RESUMO
Apoptosis is characterized by chromatin condensation, phosphatidylserine translocation, and caspase activation. Neuronal apoptotic death involves the participation of reactive oxygen species (ROS), which have also been implicated in necrotic cell death. In this study we evaluated the role of different ROS in neuronal death. Superoxide anion was produced by incubating cells with xanthine and xanthine oxidase plus catalase, singlet oxygen was generated with rose Bengal and luminic stimuli, and hydrogen peroxide was induced with the glucose and glucose oxidase. Cultured cerebellar granule neurons died with the characteristics of apoptotic death in the presence of superoxide anion or singlet oxygen. These two conditions induced caspase activation, nuclear condensation, phosphatidylserine translocation, and a decrease in intracellular calcium levels. On the other hand, hydrogen peroxide led to a necrosis-like cell death that did not induce caspase activation, phosphatidylserine translocation, or changes in calcium levels. Cell death produced by both singlet oxygen and superoxide anion, but not hydrogen peroxide, was partially reduced by an increase in intracellular calcium levels. These results suggest that formation of specific ROS can lead to different molecular cell death mechanisms (necrosis and apoptosis) and that ROS formed under different conditions could act as initiators or executioners on neuronal death.
Assuntos
Neurônios/citologia , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Bioensaio , Cálcio/metabolismo , Caspases/metabolismo , Catalase/farmacologia , Técnicas de Cultura de Células , Morte Celular/fisiologia , Sobrevivência Celular , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Ionomicina/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Sais de Tetrazólio/química , Tiazóis/química , Xantina/farmacologia , Xantina Oxidase/farmacologiaRESUMO
Available evidence for oxidative stress after angioplasty is indirect or ambiguous. We sought to characterize the pattern, time course, and possible sources of free radical generation early after arterial balloon injury. Ex vivo injury performed in arterial rings in buffer with lucigenin yielded a massive oxygen-dependent peak of luminescence that decayed exponentially and was proportional to the degree of injury. Signals for injured vs. control arteries were 207. 1 +/- 17.9 (n = 13) vs 4.1 +/- 0.7 (n = 22) cpm x 10(3)/mg/min (p <. 001). Data obtained with 0.25 mmol/l lucigenin were validated with 0. 005-0.05 mmol/l lucigenin or the novel superoxide-sensitive probe coelenterazine (5 micromol/l). Gentle removal of endothelium prior to injury scarcely affected the amount of luminescence. Lucigenin signals were amplified 5- to 20-fold by exogenous NAD(P)H, and were >85% inhibited by diphenyliodonium (DPI, a flavoenzyme inhibitor). Antagonists of several other potential free radical sources, including xanthine oxidase, nitric oxide synthase, and mitochondrial electron transport, were without effect. Overdistension of intact rabbit iliac arteries in vivo (n = 7) induced 72% fall in intracellular reduced glutathione and 68% increase in oxidized glutathione, so that GSH/GSSG ratio changed from 7.93 +/- 2.14 to 0. 81 +/- 0.16 (p <.005). There was also 28.7% loss of the glutathione pool. Further studies were performed with electron paramagnetic resonance spectroscopy. Rabbit aortas submitted to ex vivo overdistension in the presence of the spin trap DEPMPO (5-diethoxy-phosphoryl-5-methyl-1-pyrroline-N-oxide, 100 mmol/l, n = 5) showed formation of radical adduct spectra, abolished by DPI or superoxide dismutase. Computer simulation indicated a mixture of hydroxyl and carbon-centered radical adducts, likely due to decay of superoxide adduct. Electrical mobility shift assays for NF-kappaB activation were performed in nuclear protein extracts from intact or previously injured rabbit aortas. Balloon injury induced early NF-kappaB activation, which was decreased by DPI. In conclusion, our data show unambiguously that arterial injury induces an immediate profound vascular oxidative stress. Such redox imbalance is likely accounted for by activation of vessel wall NAD(P)H oxidoreductase(s), generating radical species potentially involved in tissue repair.
