3-nitrotyrosine and 3-nitropropionic-acid

There is accumulating evidence that excitotoxicity and oxidative stress resulting from excessive activation of glutamate (N-methyl-D-aspartate) NMDA receptors are major participants in striatal degeneration associated with 3-nitropropionic acid (3NP) administration. Although excitotoxic and oxidative mechanisms are implicated in 3NP toxicity, there are conflicting reports as to whether NMDA receptor antagonists attenuate or exacerbate the 3NP-induced neurodegeneration. In the present study, we investigated the involvement of NMDA receptors in striatal degeneration, protein oxidation and motor impairment following systemic 3NP administration. We examined whether NMDA receptor antagonists, memantine and ifenprodil, influence the neurotoxicity of 3NP. The development of striatal lesion and protein oxidation following 3NP administration is delayed by memantine but not affected by ifenprodil. However, in behavioral experiments, memantine failed to improve and ifenprodil exacerbated the motor deficits associated with 3NP toxicity. Together, these findings suggest caution in the application of NMDA receptor antagonists as a neuroprotective agent in neurodegenerative disorders associated with metabolic impairment.

Topics: Adenosine Diphosphate; Animals; Corpus Striatum; Dizocilpine Maleate; Drug Interactions; Male; Memantine; Motor Activity; Nerve Degeneration; Neuroprotective Agents; Nitro Compounds; Piperidines; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Propionates; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Tyrosine

3-Nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, has been shown to protect against ischemic injury in the brain and in the heart via a preconditioning-like effect; however, the cellular mechanism is not known. The aim of the present study was to investigate if 3-NP pretreatment reduces infarct size and if altered metabolism of nitric oxide and reactive oxygen species are involved.. Hearts were assigned into 3 groups: 3 intermittent cycles of 5 min no-flow ischemia separated by 5 min aerobic perfusion protocol were used to induce ischemic preconditioning as a positive control; a time-matched non-preconditioning group served as control; and 3-NP (20 mg/kg, i.p.) was injected 3 h before the perfusion protocol to induce pharmacological preconditioning. Hearts from all groups were then subjected to 30 min global ischemia followed by 120 min reperfusion.. Infarct size and lactate dehydrogenase release were significantly reduced after ischemia/reperfusion. While cardiac nitric oxide (NO) was increased, superoxide formation, nitrotyrosine level, and cardiac NADH oxidase and xanthine oxidase (XO) activities were markedly reduced by 3-NP administration. Cardiac activities of NO synthase and superoxide dismutase were not changed by 3-NP.. This is the first demonstration in the rat myocardium that 3-NP induces pharmacological preconditioning, thereby limiting infarct size, and that this effect is associated with increased NO bioavailability and reduced peroxynitrite formation due to inhibition of superoxide formation by XO and NADH oxidase.

Topics: Animals; Biomarkers; Ischemic Preconditioning, Myocardial; L-Lactate Dehydrogenase; Male; Myocardial Reperfusion Injury; Myocardium; NADPH Oxidases; Nitric Oxide; Nitric Oxide Synthase; Nitro Compounds; Peroxynitrous Acid; Propionates; Rats; Rats, Wistar; Succinate Dehydrogenase; Superoxide Dismutase; Tyrosine; Xanthine Oxidase

