Inhibition of GTP binding to Rac2 by peroxynitrite: potential role for tyrosine modification.

Peroxynitrite is a potent oxidant generated by the reaction of nitric oxide (*NO) and superoxide anion (O2*-), and both can be produced in inflammatory tissues. In the present studies, we analyzed the effects of peroxynitrite treatment on the GTP-binding activity of Rac2, a low molecular weight GTP-binding protein important in regulating a number of cellular functions. Using a fluorescent analog of GTP (methylanthraniloyl guanosine triphosphate or mant-GTP) as a reporter group, we found that treatment of Rac2 with peroxynitrite inhibited the binding of mant-GTP to Rac2 in a dose-dependent manner. Peroxynitrite was also able to react directly with free mant-GTP, resulting in a significant decrease in mant-GTP fluorescence; however, the mechanism of peroxynitrite-mediated damage to mant-GTP was different than with Rac2. In the case of mant-GTP, protection from peroxynitrite-mediated oxidation was observed in the presence of the free radical scavengers, mannitol and DMTU. In contrast, DMTU was unable to prevent peroxynitrite-mediated inhibition of mant-GTP binding to Rac2. Instead, our data demonstrates a role for peroxynitrite-mediated tyrosine modification in the inhibition of mant-GTP binding to Rac2, and we were able to demonstrate the formation of a significant level of nitrotyrosine formation in Rac2 exposed to peroxynitrite. Thus, our studies support the premise that oxidative modification of key cellular proteins, such as Rac2, plays an important role in the cytotoxic effects observed for peroxynitrite and other reactive oxidants.     

Bactericidal activity of peroxynitrite.

Peroxynitrite is a strong oxidant formed by macrophages and potentially by other cells that produce nitric oxide and superoxide. Peroxynitrite was highly bactericidal, killing Escherichia coli in direct proportion to its concentration with an LD50 of 250 microM at 37 degrees C in potassium phosphate, pH 7.4. The apparent bactericidal activity of a given concentration peroxynitrite at acidic pH was less than that at neutral and alkaline pH. However, after taking the rapid pH-dependent decomposition of peroxynitrite into account, the rate of the killing was not significantly different at pH 5 compared to pH 7.4. Metal chelators did not decrease peroxynitrite-mediated killing, indicating that exogenous transition metals were not required for toxicity. The hydroxyl radical scavengers mannitol, ethanol, and benzoate did not significantly affect toxicity while dimethyl sulfoxide enhanced peroxynitrite-mediated killing. Dimethyl sulfoxide is a more efficient hydroxyl radical scavenger than the other three scavengers and increased the formation of nitrogen dioxide from peroxynitrite. In the presence of 100 mM dimethyl sulfoxide, 60.0 +/- 0.3 microM nitrogen dioxide was formed from 250 microM peroxynitrite as compared to 2.0 +/- 0.1 microM in buffer alone. Thus, formation of nitrogen dioxide may have enhanced the toxicity of peroxynitrite decomposing in the presence of dimethyl sulfoxide.     

Rapid and irreversible inactivation of protein tyrosine phosphatases PTP1B, CD45, and LAR by peroxynitrite.

Protein tyrosine phosphatases (PTPs) contain an essential thiol in the active site which may be susceptible to attack by nitric oxide-derived biological oxidants. We assessed the effects of peroxynitrite, nitric oxide, and S-nitrosoglutathione on the activity of three human tyrosine phosphatases in vitro. The receptor-like T-cell tyrosine phosphatase (CD45), the non-receptor-like tyrosine phosphatase PTP1B, and leukocyte-antigen-related (LAR) phosphatase were all irreversibly inactivated by peroxynitrite in less than 1 s with IC(50) values of 相似文献   

Inhibitory effect of isoflavones on peroxynitrite-mediated low-density lipoprotein oxidation

Peroxynitrite, a potent oxidant formed in vivo from the reaction of nitric oxide with superoxide, can mediate low-density liprotein (LDL) oxidation which is thought to increase the risk of atherosclerosis. This study investigates the inhibitory effect of the isoflavones, genistein and daidzein, together with their glycosidic forms, genistin and daidzin, on the peroxynitrite-mediated LDL oxidation and nitration of tyrosine. Genistein and daidzein were observed to dose-dependently inhibit peroxynitrite-mediated LDL oxidation, while their glucoside conjugates showed less activity. Moreover, all the isoflavones used in this study were found to be potent peroxynitrite scavengers, preventing the nitration of tyrosine. The ability of the isoflavones at 50 microM to decrease the tyrosine nitration induced by peroxynitrite (1 mM) was in the ratios of genistein (49%), daidzein (40%), daidzin (41%) and genistin (42%) when compared to the control (tyrosine incubated only with peroxynitrite). These results suggest that an intake of isoflavones could contribute to protecting against cardiovascular diseases and chronic inflammatory diseases.     