Assuntos
Cateterismo/efeitos adversos , Endotélio Vascular/lesões , Imidazóis , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidases , Espécies Reativas de Oxigênio , Acridinas/metabolismo , Animais , Compostos de Bifenilo/farmacologia , Óxidos N-Cíclicos , Inibidores de Ciclo-Oxigenase/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Endotélio Vascular/enzimologia , Radicais Livres , Glutationa/metabolismo , Inibidores de Lipoxigenase/farmacologia , Medições Luminescentes , Masculino , Metaloporfirinas/farmacologia , NAD/metabolismo , NADP/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico Sintase/farmacologia , Oniocompostos/farmacologia , Oxirredução , Estresse Oxidativo , Consumo de Oxigênio , Pirazinas/farmacologia , Coelhos , Proteínas Recombinantes de Fusão/metabolismo , Marcadores de Spin , Superóxido Dismutase/farmacologia , Superóxidos/metabolismo , Transcrição Gênica , Cicatrização , Xantina Oxidase/farmacologiaRESUMO
Pyridoxal isonicotinoyl hydrazone (PIH) has previously been studied for use in iron chelation therapy in iron-overload diseases. It is an efficient in vitro antioxidant due to its Fe(III) complexing activity (Schulman, H. M., et al. Redox Report 1:373-378; 1995). Pathologies associated with iron-overload include hepatic and other cancers. Since oxidative alterations of DNA can be linked to the development of cancer, we decided to study whether PIH protects DNA against in vitro oxidative stress. We report here that pUC-18 plasmid DNA is damaged by *OH radicals generated from Fe(II) plus H2O2 or from Fe(II) plus hypoxanthine/xanthine oxidase. The DNA damage was quantified by determining the diminution of supercoiled DNA forms after oxidative attack using agar gel electrophoresis. Micromolar amounts of PIH (20-30 microM) were able to half-protect DNA from iron (1-7.5 microM)-mediated *OH formation. The antioxidant capacity of PIH was significantly higher than that of some of its analogs and desferrioxamine. PIH and some of its analogues could also inhibit the oxidative degradation of 2-deoxyribose caused by Fenton reagents. Since we observed that PIH enhances the Fe(II) autoxidation rate, measured by the ferrozine technique, PIH may limit *OH formation and consequently DNA damage by decreasing the amount of Fe(II) available to catalyze Fenton reactions.
Assuntos
Antioxidantes , Dano ao DNA/efeitos dos fármacos , Radical Hidroxila/farmacologia , Quelantes de Ferro/farmacologia , Isoniazida/análogos & derivados , Plasmídeos/genética , Piridoxal/análogos & derivados , Compostos Ferrosos/química , Radical Hidroxila/metabolismo , Hipoxantina/farmacologia , Isoniazida/farmacologia , Oxirredução , Piridoxal/farmacologia , Xantina Oxidase/farmacologiaRESUMO
Time courses of total (GSH-t), disulfide (GSSG), and mixed disulfide (PSSG) forms of glutathione were studied in chicken blood submitted to oxidative stress induced by diamide or by the reactive oxygen species (ROS)-producing system xanthine/xanthine oxidase (X/XO). Diamide-treated blood induced an immediate increase in GSSG and PSSG, while X/XO produced a slow and sustained stress with increased values of GSSG and PSSG only after 30 and/or 60 min of incubation. Both total protein S-thiolation (mixed disulfide with glutathione) and dethiolation and hemoglobin A S-thiolation and dethiolation were clearly observed. Hemoglobin A (Hb A) was the major S-thiolated protein. We further characterized chicken Hb S-thiolation through the reaction of Hb with GSSG or the GSH/GSSG redox couple. Methemoglobin levels did not change with diamide or with X/XO treatment. Present results suggest that the most reactive cysteine pair of Hb A, the major chicken Hb, might function as an antioxidant under in vivo oxidative stress conditions.
