H2O2-Induced Cell Death in Human Glioma Cells: Role of Lipid Peroxidation and PARP Activation

underlying mechanism of ROS-induced cell injury remains to be defined. This study was undertaken to examine the role of lipid peroxidation and poly (ADP-ribose) polymerase (PARP) activation in H₂O₂-induced cell death in A172 cells, a human glioma cell line. H₂O₂ induced a dose- and time-dependent cell death. The cell death was prevented by thiols (dithiothreitol and glutathione), iron chelators (deferoxamine and phenanthroline), H₂O₂ scavengers (catalase and pyruvate), and a hydroxyl radical scavenger (dimethylthiourea). Antioxidants N,N-diphenyl-p-phenylenediamine (DPPD) and Trolox had no effect on the H₂O₂-induced cell death. Lipid peroxidation did not increase in human glioma cells exposed to H₂O₂. The PARP inhibitor 3-aminobenzamide prevented the cell death induced by H₂O₂. The PARP activity was increased by H₂O₂ and the H₂O₂ effect was prevented by 3-aminobenzamide, dithiothreitol, and phenanthroline. The ATP depletion induced by H₂O₂ was prevented by catalase, dithiothreitol, phenanthroline, and 3-aminobenzamide, but not by DPPD. These results indicate that the H₂O₂-induced cell death is mediated by PARP activation but not by lipid peroxidation in human glioma cells.     

Role of ERK in Hydrogen Peroxide-Induced Cell Death of Human Glioma Cells

Oxidative stress is known to induce cell death in a wide variety of cell types, apparently by modulating intracellular signaling pathways. Activation of extracellular signal-regulated kinase (ERK) in oxidative stress remains controversial. In some cellular systems, the ERK activation is associated with protection against oxidative stress, while in other system, the ERK activation is involved in apoptotic cell death. The present study was undertaken to examine the role of ERK activation in H₂O₂-induced cell death of human glioma (A172) cells. H₂O₂ resulted in a time- and dose-dependent cell death, which was largely attributed to apoptosis. H₂O₂ treatment caused marked sustained activation of ERK. The ERK activation and cell death induced by H₂O₂ was prevented by catalase, the hydrogen peroxide scavenger, and U0126, an inhibitor of ERK upstream kinase MEK1/2. Transient transfection with constitutive active MEK1, an upstream activator of ERK1/2, increased H₂O₂-induced cell death, whereas transfection with dominant-negative mutants of MEK1 decreased the cell death. The ERK activation and cell death caused by H₂O₂ was inhibited by antioxidants (N-acetylcysteine and trolox), Ras inhibitor, and suramin. H₂O₂ produced depolarization of mitochondrial membrane potential and its effect was prevented by catalase and U0126. Taken together, these findings suggest that growth factor receptor/Ras/MEK/ERK signaling pathway plays an active role in mediating H₂O₂-induced apoptosis of human glioma cells and functions upstream of mitochondria-dependent pathway to initiate the apoptotic signal.     

Beta-nodavirus B2 protein induces hydrogen peroxide production,leading to Drp1-recruited mitochondrial fragmentation and cell death via mitochondrial targeting

Because the role of the viral B2 protein in the pathogenesis of nervous necrosis virus infection remains unknown, the aim of the present study was to determine the effects of B2 protein on hydrogen peroxide (H₂O₂)-mediated cell death via mitochondrial targeting. Using a B2 deletion mutant, the B2 mitochondrial targeting signal sequence (⁴¹RTFVISAHAA⁵⁰) correlated with mitochondrial free radical production and cell death in fish cells, embryonic zebrafish, and human cancer cells. After treatment of grouper fin cells (GF-1) overexpressing B2 protein with the anti-oxidant drug, N-acetylcysteine (NAC), and overexpression of the antioxidant enzymes, zfCu/Zn superoxide dismutase (SOD) and zfCatalase, decreased H₂O₂ production and cell death were observed. To investigate the correlation between B2 cytotoxicity and H₂O₂ production in vivo, B2 was injected into zebrafish embryos. Cell damage, as assessed by the acridine orange assay, gradually increased over 24 h post-fertilization, and was accompanied by marked increases in H₂O₂ production and embryonic death. Increased oxidative stress, as evidenced by the up-regulation of Mn SOD, catalase, and Nrf2, was also observed during this period. Finally, B2-induced dynamin-related protein 1 (Drp1)-mediated mitochondrial fragmentation and cell death could be reversed by NAC and inhibitors of Drp1 and Mdivi in GF-1 cells. Taken together, betanodavirus B2 induces H₂O₂ production via targeting the mitochondria, where it inhibits complex II function. H₂O₂ activates Drp1, resulting in its association with the mitochondria, mitochondrial fission and cell death in vitro and in vivo.     

