Singlet oxygen generation by the reaction of acrolein with peroxynitrite via a 2-hydroxyvinyl radical intermediate.

Acrolein (2-propenal) is an environmental pollutant, food contaminant, and endogenous toxic by-product formed in the thermal decomposition and peroxidation of lipids, proteins, and carbohydrates. Like other α,ß-unsaturated aldehydes, acrolein undergoes Michael addition of nucleophiles such as basic amino acids residues of proteins and nucleobases, triggering aging associated disorders. Here, we show that acrolein is also a potential target of the potent biological oxidant, nitrosating and nitrating agent peroxynitrite. In vitro studies revealed the occurrence of 1,4-addition of peroxynitrite (k2 = 6 × 103 M-1 s-1, pH 7.2, 25 °C) to acrolein in air-equilibrated phosphate buffer. This is attested by acrolein concentration-dependent oxygen uptake, peroxynitrite consumption, and generation of formaldehyde and glyoxal as final products. These products are predicted to be originated from the Russell termination of •OOCH=CH(OH) radical which also includes molecular oxygen at the singlet delta state (O21Δg). Accordingly, EPR spin trapping studies with the 2,6-nitrosobenzene-4-sulfonate ion (DBNBS) revealed a 6-line spectrum attributable to the 2-hydroxyvinyl radical adduct. Singlet oxygen was identified by its characteristic monomolecular IR emission at 1,270 nm in deuterated buffer, which was expectedly quenched upon addition of water and sodium azide. These data represent the first report on singlet oxygen creation from a vinylperoxyl radical, previously reported for alkyl- and formylperoxyl radicals, and may contribute to better understand the adverse acrolein behavior in vivo.

The labile iron pool attenuates peroxynitrite-dependent damage and can no longer be considered solely a pro-oxidative cellular iron source.

The ubiquitous cellular labile iron pool (LIP) is often associated with the production of the highly reactive hydroxyl radical, which forms through a redox reaction with hydrogen peroxide. Peroxynitrite is a biologically relevant peroxide produced by the recombination of nitric oxide and superoxide. It is a strong oxidant that may be involved in multiple pathological conditions, but whether and how it interacts with the LIP are unclear. Here, using fluorescence spectroscopy, we investigated the interaction between the LIP and peroxynitrite by monitoring peroxynitrite-dependent accumulation of nitrosated and oxidized fluorescent intracellular indicators. We found that, in murine macrophages, removal of the LIP with membrane-permeable iron chelators sustainably accelerates the peroxynitrite-dependent oxidation and nitrosation of these indicators. These observations could not be reproduced in cell-free assays, indicating that the chelator-enhancing effect on peroxynitrite-dependent modifications of the indicators depended on cell constituents, presumably including LIP, that react with these chelators. Moreover, neither free nor ferrous-complexed chelators stimulated intracellular or extracellular oxidative and nitrosative chemistries. On the basis of these results, LIP appears to be a relevant and competitive cellular target of peroxynitrite or its derived oxidants, and thereby it reduces oxidative processes, an observation that may change the conventional notion that the LIP is simply a cellular source of pro-oxidant iron.

Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP).

The Trypanosoma cruzi ascorbate peroxidase is, by sequence analysis, a hybrid type A member of class I heme peroxidases [TcAPx-cytochrome c peroxidase (CcP)], suggesting both ascorbate (Asc) and cytochrome c (Cc) peroxidase activity. Here, we show that the enzyme reacts fast with H2O2 (k = 2.9 × 107 M-1⋅s-1) and catalytically decomposes H2O2 using Cc as the reducing substrate with higher efficiency than Asc (kcat/Km = 2.1 × 105 versus 3.5 × 104 M-1⋅s-1, respectively). Visible-absorption spectra of purified recombinant TcAPx-CcP after H2O2 reaction denote the formation of a compound I-like product, characteristic of the generation of a tryptophanyl radical-cation (Trp233•+). Mutation of Trp233 to phenylalanine (W233F) completely abolishes the Cc-dependent peroxidase activity. In addition to Trp233•+, a Cys222-derived radical was identified by electron paramagnetic resonance spin trapping, immunospin trapping, and MS analysis after equimolar H2O2 addition, supporting an alternative electron transfer (ET) pathway from the heme. Molecular dynamics studies revealed that ET between Trp233 and Cys222 is possible and likely to participate in the catalytic cycle. Recognizing the ability of TcAPx-CcP to use alternative reducing substrates, we searched for its subcellular localization in the infective parasite stages (intracellular amastigotes and extracellular trypomastigotes). TcAPx-CcP was found closely associated with mitochondrial membranes and, most interestingly, with the outer leaflet of the plasma membrane, suggesting a role at the host-parasite interface. TcAPx-CcP overexpressers were significantly more infective to macrophages and cardiomyocytes, as well as in the mouse model of Chagas disease, supporting the involvement of TcAPx-CcP in pathogen virulence as part of the parasite antioxidant armamentarium.