Assuntos
Diamida/farmacologia , Hemoglobina A/metabolismo , Líquido Intracelular/metabolismo , Estresse Oxidativo , Compostos de Sulfidrila/sangue , Xantina Oxidase/farmacologia , Animais , Galinhas , Eritrócitos/metabolismo , Glutationa/sangue , Dissulfeto de Glutationa/sangue , Hemoglobina A/química , Oxirredução/efeitos dos fármacosRESUMO
The relationship between the metal-binding properties of metallothionein (MT) and its ability to interact with peroxides and free radicals was explored in vitro. The binding of 109Cd to MT and the thiol density of the protein were determined after incubation of a purified Zn/Cd-metallothionein preparation with either hydrogen peroxide alone, or with a number of free radical generating systems. Exposure of MT to H2O2, whether in the presence or absence of Fe2+, resulted in the progressive loss of the thiol residues of the protein and led to a parallel decrease of its 109Cd-binding capacity. These changes correlated with r values of 0.999 (P = 0.001) and 0.998 (P = 0.001), in the absence and presence of iron, respectively. The effects of H2O2, alone or plus Fe2+, on MT were completely prevented by catalase, but totally unaffected by superoxide dismutase or desferrioxamine. Exposure of MT to xanthine/xanthine oxidase also led to thiol oxidation and to a concomitant loss of the Cd-binding properties. In this system, both changes correlated with an r of 0.993 (P = 0.001) and were completely inhibited by superoxide dismutase. Exposure of MT to the peroxyl radical generator, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), resulted in the progressive loss of its the metal-binding properties and its thiol residues, both changes correlating with an r of 0.986 (P = 0.002). The ability of MT to bind 109Cd, lost as a result of its prior exposure to either H2O2 alone, H2O2 plus Fe2+, xanthine/xanthine oxidase, or to AAPH was, in all cases, completely recovered after incubation of the modified protein with dithiothreitol. These results indicate that H2O2 alone, and/or the oxygen-derived species, superoxide anion and peroxyl radicals, can all directly interact in vitro with MT to modify the protein oxidatively, and suggest that, under in vivo conditions, these species may be implicated as modifying factors of the metal-binding capacity of metallothionein.
Assuntos
Radioisótopos de Cádmio/metabolismo , Peróxido de Hidrogênio/farmacologia , Metalotioneína/metabolismo , Oxidantes/farmacologia , Amidinas/farmacologia , Ditiotreitol/farmacologia , Radicais Livres/farmacologia , Técnicas In Vitro , Ligação Proteica/efeitos dos fármacos , Compostos de Sulfidrila/química , Xantina , Xantina Oxidase/farmacologia , Xantinas/farmacologiaRESUMO
Several studies indicate that reactive oxygen species (ROS) are involved in defective sperm function pathophysiology. In this study we attempted to determine differentially the effects of xanthine (0.12 mM) plus xanthine oxidase (0.035 U/mL) (X+XO, a ROS promoter system), ROS scavengers (Tiron (TIR, 15 mM); catalase (CAT, 10 micrograms/mL); dimethylsulfoxide (DMSO, 140 mM)), and X+XO plus scavengers on several epididymal mouse spermatozoa functional parameters, incubated in NTPC medium, for 29 min. In the presence of X+XO, progressive gametes significantly diminished. TIR or CAT attenuated this effect, but DMSO did not. Inversely, X+XO increased the bending-forms population; only TIR reversed this phenomenon. The ROS promoter system diminished the viable cell population; all scavengers assayed maintained sperm viability at levels similar to control ones. When exposed to hypoosmotic shock after 29 min incubation with X+XO, the percentage of swollen cells decreased; TIR, CAT, or DMSO did not prevent this effect. Our experiments demonstrate that it is possible to differentiate the deleterious ROS effects upon sperm functional activity. O-2. and H2O2 preferentially seem to modify sperm motility, O-2. exhibiting the greatest ability for generating bending-form gametes, OH-being the most lethal ROS. In addition, sperm membrane clearly appears as the most damaged structure.
