Over expression of glutamate cysteine ligase increases cellular resistance to H2O2-induced DNA single-strand breaks.

Hydrogen peroxide (H2O2) can cause single strand DNA breaks (ssDNA) in cells when the mechanisms normally in place to reduce it are overwhelmed. Such mechanisms include catalase, glutathione peroxidases (GPx), and peroxiredoxins. The relative importance of these enzymes in H2O2 reduction varies with cell and tissue type. The role of the GPx cofactor glutathione (GSH) in oxidative defense can be further understood by modulating its synthesis. The first and rate-limiting enzyme in GSH synthesis is glutamate-cysteine ligase (GCL), which has a catalytic subunit (Gclc) and a modifier subunit (Gclm). Using mouse hepatoma cells we evaluated the effects of GCL over expression on H2O2-induced changes in GSH and ssDNA break formation with the single cell gel electrophoresis assay (SCG or comet assay), and the acridine orange DNA unwinding flow cytometry assay (AO unwinding assay). Cells over expressing GCL had higher GSH content than control cells, and both SCG and AO unwinding assays revealed that cells over expressing GCL were significantly more resistant to H2O2-induced ssDNA break formation. Furthermore, using the AO unwinding assay, the prevalence of H2O2-induced breaks in different phases of the cell cycle was not different, and the degree of protection afforded by GCL over expression was also not cell cycle phase dependent. Our results support the hypothesis that GCL over expression enhanced GSH biosynthesis and protected cells from H2O2-induced DNA breaks. These results also suggest that genetic polymorphisms that affect GCL expression may be important determinants of oxidative DNA damage and cancer.     

Antioxidant mechanisms of the Nereidid Laeonereis acuta (Anelida: Polychaeta) to cope with environmental hydrogen peroxide

Hydrogen peroxide (H(2)O(2)) is a naturally occurring prooxidant molecule, and its effects in the macroinvertebrate infauna were previously observed. The existence of a gradient of antioxidant enzymes activity (catalase [CAT], glutathione peroxidase [GPx], superoxide dismutase [SOD], and glutathione-S-transferase [GST]) and/or oxidative damage along the body of the estuarine polychaeta Laeonereis acuta (Polychaeta, Nereididae) was analyzed after exposure to H(2)O(2). Because this species secretes conspicuous amounts of mucus, its capability in degrading H(2)O(2) was studied. The results suggest that L. acuta deal with the generation of oxidative stress with different strategies along the body. In the posterior region, higher CAT and SOD activities ensure the degradation of inductors of lipid peroxidation such as H(2)O(2) and superoxide anion (O(2)(.-)). The higher GST activity in anterior region aids to conjugate lipid peroxides products. In the middle region, the lack of high CAT, SOD, or GST activities correlates with the higher lipid hydroperoxide levels found after H(2)O(2) exposure. Ten days of exposure to H(2)O(2) also induced oxidative stress (lipid peroxidation and DNA damage) in the whole animal paralleled by a lack of CAT induction. The mucus production contributes substantially to H(2)O(2) degradation, suggesting that bacteria that grow in this secretion provide this capability.     

胶原蛋白肽经由Nrf2信号通路对H_2O_2诱导的肝细胞氧化损伤的保护作用(英文)

目的:氧化应激在肝脏疾病中扮演着重要的角色。胶原蛋白肽是天然的抗氧化剂,其在动物实验中已经被证实有抑制氧化应激的作用。最新研究证实胶原蛋白肽将有可能被应用在肝脏疾病的预防中,但是很少有研究报道其分子作用机制。因此本研究在胶原蛋白肽是对H2O2诱导的正常人的肝细胞系HL7702氧化损伤有保护作用的基础上,并探索其分子作用机制。方法:实验设空白对照组,H2O2模型组,胶原蛋白肽低、中、高剂量组(10,100,200μg/ml)。胶原蛋白肽各组加入相应浓度的药物预处理12 h后,与模型组一起加入300μM H2O2的H2O2共同培养12 h,空白对照组正常培养。细胞毒性是由CCK8和乳酸脱氢酶(LDH)的释放检测。抗氧化试剂盒检测细胞内活性氧的水平,超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性和丙二醛(MDA)含量的变化。Western blot检测细胞内Nrf2蛋白的表达水平。结果:胶原蛋白肽对H2O2诱导的正常人的肝细胞系HL7702氧化损伤有保护作用。胶原蛋白肽能够及时清除细胞内的活性氧,增加Nrf2的蛋白表达水平,提高超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的活性,减轻脂质过氧化反应,从而保护正常人的肝细胞系HL7702。结论:总之,胶原蛋白肽通过增加Nrf2的蛋白表达水平,提高抗氧化活性,对H2O2诱导损伤的肝细胞发挥保护作用。本研究为胶原蛋白肽的分子作用机制提供了新的证据,将有助于预防氧化应激所致的肝损伤。     

