The protein interaction of Saccharomyces cerevisiae cytoplasmic thiol peroxidase II with SFH2p and its in vivo function

Previously, we reported that the yeast cytoplasmic thiol peroxidase type II isoform (cTPx II), a member of the TSA/AhpC family, showed a very low peroxidase activity when compared with other cytoplasmic yeast isoforms, and that cTPx II mutant (cTPx II Delta) showed a severe growth retardation compared with that of the wild-type cells. To reveal the physiological function of cTPx II in yeast cell growth, we searched for proteins which react with cTPx II. In this study, we identified a novel interaction between cTPx II and CSR1p using the yeast two-hybrid system. CSR1p (SFH2p) has been known to be one member of Sec14 homologous (SFH2) proteins. SFH2p exhibits phosphatidylinositol transfer protein activity. Interestingly, we found that cTPx II selectively bound to SFH2p among the five types of SFH proteins and Sec14p. The interaction required the dimerization of cTPx II. In addition, SFH2p also specifically bound to cTPx II among the yeast thiol peroxidase isoforms. The selective interaction of the dimer form of cTPx II (the oxidized form) with SFH2p was also confirmed by glutathione S-transferase pull-down and immunoprecipitation assays. The growth retardation, clearly reflected by the length of the lag phase, of cTPx II Delta was rescued by deleting SFH2p in the cTPx II Delta strain. The SFH2 Delta strain did not show any growth retardation. In addition, the double mutant showed a higher susceptibility to oxidative stress. This finding provides the first in vivo demonstration of the specific interaction of cTPx II with SFH2p in an oxidative stress-sensitive manner and a novel physiological function of the complex of cTPx II and SFH2p.     

Trichostatin a modulates intracellular reactive oxygen species through SOD2 and FOXO1 in human bone marrow‐mesenchymal stem cells

Engraft cells are often exposed to oxidative stress and inflammation; therefore, any factor that can provide the stem cells resistance to these stresses may yield better efficacy in stem cell therapy. Studies indicate that histone deacetylase (HDACs) inhibitors alleviate damage induced by oxidative stress. In this study, we investigated whether regulation of reactive oxygen species (ROS) occurs through the HDAC inhibitor trichostatin A (TSA) in human bone marrow‐mesenchymal stem cells (hBM‐MSCs). Intracellular ROS levels increased following exposure to hydrogen peroxide (H₂O₂), and were suppressed by TSA treatment. Levels of the antioxidant enzyme superoxide dismutase 2 (SOD2) increased following treatment with 200 nM TSA and to a lesser level at 1–5 μM TSA. Cell protective effects against oxidative stress were significantly increased in TSA‐MSCs after treatment with low doses of TSA (50–500 nM) and decreased with high doses of TSA (5–10 μM). Consistent results were obtained with immunoblot analysis for caspase3. Investigation of Forkhead box O1 (FOXO1), superoxide dismutase 2 (SOD2), and p53 levels to determine intracellular signaling by TSA in oxidative stress‐induced MSCs demonstrated that expression of phosphorylated‐FOXO1 and phosphorylated‐SOD2 decreased in H₂O₂‐treated MSCs while levels of p53 increased. These effects were reversed by the treatment of 200 nM TSA. These results suggest that the main function of ROS modulation by TSA is activated through SOD2 and FOXO1. Thus, optimal treatment with TSA may protect hBM‐MSCs against oxidative stress. Copyright © 2014 John Wiley & Sons, Ltd.     

