2-phenyl-4-4-5-5-tetramethylimidazoline-1-oxyl-3-oxide and peroxynitric-acid

A short-term exposure to tert-butylhydroperoxide (tB-OOH) promoted a concentration-dependent formation of DNA single-strand breaks in PC12 cells. These events were paralleled by an increase in the cytosolic concentration of Ca2+ that was in part cleared by the mitochondria. Unlike the extent of Ca2+ mobilization and/or mitochondrial Ca2+ clearance, the DNA strand scission evoked by the hydroperoxide was markedly reduced by the nitric oxide (NO) scavenger 2-phenyl-4,4,5,5-tetramethylimidazolin-1-oxyl-3-oxide (PTIO) or by the NO synthase inhibitor N-nitro-L-arginine methylester (L-NAME). Inhibitors of electron transport (rotenone and myxothiazol), ruthenium red (RR, a polycation which inhibits the calcium uniporter of mitochondria), or peroxynitrite scavengers (Trolox and L-methionine) were as effective as PTIO or L-NAME in inhibiting the DNA-damaging response mediated by tB-OOH. Rotenone, RR or peroxynitrite scavengers did not further reduce the residual DNA cleavage observed following treatment with tB-OOH in L-NAME-supplemented cells. Exogenous NO also increased the DNA damage caused by tB-OOH in L-NAME-supplemented cells and this response was blunted by RR or by inhibitors of electron transport but was insensitive to peroxynitrite scavengers. We conclude that both endogenous and exogenous NO enhance the DNA cleavage generated by tB-OOH in PC12 cells. However, only endogenous NO set the bases for an involvement of peroxynitrite in this DNA-damaging response.

Topics: Animals; Calcium; Chromans; Cyclic N-Oxides; DNA Damage; DNA, Single-Stranded; Electron Transport; Free Radical Scavengers; Imidazoles; Methacrylates; Methionine; Mitochondria; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide; Nitric Oxide Donors; Oxidants; PC12 Cells; Penicillamine; Rats; Rotenone; Ruthenium Red; S-Nitroso-N-Acetylpenicillamine; tert-Butylhydroperoxide; Thiazoles

SIN-1 has been used, in vitro, to simultaneously generate nitric oxide (*NO) and superoxide (O*-2). However, the pharmacological activity of SIN-1 resembles that of a *NO donor. SIN-1 decays by a three-step mechanism. After initial isomerization to an open ring form, SIN-1A reduces oxygen by a one-electron transfer reaction to give O*-2 and the SIN-1 cation radical, which decomposes to form SIN-1C and *NO. Here we report that one-electron oxidizing agents, in addition to oxygen, can oxidize SIN-1A, resulting in the release of *NO without the concomitant formation of O*-2. We demonstrate that easily reducible nitroxides, such as the nitronyl and imino nitroxides, are able to oxidize SIN-1. Biological oxidizing agents such as ferricytochrome c also stimulate *NO production from SIN-1. In addition, decomposition of SIN-1 by human plasma or by the homogenate of rat liver, kidney, and heart tissues results in the formation of *NO. Our findings suggest that SIN-1 may react with heme proteins and other electron acceptors in biological systems to produce *NO. Thus, at the relatively low in vivo oxygen concentrations, SIN-1 is likely to behave more like an *NO donor than a peroxynitrite donor. The relevance of this reaction to myocardial protection afforded by SIN-1 in ischemia/reperfusion-induced injury is discussed.

Topics: Animals; Cyclic N-Oxides; Free Radical Scavengers; Hemeproteins; Humans; Imidazoles; Kidney; Liver; Molsidomine; Myocardium; Nitrates; Nitric Oxide Donors; Oxidants; Rats