Regulation of reactive oxygen species by nerve growth factor but not Bcl-2 as a novel mechanism of protection of PC12 cells from superoxide anion-induced death

Although neurotrophins protect PC12 cells and neurons from oxidative stress-induced death, the molecular mechanism of this effect is largely unknown. Xanthine (XA)+xanthine oxidase (XO) increased the production of the superoxide anion (O2-) and hydrogen peroxide (H2O2), and the death of PC12 cells. Catalase but not superoxide dismutase (SOD) nor a NO scavenger protected PC12 cells from death, indicating that H2O2 is the main effector responsible for this cell death. Both nerve growth factor (NGF) and Bcl-2 protected PC12 cells from O2--induced toxicity. NGF enhanced the production of O2- and suppressed that of H2O2, suggesting that it inhibits the conversion of O2- to H2O2, while Bcl-2 had no such effect. These results suggested that NGF protected the cells from oxidative stress by altering the composition of the reactive oxygen species (ROS) without affecting their total level.     

A new strategy to enhance glutathione production by multiple H2O2 induced oxidative stresses in Candida utilis

Effect of H(2)O(2)-induced oxidative stress on glutathione (GSH) production in Candida utilis was investigated. Based on the results that H(2)O(2) can effectively stimulate GSH accumulation but inhibit cell growth simultaneously, a novel strategy of multiple H(2)O(2) stresses with different concentrations (1 mmol/L at 4h, 2 mmol/L at 8h, and 4 mmol/L at 12h) were developed to maximize GSH production. As a result, a maximal GSH yield of 218 mg/L was achieved and a corresponding intracellular GSH content was 2.15%, which were 54.6% and 58.1% higher than the control. By further applying this strategy to 7 L fermentor, GSH yield and intracellular GSH content were 328 mg/L and 2.30%. Moreover, increased activities of catalase (CAT) and GSH reductase (GR) indicated that GSH and CAT were directly involved in protecting cell against oxidative stress by H(2)O(2).     

Induced resistance in cells exposed to repeated low doses of H2O2 involves enhanced activity of antioxidant enzymes

We have derived cells from the Chinese hamster V79 cell line by conditioning them with repeated low doses of hydrogen peroxide (H(2)O(2)). This mimics the physiological condition where cells are repeatedly exposed to low levels of oxidants. In an attempt to characterize such cells, we have exposed both conditioned cells (V79(C)) and the parental V79 cells (V79(P)) to different types of cytotoxic agents and compared their sensitivity to cell killing. The V79(C) cells were found to be stably resistant to killing by agents that produced toxicity through oxidative stress, e.g. H(2)O(2) and cisplatin. It was also found that the lipid peroxidation produced by these agents were considerably lower in the V79(C) cells. Thus, the difference in sensitivity could be due to lesser extent of damage to these cells. V79(C) cells had greater antioxidant defense through higher GSH content and greater activity of enzymes such as Cu-Zn superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), which provided protection from damage. Enzyme activities were also assayed at different times after treatment with various cytotoxic agents; there was a relatively large increase in SOD activity which perhaps plays a key role in determining the resistance of the V79(C) cells to killing.     

