RESUMO
Oxidative stress occurs when oxidant production exceeds the antioxidant capacity to detoxify the reactive intermediates or to repair the resulting damage. Feed efficiency has been associated with mitochondrial function due to its impact on cell energy metabolism. However, mitochondria are also recognized as a major source of oxidants. The aim of this study was to determine lipid and protein oxidative stress markers, and gene and protein expression as well as activity of antioxidant enzymes in the liver of steers of divergent residual feed intake (RFI) phenotypes. Hereford steers (n = 111) were evaluated in post-weaning 70 days standard test for RFI. Eighteen steers exhibiting the greatest (n = 9; high-RFI) and the lowest (n = 9; low-RFI) RFI values were selected for this study. After the test, steers were managed together under grazing conditions until slaughter when they reached the slaughter body weight. At slaughter, hepatic samples were obtained, were snap-frozen in liquid nitrogen and stored at -80°C until analyses. Hepatic thiobarbituric acid reactive species and protein carbonyls were greater (P = 0.05) and hepatic 4-hydroxynonenal protein adducts tended (P = 0.10) to be greater for high- than low-RFI steers. Hepatic gene expression glutathione peroxidase 4, glutamate-cysteine ligase catalytic subunit and peroxiredoxin 5 mRNA was greater (P ≤ 0.05) and glutathione peroxidase 3 mRNA tended (P = 0.10) to be greater in low- than high-RFI steers. Hepatic protein expression and enzyme activity of manganese superoxide dismutase and glutathione peroxidase enzyme activity tended (P ≤ 0.10) to be greater for low- than high-RFI steers. High-efficiency steers (low-RFI) probably had better hepatic oxidative status which was strongly associated with greater antioxidant ability near to the oxidant production site and, therefore, reduced oxidative stress of the liver. Decreased hepatic oxidative stress would reduce maintenance requirements due to a lower protein and lipid turnover and better efficiency in the use of energy.
Assuntos
Ração Animal/análise , Bovinos/fisiologia , Metabolismo Energético , Fígado/metabolismo , Estresse Oxidativo , Animais , Bovinos/genética , Masculino , Oxirredução , FenótipoRESUMO
Increased production of reactive nitrogen (RNS) and oxygen (ROS) species and its detrimental effect to mitochondria are associated with endothelial dysfunction. This study was designed to determine the effect of a peroxynitrite flux, promoted by 1,3-morpholinosydnonimine (SIN-1), in mitochondrial function and some redox homeostasis parameters in bovine aortic endothelial cells (BAEC). Moreover, the effect of diphenyl diselenide (PhSe)2, a simple organic selenium compound, in preventing peroxynitrite-mediated cytotoxicity was also investigated. Our results showed that overnight exposure to SIN-1 (250 µM) caused a profound impairment of oxygen consumption, energy generation and reserve capacity in mitochondria of BAEC. Mitochondrial dysfunction resulted in an additional intracellular production of peroxynitrite, amplifying the phenomenon and leading to changes in redox homeostasis. Moreover, we observed an extensive decline in mitochondrial membrane potential (ΔΨm) induced by peroxynitrite and this event was associated with apoptotic-type cell death. Alternatively, the pretreatment of BAEC with (PhSe)2, hindered peroxynitrite-mediated cell damage by preserving mitochondrial and endothelial function and consequently preventing apoptosis. The protective effect of (PhSe)2 was related to its ability to improve the intracellular redox state by increasing the expression of different isoforms of peroxiredoxins (Prx-1-3), efficient enzymes in peroxynitrite detoxification.