Oxidative stress and excitotoxicity have been implicated in selective striatal vulnerability caused by the mitochondrial toxin, 3-nitropropionic acid (3-NP), which may simulate Huntington's disease in animals and humans. The detailed mechanism of the role of superoxide in striatal vulnerability induced by 3-NP is still unknown. The authors investigated oxidative cellular injury and DNA fragmentation after systemic 3-NP injection in wild-type (Wt) mice and mutant mice with a deficiency in manganese superoxide dismutase (MnSOD; Sod2 -/+). Furthermore, they investigated the effects of decortication after 3-NP treatment in Sod2 -/+ mice, and copper/zinc SOD (CuZnSOD) treatment in recently developed Sod2 -/+ mice that overexpress CuZnSOD (SOD1 +/- / Sod2 -/+ mice). Oxidized hydroethidine, 8-hydroxyguanosine immunoreactivity, and nitrotyrosine immunoreactivity were increased in the Sod2 -/+ mice compared with the Wt mice after 3-NP treatment (P < 0.001). Decortication completely abolished oxidative striatal damage after 3-NP treatment in the Sod2 -/+ mice. Increased CuZnSOD attenuated DNA fragmentation and striatal lesion volume after 3-NP treatment in the Sod2 -/+ mice (P < 0.001). These data suggest that production of superoxide may be a critical step to excitotoxicity and subsequent DNA fragmentation in selective striatal vulnerability after 3-NP treatment.

Topics: Animals; Cerebral Decortication; Corpus Striatum; DNA Damage; DNA Fragmentation; Gene Expression; Mice; Mice, Knockout; Neurotoxins; Nitro Compounds; Oxidation-Reduction; Phenanthridines; Propionates; Superoxide Dismutase; Superoxides; Tyrosine

The present study investigated whether 3-nitrotyrosine is an early marker for neurodegenerative processes involving oxidative stress. We characterized the 3-nitrotyrosine formation after 3-nitropropionic acid (3-NP) exposure in the whole brain spheroid culture model and in a rat model, using Lewis and Wistar rats. Increased 3-nitrotyrosine concentration in spheroid cultures from Lewis rats was observed at lower dose of and shorter exposure time to 3-NP as compared to alterations in glial fibrillary acidic protein concentration, decrease in glutamine synthetase activity or cell loss. Five days of exposure to 3-NP (5 mM) resulted in decreased staining of GABAergic processes, while neuronal nitric oxide synthase staining was preserved. In addition, staining of EAAC1, anti-2',3'-cyclic nucleotide 3'-phosphohydrolase and ED1 was diminished after treatment of spheroid cultures with 3-nitropropionic acid (5 mM), while isolectin B4 staining was increased. Dithiothreitol and vitamin E inhibited the increased formation of 3-nitrotyrosine. Interestingly, N(G)-nitro-L-arginine methyl ester increased the 3-nitrotyrosine formation. No increased 3-nitrotyrosine concentration was shown after exposure to 3-nitropropionic acid during 5 days in spheroid cultures obtained from Wistar rats. In the striatum of 3-NP-exposed Lewis and Wistar rats, no change in 3-nitrotyrosine concentration was observed, whereas only in Wistar rats the glial fibrillary acidic protein concentration was increased in addition to activation of microglial cells. It is concluded that 3-nitrotyrosine was a more sensitive marker for oxidative stress-induced neurodegeneration than glial fibrillary acidic protein and glutamine synthase in spheroid cell cultures of Lewis rats. Finally, the similarities between the 3-NP spheroid model and the vivo model indicate that the spheroid cultures provide a good alternative for chronic exposure of animals to neurotoxins.

Topics: Animals; Antibodies, Monoclonal; Behavior, Animal; Biomarkers; Brain Chemistry; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Fluorescent Antibody Technique; Free Radicals; Glial Fibrillary Acidic Protein; Image Processing, Computer-Assisted; Immunohistochemistry; Neurotoxins; NG-Nitroarginine Methyl Ester; Nitro Compounds; Oxidative Stress; Pregnancy; Propionates; Rats; Rats, Inbred Lew; Rats, Wistar; Species Specificity; Tyrosine

