Critical role of free cytosolic calcium, but not uncoupling, in mitochondrial permeability transition and cell death induced by diclofenac oxidative metabolites in immortalized human hepatocytes
Critical role of free cytosolic calcium, but not uncoupling, in mitochondrial permeability transition and cell death induced by diclofenac oxidative metabolites in immortalized human hepatocytes
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DOI：
10.1016/j.taap.2006.09.012
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发表时间：
2006-12-15
影响因子：
3.8
通讯作者：
Boelsterli, Urs A.
中科院分区：
文献类型：
--
作者：
Lim, Miao Shan;Lim, Priscilla L. K.;Boelsterli, Urs A.
双氯芬酸是一种广泛使用的非甾体抗炎药，与罕见但严重的肝毒性相关。实验证据表明，双氯芬酸靶向线粒体并诱导通透性转换（mPT），导致肝细胞凋亡。虽然下游效应机制已得到很好的表征，但导致mPT的更近端途径尚不清楚。本研究旨在探讨胞浆游离钙（Ca-c（2+））在甘草酸诱导的永生化人肝细胞损伤中的作用。我们发现，暴露于双氯芬酸引起的时间和浓度依赖性细胞损伤，这是由特定的mPT抑制剂环孢素A（CsA，5 μ M）。在8 h时，双氯芬酸导致[Ca 2 +]（c）（Fluo-4荧光）增加，这不受CsA的影响。联合暴露于双氯芬酸/BAPTA（Ca 2+螯合剂）可抑制细胞损伤，表明Ca 2+在沉淀mPT中起关键作用。即使在存在CsA或BAPTA的情况下，双氯芬酸也可降低线粒体膜电位Δ psi（m）（JC-1荧光），表明线粒体去极化不是mPT或[Ca 2 +]升高的结果（c）。CYP 2C 9抑制剂磺胺苯吡唑（10 μ M）可保护细胞免受双羟萘酸诱导的细胞损伤，并防止[Ca 2 +]（c）增加，但对Delta psi（m）的消散无影响。最后，双氯芬酸暴露大大增加了继发于[Ca 2 +]增加的双氯芬酸选择性超氧化物水平（c）。总之，这些数据表明，双氯芬酸对线粒体具有直接去极化作用，不会导致细胞损伤，而CYP 2C 9介导的生物活化导致[Ca 2 +]（c）增加，触发mPT并促使细胞死亡。(c)2006年爱思唯尔公司All rights reserved.
Diclofenac is a widely used nonsteroidal anti-inflammatory drug that has been associated with rare but serious hepatotoxicity. Experimental evidence indicates that diclofenac targets mitochondria and induces the permeability transition (mPT) which leads to apoptotic cell death in hepatocytes. While the downstream effector mechanisms have been well characterized, the more proximal pathways leading to the mPT are not known. The purpose of this study was to explore the role of free cytosolic calcium (Ca-c(2+)) in diclofenac-induced cell injury in immortalized human hepatocytes. We show that exposure to diclofenac caused time- and concentration-dependent cell injury, which was prevented by the specific mPT inhibitor cyclosporin A (CsA, 5 mu M). At 8 h, diclofenac caused increases in [Ca2+](c) (Fluo-4 fluorescence), which was unaffected by CsA. Combined exposure to diclofenac/BAPTA (Ca2+ chelator) inhibited cell injury, indicating that Ca2+ plays a critical role in precipitating mPT. Diclofenac decreased the mitochondrial membrane potential, Delta psi(m), (JC-1 fluorescence), even in the presence of CsA or BAPTA, indicating that mitochondrial depolarization was not a consequence of the mPT or elevated [Ca2+](c). The CYP2C9 inhibitor sulphaphenazole (10 mu M) protected from diclofenac-induced cell injury and prevented increases in [Ca2+](c) while it had no effect on the dissipation of the Delta psi(m). Finally, diclofenac exposure greatly increased the mitochondria-selective superoxide levels secondary to the increases in [Ca2+](c). In conclusion, these data demonstrate that diclofenac has direct depolarizing effects on mitochondria which does not lead to cell injury, while CYP2C9-mediated bioactivation causes increases in [Ca2+](c), triggering the mPT and precipitating cell death. (c) 2006 Elsevier Inc. All rights reserved.