Aromatic thiol-mediated cleavage of N-O bonds enables chemical ubiquitylation of folded proteins.

Access to protein substrates homogenously modified by ubiquitin (Ub) is critical for biophysical and biochemical investigations aimed at deconvoluting the myriad biological roles for Ub. Current chemical strategies for protein ubiquitylation, however, employ temporary ligation auxiliaries that are removed under harsh denaturing conditions and have limited applicability. We report an unprecedented aromatic thiol-mediated N-O bond cleavage and its application towards native chemical ubiquitylation with the ligation auxiliary 2-aminooxyethanethiol. Our interrogation of the reaction mechanism suggests a disulfide radical anion as the active species capable of cleaving the N-O bond. The successful semisynthesis of full-length histone H2B modified by the small ubiquitin-like modifier-3 (SUMO-3) protein further demonstrates the generalizability and compatibility of our strategy with folded proteins.

Effects of hyperbaric oxygen on Pseudomonas aeruginosa susceptibility to imipenem and macrophages.

BACKGROUND: The seriousness to treat burn wounds infected with Pseudomonas aeruginosa led us to examine whether the effect of the carbapenem antibiotic imipenem is enhanced by hyperbaric oxygen (HBO). MATERIALS & METHODS: The effects of HBO (100% O2, 3 ATA, 5 h) in combination with imipenen on bacterial counts of six isolates of P. aeruginosa and bacterial ultrastructure were investigated. Infected macrophages were exposed to HBO (100% O2, 3 ATA, 90 min) and the production of reactive oxygen species monitored. RESULTS: HBO enhanced the effects of imipenen. HBO increased superoxide anion production by macrophages and likely kills bacteria by oxidative mechanisms. CONCLUSION: HBO in combination with imipenem can be used to kill P. aeruginosa in vitro and such treatment may be beneficial for the patients with injuries containing the P. aeruginosa.

Oxidation of the tryptophan 32 residue of human superoxide dismutase 1 caused by its bicarbonate-dependent peroxidase activity triggers the non-amyloid aggregation of the enzyme.

The role of oxidative post-translational modifications of human superoxide dismutase 1 (hSOD1) in the amyotrophic lateral sclerosis (ALS) pathology is an attractive hypothesis to explore based on several lines of evidence. Among them, the remarkable stability of hSOD1(WT) and several of its ALS-associated mutants suggests that hSOD1 oxidation may precede its conversion to the unfolded and aggregated forms found in ALS patients. The bicarbonate-dependent peroxidase activity of hSOD1 causes oxidation of its own solvent-exposed Trp(32) residue. The resulting products are apparently different from those produced in the absence of bicarbonate and are most likely specific for simian SOD1s, which contain the Trp(32) residue. The aims of this work were to examine whether the bicarbonate-dependent peroxidase activity of hSOD1 (hSOD1(WT) and hSOD1(G93A) mutant) triggers aggregation of the enzyme and to comprehend the role of the Trp(32) residue in the process. The results showed that Trp(32) residues of both enzymes are oxidized to a similar extent to hSOD1-derived tryptophanyl radicals. These radicals decayed to hSOD1-N-formylkynurenine and hSOD1-kynurenine or to a hSOD1 covalent dimer cross-linked by a ditryptophan bond, causing hSOD1 unfolding, oligomerization, and non-amyloid aggregation. The latter process was inhibited by tempol, which recombines with the hSOD1-derived tryptophanyl radical, and did not occur in the absence of bicarbonate or with enzymes that lack the Trp(32) residue (bovine SOD1 and hSOD1(W32F) mutant). The results support a role for the oxidation products of the hSOD1-Trp(32) residue, particularly the covalent dimer, in triggering the non-amyloid aggregation of hSOD1.