Peroxynitrite Induces Tyrosine Nitration and Modulates Tyrosine Phosphorylation of Synaptic Proteins

Peroxynitrite, the product of the radical-radical reaction between nitric oxide and superoxide anion, is a potent oxidant involved in tissue damage in neurodegenerative disorders. We investigated the modifications induced by peroxynitrite in tyrosine residues of proteins from synaptosomes. Peroxynitrite treatment (> or =50 microM) induced tyrosine nitration and increased tyrosine phosphorylation. Synaptophysin was identified as one of the major nitrated proteins and pp60src kinase as one of the major phosphorylated substrates. Further fractionation of synaptosomes revealed nitrated synaptophysin in the synaptic vesicles, whereas phosphorylated pp60src was enriched in the postsynaptic density fraction. Tyrosine phosphorylation was increased by treatment with 50-500 microM peroxynitrite and decreased by higher concentrations, suggesting a possible activation/inactivation of kinases. Immunocomplex kinase assay proved that peroxynitrite treatment of synaptosomes modulated the pp60src autophosphorylation activity. The addition of bicarbonate (CO2 1.3 mM) produced a moderate enhancing effect on some nitrated proteins but significantly protected the activity of pp60src against peroxynitrite-mediated inhibition so that at 1 mM peroxynitrite, the kinase was still more active than in untreated synaptosomes. The phosphotyrosine phosphatase activity of synaptosomes was inhibited by peroxynitrite (> or =50 microM) but significantly protected by CO2. Thus, the increase of phosphorylation cannot be attributed to peroxynitrite-mediated inhibition of phosphatases. We suggest that peroxynitrite may regulate the posttranslational modification of tyrosine residues in pre- and postsynaptic proteins. Identification of the major protein targets gives insight into the pathways possibly involved in neuronal degeneration associated with peroxynitrite overproduction.     

Ethanol induces peroxynitrite-mediated toxicity through inactivation of NADP+-dependent isocitrate dehydrogenase and superoxide dismutase

It has been reported that chronic alcohol administration increases peroxynitrite hepatotoxicity by enhancing concomitant production of nitric oxide and superoxide. Several studies have shown the importance of superoxide dismutase (SOD) in protecting cells against ethanol-induced oxidative stress. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. In this report, we demonstrate that ethanol induces the peroxynitrite-mediated cytotoxicity in HepG2 cells through inactivation of antioxidant enzymes such as ICDH and SOD. Upon exposure to 100mM ethanol for 3days to HepG2 cells, a significant decrease in the viability and activities of ICDH and SOD was observed. The ethanol-induced inactivation of antioxidant enzymes resulted in the cellular oxidative damage and modulation of redox status as well as mitochondrial dysfunction in HepG2 cells. The cytoxicity of ethanol and inactivation of antioxidant enzymes were effectively protected by manganeses(III) tetrakis(N-methyl-2-pyridyl) porphyrin, a manganese SOD mimetic, and N'-monomethyl-l-arginine, a nitric oxide synthase inhibitor. These results indicate that ethanol toxicity is mediated by peroxynitrite and the peroxynitrite-mediated damage to ICDH and SOD may be resulted in the perturbation of the cellular antioxidant defense systems and subsequently lead to a pro-oxidant condition.     

Nitric oxide in paraquat-mediated toxicity: A review

Paraquat, a cationic herbicide, produces degenerative lesions in the lung and in the nervous system after systemic administration to man and animals. Many cases of acute poisoning and death have been reported over the past few decades. Although a definitive mechanism of toxicity of paraquat has not been delineated, a cyclic single electron reduction/oxidation is a critical mechanistic event. The redox cycling of paraquat has two potentially important consequences relevant to the development of toxicity: the generation of the superoxide anion, which can lead to the formation of more toxic reactive oxygen species which are highly reactive to cellular macromolecules; and the oxidation of reducing equivalents (e.g., NADPH, reduced glutathione), which results in the disruption of important NADPH-requiring biochemical processes necessary for normal cell function. Nitric oxide is an important signaling molecule that reacts with superoxide derived from the paraquat redox cycle, to form the potent oxidant peroxynitrite, which causes serious cell damage. Although nitric oxide has been involved in the mechanism of paraquat-mediated toxicity, the role of nitric oxide has been controversial as both protective and harmful effects have been described. The present review summarizes recent findings in the field and describes new knowledge on the role of nitric oxide in the paraquat-mediated toxicity.     

