxidase, manganese superoxide dismutase, and copper-zinc superoxide dismutase activities at the time of 4HNE treatment in the O₂-preexposed cells relative to density-matched control. The results demonstrate that in vitro tolerance to the cytotoxic effects of hyperoxia can be achieved through media replacement during O₂ exposure. Tolerance to oxygen toxicity conferred resistance to the cytotoxic effects of 4HNE, possibly through GST-catalyzed detoxification. These results provide further support for the hypothesis that toxic aldehydic byproducts of lipid peroxidation contribute to hyperoxic injury.     

Protective effect of sesaminol from Sesamum indicum Linn. against oxidative damage in PC12 cells

Sesaminol is one component of sesame oil and has been widely used as the stabilizer to extend the storage period of food oil in China. In this study, we tried to investigate the antioxidant activity of sesaminol on rat pheochromocytoma (PC12) cells oxidative damaged by H₂O₂. Cell viability, LDH level and apoptosis of the PC12 cells were assayed after treatment with sesaminol for 3 h and exposure to H₂O₂. Furthermore, superoxide (SOD), catalase (CAT), glutathione peroxidase (GSH‐Px) and intracellular ROS were assayed after exposure of the PC12 cells to H₂O₂. The results showed that pre‐treatment with sesaminol prior to H₂O₂ exposure significantly elevated cell survival rate and SOD, CAT and GSH‐Px activity. Meanwhile, sesaminol declined the secreted LDH level, apoptosis rate and ROS level of H₂O₂ exposed cells. Thus, sesaminol may protect PC12 against oxidative injury. Copyright © 2012 John Wiley & Sons, Ltd.     

Role of glycolysis and antioxidant enzymes in the toxicity of amyloid beta peptide Aβ<Subscript>25–35</Subscript> to erythrocytes

The role of glycolysis and antioxidant enzymes in amyloid beta peptide Aβ_25–35 toxicity to human and rat erythrocytes was studied. The erythrotoxicity of Aβ_25–35 was shown to increase two-to fourfold both in the absence of glucose in the incubation medium and upon the addition of sodium fluoride, an enolase inhibitor. Potassium cyanide, a Cu,Zn-superoxide dismutase inhibitor, abolishes the toxic effect of Aβ_25–35 to erythrocytes, whereas mercaptosuccinate, a glutathione peroxidase inhibitor, and ouabain, a Na⁺,K⁺-ATPase inhibitor, promote it. Sodium azide, a catalase inhibitor, did not affect the cell lysis under the action of Aβ_25–35. The results support the hypothesis that H₂O₂, Cu,Zn superoxide dismutase, and glutathione peroxidase are involved in the toxicity mechanism rather than superoxide radical. Glycolysis and Na⁺,K⁺-ATPase play a substantial protective role. Fullerene C₆₀ nanoparticles are toxic to erythrocytes of both types; their toxicity is not related to enhanced oxidative stress and the mechanism of toxicity differs from that of Aβ_25–35.     

Noninvasive assessment of the relative roles of cerebral antioxidant enzymes by quantitation of pentose phosphate pathway activity

Cerebral pentose phosphate pathway (PPP) plays a role in the biosynthesis of macromolecules, antioxidant defense and neurotransmitter metabolism. Studies on this potentially important pathway have been hampered by the inability to easily quantitate its activity, particularly in vivo. In this study we review the use of [1,6-¹³C₂,6,6-²H₂]glucose for measuring the relative activities of the PPP and glycolysis in a single incubation in cultured neurons and in vivo, when combined with microdialysis techniques. PPP activity in primary cerebrocortical cultures and in the caudate putamem of the rat in vivo was quantitated from data obtained by GC/MS analysis of released labeled lactate following metabolic degradation of [1,6-¹³C₂,6,6-²H₂]glucose. Exposure of cultures to H₂O₂ resulted in stimulation of PPP activity in a concentration-dependent fashion and subsequent cell death. Chelation of iron during H₂O₂ exposure exerted a protective effect thus implicating the participation of the Fenton reaction in mediating damage caused by the oxidative insult. Partial inhibition of glutathione peroxidase, but not catalase, was extremely toxic to the cultures reflecting the pivotal role of GPx in H₂O₂ detoxification. These results demonstrate the ability to dynamically monitor PPP activity and its response to oxidative challenges and should assist in facilitating our understanding of antioxidant pathways in the CNS. Special issue dedicated to Dr. Herman Bachelard.     