Tempol moderately extends survival in a hSOD1(G93A) ALS rat model by inhibiting neuronal cell loss, oxidative damage and levels of non-native hSOD1(G93A) forms.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive dysfunction and death of motor neurons by mechanisms that remain unclear. Evidence indicates that oxidative mechanisms contribute to ALS pathology, but classical antioxidants have not performed well in clinical trials. Cyclic nitroxides are an alternative worth exploring because they are multifunctional antioxidants that display low toxicity in vivo. Here, we examine the effects of the cyclic nitroxide tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl) on ALS onset and progression in transgenic female rats over-expressing the mutant hSOD1(G93A) . Starting at 7 weeks of age, a high dose of tempol (155 mg/day/rat) in the rat´s drinking water had marginal effects on the disease onset but decelerated disease progression and extended survival by 9 days. In addition, tempol protected spinal cord tissues as monitored by the number of neuronal cells, and the reducing capability and levels of carbonylated proteins and non-native hSOD1 forms in spinal cord homogenates. Intraperitoneal tempol (26 mg/rat, 3 times/week) extended survival by 17 days. This group of rats, however, diverted to a decelerated disease progression. Therefore, it was inconclusive whether the higher protective effect of the lower i.p. dose was due to higher tempol bioavailability, decelerated disease development or both. Collectively, the results show that tempol moderately extends the survival of ALS rats while protecting their cellular and molecular structures against damage. Thus, the results provide proof that cyclic nitroxides are alternatives worth to be further tested in animal models of ALS.

Regulatory effects of nitric oxide on Src kinase, FAK, p130Cas, and receptor protein tyrosine phosphatase alpha (PTP-alpha): a role for the cellular redox environment.

The role of NO in regulating the focal adhesion proteins, Src, FAK, p130 Cas, and PTP-alpha, was investigated. Fibroblasts expressing PTP-alpha (PTP-alpha(WT) cells), fibroblasts "knockout" for PTP-alpha (PTP-alpha(-/-) cells), and "rescued" "knockout" fibroblasts (PTP-alpha A5/3 cells) were stimulated with either S-nitroso-N-acetylpenicillamine (SNAP) or fetal bovine serum (FBS). FBS increased inducible NO synthase in both cell lines. Activation of Src mediated either by SNAP or by FBS occurred independent of dephosphorylation of Tyr527 in PTP-alpha(-/-) cells. Both stimuli promoted dephosphorylation of Tyr527 and activation of Src kinase in PTP-alpha(WT) cells. NO-mediated activation of Src kinase affected the activities of FAK and p130Cas and was dependent on the expression of PTP-alpha. Analogous to tyrosine phosphorylation, SNAP and FBS stimulated differential generation of NO and S-nitrosylation of Src kinase in both cell lines. Incubation with SNAP resulted in higher levels of NO and S-nitrosylation of immunoprecipitated Src in PTP-alpha(-/-) cells (oxidizing redox environment) as compared with the levels of NO and S-nitrosylated Src in PTP-alpha(WT) cells (reducing redox environment). SNAP differentially stimulated cell proliferation of both cell lines is dependent on the intracellular redox environment, Src activity, and PTP-alpha expression. This dependence also is observed with FBS-stimulated cell migration.

Tempol ameliorates murine viral encephalomyelitis by preserving the blood-brain barrier, reducing viral load, and lessening inflammation.

Multiple sclerosis (MS) is a progressive inflammatory and/or demyelinating disease of the human central nervous system (CNS). Most of the knowledge about the pathogenesis of MS has been derived from murine models, such as experimental autoimmune encephalomyelitis and viral encephalomyelitis. Here, we infected female C57BL/6 mice with a neurotropic strain of the mouse hepatitis virus (MHV-59A) to evaluate whether treatment with the multifunctional antioxidant tempol (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy) affects the ensuing encephalomyelitis. In untreated animals, neurological symptoms developed quickly: 90% of infected mice died 10 days after virus inoculation and the few survivors presented neurological deficits. Treatment with tempol (24 mg/kg, ip, two doses on the first day and daily doses for 7 days plus 2 mM tempol in the drinking water ad libitum) profoundly altered the disease outcome: neurological symptoms were attenuated, mouse survival increased up to 70%, and half of the survivors behaved as normal mice. Not surprisingly, tempol substantially preserved the integrity of the CNS, including the blood-brain barrier. Furthermore, treatment with tempol decreased CNS viral titers, macrophage and T lymphocyte infiltration, and levels of markers of inflammation, such as expression of inducible nitric oxide synthase, transcription of tumor necrosis factor-alpha and interferon-gamma, and protein nitration. The results indicate that tempol ameliorates murine viral encephalomyelitis by altering the redox status of the infectious environment that contributes to an attenuated CNS inflammatory response. Overall, our study supports the development of therapeutic strategies based on nitroxides to manage neuroinflammatory diseases, including MS.