Assuntos
Derivados de Benzeno/farmacologia , Células Endoteliais/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Molsidomina/análogos & derivados , Compostos Organosselênicos/farmacologia , Peroxirredoxinas/metabolismo , Ácido Peroxinitroso/metabolismo , Animais , Aorta/citologia , Bovinos , Células Endoteliais/enzimologia , Homeostase/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/enzimologia , Molsidomina/química , Oxirredução , Ácido Peroxinitroso/química , Ácido Peroxinitroso/toxicidadeRESUMO
3-Hydroxyglutaric acid (3HGA) accumulates in the inherited neurometabolic disorder known as glutaryl-CoA dehydrogenase deficiency. The disease is clinically characterized by severe neurological symptoms, frontotemporal atrophy and striatum degeneration. Because of the pathophysiology of the brain damage in glutaryl-CoA dehydrogenase deficiency is not completed clear, we investigated the in vitro effect of 3HGA (0.01-5.0mM) on critical enzyme activities of energy metabolism, including the respiratory chain complexes I-V, creatine kinase isoforms and Na(+),K(+)-ATPase in cerebral cortex and striatum from 30-day-old rats. Complex II activity was also studied in rat C6-glioma cells exposed to 3HGA. The effect of 3HGA was further investigated on the rate of oxygen consumption in mitochondria from rat cerebrum. We observed that 1.0mM 3HGA significantly inhibited complex II in cerebral cortex and C6 cells but not the other activities of the respiratory chain complexes. Creatine kinase isoforms and Na(+),K(+)-ATPase were also not affected by the acid. Furthermore, no inhibition of complex II activity occurred when mitochondrial preparations from cerebral cortex or striatum homogenates were used. In addition, 3HGA significantly lowered the respiratory control ratio in the presence of glutamate/malate and succinate under stressful conditions or when mitochondria were permeabilized with digitonin. Since 3HGA stimulated oxygen consumption in state IV and compromised ATP formation, it can be presumed that this organic acid might act as an endogenous uncoupler of mitochondria respiration. Finally, we observed that 3HGA changed C6 cell morphology from a round flat to a spindle-differentiated shape, but did not alter cell viability neither induced apoptosis. The data provide evidence that 3HGA provokes a moderate impairment of brain energy metabolism and do not support the view that 3HGA-induced energy failure would solely explain the characteristic brain degeneration observed in glutaryl-CoA dehydrogenase deficiency patients.
Assuntos
Química Encefálica/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Glutaratos/farmacologia , Animais , ATPase de Ca(2+) e Mg(2+)/metabolismo , Linhagem Celular Tumoral/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Sobrevivência Celular/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Córtex Cerebral/metabolismo , Creatina Quinase/metabolismo , Citosol/enzimologia , Transporte de Elétrons/efeitos dos fármacos , Glioma/metabolismo , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Consumo de Oxigênio/efeitos dos fármacos , Ratos , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Peroxynitrite (ONOO(-)), the product of superoxide (O(2)) and nitric oxide (.NO) reaction, inhibits mitochondrial respiration and can stimulate apoptosis. Cytochrome c, a mediator of these two aspects of mitochondrial function, thus represents an important potential target of ONOO(-) during conditions involving accelerated rates of oxygen radical and.NO generation. Horse heart cytochrome c(3+) was nitrated by ONOO(-), as indicated by spectral changes, Western blot analysis, and mass spectrometry. A dose-dependent loss of cytochrome c(3+) 695 nm absorption occurred, inferring that nitration of a critical heme-vicinal tyrosine (Tyr-67) promoted a conformational change, displacing the Met-80 heme ligand. Nitration was confirmed by cross-reactivity with a specific antibody against 3-nitrotyrosine and by increased molecular mass compatible with the addition of a nitro-(-NO(2)) group. Mass analysis of tryptic digests indicated the preferential nitration of Tyr-67 among the four conserved tyrosine residues in cytochrome c. Cytochrome c(3+) was more extensively nitrated than cytochrome c(2+) because of the preferential oxidation of the reduced heme by ONOO(-). Similar protein nitration patterns were obtained by ONOO(-) reaction in the presence of carbon dioxide, whereupon secondary nitrating species arise from the decomposition of the nitroso-peroxocarboxylate (ONOOCO(2)(-)) intermediate. Peroxynitrite-nitrated cytochrome c displayed significant changes in redox properties, including (a) increased peroxidatic activity, (b) resistance to reduction by ascorbate, and (c) impaired support of state 4-dependent respiration in intact rat heart mitochondria. These results indicate that cytochrome c nitration may represent both oxidative and signaling events occurring during .NO- and ONOO(-)-mediated cell injury.