The role of intracellular zinc in H2O2-induced oxidative stress in human erythrocytes

In vitro exposure of human erythrocytes to H₂O₂ at concentrations of 30–1000 μM resulted in a dose-dependent increase of the intracellular levels of Zn²⁺ and inhibition of the cytosolic esterase activity, which is a major marker of erythrocyte viability. The observed effect depended on the concentration of H₂O₂ and the duration of exposure of the cells to this compound. An inverse relationship between the changes in the intracellular level of labile zinc ions and esterase activity in the cells exposed to hydrogen peroxide was detected; this was indicative of the role of Zn²⁺ in the programmed death of red blood cells. The combined action of hydrogen peroxide and N',N'-tetrakis-(2-pyridyl-methyl)-ethylenediamine, an intracellular zinc ion chelator, has been found to eliminate the cytotoxic effect of H₂O₂, whereas the addition of Zn²⁺ to the erythrocyte incubation medium enhanced the effects of hydrogen peroxide. The reduction of the concentration of non-protein thiol groups due to a decrease of the level of reduced glutathione was shown to contribute to the release of Zn²⁺ from the intracellular binding sites during oxidative stress induced by H₂O₂ in human erythrocytes.

     

MicroRNA‐328 is involved in the effect of selenium on hydrogen peroxide‐induced injury in H9c2 cells

Oxidative stress induces apoptosis in cardiac cells, and antioxidants attenuate the injury. MicroRNAs (miRNAs) are also involved in cell death; therefore, this study aimed to investigate the role of miRNAs in the effect of selenium on oxidative stress‐induced apoptosis. The effects of sodium selenite were analyzed via cell viability, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) concentration. Flow cytometry was used to evaluate cell apoptosis. Fura‐2AM was used to calculate intracellular Ca²⁺ concentration. Sodium selenite could ameliorate hydrogen peroxide (H₂O₂)‐induced cell apoptosis and improve expression levels of glutathione peroxidase and thioredoxin reductase. Pretreatment with sodium selenite improved SOD activity and reduced MDA concentration. Treatments with H₂O₂ or sodium selenite decreased miR‐328 levels. MiR‐328 overexpression enhanced cell apoptosis, reduced ATP2A2 levels, and increased intracellular Ca²⁺ concentration, while inhibition produced opposite effects. MiR‐328 might be involved in the effect of sodium selenite on H₂O₂‐induced cell death in H9c2 cells.     

Intracellular catalase function: analysis of the catalatic activity by product formation in isolated liver cells

Intracellular catalase activity was measured in isolated rat hepatocytes by adding H₂O₂ under anaerobic conditions and measuring O₂ evolution. Hydrogen peroxide was introduced either by continuous infusion or by pulse injection. Continuous infusion at a rate similar to the endogenous H₂O₂ production rate provided results that 60–70% of the H₂O₂ was metabolized by the catalatic reaction. Comparison of rates of O₂ evolution to estimated rates of H₂O₂ metabolism obtained by the methanol-titration method (H. Sies and B. Chance, 1970, FEBS Lett.11, 172–176) indicated that the contribution of the peroxidatic reaction of catalase was small. The intracellular activity of glutathione peroxidase was estimated as the catalase-independent metabolism and used to determine the rate of intracellular H₂O₂ metabolism by the peroxidase. The results provide a quantitative basis for analysis of the physiological and toxicological aspects of H₂O₂ metabolism by liver.     

PI3Kγ contributes to MEK1/2 activation in oxidative glutamate toxicity via PDK1

The role of phosphoinositide 3‐kinase (PI3K) in oxidative glutamate toxicity is not clear. Here, we investigate its role in HT22 mouse hippocampal cells and primary cortical neuronal cultures, showing that inhibitors of PI3K, LY294002, and wortmannin suppress extracellular hydrogen peroxide (H₂O₂) generation and increase cell survival during glutamate toxicity in HT22 cells. The mitogen‐activated protein kinase kinase (MEK) inhibitor U0126 also reduced glutamate‐induced H₂O₂ generation and inhibited phosphorylation of extracellular signal‐regulated kinase (ERK) 1/2. LY294002 was seen to abolish phosphorylation of both ERK1/2 and Akt. A small interfering RNA (siRNA) study showed that PI3Kβ and PI3Kγ, rather than PI3Kα and PI3Kδ, contribute to glutamate‐induced H₂O₂ generation and cell death. PI3Kγ knockdown also inhibited glutamate‐induced ERK1/2 phosphorylation, whereas transfection with the constitutively active form of human PI3Kγ (PI3Kγ‐CAAX) triggered MEK1/2 and ERK1/2 phosphorylation and H₂O₂ generation without glutamate exposure. This H₂O₂ generation was reduced by inhibition of MEK. Transfection with kinase‐dead 3‐phosphoinositide‐dependent protein kinase 1 (PDK1‐KD) reduced glutamate‐induced ERK1/2 phosphorylation and H₂O₂ generation. Accordingly, cotransfection of cells with PDK1‐KD and PI3Kγ‐CAAX suppressed PI3Kγ‐CAAX‐triggered ERK1/2 phosphorylation and H₂O₂ generation. These results suggest that activation of PI3Kγ induces ERK1/2 phosphorylation, leading to extracellular H₂O₂ generation via PDK1 in oxidative glutamate toxicity.