Melatonin is a scavenger of peroxynitrite

Peroxynitrite is a toxic oxidant formed from the reaction of Superoxide and nitric oxide under conditions of inflammation and oxidant stress. Here we demonstrate that the pineal neurohormone melatonin inhibits peroxynitrite-mediated oxidant processes. Melatonin caused a dose-dependent inhibition of the oxidation of dihydrorhodamine 123 to rhodamine in vitro. Moreover, in cultured J774 macrophages, melatonin inhibited the development of DNA single strand breakage in response to peroxynitrite and reduced the suppression of mitochondrial respiration. Thus, melatonin appears to be a scavenger of peroxynitrite. This action may contribute to the antioxidant and antiinflammatory effects of melatonin in various pathophysiological conditions.     

Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide.

Peroxynitrite anion (ONOO-) is a potent oxidant that mediates oxidation of both nonprotein and protein sulfhydryls. Endothelial cells, macrophages, and neutrophils can generate superoxide as well as nitric oxide, leading to the production of peroxynitrite anion in vivo. Apparent second order rate constants were 5,900 M-1.s-1 and 2,600-2,800 M-1.s-1 for the reaction of peroxynitrite anion with free cysteine and the single thiol of albumin, respectively, at pH 7.4 and 37 degrees C. These rate constants are 3 orders of magnitude greater than the corresponding rate constants for the reaction of hydrogen peroxide with sulfhydryls at pH 7.4. Unlike hydrogen peroxide, which oxidizes thiolate anion, peroxynitrite anion reacts preferentially with the undissociated form of the thiol group. Peroxynitrite oxidizes cysteine to cystine and the bovine serum albumin thiol group to an arsenite nonreducible product, suggesting oxidation beyond sulfenic acid. Peroxynitrous acid was a less effective thiol-oxidizing agent than its anion, with oxidation presumably mediated by the decomposition products, hydroxyl radical and nitrogen dioxide. The reactive peroxynitrite anion may exert cytotoxic effects in part by oxidizing tissue sulfhydryls.     

Peroxynitrite-mediated mitochondrial dysfunction

Peroxynitrite anion (ONOO(-)) is a potent biological oxidant produced by the near diffusion-limited reaction of superoxide and nitric oxide. Peroxynitrite has been implicated in diverse forms of free radical-induced tissue injury. Experimental evidence showed that exogenous and endogenous peroxynitrite causes alterations of the structure and function of mitochondrial proteins, leading to mitochondrial dysfunction and cellular or organ injury. These data are discussed along with its physiopathological implications.     

Human sensitive to apoptosis gene protein inhibits peroxynitrite-induced DNA damage

Sensitive to apoptosis gene (SAG) protein, a novel zinc RING finger protein, which is redox responsive and protects mammalian cells from apoptosis, is a metal chelator and a potential reactive oxygen species scavenger, but its antioxidant properties have not been completely defined. The present study was undertaken to test the hypothesis that human SAG protects from DNA damage induced by peroxynitrite, a potent physiological inorganic toxin. The present study has shown that SAG significantly inhibits single strand breaks in supercoiled plasmid DNA induced by synthesized peroxynitrite (ONOO(-)) and 3-morpholinosydnomine N-ethylcarbamide (SIN-1), a generator of peroxynitrite through the reaction between nitric oxide and superoxide anion. The formation of 8-hydroxy-2(')-deoxyguanosine in calf thymus DNA by peroxynitrite and SIN-1 was also significantly inhibited by SAG. The protective effect on peroxynitrite-mediated DNA damage was completely abolished by the reaction of SAG with N-ethylmaleimide, a chemical modification agent for the sulfhydryl group of proteins. These observations suggested that the sulfhydryl group of cysteines in SAG could react directly with peroxynitrite to prevent DNA damage.     