Hydrogen peroxide production and mitochondrial dysfunction contribute to the fusaric acid-induced programmed cell death in tobacco cells

Fusaric acid (FA), a non-specific toxin produced mainly by Fusarium spp., can cause programmed cell death (PCD) in tobacco suspension cells. The mechanism underlying the FA-induced PCD was not well understood. In this study, we analyzed the roles of hydrogen peroxide (H₂O₂) and mitochondrial function in the FA-induced PCD. Tobacco suspension cells were treated with 100 μM FA and then analyzed for H₂O₂ accumulation and mitochondrial functions. Here we demonstrate that cells undergoing FA-induced PCD exhibited H₂O₂ production, lipid peroxidation, and a decrease of the catalase and ascorbate peroxidase activities. Pre-treatment of tobacco suspension cells with antioxidant ascorbic acid and NADPH oxidase inhibitor diphenyl iodonium significantly reduced the rate of FA-induced cell death as well as the caspase-3-like protease activity. Moreover, FA treatment of tobacco cells decreased the mitochondrial membrane potential and ATP content. Oligomycin and cyclosporine A, inhibitors of the mitochondrial ATP synthase and the mitochondrial permeability transition pore, respectively, could also reduce the rate of FA-induced cell death significantly. Taken together, the results presented in this paper demonstrate that H₂O₂ accumulation and mitochondrial dysfunction are the crucial events during the FA-induced PCD in tobacco suspension cells.     

Silencing of miR-23a attenuates hydrogen peroxide (H2O2) induced oxidative damages in ARPE-19 cells by upregulating GLS1: an in vitro study

BackgroundOxidative damages contributes to age-related macular degeneration (AMD) caused vision blindness, but the molecular mechanisms are still largely unknown.ObjectivesThis study managed to investigate this issue by conducting in vitro experiments.MethodsOxidative stress were evaluated by L-012 dye, DHE staining and MDA assay. CCK-8 and colony formation assay were conducted to examine cell proliferation. Cell death was evaluated by trypan blue staining and Annexin V-FITC/PI double staining method through flow cytometry (FCM). The binding sites of miR-23a and GLS1 mRNA were predicted by online miRDB database and validated by dual-luciferase reporter gene system. Real-Time qPCR for miR-23a levels and Western Blot for protein expressions.ResultsThe retinal pigment epithelial (RPE) cells (ARPE-19) were subjected to hydrogen peroxide (H₂O₂) stimulation to simulate AMD progression in vitro, and we identified a novel miR-23a/glutaminase-1 (GLS1) pathway that regulated H₂O₂ induced oxidative damages in ARPE-19 cells. Mechanistically, H₂O₂ induced oxidative stress, inhibited cell proliferation and induced cell death in ARPE-19 cells in a dose- and time-dependent manner. Also, H₂O₂ stimulation hindered cell invasion, migration and glutamine uptake in ARPE-19 cells. Interestingly, we proved that H₂O₂ increased miR-23a levels, while downregulated glutaminase-1 (GLS1) in ARPE-19 cells, and miR-23a targeted 3′ untranslated region (3′UTR) of GLS1 mRNA for GLS1 degradation. Finally, our data suggested that silencing miR-23a upregulated GLS1 to reverse the detrimental effects of H₂O₂ treatment on ARPE-19 cells.ConclusionsIn general, analysis of the data suggested that miR-23a ablation upregulated GLS1 to attenuate H₂O₂ stimulation induced oxidative damages in ARPE-19 cells in vitro, and this study broadened our knowledge in this field, which might help to provide novel theranostic signatures for AMD.     