Tempol ameliorates murine viral encephalomyelitis by preserving the blood–brainbarrier, reducing viral load, and lessening inflammation

Multiple sclerosis (MS) is a progressive inflammatory and/or demyelinating disease of the human centralnervous system (CNS). Most of the knowledge about the pathogenesis of MS has been derived from murinemodels, such as experimental autoimmune encephalomyelitis and viral encephalomyelitis. Here, we infectedfemale C57BL/6 mice with a neurotropic strain of the mouse hepatitis virus (MHV-59A) to evaluate whethertreatment with the multifunctional antioxidant tempol (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy)affects the ensuing encephalomyelitis. In untreated animals, neurological symptoms developed quickly: 90%of infected mice died 10 days after virus inoculation and the few survivors presented neurological deficits.Treatment with tempol (24 mg/kg, ip, two doses on the first day and daily doses for 7 days plus 2 mMtempol in the drinking water ad libitum) profoundly altered the disease outcome: neurological symptomswere attenuated, mouse survival increased up to 70%, and half of the survivors behaved as normal mice. Notsurprisingly, tempol substantially preserved the integrity of the CNS, including the blood–brain barrier.Furthermore, treatment with tempol decreased CNS viral titers, macrophage and T lymphocyte infiltration,and levels of markers of inflammation, such as expression of inducible nitric oxide synthase, transcription oftumor necrosis factor-á and interferon-ã, and protein nitration. The results indicate that tempol amelioratesmurine viral encephalomyelitis by altering the redox status of the infectious environment that contributes toan attenuated CNS inflammatory response. Overall, our study supports the development of therapeuticstrategies based on nitroxides to manage neuroinflammatory diseases, including MS.

Argininosuccinate synthetase is a functional target for a snake venom anti-hypertensive peptide: role in arginine and nitric oxide production.

Bj-BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that Bj-BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological effect independent of angiotensin-converting enzyme inhibition. Therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for Bj-BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-de pend ent manner. AsS is recognized as an important player of the citrulline-NO cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of Bj-BPP-10c and AsS was evidenced by the following effects promoted by the peptide: (i) increase of NO metabolite production in human umbilical vein endothelial cell culture and of arginine in human embryonic kidney cells and (ii) increase of arginine plasma concentration in SHR. Moreover, alpha-methyl-dl-aspartic acid, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of Bj-BPP-10c in SHR. Taken together, these results suggest that AsS plays a role in the anti-hypertensive action of Bj-BPP-10c. Therefore, we propose the activation of AsS as a new mechanism for the anti-hypertensive effect of Bj-BPP-10c in SHR and AsS as a novel target for the therapy of hypertension-related diseases.

Evidence of a Ca(2+)-(*)NO-cGMP signaling pathway controlling zoospore biogenesis in the aquatic fungus Blastocladiella emersonii.