Assuntos
Grupo dos Citocromos c/química , Grupo dos Citocromos c/metabolismo , Mitocôndrias Cardíacas/metabolismo , Nitratos/química , Animais , Sítios de Ligação , Cavalos , Cinética , Espectrometria de Massas , Modelos Moleculares , Conformação Molecular , Nitratos/farmacologia , Oxidantes/química , Oxidantes/farmacologia , Mapeamento de Peptídeos , Conformação Proteica , Ratos , Espectrofotometria , TripsinaRESUMO
Various authors have suggested that nitric oxide (.NO) exerts cytotoxic effects through the inhibition of cellular respiration. Indeed, in intact cells .NO inhibits glutamate-malate (complex I) as well as succinate (complex II)-supported mitochondrial electron transport, without affecting TMPD/ascorbate (complex IV)-dependent respiration. However, experiments in our lab using isolated rat heart mitochondria indicated that authentic .NO inhibited electron transport mostly by reversible binding to the terminal oxidase, cytochrome a3, having a less significant effect on complex II- and no effect on complex I-electron transport components. The inhibitory action of .NO was more profound at lower oxygen tensions and resulted in differential spectra similar to that observed in dithionite-treated mitochondria. On the other hand, continuous fluxes of .NO plus superoxide (O.(2)(-)), which lead to formation of micromolar steady-state levels of peroxynitrite anion (ONOO-), caused a strong inhibition of complex I- and complex II-dependent mitochondrial oxygen consumption and significantly inhibited the activities of succinate dehydrogenase and ATPase, without affecting complex IV-dependent respiration and cytochrome c oxidase activity. In conclusion, even though nitric oxide can directly cause a transient inhibition of electron transport, the inhibition pattern of mitochondrial respiration observed in the presence of peroxynitrite is the one that closely resembles that found secondary to .NO interactions with intact cells and strongly points to peroxynitrite as the ultimate reactive intermediate accounting for nitric oxide-dependent inactivation of electron transport components and ATPase in living cells and tissues.
Assuntos
Mitocôndrias Cardíacas/metabolismo , Nitratos/farmacologia , Óxido Nítrico/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Animais , Ditionita/farmacologia , Transporte de Elétrons/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Glutationa/análogos & derivados , Glutationa/farmacologia , Cinética , Mitocôndrias Cardíacas/efeitos dos fármacos , NADH Desidrogenase/metabolismo , Compostos Nitrosos/farmacologia , Penicilamina/análogos & derivados , Penicilamina/farmacologia , Ratos , S-Nitroso-N-Acetilpenicilamina , S-Nitrosoglutationa , Succinato Desidrogenase/metabolismo , Superóxidos/farmacologiaRESUMO
A recent report (Radi et al., J. Biol. Chem. 266:22028-22034, 1991) showed that rat heart mitochondria contain catalase. The protective role of mitochondrial catalase was tested by exposing heart or kidney mitochondria and mitoplasts to two oxidants (H2O2) or tert-butyl hydroperoxide, t-BOOH), estimating lipid peroxidation (as thiobarbituric acid-reactive substances, TBARS) and overall oxidative stress (as chemiluminescence). Additional controls included heart and kidney preparations from aminotriazole-treated (catalase-depleted) rats. Both oxidants increased TBARS in catalase-free preparations to similar extents over their respective controls (between 200 to 350%). In catalase-containing preparations, H2O2 lipid peroxidation increased by only 40 to 96% over controls. Similar qualitative results were obtained when measuring chemiluminescence. The catalytic role of cytochrome c in mitochondrial lipid peroxidation was investigated by exposing either control or cytochrome-c-depleted kidney mitoplasts (catalase free) to either H2O2 or t-BOOH. Hydrogen-peroxide-dependent mitochondrial lipid peroxidation varied with cytochrome c concentration, remaining close to controls when cytochrome c concentration decreased by 66%, even though there was no catalase present. Tert-butyl hydroperoxide-dependent lipid peroxidation was less affected by cytochrome c remaining 2.3-fold above controls under the same conditions, suggesting that organic peroxides are more likely to remain in the less polar membrane environment being decomposed by heme or nonheme iron imbedded in the inner mitochondrial membrane. Chemiluminescence was less affected by cytochrome c depletion. Comparing control and cytochrome-c-deficient mitochondria, chemiluminescence was 1.7-fold and 2.8-fold higher when control preparations were challenged with t-BOOH or H2O2, respectively.