Masubuchi Yasuhiro, Nakayama Shintaro, Horie Toshiharu
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
Hepatology. 2002 Mar;35(3):544-51. doi: 10.1053/jhep.2002.31871.
Hepatotoxicity of diclofenac has been known in experimental animals and humans but its mechanism has not been fully understood. The present study examined the role of mitochondrial permeability transition (MPT) in the pathogenesis of diclofenac-induced hepatocyte injury by using isolated mitochondria and primary culture hepatocytes from rats. Incubation of energized mitochondria with succinate in the presence of Ca(2+) and diclofenac resulted in mitochondrial swelling, leakage of accumulated Ca(2+), membrane depolarization, and oxidation of nicotinamide adenine dinucleotide phosphate and protein thiol. All of these phenomena were suppressed by coincubation of the mitochondria with cyclosporin A, a typical inhibitor of MPT, showing that diclofenac opened the MPT pore. It was also suggested that reactive oxygen species probably generated during mitochondrial respiration and/or voltage-dependent mechanism was involved in MPT, which are proposed as mechanisms of MPT by uncouplers of mitochondrial oxidative phosphorylation. Culture of hepatocytes for 24 hours with diclofenac caused a decrease in cellular ATP, leakage of lactate dehydrogenase and membrane depolarization. The hepatocyte toxicity thus observed was attenuated by coincubation of the hepatocytes with cyclosporin A and verapamil, a Ca(2+) channel blocker. In conclusion, these results showed the important role of MPT in pathogenesis of hepatocyte injury induced by diclofenac and its possible contribution to human idiosyncratic hepatotoxicity.
双氯芬酸的肝毒性在实验动物和人类中是已知的，但其机制尚未完全明确。本研究通过使用大鼠分离的线粒体和原代培养肝细胞，探讨线粒体通透性转换（MPT）在双氯芬酸诱导的肝细胞损伤发病机制中的作用。在存在Ca(2+)和双氯芬酸的情况下，用琥珀酸孵育供能线粒体，导致线粒体肿胀、积累的Ca(2+)泄漏、膜去极化以及烟酰胺腺嘌呤二核苷酸磷酸和蛋白质硫醇的氧化。线粒体与MPT的典型抑制剂环孢素A共同孵育可抑制所有这些现象，表明双氯芬酸打开了MPT孔。还表明，线粒体呼吸过程中可能产生的活性氧物种和/或电压依赖性机制参与了MPT，这被认为是线粒体氧化磷酸化解偶联剂导致MPT的机制。用双氯芬酸培养肝细胞24小时会导致细胞ATP减少、乳酸脱氢酶泄漏和膜去极化。肝细胞与环孢素A和维拉帕米（一种Ca(2+)通道阻滞剂）共同孵育可减轻由此观察到的肝细胞毒性。总之，这些结果表明MPT在双氯芬酸诱导的肝细胞损伤发病机制中起重要作用，及其对人类特异质性肝毒性的可能影响。