Hydrogen peroxide-induced carbonylation of key metabolic enzymes in Saccharomyces cerevisiae: the involvement of the oxidative stress response regulators Yap1 and Skn7

H(2)O(2) induces a specific protein oxidation in yeast cells, and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (Tdh) is a major target. Using a 2D-gel system to study protein carbonylation, it is shown in this work that both Tdh2p and Tdh3p isozymes were oxidized during exposure to H(2)O(2). In addition, we identified two other proteins carbonylated and inactivated: Cu,Zn-superoxide dismutase and phosphoglycerate mutase. The oxidative inactivation of Cu,Zn-superoxide dismutase decreases the antioxidant capacity of yeast cells and probably contributes to H(2)O(2)-induced cell death. Cyclophilin 1 was also carbonylated, but CPH1 gene disruption did not affect peroxide stress sensitivity. The correlation between H(2)O(2) sensitivity and the accumulation of oxidized proteins was evaluated by assaying protein carbonyls in mutants deficient in the stress response regulators Yap1p and Skn7p. The results show that the high sensitivity of yap1delta and skn7delta mutants to H(2)O(2) was correlated with an increased induction of protein carbonylation. In wild-type cells, the acquisition of stress resistance by pre-exposure to a sublethal H(2)O(2) stress was associated with a lower accumulation of oxidized proteins. However, pre-exposure of yap1delta and skn7delta cells to 0.4 mM H(2)O(2) decreased protein carbonylation induced by 1.5 mM H(2)O(2), indicating that the adaptive mechanism involved in the protection of proteins from carbonylation is Yap1p- and Skn7p-independent.     

Neuroprotective effects of 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone on ischaemia/reperfusion‐induced neuronal injury by activating the Nrf2/HO‐1 pathway

1‐O‐Hexyl‐2,3,5‐trimethylhydroquinone (HTHQ), a lipophilic phenolic agent, has an antioxidant activity and reactive oxygen species (ROS) scavenging property. However, the role of HTHQ on cerebral ischaemic/reperfusion (I/R) injury and the underlying mechanisms remain poorly understood. In the present study, we demonstrated that HTHQ treatment ameliorated cerebral I/R injury in vivo, as demonstrated by the decreased infarct volume ration, neurological deficits, oxidative stress and neuronal apoptosis. HTHQ treatment increased the levels of nuclear factor erythroid 2–related factor 2 (Nrf2) and its downstream antioxidant protein, haeme oxygenase‐1 (HO‐1). In addition, HTHQ treatment decreases oxidative stress and neuronal apoptosis of PC12 cells following hypoxia and reperfusion (H/R) in vitro. Moreover, we provided evidence that PC12 cells were more vulnerable to H/R‐induced oxidative stress after si‐Nrf2 transfection, and the HTHQ‐mediated protection was lost in PC12 cells transfected with siNrf2. In conclusion, these results suggested that HTHQ possesses neuroprotective effects against oxidative stress and apoptosis after cerebral I/R injury via activation of the Nrf2/HO‐1 pathway.     

Hypoxia induces H2O2 production and activates antioxidant defence system in grapevine buds through mediation of H2O2 and ethylene

Paradoxically, in eukaryotic cells, hydrogen peroxide (H(2)O(2)) accumulates in response to oxygen deprivation (hypoxia). The source of H(2)O(2) under hypoxia varies according to the species, organs, and tissue. In non-photosynthetic tissues, H(2)O(2) is mainly produced by activation of NAD(P)H-oxidases or by disruption of the mitochondrial electron transport chain (m-ETC). This study showed that hypoxia, and inhibitors of respiration like potassium cyanide (KCN) and sodium nitroprusside (SNP), trigger the production of H(2)O(2) in grapevine buds. However, diphenyleneiodonium, an inhibitor of NAD(P)H-oxidase, did not reduce the H(2)O(2) levels induced by KCN, suggesting that, under respiratory stress, H(2)O(2) is mainly produced by disruption of the m-ETC. On the other hand, γ-aminobutyric acid (GABA), a metabolite that in plants alleviates oxidative stress by activating antioxidant enzymes, reduced significantly the levels of H(2)O(2) induced by KCN and, surprisingly, repressed the expression of genes encoding antioxidant enzymes such as ASCORBATE PEROXIDASE (VvAPX), GLUTATHIONE PEROXIDASE (VvGLPX), SUPEROXIDE DISMUTASE (VvSOD), and one of the CATALASE isoforms (VvCAT1), while VvCAT2 was upregulated. In contrast to GABA, hypoxia, H(2)O(2), and ethylene increased dramatically the expression of genes encoding antioxidant enzymes and enzymes of the alternative respiratory pathway such as ALTERNATIVE NADH-DEHYDROGENASES (VvaNDs) and ALTERNATIVE OXIDASES (VvAOXs). Hence, it is concluded that H(2)O(2) production is stimulated by respiratory stress in grapevine buds, that H(2)O(2) and ethylene act as signalling molecules and activate genes related to the antioxidant defence system, and finally that GABA reduces H(2)O(2) levels by up-regulating the expression of VvCAT2.     