Glutathione peroxidase (GSHPx) is a critical intracellular enzyme involved in detoxification of hydrogen peroxide (H(2)O(2)) to water. In the present study we examined the susceptibility of mice with a disruption of the glutathione peroxidase gene to the neurotoxic effects of malonate, 3-nitropropionic acid (3-NP), and 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). Glutathione peroxidase knock-out mice showed no evidence of neuropathological or behavioral abnormalities at 2-3 months of age. Intrastriatal injections of malonate resulted in a significant twofold increase in lesion volume in homozygote GSHPx knock-out mice as compared to both heterozygote GSHPx knock-out and wild-type control mice. Malonate-induced increases in conversion of salicylate to 2,3- and 2, 5-dihydroxybenzoic acid, an index of hydroxyl radical generation, were greater in homozygote GSHPx knock-out mice as compared with both heterozygote GSHPx knock-out and wild-type control mice. Administration of MPTP resulted in significantly greater depletions of dopamine, 3,4-dihydroxybenzoic acid, and homovanillic acid in GSHPx knock-out mice than those seen in wild-type control mice. Striatal 3-nitrotyrosine (3-NT) concentrations after MPTP were significantly increased in GSHPx knock-out mice as compared with wild-type control mice. Systemic 3-NP administration resulted in significantly greater striatal damage and increases in 3-NT in GSHPx knock-out mice as compared to wild-type control mice. The present results indicate that a knock-out of GSHPx may be adequately compensated under nonstressed conditions, but that after administration of mitochondrial toxins GSHPx plays an important role in detoxifying increases in oxygen radicals.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; 3,4-Dihydroxyphenylacetic Acid; Animals; Brain Chemistry; Catechols; Convulsants; Corpus Striatum; Disease Models, Animal; Dopamine Agents; Female; Free Radicals; Glutathione; Glutathione Peroxidase; Heterozygote; Homovanillic Acid; Homozygote; Huntington Disease; Male; Malonates; Mice; Mice, Inbred Strains; Mice, Knockout; MPTP Poisoning; Nitro Compounds; Oxidative Stress; Parkinson Disease, Secondary; Propionates; Tyrosine

The gene defect in Huntington's disease (HD) may result in an impairment of energy metabolism. Malonate and 3-nitropropionic acid (3-NP) are inhibitors of succinate dehydrogenase that produce energy depletion and lesions that closely resemble those of HD. Oral supplementation with creatine or cyclocreatine, which are substrates for the enzyme creatine kinase, may increase phosphocreatine (PCr) or phosphocyclocreatine (PCCr) levels and ATP generation and thereby may exert neuroprotective effects. We found that oral supplementation with either creatine or cyclocreatine produced significant protection against malonate lesions, and that creatine but not cyclocreatine supplementation significantly protected against 3-NP neurotoxicity. Creatine and cyclocreatine increased brain concentrations of PCr and PCCr, respectively, and creatine protected against depletions of PCr and ATP produced by 3-NP. Creatine supplementation protected against 3-NP induced increases in striatal lactate concentrations in vivo as assessed by 1H magnetic resonance spectroscopy. Creatine and cyclocreatine protected against malonate-induced increases in the conversion of salicylate to 2,3- and 2,5-dihydroxybenzoic acid, biochemical markers of hydroxyl radical generation. Creatine administration protected against 3-NP-induced increases in 3-nitrotyrosine concentrations, a marker of peroxynitrite-mediated oxidative injury. Oral supplementation with creatine or cyclocreatine results in neuroprotective effects in vivo, which may represent a novel therapeutic strategy for HD and other neurodegenerative diseases.

Topics: Adenosine Triphosphate; Animals; Antihypertensive Agents; Antineoplastic Agents; Creatine; Creatinine; Disease Models, Animal; Energy Metabolism; Free Radicals; Huntington Disease; Lactates; Male; Malonates; Neostriatum; Neuroprotective Agents; Neurotoxins; Nitro Compounds; Oxidative Stress; Propionates; Rats; Rats, Sprague-Dawley; Tyrosine

