Role of mitochondrial calcium in hypochlorite induced oxidative damage of cells.

Hypochlorite (HOCl) is one of the most important mediators of inflammatory processes. Recent evidence demonstrates that changes in intracellular calcium pool play a significant role in the damaging effects of hypochlorite and other oxidants. Mitochondria are shown to be one of the intracellular targets of hypochlorite. But little is known about the mitochondrial calcium pool changes in HOCl-induced mitochondrial dysfunction. Using isolated rat liver mitochondria, we showed the oxidative damage of mitochondria (GSH oxidation and mixed protein-glutathione formation without membrane lipid peroxidation) and alterations in the mitochondrial functional parameters (decrease of respiratory activity and efficiency of oxidative phosphorylation, NADH and FADH coenzyme levels, and membrane potential) under hypochlorite action (50-300 µM). Simultaneously, the mitochondrial calcium release and swelling were demonstrated. In the presence of EGTA, the damaging effects of HOCl were less pronounced, reflecting direct involvement of mitochondrial Ca2+ in mechanisms of oxidant-induced injury. Furthermore, exposure of HeLa cells to hypochlorite resulted in a considerable increase in cytoplasmic calcium concentrations and a decrease in mitochondrial ones. Applying specific inhibitors of calcium transfer systems, we demonstrated that mitochondria play a key role in the redistribution of cytoplasmic Ca2+ ions under hypochlorite action and act as mediators of calcium release from the endoplasmic reticulum into the cytoplasm.

Position Impact of Hydroxy Groups on Spectral, Acid-Base Profiles and DNA Interactions of Several Monohydroxy Flavanones.

Structure-related biological activities of flavanones are still considered largely unexplored. Since they exhibit various medicinal activities, it is intriguing to enter deeper into their chemical structures, electronic transitions or interactions with some biomolecules in order to find properties that allow us to better understand their effects. Little information is available on biological activity of flavanone and its monohydroxy derivatives in relation to their physicochemical properties as spectral profiles, existence of protonated/deprotonated species under pH changes or interaction with Calf Thymus DNA. We devoted this work to research demonstrating differences in the physicochemical properties of the four flavanones: flavanone, 2'-hydroxyflavanone, 6-hydroxyflavanone and 7-hydroxyflavanone and linking them to their biological activity. Potentiometric titration, UV-Vis spectroscopy were used to investigate influence of pH on acid-base and spectral profiles and to propose the mode of interaction with DNA. Cyclic voltammetry was applied to evaluate antioxidant potentiality and additionally, theoretical DFT(B3LYP) method to disclose electronic structure and properties of the compounds. Molecular geometries, proton affinities and pKa values have been determined. According to computational and cyclic voltammetry results we could predict higher antioxidant activity of 6-hydroxyflavanone with respect to other compounds. The values of Kb intrinsic binding constants of the flavanones indicated weak interactions with DNA. Structure-activity relationships observed for antioxidant activity and DNA interactions suggest that 6-hydroxyflavanone can protect DNA against oxidative damage most effectively than flavanone, 2'-hydroxyflavanone or 7-hydroxyflavanone.

Ferutinin Induces Membrane Depolarization, Permeability Transition Pore Formation, and Respiration Uncoupling in Isolated Rat Liver Mitochondria by Stimulation of Ca2+-Permeability.

It is well known that the terpenoid ferutinin (4-oxy-6-(4-oxybenzoyloxy) dauc-8,9-en), isolated from the plant Ferula tenuisecta, considerably increases the permeability of artificial and cellular membranes to Ca2+-ions and produces apoptotic cell death in different cell lines in a mitochondria-dependent manner. The present study was designed for further evaluation of the mechanism(s) of mitochondrial effects of ferutinin using isolated rat liver mitochondria. Our findings provide evidence for ferutinin at concentrations of 5-27 µM to decrease state 3 respiration and the acceptor control ratio in the case of glutamate/malate as substrates. Ferutinin alone (10-60 µM) also dose-dependently dissipated membrane potential. In the presence of Ca2+-ions, ferutinin (10-60 µM) induced considerable depolarization of the inner mitochondrial membrane, which was partially inhibited by EGTA, and permeability transition pore formation, which was diminished partly by cyclosporin A, and did not influence markedly the effect of Ca2+ on mitochondrial respiration. Ruthenium Red, a specific inhibitor of mitochondrial calcium uniporter, completely inhibited Ca2+-induced mitochondria swelling and membrane depolarization, but did not affect markedly the stimulation of these Ca2+-dependent processes by ferutinin. We concluded that the mitochondrial effects of ferutinin might be primarily induced by stimulation of mitochondrial membrane Ca2+-permeability, but other mechanisms, such as driving of univalent cations, might be involved.