Inhibiting catalase activity sensitizes 36B10 rat glioma cells to oxidative stress

Gliomas are extremely resistant to anticancer therapies resulting in poor patient survival, due, in part, to altered expression of antioxidant enzymes. The primary antioxidant enzyme, catalase, is elevated constitutively in gliomas compared to normal astrocytes. We hypothesized that downregulating catalase in glioma cells would sensitize these cells to oxidative stress. To test this hypothesis, we implemented two approaches. The first, a pharmacological approach, used 3-amino-1,2,4-triazole, an irreversible inhibitor that reduced catalase enzymatic activity by 75%. Pharmacological inhibition of catalase was not associated with a reduction in rat 36B10 glioma cell viability until the cells were challenged with additional oxidative stress, i.e., ionizing radiation or hydrogen peroxide (H(2)O(2)). In the second molecular approach, we generated 36B10 glioma cells stably expressing catalase shRNA; a stable cell line displayed a 75% reduction in catalase immunoreactive protein and enzymatic activity. This was accompanied by an increase in intracellular reactive oxygen species and extracellular H(2)O(2). These cells exhibited increased sensitivity to radiation and H(2)O(2), which was rescued by the antioxidant, N-acetylcysteine. These results support the hypothesis that catalase is a major participant in the defense of 36B10 glioma cells against oxidative stress mediated by anticancer agents capable of increasing steady-state levels of H(2)O(2).     

Mitochondria deficient in complex I activity are depolarized by hydrogen peroxide in nerve terminals: relevance to Parkinson's disease

Deficiency of complex I in the respiratory chain and oxidative stress induced by hydrogen peroxide occur simultaneously in dopaminergic neurones in Parkinson's disease. Here we demonstrate that the membrane potential of in situ mitochondria (Delta Psi m), as measured by the fluorescence change of JC-l (5,5',6,6'-tetrachloro-1,1,3,3'-tetraethylbezimidazolyl-carbocyani ne iodide), collapses when isolated nerve terminals are exposed to hydrogen peroxide (H(2)O(2), 100 and 500 microM) in combination with the inhibition of complex I by rotenone (5 nM-1 microM). H(2)O(2) reduced the activity of complex I by 17%, and the effect of H(2)O(2) and rotenone on the enzyme was found to be additive. A decrease in Delta Psi m induced by H(2)O(2) was significant when the activity of complex I was reduced to a similar extent as found in Parkinson's disease (26%). The loss of Delta Psi m observed in the combined presence of complex I deficiency and H(2)O(2) indicates that when complex I is partially inhibited, mitochondria in nerve terminals become more vulnerable to H(2)O(2)-induced oxidative stress. This mechanism could be crucial in the development of bioenergetic failure in Parkinson's disease.     

OXR1 signaling pathway as a possible mechanism of elastase-induced oxidative damage in pulmonary cells: the protective role of ellagic acid

Background and objective

Oxidative stress is a process that occurs through free radicals on the cell membranes which causes damage to the cell and intracellular organelles, especially mitochondria membranes. H2O2 induced oxidative stress in human cells is of interest in toxicological research since oxidative stress plays a main role in the etiology of several pathological conditions. Neutrophil Elastase (Serine proteinase) is involved in the pathology process of emphysema as a respiratory disease through lung inflammation, and destruction of alveolar walls. The present study investigated the direct oxidative stress effects of Elastase in comparison with H2O2 on human lung epithelial cells (A549 cells) concerning the generation of reactive oxygen species (ROS) and modulation of oxidation resistance 1 (OXR1) and its downstream pathway using the well-known antioxidant Ellagic acid as an activator of antioxidant genes.

