Ratra G S, Morgan W A, Mullervy J, Powell C J, Wright M C
Department of Toxicology, St Bartholomew's and the Royal London School of Medicine and Dentistry, UK.
Toxicology. 1998 Sep 15;130(2-3):79-93. doi: 10.1016/s0300-483x(98)00096-1.
Methapyrilene (MP) is an unusual hepatotoxin in that it causes periportal necrosis in rats. The mechanism of acute methapyrilene hepatotoxicity has, therefore, been investigated in cultured male rat hepatocytes. Addition of methapyrilene to rat hepatocytes resulted in a time- and dose-dependent loss in cell viability between 4 and 8 h of incubation as judged by cellular enzyme leakage. The cytochrome P450 (CYP) inhibitor metyrapone protected against methapyrilene-mediated toxicity suggesting that MP is metabolised by CYP for toxicity. The concentration-dependent protection from methapyrilene toxicity afforded by metyrapone correlated with an inhibition of microsomal CYP2C11-associated androstenedione 16alpha hydroxylase activity, and hepatocytes prepared from hypophysectomised rats (containing reduced levels of microsomal immunodetectable CYP2C11 and associated androstenedione 16alpha hydroxylase activity) showed resistance to the toxic effects of methapyrilene. These data suggest that the toxicity of methapyrilene is predominantly dependent on the CYP2C11 isoform. Treatment of hepatocytes with a toxic concentration of MP caused oxidative stress as indicated by increases in NADP+ levels within 2 h and cellular thiol oxidation as evidenced by a reduction--but not complete loss--in glutathione levels. Methapyrilene hepatotoxicity was associated with an early loss in mitochondrial function, as indicated by mitochondrial swelling and significant losses in cellular ATP within 2 h. Co-incubation of methapyrilene-treated hepatocytes with inhibitors of inner mitochondrial transition permeability pore opening--cyclosporin A or the thiol reductant dithiothreitol--abrogated cell death suggesting that pore opening and loss of mitochondrial Ca2+ homeostasis play a significant role in methapyrilene-mediated cell death. Co-incubation of methapyrilene-treated hepatocytes with the phenylalkylamine calcium channel blocker verapamil--but not by treating cells in a nominally calcium-free medium--also abrogated cell death, suggesting that if Ca2+ is involved in cell killing then it is dependent on an intracellular Ca2+ pool. Pre-treatment of hepatocytes for 1 h with verapamil--to inhibit intracellular Ca2+ pool filling--increased the potency of verapamil protection against methapyrilene toxicity by approximately 100-fold. Taken together, these data indicate that methapyrilene intoxication leads to mitochondrial disfunction and suggest a critical role for a loss of mitochondrial Ca2+ homeostasis in this model of hepatocyte death.
甲吡咯烷（MP）是一种不同寻常的肝毒素，它会导致大鼠肝门周围坏死。因此，人们在培养的雄性大鼠肝细胞中研究了急性甲吡咯烷肝毒性的机制。将甲吡咯烷添加到大鼠肝细胞中，在孵育4至8小时后，根据细胞酶泄漏判断，细胞活力出现了时间和剂量依赖性的丧失。细胞色素P450（CYP）抑制剂美替拉酮可保护细胞免受甲吡咯烷介导的毒性，这表明MP通过CYP代谢产生毒性。美替拉酮对甲吡咯烷毒性的浓度依赖性保护作用与微粒体CYP2C11相关的雄烯二酮16α羟化酶活性的抑制相关，并且从垂体切除大鼠制备的肝细胞（微粒体免疫可检测的CYP2C11和相关的雄烯二酮16α羟化酶活性水平降低）对甲吡咯烷的毒性表现出抗性。这些数据表明，甲吡咯烷的毒性主要取决于CYP2C11同工型。用有毒浓度的MP处理肝细胞会导致氧化应激，表现为2小时内NADP +水平升高，以及细胞硫醇氧化，表现为谷胱甘肽水平降低但未完全丧失。甲吡咯烷肝毒性与线粒体功能的早期丧失有关，表现为线粒体肿胀和2小时内细胞ATP显著损失。将用甲吡咯烷处理的肝细胞与线粒体内膜通透性转换孔开放抑制剂环孢素A或硫醇还原剂二硫苏糖醇共同孵育可消除细胞死亡，这表明孔开放和线粒体Ca2 +稳态丧失在甲吡咯烷介导的细胞死亡中起重要作用。将用甲吡咯烷处理的肝细胞与苯烷基胺钙通道阻滞剂维拉帕米共同孵育（但不是在名义上无钙的培养基中处理细胞）也可消除细胞死亡，这表明如果Ca2 +参与细胞杀伤，那么它依赖于细胞内Ca2 +池。用维拉帕米预处理肝细胞1小时以抑制细胞内Ca2 +池填充，可使维拉帕米对甲吡咯烷毒性的保护效力提高约100倍。综上所述，这些数据表明甲吡咯烷中毒会导致线粒体功能障碍，并表明线粒体Ca2 +稳态丧失在这种肝细胞死亡模型中起关键作用。