Assuntos
Espécies Reativas de Oxigênio , Espermatozoides/fisiologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Epididimo/citologia , Sequestradores de Radicais Livres/farmacologia , Técnicas In Vitro , Masculino , Camundongos , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Xantina , Xantina Oxidase/farmacologia , Xantinas/farmacologiaRESUMO
Xanthine oxidase was covalently immobilized on polyacrylamide gel beads, polyamide-11 and dacron. Hypoxanthine (15 ml of 200 microM), prepared in 0.1 M phosphate buffer, pH 8.0, was circulated through a column containing 1.0 g derivatized enzyme at a flow rate of 1.0 ml/min at 28 degrees C. Specific activities of 0.660, 0.072 and 0.016 Units/mg of protein were demonstrable for the polyacrylamide gel beads, dacron and polyamide-11 derivatives, respectively. The action of these water insoluble enzyme derivatives on 6-mercaptopurine (15 ml of 660 microM) was also investigated, under the same experimental conditions, showing specific activities of 0.063 Units/mg, 0.574 muUnits/mg and 0.118 muUnits/mg, respectively. The 6-mercaptopurine oxidative pathway catalyzed by immobilized xanthine oxidase on dacron stopped at the intermediate compound, 6-mercapto-8-hydroxypurine, so that no 6-thiouric acid was produced, whereas the immobilized preparations using polyacrylamide gel beads and polyamide-11 behaved like the soluble enzyme, namely, 6-thiouric acid was the final product. The behavior of dacron-xanthine oxidase compound was similar to that previously described for the derivatives obtained with carboxymethylcellulose and chitosan. The hypoxanthine oxidative pathway catalyzed by xanthine oxidase immobilized on these three supports was similar to the soluble enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Enzimas Imobilizadas/farmacologia , Mercaptopurina , Xantina Oxidase/farmacologia , Enzimas Imobilizadas/metabolismo , Xantina Oxidase/metabolismoRESUMO
Xanthine oxidase was covalently immbolized on polyacrylamide gel beads, polyamide- 11 and dacron. Hypoxanthine (15 ml of 200 µM), prepared in 0.1 M phosphate buffer, pH 8.0, was circulated through a column containing 1.0g derivatized enzyme at a flow rate of 1.0 ml/min at 28§C. Specific activities of 0.660, 0.072 and 0.016 Units/mg of protein were demonstrable for the polyacrylamide gel beads, dacron and polyamide-11 derivatives, respectively. The action of these water insoluble enzyme derivatives on 6 mercaptopurine (15 ml of 660 µM) was also investigated, under the same experimental conditions, showing specific activites of 0.063 Units/mg, 0.574 µUnits/mg and 0.118 µUnitis/mg, respectively. The 6-mercaptopurine oxidative pathway catalyzed by immobilized xanthine oxidase on dacron stopped at the intermediate compound 6-mercaptopurine oxidative on dracon stopped at the intermediate compound, 6-mercapto-8-hydroxypurine, so that no 6-thiouric acid was produced, whereas the immobilized preparations using polyacrylamide gel beads and polyamide-11 behaved like the soluble enzyme, namely, 6-thiouric acid was the final product. The behavior of dracon-xanthine oxidase immobilized on these three supports was similar to the soluble enzyme. However, although its oxidation is stoichiometric for polyacrylamide gel beads and polyamide- 11 derivatives, and no xanthine formation is observed (steady-state equilibrium), under the action of the enzymedacron derivative the xanthine formation rate (0.164 µUnits/mg) is higher than the uric acid formation rate (0.017 µUnits/mg) compared to the hypoxanthine consumption (0.072 µUnits/mg). These findings suggest again that xanthine oxidase-dacron derivative is limited to the catalysis of oxidation of hypoxanthine carbon atom number 2 as in 6-mercaptopurine
Assuntos
Mercaptopurina/química , Enzimas Imobilizadas/química , Xantina Oxidase/química , Enzimas Imobilizadas/metabolismo , Enzimas Imobilizadas/farmacologia , Xantina Oxidase/metabolismo , Xantina Oxidase/farmacologiaRESUMO
Phenazine methosulfate, a cationic electron carrier, inhibits the extracellular growth of promastigotes and the conversion of amastigotes into promastigote forms of Leishmania mexicana amazonensis. Growth inhibition and damage of extracellular parasites by PMS was counteracted by superoxide dismutase, a scavenger of the superoxide anion (O2-), and to a lesser extent, by catalase, a scavenger of hydrogen peroxide (H2O2). Inactivated dismutase and catalase were ineffective. Thus, damage of isolated L.m. amazonensis by phenazine methosulfate, involves the participation of O2- and H2O2. The role of the oxygen metabolites in the toxicity of phenazine methosulfate remains unknown. That O2- can damage the parasites is supported by the finding that superoxide dismutase also protected promastigotes from damage induced by oxygen intermediates generated by a xanthine-xanthine oxidase system. Killing of the parasites by crystal violet, a triphenylmethane, or basic blue 24, a phenothiazine, was not inhibited by superoxide dismutase.