Materials and methods

The human pulmonary epithelial cells (A549) were divided into the nine groups including Negative control, Positive control (H2O2), Elastase (15, 30, and 60 mU/mL), Ellagic acid (10 μmol/L), and Elastase?+?Ellagic acid. Cytotoxicity, ROS generation, oxidative stress profile, level of reactive metabolites, and gene expression of OXR1 and its downstream genes were measured in all groups.

Results

The obtained data demonstrated that Elastase exposure caused oxidative stress damage in a dose-depended manner which was associated with decreases in antioxidant defense system genes. Conversely, treatment with Ellagic acid as a potent antioxidant showed improved antioxidant enzyme activity and content which was in line with the upregulation of OXR1 signaling pathway genes.

Conclusions

The present findings can highlight the novel mechanism underlying the oxidative stress induced by Neutrophil Elastase through OXR1 and related genes. Moreover, the benefit of Ellagic acid on cytoprotection, resulting from its antioxidant properties was documented.

     

Cytosolic thioredoxin peroxidase I and II are important defenses of yeast against organic hydroperoxide insult: catalases and peroxiredoxins cooperate in the decomposition of H2O2 by yeast

The cytosolic thioredoxin peroxidase II (cTPxII/Tsa2p) from Saccharomyces cerevisiae shares 86% identity with the relatively well characterized cytosolic thioredoxin peroxidase I (cTPxI/Tsa1p). In contrast to cTPxI protein, cTPxII is not abundant and is highly inducible by peroxides. Here, we describe a unique phenotype for DeltacTPxII strain; these cells were highly sensitive to tert-butylhydroperoxide (TBHP) but presented resistance to H(2)O(2) in fermentative and respiratory conditions. In contrast, DeltacTPxI strain was very sensitive to both TBHP and H(2)O(2), whatever the carbon source present in the media. These differences in the response of mutant cells to the different kinds of peroxide insult could not be attributed to enzymatic properties of cTPxI and cTPxII since the recombinant proteins showed similar in vitro efficiencies (K(cat) /K(m)) in the removals of both kinds of peroxide. This specific sensitivity of DeltacTPxII cells to TBHP could not be related to the expression pattern of TSA2 (cytosolic thioredoxin peroxidase II gene) either, since this gene is highly inducible by both H(2)O(2) and TBHP when cells were grown in different conditions. Finally, peroxide-removing assays were performed and showed that catalase activity increased significantly only in DeltacTPxII cells, which appear to be related with the resistance of this strain to H(2)O(2). Taken together, present data indicate that cTPxII and cTPxI are key components of the yeast defense system against organic peroxide insult. In regard to the stress induced by H(2)O(2), catalases (peroxisomal and/or cytosolic) and cTPxII seemed to cooperate with cTPxI in the defense of yeast against this oxidant.     

Loss of vacuolar proton-translocating ATPase activity in yeast results in chronic oxidative stress

Yeast mutants lacking vacuolar proton-translocating ATPase (V-ATPase) subunits (vma mutants) were sensitive to several different oxidants in a recent genomic screen (Thorpe, G. W., Fong, C. S., Alic, N., Higgins, V. J., and Dawes, I. W. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 6564-6569). We confirmed that mutants lacking a V(1) subunit (vma2Delta), V(o) subunit, or either of the two V(o) a subunit isoforms are acutely sensitive to H(2)O(2) and more sensitive to menadione and diamide than wild-type cells. The vma2Delta mutant contains elevated levels of reactive oxygen species and high levels of oxidative protein damage even in the absence of an applied oxidant, suggesting an endogenous source of oxidative stress. vma2Delta mutants lacking mitochondrial DNA showed neither improved growth nor decreased sensitivity to peroxide, excluding respiration as the major source of the endogenous reactive oxygen species in the mutant. Double mutants lacking both VMA2 and components of the major cytosolic defense systems exhibited synthetic sensitivity to H(2)O(2). Microarray analysis comparing wild-type and vma2Delta mutant cells grown at pH 5, permissive conditions for the vma2Delta mutant, indicated high level up-regulation of several iron uptake and metabolism genes that are part of the Aft1/Aft2 regulon. TSA2, which encodes an isoform of the cytosolic thioredoxin peroxidase, was strongly induced, but other oxidative stress defense systems were not induced. The results indicate that V-ATPase activity helps to protect cells from endogenous oxidative stress.     