The interaction of resveratrol with ferrylmyoglobin and peroxynitrite; protection against LDL oxidation

Resveratrol (3,4',5-trihydroxystilbene) is a natural phytoalexin synthesized in response to injury or fungal attack, found in the grape skin and wine, specially red wine. A large number of studies have demonstrated that resveratrol regulates many biological activities, namely protection against atherosclerosis by a set of pharmacological properties, including the antioxidant activity. In this study, we explored the capacity of resveratrol in protecting low density lipoproteins (LDL) against either ferrylmyoglobin- or peroxynitrite-mediated oxidation and the underlying mechanisms of its antioxidant potential. Resveratrol efficiently decreases the accumulation of hydroperoxides in LDL promoted by ferrylmyoglobin, a potent oxidant formed by the reaction of metmyoglobin with hydrogen peroxide, in a concentration-dependent manner, promptly reducing the oxoferryl complex to metmyoglobin. Simultaneously, resveratrol is consumed as detected by the rapid decrease in the characteristic peak at 310 nm, in a similar way to that observed upon its reaction with peroxidase/H 2 O 2 , pointing to a mechanism of one-electron oxidation and subsequent resveratrol dimer formation. On the other hand, resveratrol inhibits LDL apoprotein modifications induced by peroxynitrite, another potent oxidant formed by the reaction between superoxide and nitric oxide, as assessed by the decrease in apo-B net charge alterations and in carbonyl groups formation mediated by that oxidant. Resveratrol also interacts with peroxynitrite in a similar way to that observed with laccases, suggesting a mechanism of resveratrol oxidation rather than a nitration one. These mechanisms are discussed. Considering that either ferrylmyoglobin or peroxynitrite are physiologically relevant oxidants implicated in several pathologies, including atherosclerosis, our results certainly contribute to the understanding of the antioxidant action of resveratrol and consequently provide a new approach for the cardiovascular benefits associated with moderate consumption of red wine.     

Increased peroxynitrite activity in AIDS dementia complex: implications for the neuropathogenesis of HIV-1 infection

Oxidative stress is suggested to be involved in several neurodegenerative diseases. One mechanism of oxidative damage is mediated by peroxynitrite, a neurotoxic reaction product of superoxide anion and nitric oxide. Expression of two cytokines and two key enzymes that are indicative of the presence of reactive oxygen intermediates and peroxynitrite was investigated in brain tissue of AIDS patients with and without AIDS dementia complex and HIV-seronegative controls. RNA expression of IL-1beta, IL-10, inducible nitric oxide synthase, and superoxide dismutase (SOD) was found to be significantly higher in demented compared with nondemented patients. Immunohistochemical analysis showed that SOD was expressed in CD68-positive microglial cells while inducible nitric oxide synthase was detected in glial fibrillary acidic protein (GFAP)-positive astrocytes and in equal amounts in microglial cells. Approximately 70% of the HIV p24-Ag-positive macrophages did express SOD, suggesting a direct HIV-induced intracellular event. HIV-1 infection of macrophages resulted in both increased superoxide anion production and elevated SOD mRNA levels, compared with uninfected macrophages. Finally, we show that nitrotyrosine, the footprint of peroxynitrite, was found more intense and frequent in brain sections of demented patients compared with nondemented patients. These results indicate that, as a result of simultaneous production of superoxide anion and nitric oxide, peroxynitrite may contribute to the neuropathogenesis of HIV-1 infection.     

Metallothionein inhibits peroxynitrite-induced DNA and lipoprotein damage

Previous studies have demonstrated that metallothionein functions as an antioxidant that protects against oxidative DNA, protein, and lipid damage induced by superoxide anion, hydrogen peroxide, hydroxyl radical, and nitric oxide. The present study was undertaken to test the hypothesis that metallothionein also protects from DNA and lipoprotein damage induced by peroxynitrite, an important reactive nitrogen species that causes a diversity of pathological processes. A cell-free system was used. DNA damage was detected by the mobility of plasmid DNA in electrophoresis. Oxidation of low density lipoprotein was measured by a thiobarbituric acid-reactive substance, which was confirmed by lipid hydroperoxide assay. Plasmid DNA damage and low density lipoprotein oxidation were induced by 3-morpholinosydnomine, which produces peroxynitrite through the reaction between nitric oxide and superoxide anion or by synthesized peroxynitrite directly. DNA damage by 3-morpholinosydnomine was prevented by both metallothionein and superoxide dismutase, whereas the damage caused by peroxynitrite was prevented by metallothionein only. The oxidation of low density lipoprotein by 3-morpholinosydnomine and peroxynitrite was also significantly inhibited by metallothionein. This study thus demonstrates that metallothionein may react directly with peroxynitrite to prevent DNA and lipoprotein damage induced by this pathological reactive nitrogen species.     