The sporulation stage of the aquatic fungus Blastocladiella emersonii culminates with the formation and release to the medium of a number of zoospores, which are motile cells responsible for the dispersal of the fungus. The presence in the sporulation solution of 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a potent and selective inhibitor of nitric oxide-sensitive guanylyl cyclases, completely prevented biogenesis of the zoospores. In addition, this compound was able to significantly reduce cGMP levels, which increase drastically during late sporulation, suggesting the existence of a nitric oxide-dependent mechanism for cGMP synthesis. Furthermore, increased levels of nitric oxide-derived products were detected during sporulation by fluorescence assays using DAF-2 DA, whose signal was drastically reduced in the presence of the nitric oxide synthase inhibitor Nomega-Nitro-L-arginine methyl ester (L-NAME). These results were confirmed by quantitative chemiluminescent determination of the intracellular levels of nitric oxide-derived products. A putative nitric oxide synthase (NOS) activity was detected throughout sporulation, and this enzyme activity decreased significantly when L-NAME and 1-[2-(Trifluoromethyl)phenyl]imidazole (TRIM) were added to the assays. NOS assays carried out in the presence of EGTA showed decreased enzyme activity, suggesting the involvement of calcium ions in enzyme activation. Additionally, expressed sequence tags (ESTs) encoding putative guanylyl cyclases and a cGMP-phosphodiesterase were found in B. emersonii EST database (http://blasto.iq.usp.br), and the mRNA levels of the corresponding genes were observed to increase during sporulation. Altogether, data presented here revealed the presence and expression of guanylyl cyclase and cGMP phosphodiesterase genes in B. emersonii and provided evidence of a Ca(2+)-(*)NO-cGMP signaling pathway playing a role in zoospore biogenesis.

Argininosuccinate Synthetase Is a Functional Target for aSnake Venom Anti-hypertensive Peptide: Role in arginine and nitric oxide production

Bj-BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that Bj-BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological effect independent of angiotensin-converting enzyme inhibition. Therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for Bj-BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-de pend ent manner. AsS is recognized as an important player of the citrulline-NO cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of Bj-BPP-10c and AsS was evidenced by the following effects promoted by the peptide: (i) increase of NO metabolite production in human umbilical vein endothelial cell culture and of arginine in human embryonic kidney cells and (ii) increase of arginine plasma concentration in SHR. Moreover, á-methyl-dl-aspartic acid, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of Bj-BPP-10c in SHR. Taken together, these results suggest that AsS plays a role in the anti-hypertensive action of Bj-BPP-10c. Therefore, we propose the activation of AsS as a new mechanism for the anti-hypertensive effect of Bj-BPP-10c in SHR and AsS as a novel target for the therapy of hypertension-related diseases.

DNA damage by sulfite autoxidation catalyzed by cobalt complexes.

DNA damage was investigated in the presence of sulfite, dissolved oxygen and cobalt(II) complexes with glycylglycylhistidine, glycylhistidyllysine, glycylglycyltyrosylarginine and tetraglycine. These studies indicated that only Co(II) complexed with glycylglycylhistidine (GGH) induced DNA strand breaks at low sulfite concentrations (1-80 microM) via strong oxidants formed in the reaction. In the presence of the other complexes, some damage occurred only in the presence of high sulfite concentrations (0.1-2.0 mM) after incubation for 4 h. In the presence of GGH, Co(II) and dissolved O2, DNA damage must involve a reactive high-valent cobalt complex. The damaging effect was increased by adding S(IV), due to the oxysulfur radicals formed as intermediates in S(IV) autoxidation catalyzed by the complex. SO3 -, HO and H radicals were detected by EPR-spin trapping experiments with DMPO (5,5-dimethyl-1-pyrroline N-oxide). The results indicate that Co(II) binds O2 in the presence of GGH, and leads to the formation of a DMPO-HO adduct without first forming free superoxide or hydroxyl radical, supporting the participation of a reactive high-valent cobalt complex.

Inhibition of in vivo leishmanicidal mechanisms by tempol: nitric oxide down-regulation and oxidant scavenging.

Tempol (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy) has long been known to protect experimental animals from the injury associated with oxidative and inflammatory conditions. In the latter case, a parallel decrease in tissue protein nitration levels has been observed. Protein nitration represents a shift in nitric oxide actions from physiological to pathophysiological and potentially damaging pathways involving its derived oxidants such as nitrogen dioxide and peroxynitrite. In infectious diseases, protein tyrosine nitration of tissues and cells has been taken as evidence for the involvement of nitric oxide-derived oxidants in microbicidal mechanisms. To examine whether tempol inhibits the microbicidal action of macrophages, we investigated its effects on Leishmania amazonensis infection in vitro (RAW 264.7 murine macrophages) and in vivo (C57Bl/6 mice). Tempol was administered in the drinking water at 2 mM throughout the experiments and shown to reach infected footpads as the nitroxide plus the hydroxylamine derivative by EPR analysis. At the time of maximum infection (6 weeks), tempol increased footpad lesion size (120%) and parasite burden (150%). In lesion extracts, tempol decreased overall nitric oxide products and expression of inducible nitric oxide synthase to about 80% of the levels in control animals. Nitric oxide-derived products produced by radical mechanisms, such as 3-nitrotyrosine and nitrosothiol, decreased to about 40% of the levels in control mice. The results indicate that tempol worsened L. amazonensis infection by a dual mechanism involving down-regulation of iNOS expression and scavenging of nitric oxide-derived oxidants. Thus, the development of therapeutic strategies based on nitroxides should take into account the potential risk of altering host resistance to parasite infection.