Nrf2/HO-1信号通路在人诱导性多能干细胞氧化应激中的作用

氧化应激是诱导性多能干细胞(induced pluripotent stem cell, iPSC)在培养和应用中遇到的一个关键问题,探讨其作用机制具有重要的理论和实践意义。目前有关iPSC氧化应激的研究相对较少,Nrf2/HO-1信号通路在其中的作用尚不明了。因此,本研究以不同浓度的H2O2(100、200、300、400 μmol/L)处理人iPSC(hiPSC),分别在4 h和24 h于倒置显微镜下观察hiPSC及其饲养层细胞SNL氧化损伤的程度,通过碱性磷酸酶（alkaline phosphatase, AP）试剂盒和超氧化物阴离子荧光探针,分别检测hiPSC多能性和细胞活性氧(reactive oxygen species, ROS)水平,并通过qRT-PCR检测H2O2处理4 h后早期应激状态下Nrf2和HO 1 mRNA的表达水平,免疫细胞化学和Western印迹检测p-Nrf2和HO-1蛋白质的表达量。结果表明：hiPSC和SNL细胞的ROS水平呈H2O2剂量依赖性升高。除了100 μmol/L H2O2组hiPSC的细胞形态和多能性保持较好外,其余浓度H2O2均导致hiPSC出现不同程度损伤和死亡。但与SNL细胞相比,hiPSC中ROS水平相对较低,细胞状态也相对较好。SNL细胞中Nrf2和HO-1-mRNA表达的变化幅度与H2O2浓度呈线性相关,而hiPSC中Nrf2和HO-1表达的变化幅度与H2O2浓度之间并未呈现线性相关,其中Nrf2在100 μmol/L H2O2组表达量最高,而HO-1在200 μmol/L H2O2组表达量最高,意味着hiPSC氧化应激调控机制的复杂性。综上结果表明,hiPSC具有较好的抗氧化能力,其相关机制与Nrf2/HO-1信号通路有关,同时也可能涉及到其它相关通路的交互作用。     

Activation of hypoxia-inducible factor-1 protects airway epithelium against oxidant-induced barrier dysfunction

The respiratory epithelium forms an important barrier against inhaled pollutants and microorganisms, and its barrier function is often compromised during inflammatory airway diseases. Epithelial activation of hypoxia-inducible factor-1 (HIF-1) represents one feature of airway inflammation, but the functional importance of HIF-1 within the respiratory epithelium is largely unknown. Using primary mouse tracheal epithelial (MTE) cells or immortalized human bronchial epithelial cells (16HBE14o-), we evaluated the impact of HIF-1 activation on loss of epithelial barrier function during oxidative stress. Exposure of either 16HBE14o- or MTE cells to H(2)O(2) resulted in significant loss of transepithelial electrical resistance and increased permeability to fluorescein isothiocyanate-dextran (4 kDa), and this was attenuated significantly after prior activation of HIF-1 by preexposure to hypoxia (2% O(2); 6 h) or the hypoxia mimics CoCl(2) or dimethyloxalylglycine (DMOG). Oxidative barrier loss was associated with reduced levels of the tight junction protein occludin and with hyperoxidation of the antioxidant enzyme peroxiredoxin (Prx-SO(2)H), events that were also attenuated by prior activation of HIF-1. Involvement of HIF-1 in these protective effects was confirmed using the pharmacological inhibitor YC-1 and by short-hairpin RNA knockdown of HIF-1α. The protective effects of HIF-1 were associated with induction of sestrin-2, a hypoxia-inducible enzyme known to reduce oxidative stress and minimize Prx hyperoxidation. Together, our results suggest that loss of epithelial barrier integrity by oxidative stress is minimized by activation of HIF-1, in part by induction of sestrin-2.     