We employed a mouse model of ALS, in which overexpression of a familial ALS-linked Cu/Zn-SOD mutation leads to progressive MN loss and a clinical phenotype remarkably similar to that of human ALS patients, to directly test the excitotoxicity hypothesis of ALS. Under basal culture conditions, MNs in mixed spinal cord cultures from the Cu/Zn-SOD mutant mice exhibited enhanced oxyradical production, lipid peroxidation, increased intracellular calcium levels, decreased intramitochondrial calcium levels, and mitochondrial dysfunction. MNs from the Cu/Zn-SOD mutant mice exhibited greatly increased vulnerability to glutamate toxicity mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors. The increased vulnerability of MNs from Cu/Zn-SOD mutant mice to glutamate toxicity was associated with enhanced oxyradical production, sustained elevations of intracellular calcium levels, and mitochondrial dysfunction. Pretreatment of cultures with vitamin E, nitric oxide-suppressing agents, peroxynitrite scavengers, and estrogen protected MNs from Cu/Zn-SOD mutant mice against excitotoxicity. Excitotoxin-induced degeneration of spinal cord MNs in adult mice was more extensive in Cu/Zn-SOD mutant mice than in wild-type mice. The mitochondrial dysfunction associated with Cu/Zn-SOD mutations may play an important role in disturbing calcium homeostasis and increasing oxyradical production, thereby increasing the vulnerability of MNs to excitotoxicity.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Antioxidants; Calcium; Cells, Cultured; Cyclic N-Oxides; Estradiol; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Fluorescent Dyes; Free Radicals; Glutamic Acid; Homeostasis; Humans; Imidazoles; Lipid Peroxidation; Mice; Mice, Transgenic; Mitochondria; Motor Neuron Disease; Motor Neurons; Neurotoxins; NG-Nitroarginine Methyl Ester; Nitrates; Oxidative Stress; Rhodamine 123; Spinal Cord; Superoxide Dismutase; Superoxides; Vitamin E

In this work, we addressed the issue of whether exogenous NO and ONOO- (peroxynitrite) are able to alter growth, viability and/or differentiation of normal epithelial cells using cultured normal human keratinocytes as a model. 3-Morpholino-sydnonimine (SIN-1), a donor of both NO and O2(-)., leading to the production of ONOO-, dose-dependently inhibited growth of human keratinocytes without loss of viability. This inhibitory effect was lowered when SIN-1 was transformed into a pure NO donor by scavenging O2(-). with superoxide dismutase/catalase. Finally, scavenging NO release from SIN-1 with carboxy-1H-imidazol-1-yloxy,2-(4-carboxyp henyl)-4,5-dihydro-4,4,5,5 -tetramethyl-3-oxide (PTIO) resulted in a loss of the inhibitory effect of SIN-1. Together these findings suggest that both ONOO- and NO exert a growth inhibitory effect on human keratinocytes without cytotoxicity. Further support for this conclusion came from the treatment of human keratinocytes with the NO. donor propanamine 3-(2-hydroxy-2-nitroso-1-propyl hydrazino) or with authentic peroxynitrite. Moreover, only SIN-1 or peroxynitrite, and not NO, was able to trigger the expression of markers of terminal differentiation in human keratinocytes. From a physiological perspective, the ability of peroxynitrite, a known genotoxic and potentially carcinogenic agent, to direct proliferating keratinocytes towards terminal differentiation may be crucial to protect the genomic stability of this barrier epithelium.

Topics: Catalase; Cell Differentiation; Cell Division; Cyclic N-Oxides; Fluorescent Antibody Technique; Free Radical Scavengers; Humans; Hydrazines; Imidazoles; Keratinocytes; Molsidomine; Nitrates; Nitric Oxide; Superoxide Dismutase; Thymidine

In this study we provide further evidence associating activated cells of the monocyte lineage with the lesions of multiple sclerosis (MS). Using a combination of immunohistochemistry and reverse transcriptase-dependent in situ polymerase chain reaction analysis, we have identified monocytes expressing inducible nitric oxide synthase (iNOS) to be prevalent in the plaque areas of post mortem brain tissue from patients with MS. In addition, we have obtained evidence of the nitration of tyrosine residues in brain areas local to accumulations of iNOS-positive cells. In parallel studies we have assessed the effects of inhibitors of iNOS induction, as well as scavengers of nitric oxide and peroxynitrite in the experimental allergic encephalomyelitis model. Significant therapeutic effects were seen with the inhibitor of iNOS induction, tricyclodecan-9-xyl-xanthogenate, a nitric oxide scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and a peroxynitrite scavenger, uric acid. In particular, treatment with high doses of uric acid virtually prevented clinical symptoms of the disease. Together with our demonstration of the presence of activated macrophages expressing high levels of iNOS and evidence of peroxynitrite formation in brain tissue from patients with MS, these findings are of importance in the development of approaches to treat this disease.

Topics: Animals; Brain; Bridged-Ring Compounds; Cyclic N-Oxides; Encephalomyelitis, Autoimmune, Experimental; Enzyme Induction; Female; Free Radical Scavengers; Humans; Imidazoles; Mice; Mice, Inbred Strains; Monocytes; Multiple Sclerosis; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Norbornanes; Polymerase Chain Reaction; RNA, Messenger; Spinal Cord; Thiocarbamates; Thiones; Transcription, Genetic; Uric Acid