The antioxidant effects of ribonuclease inhibitor

Ribonuclease inhibitor (RI) is an acidic cytosolic glycoprotein with molecular weight of about 50 kDa, which contains 32 cysteine residues. It is possibly that RI may have antioxidant effect by thiol-disulfide exchange reaction. We studied the effects of RI over-expression on the rat glial cell line C6 injured with H2O2. The transfected C6 cells with RI cDNA (C6') had higher viability, less LDH leakage and MDA contents, but more GSH contents compare that in the control C6 cells. In transfected C6 cells, the activities of CAT and GST were higher than that in the control C6 cells. Without H202 stress, the activities of CAT and GST in the C6' cells were 1.73 and 3.62 times that in the control C6 cells, respectively; With 1.00 mmol/L H2O2 stress, the activities of CATand GSTin the C6' cells were 3.38 and 2.11 times that in the C6 cells, respectively. These results suggest that the over-expression RI has antioxidant activity and it is able to protect cells from per-oxidative injuries. Moreover, we investigated whether RI has a protective role against mouse hepatic damage in vivo. The mice pretreated with different doses of human RI were injected by CC14. The results show that the SOD activities of therapy groups were significantly higher than that of the control group (p < 0.01), while the contents of MOD and activities of ALT and AST in blood were remarkably lower than that of the control group (p < 0.01). Pathological examination shows that the degree of damage was alleviated with RI therapy. These results suggest that RI has the protective role against mouse hepatic damage induced by CC14. The anti-oxidative effects of RI may play an important role in cell protection from per-oxidative injuries.     

N-acetylcysteine increases apoptosis induced by H(2)O(2) and mo-antiFas triggering in a 3DO hybridoma cell line

N-Acetylcysteine (NAC) has been used as an antioxidant to prevent apoptosis triggered by different stimuli in different cell types. It is common opinion that cellular redox, which is largely determined by the ratio of oxidized and reduced glutathione (GSH), plays a significant role in the propensity of cells to undergo apoptosis. However, there are also contrasting opinions stating that intracellular GSH depletion or supplemented GSH alone are not sufficient to lead cells to apoptosis or conversely protect them. Unexpectedly, this study shows that NAC, even if it maintains the peculiar characteristics of an agent capable of reducing cell proliferation and increasing intracellular GSH content, increases apoptosis induced by H(2)O(2) treatment and mo-antiFas triggering in a 3DO cell line. We found that 24 h of NAC pre-treatment can shift cellular death from necrotic to apoptotic and determine an early expression of FasL in a 3DO cell line treated with H(2)O(2).     

Enhancement of intracellular glutathione protects endothelial cells against oxidant damage

We studied the role of glutathione in the endothelial cell defense against H2O2 damage. Treatment of endothelial cells with buthionine sulfoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, depleted the cells of GSH, while L-2-oxothiazolidine-4-carboxylate, an effective intracellular cysteine delivery agent, markedly enhanced endothelial cell GSH concentration. Depletion of intracellular GSH sensitized the endothelial cells to injury by H2O2 either preformed or generated by the glucose-glucose oxidase system. In contrast, an increase of intracellular GSH protected the cells from H2O2 damage. There was an inverse, linear relationship between the intracellular GSH concentrations and killing of endothelial cells by H2O2. Our results suggest that enhancement of endothelial cell GSH may be an alternative approach toward the prevention of oxidant-induced endothelial damage such as adult respiratory distress syndrome.     

Role of TRPM2 in H(2)O(2)-Induced Cell Apoptosis in Endothelial Cells

Melastatin-like transient receptor potential channel 2 (TRPM2) is an oxidant-sensitive and cationic non-selective channel that is expressed in mammalian vascular endothelium. Here we investigated the functional role of TRPM2 channels in hydrogen peroxide (H(2)O(2))-induced cytosolic Ca(2+) ([Ca(2+)](i)) elavation, whole-cell current increase, and apoptotic cell death in murine heart microvessel endothelial cell line H5V. A TRPM2 blocking antibody (TM2E3), which targets the E3 region near the ion permeation pore of TRPM2, was developed. Treatment of H5V cells with TM2E3 reduced the [Ca(2+)](i) rise and whole-cell current change in response to H(2)O(2). Suppressing TRPM2 expression using TRPM2-specific short hairpin RNA (shRNA) had similar inhibitory effect. H(2)O(2)-induced apoptotic cell death in H5V cells was examined using MTT assay, DNA ladder formation analysis, and DAPI-based nuclear DNA condensation assay. Based on these assays, TM2E3 and TRPM2-specific shRNA both showed protective effect against H(2)O(2)-induced apoptotic cell death. TM2E3 and TRPM2-specific shRNA also protect the cells from tumor necrosis factor (TNF)-α-induced cell death in MTT assay. In contrast, overexpression of TRPM2 in H5V cells resulted in an increased response in [Ca(2+)](i) and whole-cell currents to H(2)O(2). TRPM2 overexpression also aggravated the H(2)O(2)-induced apoptotic cell death. Downstream pathways following TRPM2 activation was examined. Results showed that TRPM2 activity stimulated caspase-8, caspase-9 and caspase-3. These findings strongly suggest that TRPM2 channel mediates cellular Ca(2+) overload in response to H(2)O(2) and contribute to oxidant-induced apoptotic cell death in vascular endothelial cells. Down-regulating endogenous TRPM2 could be a means to protect the vascular endothelial cells from apoptotic cell death.     