Bovine pituitary extract provides remarkable protection against oxidative stress in human prostate epithelial cells

Bovine pituitary extract (BPE) is routinely used as a mitogenic supplement in serum-free growth medium. In addition to its mitogenic activity, BPE contains a variety of growth factors and hormones with reported antioxidant activity. This study examines the antioxidant potential of BPE in nontumorigenic human prostate epithelial cells (RWPE-1). Treatment of RWPE-1 cells with BPE (50 microg/ml) provided significant protection against H(2)O(2)-induced cell death, deoxyribonucleic acid fragmentation, protein oxidation, and membrane damage. Treatment with heat (71 degrees C, 10 min) and proteolytic enzymes reduced the antioxidant activity of BPE, suggesting that proteins present in BPE may be responsible for the antioxidant activity. Residual catalase activity present in BPE was responsible for a portion (30%) of the antioxidant activity. Interestingly, RWPE-1 cells treated with BPE and H(2)O(2) rapidly accumulated intracellular reactive oxygen species (ROS) to a greater extent than cells receiving only H(2)O(2). Pretreatment of RWPE-1 cells with tyrosine kinase inhibitors (genistein, tyrphostin 47, and AG-1296) before the addition of H(2)O(2) diminished BPE protection against H(2)O(2)-induced cell death, whereas treatment with purified mitogens commonly found in BPE, growth hormone and basic fibroblast growth factor, did not protect against oxidative damage. Taken together, these data suggest that BPE contains proteins or protein complexes with remarkable antioxidant activity. These yet unidentified compounds appear to confer protection against H(2)O(2)-induced cell death by tyrosine kinase-dependent pathways that increase intracellular ROS generation. The antioxidant activity of BPE may represent a confounding variable when studying oxidative stress in cells maintained in BPE-supplemented serum-free medium.     

Mechanism of vitamin C inhibition of cell death induced by oxidative stress in glutathione-depleted HL-60 cells

Vitamin C is a well known antioxidant whose precise role in protecting cells from oxidative challenge is uncertain. In vitro results have been confounded by pro-oxidant effects of ascorbic acid and an overlapping role of glutathione. We used HL-60 cells as a model to determine the precise and independent role of vitamin C in cellular protection against cell death induced by oxidative stress. HL-60 cells do not depend on glutathione to transport or reduce dehydroascorbic acid. Depletion of glutathione rendered the HL-60 cells highly sensitive to cell death induced by H2O2, an effect that was not mediated by changes in the activities of glutathione reductase, glutathione peroxidase, catalase, or superoxide dismutase. The increased sensitivity to oxidative stress was largely reversed when glutathione-depleted cells were preloaded with ascorbic acid by exposure to dehydroascorbic acid. Resistance to H2O2 treatment in cells loaded with vitamin C was accompanied by intracellular consumption of ascorbic acid, generation of dehydroascorbic acid, and a decrease in the cellular content of reactive oxygen species. Some of the dehydroascorbic acid generated was exported out of the cells via the glucose transporters. Our data indicate that vitamin C is an important independent antioxidant in protecting cells against death from oxidative stress.     

DLPC decreases TGF-beta1-induced collagen mRNA by inhibiting p38 MAPK in hepatic stellate cells