Over-expression of Trxo1 increases the viability of tobacco BY-2 cells under H2O2 treatment

Background and Aims Reactive oxygen species (ROS), especially hydrogen peroxide, play a critical role in the regulation of plant development and in the induction of plant defence responses during stress adaptation, as well as in plant cell death. The antioxidant system is responsible for controlling ROS levels in these processes but redox homeostasis is also a key factor in plant cell metabolism under normal and stress situations. Thioredoxins (Trxs) are ubiquitous small proteins found in different cell compartments, including mitochondria and nuclei (Trxo1), and are involved in the regulation of target proteins through reduction of disulphide bonds, although their role under oxidative stress has been less well studied. This study describes over-expression of a Trxo1 for the first time, using a cell-culture model subjected to an oxidative treatment provoked by H₂O₂.Methods Control and over-expressing PsTrxo1 tobacco (Nicotiana tabacum) BY-2 cells were treated with 35 mm H₂O₂ and the effects were analysed by studying the growth dynamics of the cultures together with oxidative stress parameters, as well as several components of the antioxidant systems involved in the metabolism of H₂O₂. Analysis of different hallmarks of programmed cell death was also carried out.Key Results Over-expression of PsTrxo1 caused significant differences in the response of TBY-2 cells to high concentrations of H₂O₂, namely higher and maintained viability in over-expressing cells, whilst the control line presented a severe decrease in viability and marked indications of oxidative stress, with generalized cell death after 3 d of treatment. In over-expressing cells, an increase in catalase activity, decreases in H₂O₂ and nitric oxide contents and maintenance of the glutathione redox state were observed.Conclusions A decreased content of endogenous H₂O₂ may be responsible in part for the delayed cell death found in over-expressing cells, in which changes in oxidative parameters and antioxidants were less extended after the oxidative treatment. It is concluded that PsTrxo1 transformation protects TBY-2 cells from exogenous H₂O₂, thus increasing their viability via a process in which not only antioxidants but also Trxo1 seem to be involved.     

Oxidative stress response of Blakeslea trispora induced by H2O2 during β-carotene biosynthesis

The cellular response of Blakeslea trispora to oxidative stress induced by H₂O₂ in shake flask culture was investigated in this study. A mild oxidative stress was created by adding 40 μm of H₂O₂ into the medium after 3 days of the fermentation. The production of β-carotene increased nearly 38 % after a 6-day culture. Under the oxidative stress induced by H₂O₂, the expressions of hmgr, ipi, carG, carRA, and carB involving the β-carotene biosynthetic pathway all increased in 3 h. The aerobic metabolism of glucose remarkably accelerated within 24 h. In addition, the specific activities of superoxide dismutase and catalase were significantly increased. These changes of B. trispora were responses for reducing cell injury, and the reasons for increasing β-carotene production caused by H₂O₂.     

Involvement of catalase in the apoptotic mechanism induced by apigenin in HepG2 human hepatoma cells

Apigenin has been reported to inhibit proliferation of cancer cells; however, the mechanism underlying its action is not completely understood. Here, we evaluated the effects of apigenin on the levels of expression and activity of antioxidant enzymes, and the involvement of ROS in the mechanism of cell death induced by apigenin in HepG2 human hepatoma cells. Upon treatment with apigenin, HepG2 cells displayed a reduction in cell viability in a dose- and time-dependent manner, and some morphological changes. In addition, apigenin treatment induced ROS generation and significantly decreased the mRNA levels and activity of catalase and levels of intracellular GSH. On the other hand, apigenin treatment did not alter the expression or activity levels of other antioxidant enzymes. Addition of exogenous catalase significantly reduced the effects of apigenin on HepG2 cell death. We also demonstrated that HepG2 cells are more sensitive to apigenin-mediated cell death than are primary cultures of mouse hepatocytes, suggesting a differential toxic effect of this agent in tumor cells. Our results suggest that apigenin-induced apoptosis in HepG2 cells may be mediated by a H₂O₂-dependent pathway via reduction of the antioxidant defenses.     

