Department of Biochemistry, Yanka Kupala Grodno State University, Blvd. Len. Kom., 50, 230017 Grodno, Belarus.
Life Sci. 2013 Jun 21;92(23):1110-7. doi: 10.1016/j.lfs.2013.04.009. Epub 2013 Apr 30.
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.
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.
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.
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.
本研究旨在进一步评估有机氢过氧化物叔丁基氢过氧化物（tBHP）诱导的肝线粒体功能障碍的生化机制，评估氧化应激过程中受损的分子靶点，研究 Ca(2+) 离子在线粒体氧化反应中的作用以及褪黑素在防止线粒体过氧化损伤中的保护作用。
通过差速离心从大鼠肝脏中分离出线粒体。测量 tBHP 暴露、EDTA、Ca(2+) 离子和褪黑素对线粒体呼吸活性、线粒体酶活性和氧化还原状态的影响。
本研究提供的证据表明，tBHP（在 0.02-0.065mM 的低浓度下，在无 EDTA 的培养基中）诱导氧化和磷酸化过程解偶联，并降低磷酸化反应的效率。这种效应取决于所使用的呼吸底物。EDTA 的存在可以防止线粒体呼吸的氧化损伤，但培养基中的 Ca(2+) 离子大大增强了氧化剂诱导的线粒体损伤。在 0.5mM EDTA 的存在下，tBHP（在高浓度下，0.5-2mM）会极大地氧化线粒体还原型谷胱甘肽，增加膜脂过氧化产物和混合蛋白-谷胱甘肽二硫化物的积累，并导致氧化戊二酸脱氢酶和琥珀酸脱氢酶的抑制。
呼吸链和线粒体基质酶复合体的直接氧化修饰、线粒体还原型谷胱甘肽耗竭、蛋白谷胱甘肽化、膜脂质过氧化和 Ca(2+) 过载是线粒体过氧化损伤的主要事件。体外实验表明，褪黑素抑制了线粒体过氧化损伤，防止了氧化还原平衡的改变和琥珀酸脱氢酶的失活。