Oxidation of tetrahydrobiopterin by peroxynitrite: implications for vascular endothelial function.

Subsaturating levels of tetrahydrobiopterin (BH(4)), an essential cofactor for nitric oxide synthase (NOS), can lead to endothelial dysfunction as a result of decreased production of nitric oxide. Furthermore, insufficient BH(4) can also result in NOS-uncoupled production of reactive oxygen intermediates, such as superoxide anion and hydrogen peroxide. Nitric oxide and superoxide react rapidly to form peroxynitrite, which may be the reactive species responsible for many of the toxic effects of nitric oxide. Here we show that BH(4) is a primary target for peroxynitrite-catalyzed oxidation because at pH 7.4, physiologically relevant concentrations of BH(4) are oxidized rapidly by low concentrations of peroxynitrite. Peroxynitrite oxidizes BH(4) to quinonoid 5,6-dihydrobiopterin and a large proportion of the quinonoid isomer readily loses its side chain to form 7,8-dihydropterin which is not a cofactor for nitric oxide synthase. Thus, abnormally low levels of BH(4) can promote a cycle of its own destruction mediated by nitric oxide synthase-dependent formation of peroxynitrite. This mechanism might contribute to vascular endothelial dysfunction induced by oxidative stress.     

Peroxynitrite affects exocytosis and SNARE complex formation and induces tyrosine nitration of synaptic proteins

The reactive species peroxynitrite, formed via the near diffusion-limited reaction of nitric oxide and superoxide anion, is a potent oxidant that contributes to tissue damage in neurodegenerative disorders. Peroxynitrite readily nitrates tyrosine residues in proteins, producing a permanent modification that can be immunologically detected. We have previously demonstrated that in the nerve terminal, nitrotyrosine immunoreactivity is primarily associated with synaptophysin. Here we identify two other presynaptic proteins nitrated by peroxynitrite, Munc-18 and SNAP25, both of which are involved in sequential steps leading to vesicle exocytosis. To investigate whether peroxynitrite affects vesicle exocytosis, we used the fluorescent dye FM1-43 to label a recycling population of secretory vesicles within the synaptosomes. Bolus addition of peroxynitrite stimulated exocytosis and glutamate release. Notably, these effects were strongly reduced in the presence of NaHCO(3), indicating that peroxynitrite acts mainly intracellularly. Furthermore, peroxynitrite enhanced the formation of the sodium dodecyl sulfate-resistant SNARE complex in a dose-dependent manner (100-1000 microm) and induced the formation of 3-nitrotyrosine in proteins of SNARE complex. These data suggest that modification(s) of synaptic vesicle proteins induced by peroxynitrite may affect protein-protein interactions in the docking/fusion steps, thus promoting exocytosis, and that, under excessive production of superoxide and nitric oxide, neurons may up-regulate neuronal signaling.     

Peroxynitrite-mediated lipid oxidation and nitration: Mechanisms and consequences

Lipid oxidation and nitration represents a novel area of research of relevance in the understanding of inflammatory processes. Peroxynitrite, the product of the diffusion-limited reaction between nitric oxide and superoxide anion, mediates oxidative modifications in lipid systems including cell membranes and lipoproteins. In this review, we discuss the mechanisms of lipid oxidation and nitration by peroxynitrite as well as the influence of physiological molecules and cell targets to redirect peroxynitrite reactivity. We also provide evidence to support that oxidation/nitration of lipids results in the formation of novel signaling modulators of key lipid-metabolizing enzymes.     