Oxidative stress response in eukaryotes: effect of glutathione, superoxide dismutase and catalase on adaptation to peroxide and menadione stresses in Saccharomyces cerevisiae

Aiming to clarify the mechanisms by which eukaryotes acquire tolerance to oxidative stress, adaptive and cross-protection responses to oxidants were investigated in Saccharomyces cerevisiae. Cells treated with sub-lethal concentrations of menadione (a source of superoxide anions) exhibited cross-protection against lethal doses of peroxide; however, cells treated with H2O2 did not acquire tolerance to a menadione stress, indicating that menadione response encompasses H2O2 adaptation. Although, deficiency in cytoplasmic superoxide dismutase (Sod1) had not interfered with response to superoxide, cells deficient in glutathione (GSH) synthesis were not able to acquire tolerance to H2O2 when pretreated with menadione. These results suggest that GSH is an inducible part of the superoxide adaptive stress response, which correlates with a decrease in the levels of intracellular oxidation. On the other hand, neither the deficiency of Sod1 nor in GSH impaired the process of acquisition of tolerance to H2O2 achieved by a mild pretreatment with peroxide. Using a strain deficient in the cytosolic catalase, we were able to conclude that the reduction in lipid peroxidation levels produced by the adaptive treatment with H2O2 was dependent on this enzyme. Corroborating these results, the pretreatment with low concentrations of H2O2 promoted an increase in catalase activity.     

Lymphokine-activated killer cell susceptibility in epirubicin-resistant and parental human non-small cell lung cancer (NSCLC)

The aim of this study was to evaluate that: (i) epirubicin-HCl (EPI) and lymphokine-activated killer (LAK) cells cytotoxicity may be mediated by free radical generation; and (ii) resistant H1299 cells may be more sensitive to combined treatment of LAK cells plus EPI than the LAK or EPI treatment alone. Viability of H1299 cells treated with EPI, LAK and LAK plus EPI was measured using the MTT test. Amount of glutathione (GSH), protein content and enzymatic activity were measured by spectrophotometer. Glutathione S-transferase (GST)-pi expression in the cells was determined by western blot analysis. LAK plus EPI combined treatment increased susceptibility of H1299 WT and H1299 EPI(R) (300-fold EPI resistant) cells to LAK cell cytotoxicity. The resistance of H1299 EPI(R) cells to EPI appears to be associated with a developed tolerance to free radicals, most likely because of a 2-fold increase in NADPH-dependent-cytochrome-P450 reductase (NADPH-CYP reductase) activity, 11-fold GST activity and 11-and 7-fold augmented selenium dependent and independent glutathione peroxidase (GSH-Px) activity, respectively. Amount of GST-pi in H1299 EPI(R) cells is statistically different from negative control and H1299 WT (p < 0.01). It is proposed that production of reactive oxygen species and hydrogen peroxide by the treatment of EPI and LAK cells can cause cytotoxicity of H1299 WT and H1299 EPI(R) cells. Superoxide dismutase, catalase, GSH-Px, GST, NADPH-CYP reductase and GSH must be considered as part of the intracellular antioxidant defense mechanism of H1299 WT and H1299 EPI(R) cells against reactive oxygen species. Combined treatment of EPI plus LAK cells caused the increasing cytotoxicity on the H1299 EPI(R) cells.     