Cyclic nitroxides inhibit the toxicity of nitric oxide-derived oxidants: mechanisms and implications.

The substantial therapeutic potential of tempol (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy) and related cyclic nitroxides as antioxidants has stimulated innumerous studies of their reactions with reactive oxygen species. In comparison, reactions of nitroxides with nitric oxide-derived oxidants have been less frequently investigated. Nevertheless, this is relevant because tempol has also been shown to protect animals from injuries associated with inflammatory conditions, which are characterized by the increased production of nitric oxide and its derived oxidants. Here, we review recent studies addressing the mechanisms by which cyclic nitroxides attenuate the toxicity of nitric oxide derived oxidants. As an example, we present data showing that tempol protects mice from acetaminophen-induced hepatotoxicity and discuss the possible protection mechanism. In view of the summarized studies, it is proposed that nitroxides attenuate tissue injury under inflammatory conditions mainly because of their ability to react rapidly with nitrogen dioxide and carbonate radical. In the process the nitroxides are oxidized to the corresponding oxammonium cation, which, in turn, can be recycled back to the nitroxides by reacting with upstream species, such as peroxynitrite and hydrogen peroxide, or with cellular reductants. An auxiliary protection mechanism may be down-regulation of inducible nitric oxide synthase expression. The possible therapeutic implications of these mechanisms are addressed.

Cyclic nitroxides inhibit the toxicity of nitric oxide-derived oxidants: mechanisms and implications

The substantial therapeutic potential of tempol (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy) and related cyclic nitroxides as antioxidants has stimulated innumerous studies of their reactions with reactive oxygen species. In comparison, reactions of nitroxides with nitric oxide-derived oxidants have been less frequently investigated. Nevertheless, this is relevant because tempol has also been shown to protect animals from injuries associated with inflammatory conditions, which are characterized by the increased production of nitric oxide and its derived oxidants. Here, we review recent studies addressing the mechanisms by which cyclic nitroxides attenuate the toxicity of nitric oxidederived oxidants. As an example, we present data showing that tempol protects mice from acetaminophen-induced hepatotoxicity and discuss the possible protection mechanism. In view of the summarized studies, it is proposed that nitroxides attenuate tissue injury under inflammatory conditions mainly because of their ability to react rapidly with nitrogen dioxide and carbonate radical. In the process the nitroxides are oxidized to the corresponding oxammonium cation, which, in turn, can be recycled back to the nitroxides by reacting with upstream species, such as peroxynitrite and hydrogen peroxide, or with cellular reductants. An auxiliary protection mechanism may be down-regulation of inducible nitric oxide synthase expression. The possible therapeutic implications of these mechanisms are addressed.

O considerável potencial terapêutico de tempol (4-hidroxi-2,2, 6,6-tetrametil-1piperiniloxila) e nitróxidos cíclicos relacionados como antioxidantes tem estimulado inúmeros estudos de suas reações com espécies reativas derivadas de oxigênio. Em comparação, as reações de nitróxidos com oxidantes derivados do óxido nítrico têm sido investigadas menos frequentemente. Todavia, essas reações são relevantes porque o tempol é também capaz de proteger animais de injúrias associadas a condições inflamatórias, as quais são caracterizadas por uma aumentada produção de óxido nítrico e derivados oxidantes. Aqui, discutimos estudos recentes abordando os mecanismos pelos quais nitróxidos cíclicos atenuam a toxicidade de oxidantes derivados do óxido nítrico. Como um exemplo, apresentamos dados que demonstram que o tempol protege camundongos do dano hepatotóxico promovido por altas doses de acetaminofeno e discutimos o possível mecanismo de proteção. Com base nos estudos sumarizados, é proposto que nitróxidos atenuam a injúria tecidual em condições inflamatórias devido principalmente a sua capacidade de reagir rapidamente com ambos, dióxido de nitrogênio e radical carbonato. Em conseqüência, os nitróxidos são oxidados ao cátion oxamônio correspondente, o qual, por sua vez, pode ser reciclado ao nitróxido através de reações com espécies precursoras, como peroxinitrito e peróxido de hidrogênio, ou com redutores celulares. Um possível mecanismo auxiliar de proteção é a regulação negativa da expressão da sintase do óxido nítrico induzível. As possíveis implicações terapêuticas desses mecanismos são abordadas.

