Division of Biochemical Toxicology, National Center for Toxicological Research, U.S. FDA, Jefferson, AR 72079, United States of America.
Division of Biochemical Toxicology, National Center for Toxicological Research, U.S. FDA, Jefferson, AR 72079, United States of America.
Toxicol In Vitro. 2020 Dec;69:104987. doi: 10.1016/j.tiv.2020.104987. Epub 2020 Aug 28.
Perhexiline is an anti-anginal drug developed in the late 1960s. Despite its therapeutic success, it caused severe hepatoxicity in selective patients, which resulted in its withdrawal from the market. In the current study we explored the molecular mechanisms underlying the cytotoxicity of perhexiline. In primary human hepatocytes, HepaRG cells, and HepG2 cells, perhexiline induced cell death in a concentration- and time-dependent manner. Perhexiline treatment also caused a significant increase in caspase 3/7 activity at 2 h and 4 h. Pretreatment with specific caspase inhibitors suggested that both intrinsic and extrinsic apoptotic pathways contributed to perhexiline-induced cytotoxicity, which was confirmed by increased expression of TNF-α, cleavage of caspase 3 and 9 upon perhexiline treatment. Moreover, perhexiline caused mitochondrial dysfunction, demonstrated by the classic glucose-galactose assay at 4 h and 24 h. Results from JC-1 staining suggested perhexiline caused loss of mitochondrial potential. Blocking mitochondrial permeability transition pore using inhibitor bongkrekic acid attenuated the cytotoxicity of perhexiline. Western blotting analysis also showed decreased expression level of pro-survival proteins Bcl-2 and Mcl-1, and increased expression of pro-apoptotic protein Bad. Direct measurement of the activity of individual components of the mitochondrial respiratory complex demonstrated that perhexiline strongly inhibited Complex IV and Complex V and moderately inhibited Complex II and Complex II + III. Overall, our data demonstrated that both mitochondrial dysfunction and apoptosis underlies perhexiline-induced hepatotoxicity.
哌克昔林是一种上世纪 60 年代末开发的抗心绞痛药物。尽管其治疗效果良好，但它会导致选择性患者严重的肝毒性，因此已从市场上撤出。在本研究中，我们探讨了哌克昔林细胞毒性的分子机制。在原代人肝细胞、HepaRG 细胞和 HepG2 细胞中，哌克昔林呈浓度和时间依赖性诱导细胞死亡。哌克昔林处理也会导致 caspase 3/7 活性在 2 小时和 4 小时显著增加。用特异性半胱天冬酶抑制剂预处理表明，内在和外在凋亡途径都导致了哌克昔林诱导的细胞毒性，这一点通过 TNF-α 的表达增加、caspase 3 和 9 的切割得到了证实。此外，哌克昔林还会导致线粒体功能障碍，在 4 小时和 24 小时的经典葡萄糖-半乳糖测定中得到证实。JC-1 染色的结果表明，哌克昔林导致线粒体膜电位丧失。用抑制剂布格克酸阻断线粒体通透性转换孔可减轻哌克昔林的细胞毒性。Western blot 分析还显示，促生存蛋白 Bcl-2 和 Mcl-1 的表达水平降低，促凋亡蛋白 Bad 的表达水平升高。对线粒体呼吸复合物各个组成部分活性的直接测量表明，哌克昔林强烈抑制复合物 IV 和复合物 V，中度抑制复合物 II 和复合物 II+III。总之，我们的数据表明，线粒体功能障碍和凋亡是哌克昔林诱导肝毒性的基础。