Induction of oxidative stress and DNA damage in cervix in acute treatment with benzo[a]pyrene

Benzo[a]pyrene [B(a)P] is one of the most prevalent environmental carcinogens and genotoxic agents. However, the mechanisms of B(a)P-induced oxidative damage in cervical tissue are still not clear. The present study was to investigate the oxidative stress and DNA damage in cervix of ICR female mice induced by acute treatment with B(a)P. Oxidative stress was assayed by analysis of malondialdehyde (MDA), superoxide anion and H(2)O(2), and antioxidant enzymes. The alkaline single-cell electrophoresis (SCGE) was used to measure DNA damage. The contents of MDA and glutathione (GSH), and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST) were significantly increased in cervix 24, 48 and 72h after B(a)P treatment of a single dose of 12.5 and 25mg/kg, while GSH, CAT, SOD and GST had no significant difference with the dose of 50mg/kg B(a)P at post-treatment time 48 and 72h except for SOD activity at 48h which was significant. The maximum values of SOD, CAT, GST and GSH were peaked at 24h and then decreased gradually while GPx activities and MDA levels persisted for up to 72h. Superoxide anion, H(2)O(2) and DNA damage changed similarly as the activity of SOD, CAT or GST. Additionally, increases of formamidopyrimidine DNA glycosylase (FPG) specific DNA damage were observed and can be greatly rescued by vitamin C pretreatment. Overall, B(a)P demonstrated a time- and dose- related oxidative stress and DNA damage in cervix.     

GRP78 Suppresses Lipid Peroxidation and Promotes Cellular Antioxidant Levels in Glial Cells following Hydrogen Peroxide Exposure

Oxidative stress, caused by the over production of reactive oxygen species (ROS), has been shown to contribute to cell damage associated with neurotrauma and neurodegenerative diseases. ROS mediates cell damage either through direct oxidation of lipids, proteins and DNA or by acting as signaling molecules to trigger cellular apoptotic pathways. The 78 kDa glucose-regulated protein (GRP78) is an ER chaperone that has been suggested to protect cells against ROS-induced damage. However, the protective mechanism of GRP78 remains unclear. In this study, we used C6 glioma cells transiently overexpressing GRP78 to investigate the protective effect of GRP78 against oxidative stress (hydrogen peroxide)-induced injury. Our results showed that the overexpression of GRP78 significantly protected cells from ROS-induced cell damage when compared to non-GRP78 overexpressing cells, which was most likely due to GRP78-overexpressing cells having higher levels of glutathione (GSH) and NAD(P)H:quinone oxidoreductase 1 (NQO1), two antioxidants that protect cells against oxidative stress. Although hydrogen peroxide treatment increased lipid peroxidation in non-GRP78 overexpressing cells, this increase was significantly reduced in GRP78-overexpressing cells. Overall, these results indicate that GRP78 plays an important role in protecting glial cells against oxidative stress via regulating the expression of GSH and NQO1.     

Absence of polo-like kinase 3 in mice stabilizes Cdc25A after DNA damage but is not sufficient to produce tumors

Oxidative stress caused by hydrogen peroxide (H(2)O(2)) plays an important role in the pathogenesis of Alzheimer's disease (AD). The prominent damages caused by H(2)O(2) include the ruin of membrane integrity, loss of intracellular neuronal glutathione (GSH), oxidative damage to DNA as well as the subsequent caspase-3 and p53 activation. Icariin is a flavonoid extracted from the traditional Chinese herb Epimedium brevicornum Maxim. We have previously reported that icariin has a good curative effect on patients with mild cognitive impairment (MCI), AD animal and cell models. However, the molecular mechanism of how icariin exerts neuroprotective effects is still not well understood. To address this question, we exposed undifferentiated neuronal cell lines (PC12 cells) to hydrogen peroxide (H(2)O(2)) and investigated the possible neuroprotective mechanisms of icariin. Vitamin E was used as a positive control. We observed that H(2)O(2) activated the JNK/p38 mitogen-activated protein kinase (MAPK) and induced PC12 cells apoptosis in a concentration-dependent manner. More over, we demonstrated that icariin protected PC12 cells by attenuating LDH leakage, reducing GSH depletion, preventing DNA oxidation damage and inhibiting subsequent activation of caspase-3 and p53, which are the main targets of H(2)O(2)-induced cell damage. In addition, we also found that icariin's neuroprotective effect may partly correlate with its inhibitory effect on JNK/p38 MAPK pathways. Therefore, our findings suggest that icariin is a candidate for a novel neuroprotective drug to against oxidative-stress induced neurodegeneration.     