Peroxynitrite-Mediated Inhibition of DOPA Synthesis in PC12 Cells

Abstract: Experimental evidence has implicated oxidative stress in the development of Parkinson's disease, amyotrophic lateral sclerosis, and other degenerative neuronal disorders. Recently, peroxynitrite, which is formed by the nearly diffusion-limited reaction of nitric oxide with superoxide, has been suggested to be a mediator of oxidant-induced cellular injury. The potential role of peroxynitrite in the pathology associated with Parkinson's disease was evaluated by examining its effect on DOPA synthesis in PC12 pheochromocytoma cells. Peroxynitrite was generated from the compound 3-morpholinosydnonimine (SIN-1), which releases superoxide and nitric oxide simultaneously. Exposure of PC12 cells to peroxynitrite for 60 min greatly diminished their ability to synthesize DOPA without apparent cell death. The inhibition was due neither to the formation of free nitrotyrosine nor the oxidation of DOPA by peroxynitrite. The inhibition in DOPA synthesis by SIN-1 was abolished when superoxide was scavenged by the addition of superoxide dismutase. These data indicated that neither nitric oxide nor hydrogen peroxide generated by the dismutation of superoxide is responsible for the SIN-1-mediated inhibition of DOPA production. The inhibition of DOPA synthesis at high concentration of SIN-1 persisted even after removal of SIN-1. The inactivation of the tyrosine hydroxylase may be responsible for the significant decline in DOPA formation by peroxynitrite. Inactivation of tyrosine hydroxylase may be part of the initial insult in oxidative damage that eventually leads to cell death.     

Crucial role of apopain in the peroxynitrite-induced apoptotic DNA fragmentation

Peroxynitrite, a cytotoxic oxidant formed in the reaction of superoxide and nitric oxide is known to cause programmed cell death. However, the mechanisms of peroxynitrite-induced apoptosis are poorly defined. The present study was designed to characterize the molecular mechanisms by which peroxynitrite induces apoptosis in HL-60 cells, with special emphasis on the role of caspases. Peroxynitrite induced the activation of apopain/caspase-3, but not ICE/caspase-1 as measured by the cleavage of fluorogenic peptides. Considering the short half-life of peroxynitrite and the kinetics of caspase-3 activation (starting 3–4 h after peroxynitrite treatment), the enzyme is not likely to become activated directly by the oxidant. Caspase-3 activation proved to be essential for DNA fragmentation, because pretreatment of the cells with the specific tetrapeptide inhibitor DEVD-fmk completely blocked peroxynitrite-induced DNA fragmentation. Peroxynitrite-induced cytotoxicity was also significantly altered by the inhibition of caspase-3, whereas phosphatidylserine exposure was unaffected by DEVD-fmk treatment. Because many of the effects of peroxynitrite are mediated by poly(ADP-ribose) synthetase (PARS) activation, we have also investigated the effect of PARS-inhibition on peroxynitrite-induced apoptosis. We have found that PARS-inhibition modulates peroxynitrite-induced apoptotic DNA fragmentation in the HL-60 cells. The effect of the PARS inhibitors, 3-aminobenzamide and 5-iodo-6-amino-1,2-benzopyrone were dependent on the concentration of peroxynitrite used. While PARS-inhibition resulted in increased DNA-fragmentation at low doses (15 μM) of peroxynitrite, a decreased DNA-fragmentation was found at high doses (60 μM) of peroxynitrite. PARS inhibition negatively affected viability as determined by flow cytometry. These data demonstrate the crucial role of caspase-3 in mediating apoptotic DNA fragmentation in HL-60 cells exposed to peroxynitrite.     

Peroxynitrite induces GADD34, 45, and 153 VIA p38 MAPK in human neuroblastoma SH-SY5Y cells

Peroxynitrite, one of the most reactive radicals, is produced from superoxide anion and nitric oxide. A peroxynitrite generator, 3-morpholinosydonimine (SIN-1), was found to induce the expression of three different growth arrest and DNA damage-inducible (GADD) mRNA, GADD34, GADD45, and GADD153, at the early phase during cell death in human neuroblastoma SH-SY5Y cells. In addition, peroxynitrite activated p38 MAPK just before induction of three GADD mRNA. A specific inhibitor of p38 MAPK, SB202190, markedly suppressed peroxynitrite-induced expression of three GADD mRNA in SH-SY5Y cells. The expression of three GADD genes and also p38 MAPK phosphorylation were suppressed by treatment with radical scavengers, superoxide dismutase plus catalase and glutathione. Glutathione depletion by L-buthionine-S, R-sulfoximine (BSO), increased the vulnerability of the cells to peroxynitrite. These findings indicate that peroxynitrite-mediated oxidative stress activated p38 MAPK to induce three GADD genes.