Ozone-Induced Cell Death Mediated with Oxidative and Calcium Signaling Pathways in Tobacco Bel-W3 and Bel-B Cell Suspension Cultures

Ozone (O₃)-induced cell death in two suspension-cultured cell lines of tobacco (Nicotiana tabacum L.) derived from Bel-W3 (hyper-sensitive to O₃) and Bel-B (highly tolerant to O₃) varieties were studied. By exposing the newly prepared cell lines to the pulse of ozonized air, we could reproduce the conditions demonstrating the difference in O₃ sensitivity as observed in their original plants, depending on the exposure time. Since O₃-induced acute cell death was observed in the dark, the requirement for photochemical reactions could be eliminated. Addition of several ROS scavengers and chelators inhibited the cell death induced by O₃, indicating that singlet oxygen (¹O₂), hydrogen peroxide (H₂O₂), hydroxyl radical and redox-active metals such as Fe²⁺ play central roles in O₃-induced acute damages to the cells. As expected, we observed the generation of ¹O₂ and H₂O₂ in the O₃-treated cells using chemiluminescent probes. On the other hand, an NADPH oxidase inhibitor, superoxide dismutase (SOD), and some SOD mimics showed no inhibitory effect. Thiols added as antioxidants unexpectedly behaved as prooxidants drastically enhancing the O₃-induced cell death. It is noteworthy that some ROS scavengers effectively rescued the cells from dying even treated after the pulse of O₃ exposure, confirming the post-ozone progress of ROS-dependent cell death mechanism. Since one of the key differences between Bel-B and Bel-W3 was suggested to be the capacity for ROS detoxification by catalase, the endogenous catalase activities were compared in vivo in two cell lines. As expected, catalase activity in Bel-B cells was ca. 7-fold greater than that in Bel-W3 cells. Interestingly, Ca²⁺ chelators added prior to (not after) the pulse of O₃ effectively inhibited the induction of cell death. In addition, increases in cytosolic Ca²⁺ concentration sensitive to Ca²⁺ chelators, ion channel blockers, and ROS scavengers were observed in the transgenic Bel-W3 cells expressing aequorin, suggesting the action of Ca²⁺ as a secondary messenger initiating the oxidative cell death. The O₃-induced calcium response in Bel-W3 cells was much greater than Bel-B cells. Based on the results, possible pathways for O₃-dependent generation of the lethal level of ROS and corresponding signaling mechanism for induction of cell death were discussed.Key Words: calcium, cell death, Nicotiana tabacum L., ozone, reactive oxygen species     

Arginase-II promotes melanoma migration and adhesion through enhancing hydrogen peroxide production and STAT3 signaling

Elevated arginase type II (Arg-II) associates with higher grade tumors. Its function and underlying molecular mechanisms in melanoma remain elusive. In the present study, we observed a significantly higher frequency of Arg-II expression in melanoma of patients with metastasis than those without metastasis. Silencing Arg-II in two human melanoma cell lines slowed down the cell growth, while overexpression of native but not a catalytically inactive Arg-II promoted cell proliferation without affecting cell death. Treatment of cells with arginase inhibitor also reduced melanoma cell number, demonstrating that Arg-II promotes melanoma cell proliferation dependently of its enzymatic activity. However, results from silencing Arg-II or overexpressing native or the inactive Arg-II as well as treatment with arginase inhibitor showed that Arg-II promotes melanoma metastasis-related processes, such as melanoma cell migration and adhesion on endothelial cells, independently of its enzymatic activity. Moreover, the treatment of the cells with STAT3 inhibitor suppressed Arg-II-promoted melanoma cell migration and adhesion. Furthermore, catalase, but not superoxide dismutase, prevented STAT3 activation as well as increased melanoma cell migration and adhesion induced by overexpressing native or the inactive Arg-II. Taken together, our study uncovers both activity-dependent and independent mechanisms of Arg-II in promoting melanoma progression. While Arg-II enhances melanoma cell proliferation through polyamine dependently of its enzymatic activity, it promotes metastasis-related processes, that is, migration and adhesion onto endothelial cell, through mitochondrial H₂O₂-STAT3 pathway independently of the enzymatic activity. Suppressing Arg-II expression rather than inhibiting its enzymatic activity may, therefore, represent a novel strategy for the treatment of melanoma.     

