cytochrome-c-t and dihydroethidium

The effects of a moderate electrical stimulation on superoxide and nitric oxide production by primary cultured skeletal muscle cells were evaluated. The involvement of the main sites of these reactive species production and the relationship between superoxide and nitric oxide production were also examined. Production of superoxide was evaluated by cytochrome c reduction and dihydroethidium oxidation assays. Electrical stimulation increased superoxide production after 1 h incubation. A xanthine oxidase inhibitor caused a partial decrease of superoxide generation and a significant amount of mitochondria-derived superoxide was also observed. Nitric oxide production was assessed by nitrite measurement and by using 4,5-diaminofluorescein diacetate (DAF-2-DA) assay. Using both methods an increased production of nitric oxide was obtained after electrical stimulation, which was also able to induce an increase of iNOS content and NF-κB activation. The participation of superoxide in nitric oxide production was investigated by incubating cells with DAF-2-DA in the presence or absence of electrical stimulation, a superoxide generator system (xanthine-xanthine oxidase), a mixture of NOS inhibitors and SOD-PEG. Our data show that the induction of muscle contraction by a moderate electrical stimulation protocol led to an increased nitric oxide production that can be controlled by superoxide generation. The cross talk between these reactive species likely plays a role in exercise-induced maintenance and adaptation by regulating muscular glucose metabolism, force of contraction, fatigue, and antioxidant systems activities.

Topics: Animals; Cells, Cultured; Cytochromes c; Electric Stimulation; Enzyme Inhibitors; Ethidium; Fluorescein; Free Radical Scavengers; Mitochondria, Muscle; Muscle Contraction; Muscle Fibers, Skeletal; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Nitrites; Oxidation-Reduction; Oxidative Stress; Primary Cell Culture; Rats; Rats, Wistar; Signal Transduction; Superoxides; Time Factors; Xanthine; Xanthine Oxidase

The bleaching of the pyrogallol red (PGR) dye mediated by superoxide anion radicals (O(2)(-)) generated from the xanthine/xanthine oxidase system (X/XO) was studied by UV-visible spectrophotometry. The absorption band (at 540 nm) of PGR quickly decreased in the presence of X/XO, implying an efficient reaction of O(2)(-) with PGR. The process was unaffected by catalase (CAT), but completely abolished by superoxide dismutase (SOD). A mechanism of the reaction involving the consumption of one PGR molecule by two O(2)(-) to generate one molecule of H(2)O(2) is proposed. PGR was used as a probe to estimate the rate of O(2)(-) generation in redox cycling reactions of a series of nitro compounds mediated by rat liver microsomes. The consumption of PGR induced by the redox cycling of nitrofurantoin was totally eliminated by the addition of SOD but unaffected by CAT. The initial rate of consumption of PGR mediated by the redox cycling of others nitro derivatives follows the order: furazolidindione > nitrofurantoin > nifurtimox > benznidazole > chloramphenicol. We concluded that PGR can be used as a probe to estimate the release of O(2)(-) from enzymatic systems or from the redox cycling of nitro compounds.

Topics: Animals; Cytochromes c; Ethidium; Hydrogen Peroxide; Male; Microsomes, Liver; Nitro Compounds; Oxidation-Reduction; Pyrogallol; Rats; Rats, Sprague-Dawley; Superoxides; Xanthine; Xanthine Oxidase

Prompted by the recently reported capacity of the physiologically occurring Cu(I)-[glutathione](2) complex (Cu(I)-[GSH)](2)) to reduce oxygen, the effect of various GSH-binding metals (Co(2+), Cd(2+), Zn(2+), Pb(2+), Al(3+), Hg(2+) and Ni(2+)) on the superoxide-generating capacity of such complex was investigated. Amongst all tested metals, only Hg(2+) was able to substantially affect the capacity of Cu(I)-[GSH](2) to generate superoxide. When Hg(2+) and Cu(I)-[GSH](2) were mixed equimolarly, the superoxide formation, assessed through the cytochrome c reduction and dihydroethidium oxidation, was increased by over 50%. Such effect was totally inhibitable by SOD. Based on the reportedly higher affinity of Hg(2+) for GSH and the observed ability of Hg(2+) to lower the concentration of Cu(I)-[GSH](2) (spectroscopically assessed), we suggest that Hg(2+) displaces Cu(I) from Cu(I)-[GSH](2), to release Cu(I) ions and form a Hg(II)-[GSH](2) complex. The latter species would account for the Hg(2+)-induced exacerbation of the superoxide production. In fact, the present study provides first time evidence that a preformed Hg(II)-[GSH](2) complex is able to concentration-dependently reduce oxygen. Such redox-activity was evidenced using cytochrome c and confirmed by EPR studies using DMPO (5,5-dimethyl-1-pyrroline N-oxide, a spin-trapping agent). Considering this novel ability of Hg(II)-[GSH](2) to generate superoxide, a further characterization of its redox-activity and its potential to affect superoxide-susceptible biological targets appears warranted.

