Galisteo M, Rissel M, Sergent O, Chevanne M, Cillard J, Guillouzo A, Lagadic-Gossmann D
Institut National de la Sant¿e et de la Recherche M¿edicale U456, D¿etoxication et R¿eparation Tissulaire, Facult¿e des Sciences Pharmaceutiques et Biologiques, Universit¿e de Rennes I, Rennes, France.
J Pharmacol Exp Ther. 2000 Jul;294(1):160-7.
Tacrine (THA), used in the treatment of Alzheimer's disease, is known to induce hepatotoxicity, the mechanisms of which remain to be fully established. We have previously shown that THA reduced intracellular glutathione concentration in rat hepatocytes in primary culture, thus pointing to a possible role for oxidative stress in THA toxicity. To test this, the effects of antioxidant molecules, namely, the flavonoids silibinin, silibinin dihydrogensuccinate, and silymarin, were evaluated on the toxicity of THA in cultured rat hepatocytes. This toxicity was investigated after a 24-h treatment over a concentration range from 0 to 1 mM, in the presence or absence of antioxidant (1 and 10 microM). We found that simultaneous treatment of hepatocytes with any of the antioxidants and THA remained ineffective on the lactate dehydrogenase release induced by THA. Then, the production of lipid-derived radicals (to estimate lipid peroxidation) was measured in THA (0.05-0.50 mM)-treated cells using a spin-trapping technique coupled to electron paramagnetic resonance (EPR) spectroscopy. No increase of the EPR signal was observed over the period of 30 min to 24 h. In contrast, treatment of cells with the spin label 12-doxyl stearic acid followed by EPR spectroscopy showed that THA (0.05 and 0.25 mM) rapidly increased hepatocyte membrane fluidity. Extracellular application of GM1 ganglioside (60 microM) both reversed this increase in fluidity and partially reduced lactate dehydrogenase release on THA exposure. In conclusion, this work indicates that early alterations of membrane fluidity, not resulting from lipid peroxidation, are likely to play an important role in the development of THA toxicity.
他克林（THA）用于治疗阿尔茨海默病，已知会诱发肝毒性，但其机制仍有待充分阐明。我们之前已表明，THA可降低原代培养的大鼠肝细胞内谷胱甘肽浓度，从而表明氧化应激在THA毒性中可能发挥作用。为验证这一点，评估了抗氧化分子（即黄酮类化合物水飞蓟宾、二氢琥珀酸水飞蓟宾和水飞蓟素）对培养的大鼠肝细胞中THA毒性的影响。在存在或不存在抗氧化剂（1和10 microM）的情况下，在0至1 mM的浓度范围内进行24小时处理后，研究了这种毒性。我们发现，将任何一种抗氧化剂与THA同时处理对THA诱导的乳酸脱氢酶释放均无效果。然后，使用与电子顺磁共振（EPR）光谱联用的自旋捕获技术，在经THA（0.05 - 0.50 mM）处理的细胞中测量脂质衍生自由基的产生（以估计脂质过氧化）。在30分钟至24小时期间未观察到EPR信号增加。相反，用自旋标记物12 - 硬脂酰氧基 - 2,2,6,6 - 四甲基哌啶 - 1 - 氧自由基处理细胞后进行EPR光谱分析表明，THA（0.05和0.25 mM）可迅速增加肝细胞膜流动性。细胞外应用GM1神经节苷脂（60 microM）既能逆转这种流动性增加，又能部分减少THA暴露时乳酸脱氢酶的释放。总之，这项工作表明，膜流动性的早期改变并非由脂质过氧化引起，可能在THA毒性发展中起重要作用。