H2O2-induced Leaf Cell Death and the Crosstalk of Reactive Nitric/Oxygen Species([F])

In plants, the chloroplast is the main reactive oxygen species (ROS) producing site under high light stress. Catalase (CAT), which decomposes hydrogen peroxide (H₂O₂), is one of the controlling enzymes that maintains leaf redox homeostasis. The catalase mutants with reduced leaf catalase activity from different plant species exhibit an H₂O₂‐induced leaf cell death phenotype. This phenotype was differently affected by light intensity or photoperiod, which may be caused by plant species, leaf redox status or growth conditions. In the rice CAT mutant nitric oxide excess 1 (noe1), higher H₂O₂ levels induced the generation of nitric oxide (NO) and higher S‐nitrosothiol (SNO) levels, suggesting that NO acts as an important endogenous mediator in H₂O₂‐induced leaf cell death. As a free radical, NO could also react with other intracellular and extracellular targets and form a series of related molecules, collectively called reactive nitrogen species (RNS). Recent studies have revealed that both RNS and ROS are important partners in plant leaf cell death. Here, we summarize the recent progress on H₂O₂‐induced leaf cell death and the crosstalk of RNS and ROS signals in the plant hypersensitive response (HR), leaf senescence, and other forms of leaf cell death triggered by diverse environmental conditions. [ Chengcai Chu (Corresponding author)]     

Neuroprotective Effects of Hydroalcoholic Extract of Ocimum sanctum Against H2O2 Induced Neuronal Cell Damage in SH-SY5Y Cells via Its Antioxidative Defence Mechanism

Oxidative stress mediates the cell damage in several ailments including neurodegenerative conditions. Ocimum sanctum is widely used in Indian ayurvedic medications to cure various ailments. The present study was carried out to investigate the antioxidant activity and neuroprotective effects of hydroalcoholic extract of O. sanctum (OSE) on hydrogen peroxide (H₂O₂)-induced oxidative challenge in SH-SY5Y human neuronal cells. The extract exhibited strong antioxidant activity against DPPH, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical and hydroxyl radicals with IC₅₀ values of 395 ± 16.2, 241 ± 11.5 and 188.6 ± 12.2 μg/ml respectively, which could be due to high amount of polyphenols and flavonoids. The observed data demonstrates 41.5 % cell survival with 100 μM H₂O₂ challenge for 24 h, which was restored to 73 % by pre-treatment with OSE for 2 h. It also decreased the lactate dehydrogenase leakage and preserved the cellular morphology. Similarly OSE inhibited lipid peroxidation, DNA damage, reactive oxygen species generation and depolarization of mitochondrial membrane. The extract restored superoxide dismutase and catalase enzyme/protein levels and further downregulated HSP-70 over-expression. These findings suggest that OSE ameliorates H₂O₂ induced neuronal damage via its antioxidant defence mechanism and might be used to treat oxidative stress mediated neuronal disorders.     

Lipopolysaccharides can protect mesenchymal stem cells (MSCs) from oxidative stress-induced apoptosis and enhance proliferation of MSCs via Toll-like receptor(TLR)-4 and PI3K/Akt

Apoptosis of implanted mesenchymal stem cells (MSCs) limits the efficiency of MSC therapy. Recent studies showed the ligands of Toll-like receptors (TLRs) could control the function of these cells. We have investigated the effect of lipopolysaccharides (LPS), a ligand of TLR4, on the survival of MSCs and explored the roles of TLR4 and PI3K/Akt. H₂O₂/serum deprivation(H₂O₂/SD) induced apoptosis of MSCs but LPS-preconditioning (1.0 μg/ml) protected MSCs from H₂O₂/SD-induced apoptosis and promoted their proliferation. Western blotting showed that 1.0 μg/ml LPS enhanced phosphorylation of both Akt at Ser 473 and nuclear factor-kappa B (NF-κB) p65 at Ser 536. However, the protective effects of LPS on survival were not observed in TLR4^lps-del MSCs. The results suggest appropriate treatments with LPS can protect MSCs from oxidative stress-induced apoptosis and improve the survival of MSCs via the TLR4 and PI3K/Akt pathway.     

