Gómez-Lechón Maria-José, Ponsoda Xavier, O'Connor Enrique, Donato Teresa, Jover R, Castell José V
Centro de Investigación, Hospital La Fe, Avda de Campanar 21, E-46009, Valencia, Spain.
Toxicol In Vitro. 2003 Oct-Dec;17(5-6):675-80. doi: 10.1016/s0887-2333(03)00105-x.
Hepatotoxicity is one of the side effects associated with the administration of diclofenac, a non-steroidal anti-inflammatory drug widely used clinically. The effect of diclofenac on the early events that trigger apoptosis cascade have been evaluated in rat hepatocytes. To do this, early and late apoptotic markers, associated with the pivotal steps of the execution phase, have been evaluated after incubation with the drug. The results show that the apoptotic effect of diclofenac occurs after exposure to sub-cytotoxic concentrations of the drug (maximal non toxic concentration, MNTC, after 24-h treatment was 450 microM), without overlapping with cell necrosis (LDH leakage evaluation). Flow cytometric analysis revealed a time- and dose-dependent increase of apoptotic nuclei with sub-diploid DNA content. Caspase 3 activation (3-5-fold control) was maximal after 12 h of exposure to 350 microM of the drug. The involvement of the mitochondrial permeability transition (MPT) in diclofenac-induced apoptosis was investigated. Cyclosporine A and decylubiquinone, MPT specific inhibitor, prevented the activation of caspase 3, thus showing that diclofenac opened the MPT pore. Treatment of hepatocytes with antioxidants (alpha-tocopherol, N,N-dimethylthiourea, superoxide dismutase) were able to prevent caspase cascade activation by diclofenac, revealing that oxidative stress at the mitochondrial level is involved in MPT induction. Finally, the differential cytotoxic and apoptotic effect produced in hepatocytes and non-metabolizing hepatoma cells suggest that CYP-mediated metabolism of diclofenac apoptosis may be related to the apoptotic effect of the drug.
肝毒性是临床广泛使用的非甾体抗炎药双氯芬酸给药相关的副作用之一。已在大鼠肝细胞中评估了双氯芬酸对触发凋亡级联反应早期事件的影响。为此，在与该药物孵育后，评估了与执行阶段关键步骤相关的早期和晚期凋亡标志物。结果表明，双氯芬酸的凋亡作用在暴露于亚细胞毒性浓度的药物后出现（24小时处理后的最大无毒浓度，MNTC，为450微摩尔），且与细胞坏死不重叠（乳酸脱氢酶泄漏评估）。流式细胞术分析显示，亚二倍体DNA含量的凋亡细胞核呈时间和剂量依赖性增加。暴露于350微摩尔该药物12小时后，半胱天冬酶3的激活（比对照高3 - 5倍）达到最大值。研究了线粒体通透性转换（MPT）在双氯芬酸诱导的凋亡中的作用。MPT特异性抑制剂环孢素A和癸基泛醌可阻止半胱天冬酶3的激活，从而表明双氯芬酸打开了MPT孔。用抗氧化剂（α - 生育酚、N，N - 二甲基硫脲、超氧化物歧化酶）处理肝细胞能够阻止双氯芬酸激活半胱天冬酶级联反应，这表明线粒体水平的氧化应激参与了MPT的诱导。最后，肝细胞和非代谢性肝癌细胞产生的不同细胞毒性和凋亡作用表明，双氯芬酸的细胞色素P450介导的代谢凋亡可能与该药物的凋亡作用有关。