Nerve growth factor (NGF)-secreting fibroblasts are able to protect against the Huntington-like striatal neurodegeneration induced by the mitochondrial toxin 3-nitropropionic acid (3-NP). In the present study, we investigated whether the neuroprotective effects of NGF are mediated through antioxidative mechanisms. Rats were grafted in the corpus callosum with NGF[+] or NGF[-] fibroblasts 7 days before administration of 3-NP. The generation of peroxynitrite was evaluated by measuring the striatal levels of 3-nitrotyrosine. NGF significantly decreased the 3-NP induced generation of 3-nitrotyrosine, presumably by decreasing peroxynitrite formation. These findings suggest that NGF might protect against neuronal death by inhibiting the production of nitric oxide or decreasing the levels of superoxide radicals, thereby decreasing the generation of oxidative agents such as peroxynitrite.

Topics: Animals; Corpus Striatum; Disease Models, Animal; Huntington Disease; Male; Nerve Growth Factors; Nitro Compounds; Propionates; Rats; Rats, Sprague-Dawley; Tyrosine

The mitochondrial toxin 3-nitropropionic acid (3-NP) produces selective striatal lesions in both experimental animals and humans. The pathogenesis of the lesions involves secondary excitotoxicity that may then lead to free radical generation. To test this further we examined the effects of 3-NP in both transgenic (Tg) mice that carry the complete sequence for the human copper/zinc superoxide dismutase (SOD) gene as well as non-Tg littermate controls. The Tg-SOD mice showed a pronounced attenuation of Nissl-stained striatal lesions compared with non-Tg mice. Systemic administration of 3-NP resulted in production of hydroxyl free radicals as assessed by the conversion of salicylate to 2,3- and 2,5-dihydroxybenzoic acid. This production was attenuated significantly in Tg-SOD mice. In a similar way, 3-NP produced significant increases in 3-nitrotyrosine/tyrosine, a marker for peroxynitrite-mediated damage, which were significantly attenuated in Tg-SOD mice. These results support that oxygen free radicals and peroxynitrite play an important role in the pathogenesis of 3-NP neurotoxicity.

Topics: Animals; Humans; Hydroxybenzoates; Hydroxyl Radical; Mice; Mice, Transgenic; Neurotoxins; Nitro Compounds; Propionates; Reference Values; Superoxide Dismutase; Tyrosine

Nitric oxide may be a key mediator of excitotoxic neuronal injury in the central nervous system. We examined the effects of the neuronal nitric oxide synthase inhibitor 7-nitroindazole (7-NI) on excitotoxic striatal lesions. 7-NI significantly attenuated lesions produced by intrastriatal injections of NMDA, but not kainic acid or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) 7-NI attenuated secondary striatal excitotoxic lesions produced by the succinate dehydrogenase inhibitor malonate, and the protection was reversed by L-arginine but not by D-arginine, 7-NI produced nearly complete protection against striatal lesions produced by systemic administration of 3-nitropropionic acid (3-NP), another succinate dehydrogenase inhibitor, 7-NI protected against malonate induced decreases in ATP, and increases in lactate, as assessed by 1H magnetic resonance spectroscopy. 7-NI had no effects on spontaneous electrophysiologic activity in the striatum in vivo, suggesting that its effects were not mediated by an interaction with excitatory amino acid receptors. 7-NI attenuated increases in hydroxyl radical, 8-hydroxy-2-deoxyguanosine and 3-nitrotyrosine generation in vivo, which may be a consequence of peroxynitrite formation. The present results implicate neuronal nitric oxide generation in the pathogenesis of both direct and secondary excitotoxic neuronal injury in vivo. As such they suggest that neuronal nitric oxide synthase inhibitors may be useful in the treatment of neurologic diseases in which excitotoxic mechanisms play a role.

Topics: Adenosine Triphosphate; Animals; Corpus Striatum; Electrophysiology; Gentisates; Hydroxybenzoates; Indazoles; Lactates; Lactic Acid; Male; Neurons; Neurotoxins; Nitric Oxide Synthase; Nitro Compounds; Propionates; Rats; Rats, Sprague-Dawley; Tyrosine