Fibroblast growth factor-8 inhibits oxidative stress-induced apoptosis in H9c2 cells

Fibroblast growth factors (FGFs) comprise a large family of signaling molecules that involve cell patterning, mobilization, differentiation, and proliferation. Various FGFs, including FGF-1, FGF-2, and FGF-5, have been shown to play a role in cytoprotection during adverse cardiac events; however, whether FGF-8 is a cytoprotective remains unclear. The current study was designed to evaluate the effect of FGF-8 treatment on oxidative stress-induced apoptosis in H9c2 cells. Cells were divided into three groups: control, H₂O₂ (400 µm H₂O₂), and H₂O₂ + FGF-8 (4 ng/ml FGF-8). Our results suggest apoptosis was significantly (p < 0.05) enhanced in the H₂O₂ group relative to control. Moreover, a significant (p < 0.05) decline in apoptosis was observed in the H₂O₂ + FGF-8 group compared to H₂O₂-treated cells as evidenced by TUNEL staining, a cell death detection ELISA, and cell viability. Levels of downstream apoptotic mediators, caspase-3 and caspase-9, were significantly (p < 0.05) upregulated following H₂O₂ treatment but were abrogated following FGF-8 application. Expression levels of Forkhead box protein O1 (FoxO-1), MnSOD, catalase, pAKT, and p-mTOR were significantly (p < 0.05) reduced in the H₂O₂ group (p < 0.05). Notably, these levels were significantly (p < 0.05) reversed following FGF-8 treatment. Our data, for the first time, suggest FGF-8 is an anti-apoptotic mediator in oxidative-stressed H9c2 cells. Furthermore, our data demonstrate that apoptotic inhibition by FGF-8 is consequent to FoxO-1 oxidative detoxification as well as augmentation to the PI3K/AKT cell survival pathway.     

Insulin increases H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced pancreatic beta cell death

Insulin resistance results, in part, from impaired insulin signaling in insulin target tissues. Consequently, increased levels of insulin are necessary to control plasma glucose levels. The effects of elevated insulin levels on pancreatic beta (β) cell function, however, are unclear. In this study, we investigated the possibility that insulin may influence survival of pancreatic β cells. Studies were conducted on RINm, RINm5F and Min-6 pancreatic β-cells. Cell death was induced by treatment with H₂O₂, and was estimated by measurements of LDH levels, viability assay (Cell-Titer Blue), propidium iodide staining and FACS analysis, and mitochondrial membrane potential (JC-1). In addition, levels of cleaved caspase-3 and caspase activity were determined. Treatment with H₂O₂ increased cell death; this effect was increased by simultaneous treatment of cells with insulin. Insulin treatment alone caused a slight increase in cell death. Inhibition of caspase-3 reduced the effect of insulin to increase H₂O₂-induced cell death. Insulin increased ROS production by pancreatic β cells and increased the effect of H₂O₂. These effects were increased by inhibition of IR signaling, indicative of an effect independent of the IR cascade. We conclude that elevated levels of insulin may act to exacerbate cell death induced by H₂O₂ and, perhaps, other inducers of apoptosis.     

Involvement of Volume-Activated Chloride Channels in H2O2 Preconditioning Against Oxidant-Induced Injury Through Modulating Cell Volume Regulation Mechanisms and Membrane Permeability in PC12 Cells