Topics: Acetaminophen; Algorithms; Copper; Cytochromes c; Electron Spin Resonance Spectroscopy; Ethidium; Glutathione; Hydrogen Peroxide; Mercury; Models, Chemical; Organometallic Compounds; Oxidation-Reduction; Protein Binding

Cu(2+) ions and GSH molecules interact swiftly to form the complex Cu(I)-glutathione. We investigated the potential capacity of such complex to reduce molecular oxygen. The addition of SOD to a solution containing Cu(I)-glutathione led to a sustained decline of the basal oxygen level. Such effect was partially reverted by the addition of catalase. The complex was able to induce the reduction of cytochrome c and the oxidation of dyhydroethidium into 2-hydroxyethidium. Both effects were totally blocked by SOD. The ability of the complex to generate superoxide radicals was confirmed by EPR spin-trapping. Cu(I)-glutathione induces no oxidation of fluorescein, a hydroxyl radical-sensitive probe. We conclude that in solutions containing the complex, oxygen is continually reduced into superoxide, and that-in absence of interceptors-the latter radicals are quantitatively re-oxidized into molecular oxygen. We suggest that, by functioning as a continuous source of superoxide, the complex could potentially affect a broad range of susceptible biological targets.

Topics: Animals; Cattle; Chromatography, High Pressure Liquid; Copper; Cytochromes c; Electron Spin Resonance Spectroscopy; Ethidium; Fluorescein; Glutathione; Oxidation-Reduction; Oxygen; Superoxides

Polyunsaturated fatty acids (PUFAs) can influence tumor growth and migration, both in vitro and in vivo. The PUFA gamma-linolenic acid (GLA) has been reported to improve the poor prognosis associated with human gliomas, although its effects at sublethal concentrations on residual cells postsurgery are poorly understood. The study investigated the effects sublethal PUFA doses (90 or 150 microM) may have on rat C6 glioma cell energy metabolism, since an adequate energy supply is essential for cell proliferation, migration, and apoptosis. Of note was the identification of mitochondrial heterogeneity in relation to the mitochondrial membrane potential (MMP), which has been suggested but unproven in previous studies. GLA and eicosapentaenoic acid (EPA) caused significant changes in cellular fatty acid composition and increased the percentage of cells with a low MMP after a 96-h exposure period. The presence of PUFAs inhibited C6 cell proliferation and migration, although apoptosis was not induced. The protein expression and activity of glucose-6-phosphate dehydrogenase was increased after 96-h incubation with 90 microM GLA and EPA and would allow redox regulation through increased NADPH production, permitting the maintenance of adequate intracellular reduced glutathione concentrations and limiting rates of lipid peroxidation and reactive oxygen species generation. Neither NADP(+)-isocitrate dehydrogenase nor NADP(+)-malate dehydrogenase activity responded to PUFAs, suggesting it is glucose-6-phosphate dehydrogenase that is the principal source of NADPH in C6 cells. These data compliment studies showing that higher concentrations of GLA induced glioma cell death and tumor regression and suggest that GLA treatment could be useful for the inhibition of residual cell proliferation and migration after surgical removal of the tumor mass.

Topics: Animals; Annexin A5; Benzimidazoles; Blotting, Western; Carbocyanines; Cell Movement; Cells, Cultured; Cytochromes c; DNA Fragmentation; Ethidium; Fatty Acids, Unsaturated; Fluorescent Antibody Technique; Fluorescent Dyes; Gas Chromatography-Mass Spectrometry; Glioma; Glucosephosphate Dehydrogenase; Membrane Potentials; Microscopy, Confocal; Mitochondria; Mitosis; Rats; Time Factors