Dilinoleoylphosphatidylcholine (DLPC), the active component of polyenylphosphatidylcholine extracted from soybeans, decreases collagen accumulation induced by TGF-beta1 in cultured hepatic stellate cells (HSCs). Because DLPC exerts antioxidant effects and TGF-beta1 generates oxidative stress, we evaluated whether the antifibrogenic effect of DLPC is linked to its antioxidant action. In passage 1 culture of rat HSCs, TGF-beta1 induced a concentration-dependent increase in procollagen-alpha(1)(I) mRNA levels and enhanced intracellular H(2)O(2) and superoxide anion formation and lipid peroxidation but decreased GSH levels. These changes were prevented by DLPC. Upregulation of collagen mRNA by TGF-beta1 was likewise inhibited by catalase and p38 MAPK inhibitor SB-203580, suggesting involvement of H(2)O(2) and p38 MAPK signaling in this process. TGF-beta1 or addition of H(2)O(2) to HSCs activated p38 MAPK with a rise in procollagen mRNA level; these changes were blocked by catalase and SB-203580 and likewise by DLPC. alpha-Smooth muscle actin abundance in HSCs was not altered by TGF-beta1 treatment (with or without DLPC), indicating that downregulation of procollagen mRNA by DLPC was not due to alteration in HSC activation. These results demonstrate that DLPC prevents TGF-beta1-induced increase in collagen mRNA by inhibiting generation of oxidative stress and associated H(2)O(2)-dependent p38 MAPK activation, which explains its antifibrogenic effect. DLPC, an innocuous phospholipid, may be considered for prevention and treatment of liver fibrosis.     

Depolarization of In Situ Mitochondria Due to Hydrogen Peroxide-Induced Oxidative Stress in Nerve Terminals

Mitochondrial membrane potential (delta psi(m)) was determined in intact isolated nerve terminals using the membrane potential-sensitive probe JC-1. Oxidative stress induced by H2O2 (0.1-1 mM) caused only a minor decrease in delta psi(m). When complex I of the respiratory chain was inhibited by rotenone (2 microM), delta psi(m) was unaltered, but on subsequent addition of H2O2, delta psi(m) started to decrease and collapsed during incubation with 0.5 mM H2O2 for 12 min. The ATP level and [ATP]/[ADP] ratio were greatly reduced in the simultaneous presence of rotenone and H2O2. H2O2 also induced a marked reduction in delta psi(m) when added after oligomycin (10 microM), an inhibitor of F0F1-ATPase. H2O2 (0.1 or 0.5 mM) inhibited alpha-ketoglutarate dehydrogenase and decreased the steady-state NAD(P)H level in nerve terminals. It is concluded that there are at least two factors that determine delta psi(m) in the presence of H2O2: (a) The NADH level reduced owing to inhibition of alpha-ketoglutarate dehydrogenase is insufficient to ensure an optimal rate of respiration, which is reflected in a fall of delta psi(m) when the F0F1-ATPase is not functional. (b) The greatly reduced ATP level in the presence of rotenone and H2O2 prevents maintenance of delta psi(m) by F0F1-ATPase. The results indicate that to maintain delta psi(m) in the nerve terminal during H2O2-induced oxidative stress, both complex I and F0F1-ATPase must be functional. Collapse of delta psi(m) could be a critical event in neuronal injury in ischemia or Parkinson's disease when H2O2 is generated in excess and complex I of the respiratory chain is simultaneously impaired.     

NF-kappa B mediates the adaptation of human U937 cells to hydrogen peroxide

Low doses of oxidative stress can induce cellular resistance to subsequent higher doses of the same stress. By using human U937 leukemia cells, we previously demonstrated that H(2)O(2) can induce such an adaptive response without elevating the cellular capacity to degrade H(2)O(2), and were able to confer the cells a cross-resistance to an H(2)O(2)-independent lethal stimulus, C(2)-ceramide. In this study, it was found that the adaptation is accompanied by the translocation of cytoplasmic NF-kappa B to the nuclei. This event was promoted or abolished when either IKK alpha or a dominant negative mutant of I kappa B, respectively, was overexpressed. The overexpression of IKK alpha also resulted in the suppression of H(2)O(2)-induced cell death and DNA fragmentation, whereas these events were accelerated by the expression of the I kappa B mutant. The protective effect of IKK alpha was accompanied neither by an elevation of protein levels of various antioxidant enzymes such as catalase, superoxide dismutase, and glutathione peroxidase, nor by an increase in the cellular capacity to consume H(2)O(2). Moreover, the overexpression of IKK alpha resulted in an enhancement of H(2)O(2)-induced resistance to C(2)-ceramide. The overall data suggest that NF-kappa B mediates the H(2)O(2) adaptation induced in a manner independent of H(2)O(2)-degrading activity.