Calcium-Induced Mitochondrial Permeability Transitions: Parameters of Ca2+ Ion Interactions with Mitochondria and Effects of Oxidative Agents.

We evaluated the parameters of Ca2+-induced mitochondrial permeability transition (MPT) pore formations, Ca2+ binding constants, stoichiometry, energy of activation, and the effect of oxidative agents, tert-butyl hydroperoxide (tBHP), and hypochlorous acid (HOCl), on Ca2+ -mediated process in rat liver mitochondria. From the Hill plot of the dependence of MPT rate on Ca2+ concentration, we determined the order of interaction of Ca2+ ions with the mitochondrial sites, n = 3, and the apparent Kd = 60 ± 12 µM. We also found the apparent Michaelis-Menten constant, Km, for Ca2+ interactions with mitochondria to be equal to 75 ± 20 µM, whereas that in the presence of 300 µM tBHP was 120 ± 20 µM. Using the Arrhenius plots of the temperature dependences of apparent mitochondrial swelling rate at various Ca2+ concentrations, we calculated the activation energy of the MPT process. ΔEa was 130 ± 20 kJ/mol at temperatures below the break point of the Arrhenius plot (30-34 °C) and 50 ± 9 kJ/mol at higher temperatures. Ca2+ ions induced rapid mitochondrial NADH depletion and membrane depolarization. Prevention of the pore formation by cyclosporin A inhibited Ca2+-dependent mitochondrial depolarization and Mg2+ ions attenuated the potential dissipation. tBHP (10-150 µM) dose-dependently enhanced the rate of MPT opening, whereas the effect of HOCl on MPT depended on the ratio of HOCl/Ca2+. The apparent Km of tBHP interaction with mitochondria in the swelling reaction was found to be Km = 11 ± 3 µM. The present study provides evidence that three calcium ions interact with mitochondrial site with high affinity during MPT. Ca2+-induced MPT pore formations due to mitochondrial membrane protein denaturation resulted in membrane potential dissipation. Oxidants with different mechanisms, tBHP and HOCl, reduced mitochondrial membrane potential and oxidized mitochondrial NADH in EDTA-free medium and had an effect on Ca2+-induced MPT onset.

Cranberry flavonoids prevent toxic rat liver mitochondrial damage in vivo and scavenge free radicals in vitro.

The present study was undertaken for further elucidation of the mechanisms of flavonoid biological activity, focusing on the antioxidative and protective effects of cranberry flavonoids in free radical-generating systems and those on mitochondrial ultrastructure during carbon tetrachloride-induced rat intoxication. Treatment of rats with cranberry flavonoids (7 mg/kg) during chronic carbon tetrachloride-induced intoxication led to prevention of mitochondrial damage, including fragmentation, rupture and local loss of the outer mitochondrial membrane. In radical-generating systems, cranberry flavonoids effectively scavenged nitric oxide (IC50 = 4.4 ± 0.4 µg/ml), superoxide anion radicals (IC50 = 2.8 ± 0.3 µg/ml) and hydroxyl radicals (IC50 = 53 ± 4 µg/ml). The IC50 for reduction of 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH) was 2.2 ± 0.3 µg/ml. Flavonoids prevented to some extent lipid peroxidation in liposomal membranes and glutathione oxidation in erythrocytes treated with UV irradiation or organic hydroperoxides as well as decreased the rigidity of the outer leaflet of the liposomal membranes. The hepatoprotective potential of cranberry flavonoids could be due to specific prevention of rat liver mitochondrial damage. The mitochondria-addressed effects of flavonoids might be related both to radical-scavenging properties and modulation of various mitochondrial events.