DNA damage and 2'-deoxyguanosine oxidation induced by S(IV) autoxidation catalyzed by copper(II) tetraglycine complexes: synergistic effect of a second metal ion.

S(IV) (SO(2),HSO(3)(-)andSO(3)(2-)) autoxidation catalyzed by Cu(II)/tetraglycine complexes in the presence of DNA or 2'-deoxyguanosine (dGuo) resulted in DNA strand breaks and formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), respectively. Ni(II), Co(II) or Mn(II) (1.0x10(-4)M) complexes had much smaller effects. Cu(II)/tetraglycine (1.0x10(-4)M) in the presence of Ni(II) or Mn(II) (10(-7)-10(-6)M) and S(IV) showed remarkable synergistic effect with these metal ions producing a higher yield of 8-oxodGuo. Oxidation of dGuo and DNA damage were attributed to oxysulfur radicals formed as intermediates in S(IV) autoxidation catalyzed by transition metal ions. SO*(3)(-) and HO* radicals were detected by EPR-spin trapping experiments with DMPO (5,5-dimethyl-1-pyrroline-N-oxide).

Association between nitric oxide synthesis and vaccination-acquired resistance to murine hepatitis virus by spf mice.

Murine hepatitis virus strain 3 (MHV-3) produces a strain-dependent pattern of disease, with A/J and BALB/c mice being considered models of resistance and susceptibility, respectively. A role for nitric oxide in controlling infection remains debatable; thus, we monitored nitric oxide levels in blood and liver of immunized and nonimmunized spf mice during infection by electron paramagnetic resonance. In parallel, liver histology, virus titers, and plasma alanine aminotransferase (ALT) activity were monitored. Nitric oxide synthesis was barely detectable in BALB/c mice, which showed a progressive increase in virus titers and ALT activity. These animals died with a shorter survival time than A/J mice. The latter displayed a less severe infection and presented detectable levels of nitric oxide as nitrosyl complexes in blood and liver at 72 hpi. Immunized mice from both strains became resistant to MHV-3 and showed comparable levels of nitrosyl complexes in blood and liver at an early time (24 hpi). Thereafter, nitric oxide levels decreased but remained detectable in blood up to 96 hpi. Immunized mice were capable of clearing the virus and clearance was inhibited by administration of a nitric oxide synthase inhibitor. Overall, the results support a role for nitric oxide in controlling MHV-3 infection.

Association between nitric oxide synthesis and vaccination-acquiredresistance to murine hepatitis virus by spf mice

Murine hepatitis virus strain 3 (MHV-3) produces a strain-dependent pattern of disease, with A/J and BALB/c mice being considered models ofresistance and susceptibility, respectively. A role for nitric oxide in controlling infection remains debatable; thus, we monitored nitric oxide levelsin blood and liver of immunized and nonimmunized spf mice during infection by electron paramagnetic resonance. In parallel, liver histology,virus titers, and plasma alanine aminotransferase (ALT) activity were monitored. Nitric oxide synthesis was barely detectable in BALB/c mice,which showed a progressive increase in virus titers and ALT activity. These animals died with a shorter survival time than A/J mice. The latterdisplayed a less severe infection and presented detectable levels of nitric oxide as nitrosyl complexes in blood and liver at 72 hpi. Immunized micefrom both strains became resistant to MHV-3 and showed comparable levels of nitrosyl complexes in blood and liver at an early time (24 hpi).Thereafter, nitric oxide levels decreased but remained detectable in blood up to 96 hpi. Immunized mice were capable of clearing the virus andclearance was inhibited by administration of a nitric oxide synthase inhibitor. Overall, the results support a role for nitric oxide in controllingMHV-3 infection.