Modulation of GST P1-1 activity by polymerization during apoptosis

Glutathione S-transferases (GSTs, EC 2.5.1.18) belong to a large family of functionally different enzymes that catalyze the S-conjugation of glutathione with a wide variety of electrophilic compounds including carcinogens and anticancer drugs. Drug resistance may result from reduction in apoptosis of neoplastic cells when exposed to antineoplastic drugs. The c-Jun N-terminal Kinase (JNK) belongs to the family of stress kinases and has been shown to be required for the maximal induction of apoptosis by DNA-damaging agents. Recently, an inhibition of JNK activity by GST P1-1, which was reversed by polymerization induced by oxidative stress, has been reported in 3T3-4A mouse fibroblast cell lines. The finding that GST P1-1 might inhibit JNK activity and that it is frequently highly expressed in tumor tissues suggests its possible implication in "apoptosis resistance" during antineoplastic therapy. We investigated the modulation of GST P1-1 during apoptosis in a neoplastic T-cell line (Jurkat) induced by hydrogen peroxide and etoposide. Apoptosis was paralleled by the appearance of a dimeric form of GST P1-1 on western blotting, associated with an increase in the Km(GSH) and a reduction in GST P1-1 specific activity toward 1-chloro-2,4-dinitrobenzene, which reached statistical significance only in H(2)O(2)-treated cells. Our data seem to suggest that H(2)O(2) and etoposide may partly act through a process of partial inactivation of the GST P1-1, possibly involving the "G" site in the process of dimerization, and thus favoring programmed cell death.     

Salidroside protects against hydrogen peroxide-induced injury in cardiac H9c2 cells via PI3K-Akt dependent pathway

Oxidative stress induces serious tissue injury in cardiovascular diseases. Salidroside, with its strong antioxidative and cytoprotective actions, is of particular interest in the development of antioxidative therapies for oxidative injury in cardiac diseases. We examined the pharmacological effects of salidroside on H9c2 rat cardiomyoblast cells under conditions of oxidative stress induced by hydrogen peroxide (H2O2) challenge. Salidroside attenuated H2O2-impaired cell viability in a concentration-dependent manner, and effectively inhibited cellular malondialdehyde production, lethal sarcolemmal disruption, cell necrosis, and apoptosis induced by H2O2 insult. Salidroside significantly augmented Akt phosphorylation at Serine 473 in the absence or presence of H2O2 stimulation; wortmannin, a specific inhibitor of PI3K, abrogated salidroside protection. Salidroside increased the intracellular mRNA expression and activities of catalase and Mn-superoxide dismutases in a PI3K-dependent manner. Our results indicated that salidroside protected cardiomyocytes against oxidative injury through activating the PI3K/Akt pathway and increasing the expression and activities of endogenous PI3K dependent antioxidant enzymes.     

Inducible nitric oxide synthase and cyclooxgenase-2 mediate protection of hydrogen peroxide preconditioning against apoptosis induced by oxidative stress in PC12 cells

The induction of inducible nitric oxide synthase (iNOS) in response to different stress is associated with simultaneous induction of cyclooxygenase-2 (COX-2) in various cell types. Both iNOS and COX-2 have been reported to mediate the late phase of cardioprotection induced by different preconditioning. However, whether both iNOS and COX-2 are mediators in the neuroprotection induced by preconditioning with hydrogen peroxide (H(2)O(2)) at low concentration is unknown. In this study, using the neurosecretory cell line-PC12 cells to set up the model of neuroprotection of preconditioning with H(2)O(2) against apoptosis, we first investigate what changes in expression of iNOS and COX-2 appear during H(2)O(2) preconditioning, then determine if both iNOS inhibitor and COX-2 inhibitor interfere with the neuroprotection elicited by preconditioning with H(2)O(2). We found that preconditioning with H(2)O(2) at 10 microM significantly protected PC12 cells against apoptosis induced by lethal H(2)O(2) (50 microM) and increased the expression of iNOS and COX-2 and that selective iNOS inhibitor, aminoguanidine (AG) and COX-2 inhibitor, NS-398 obviously blocked the protective effects induced by preconditioning with 10 microM H(2)O(2). The results of this study suggest that both iNOS and COX-2 are mediators of the neuroprotection induced by preconditioning with oxidative stress (H(2)O(2) at low concentration) in PC12 cells.