The functions of chloride channels in preconditioning-induced cell protection remain unclear. In this report, we show that the volume-activated chloride channels play a key role in hydrogen peroxide (H₂O₂) preconditioning-induced cell protection in pheochromocytoma PC12 cells. The preconditioning with 100 μM H₂O₂ for 90 min protected the cells from injury induced by long period exposure to 300 μM H₂O₂. The protective effect was attenuated by pretreatment with the chloride channel blockers, 5-nitro-2-3-phenylpropylamino benzoic acid (NPPB) and tamoxifen. H₂O₂ preconditioning directly activated a chloride current, which was moderately outward-rectified and sensitive to the chloride channel blockers and hypertonicity-induced cell shrinkage. H₂O₂ preconditioning functionally up-regulated the activities of volume-activated chloride channels and enhanced the regulatory volume decrease when exposure to extracellular hypotonic challenges. In addition, acute application of H₂O₂ showed distinctive actions on cell volume and membrane permeability in H₂O₂ preconditioned cells. In H₂O₂ preconditioned cells, acute application of 300 μM H₂O₂ first promptly induced a decrease of cell volume and enhancement of cell membrane permeability, and then, cell volume was maintained at a relatively stable level and the facilitation of membrane permeability was reduced. Conversely, in control cells, 300 μM H₂O₂ induced a slow but persistent apoptotic volume decrease (AVD) and facilitation of membrane permeability. H₂O₂ preconditioning also significantly up-regulated the expression of ClC-3 protein, the molecular candidate of the volume-activated chloride channel. These results suggest that H₂O₂ preconditioning can enhance the expression and functional activities of volume-activated chloride channels, thereby modulate cell volume and cell membrane permeability, which may contribute to neuroprotection against oxidant-induced injury.     

17β-Estradiol and steady-state concentrations of H2O2: antiapoptotic effect in endometrial cells from patients with endometriosis

Increased levels of hydrogen peroxide (H₂O₂) can initiate protective responses to limit or repair oxidative damage. However, H₂O₂ signals also fine-tune responses to growth factors and cytokines controlling cell division, differentiation, and proliferation. Because 17β-estradiol (E₂) also plays important roles in these processes, and is considered a major risk factor in the development and progression of endometriosis, this study evaluated whether E₂ has an antiapoptotic effect on oxidative stress in endometrial cells in combination with steady-state H₂O₂ levels ([H₂O₂]ss). Endometrial stromal cells were prepared from the eutopic endometrium of 18 women with and without endometriosis to produce primary cells. These cells were stimulated with E₂ for 20 h, exposed to [H₂O₂]ss, and examined for cell viability, proliferation, and apoptosis. The endometrial cells from women with endometriosis maintained the steady state for 120 min at high H₂O₂ concentrations. When they were pretreated with E₂ and exposed to [H₂O₂]ss, a decrease in apoptosis level was observed compared to the control cells (p<0.01). The endometrial cells from patients with endometriosis subjected to both E₂ and [H₂O₂]ss showed increased ERK phosphorylation. These findings suggested that H₂O₂ is a signaling molecule that downregulates apoptosis in endometrial cells, supporting the fact that endometriosis, albeit a benign disease, shares some features with cancer such as decreased catalase levels. These results link the E₂ effects on [H₂O₂]ss to resistance to apoptosis and progression of endometriosis.     

Loss of oxidative stress tolerance in hypertension is linked to reduced catalase activity and increased c-Jun NH2-terminal kinase activation

Hypertension is accompanied by increased levels of reactive oxygen species, which may contribute to progressive renal injury and dysfunction. Here we tested the hypothesis that sensitivity to exogenous hydrogen peroxide (H₂O₂) is enhanced in immortalized renal proximal tubular epithelial cells from spontaneously hypertensive rats (SHR) compared to normotensive control Wistar Kyoto rats (WKY). We found that SHR cells were more sensitive to H₂O₂-induced cell death than WKY cells. Lower survival in SHR cells correlated with increased DNA fragmentation, chromatin condensation, and caspase-3 activity, indicating apoptosis. H₂O₂ degradation was slower in SHR than in WKY cells, suggesting that reduced antioxidant enzyme activity might be the basis for their increased sensitivity. In fact, catalase activity was downregulated in SHR cells, whereas glutathione peroxidase activity was similar in both cell types. We next examined whether MAPK signaling pathways contributed to H₂O₂-mediated apoptosis. Inhibition of c-Jun NH₂-terminal kinase (JNK) with SP600125 partially rescued H₂O₂-induced apoptosis in WKY but not in SHR cells. In addition, p54 JNK2 isoform was robustly phosphorylated by H₂O₂, this effect being more pronounced in SHR cells. Together, these results suggest that the survival disadvantage of SHR cells upon exposure to H₂O₂ stems from impaired antioxidant mechanisms and activated JNK proapoptotic signaling pathways.