Oxidative damage of rat liver mitochondria during exposure to t-butyl hydroperoxide. Role of Ca²âº ions in oxidative processes.

AIMS: The present study was designed for further evaluation of the biochemical mechanism of hepatic mitochondrial dysfunction under oxidative damages induced by organic hydroperoxide, tert-butyl hydroperoxide (tBHP), for estimation of the molecular targets impaired during oxidative stress, and for investigation of the role of Ca(2+) ions in mitochondrial oxidative reactions and of the protective effect of melatonin during mitochondrial peroxidative damage. MAIN METHODS: Mitochondria were isolated by differential centrifugation from the rat liver. The effects of tBHP exposure, EDTA, Ca(2+) ions and melatonin on mitochondrial respiratory activity, mitochondrial enzyme activities and redox status were measured. KEY FINDINGS: The present study provides evidence that tBHP (at low concentrations of 0.02-0.065mM, in EDTA-free medium) induced uncoupling of the oxidation and phosphorylation processes and decreased the efficiency of the phosphorylation reaction. This effect depended on the respiratory substrate used. The presence of EDTA prevented oxidative impairment of mitochondrial respiration, but Ca(2+) ions in the medium enhanced oxidant-induced mitochondrial damage considerably. In the presence of 0.5mM EDTA, tBHP (at high concentrations, 0.5-2mM) considerably oxidized mitochondrial reduced glutathione, enhanced accumulation of membrane lipid peroxidation products and mixed protein-glutathione disulfides and led to an inhibition of oxoglutarate dehydrogenase and succinate dehydrogenase. SIGNIFICANCE: Direct oxidative modification of enzymatic complexes of the respiratory chain and mitochondrial matrix, mitochondrial reduced glutathione depletion, protein glutathionylation, membrane lipid peroxidation and Ca(2+) overload are the main events of mitochondrial peroxidative damages. Experiments in vitro demonstrated that melatonin inhibited the mitochondrial peroxidative damage, preventing redox-balance changes and succinate dehydrogenase inactivation.

Corrections by melatonin of liver mitochondrial disorders under diabetes and acute intoxication in rats.

The aim of the present work was to investigate the mechanisms of oxidative damage of the liver mitochondria under diabetes and intoxication in rats as well as to evaluate the possibility of corrections of mitochondrial disorders by pharmacological doses of melatonin. The experimental (30 days) streptozotocin-induced diabetes mellitus caused a significant damage of the respiratory activity in rat liver mitochondria. In the case of succinate as a respiratory substrate, the ADP-stimulated respiration rate V3 considerably decreased (by 25%, p < 0·05) as well as the acceptor control ratio (ACR) V3/V2 markedly diminished (by 25%, p < 0·01). We observed a decrease of the ADP-stimulated respiration rate V3 by 35% (p < 0·05), with glutamate as substrate. In this case, ACR also decreased (by 20%, p < 0·05). Surprisingly, the phosphorylation coefficient ADP/O did not change under diabetic liver damage. Acute rat carbon tetrachloride-induced intoxication resulted in considerable decrease of the phosphorylation coefficient because of uncoupling of the oxidation and phosphorylation processes in the liver mitochondria. The melatonin administration during diabetes (10 mg·kg⁻¹ body weight, 30 days, daily) showed a considerable protective effect on the liver mitochondrial function, reversing the decreased respiration rate V3 and the diminished ACR to the control values both for succinate-dependent respiration and for glutamate-dependent respiration. The melatonin administration to intoxicated animals (10 mg·kg⁻¹ body weight, three times) partially increased the rate of succinate-dependent respiration coupled with phosphorylation. The impairment of mitochondrial respiratory plays a key role in the development of liver injury under diabetes and intoxication. Melatonin might be considered as an effector that regulates the mitochondrial function under diabetes.