Mechanism of clofibrate hepatotoxicity: Mitochondrial damage and oxidativestress in hepatocytes

Peroxisome proliferators have been found to induce hepatocarcinogenesis in rodents, and may cause mitochondrial damage. Consistent with this, clofibra te increased hepatic mitochondrial oxidative DNA and protein damage in mice . The present investigation aimed to study the mechanism by which this migh t occur by examining the effect of clofibrate on freshly isolated mouse liv er mitochondria and a cultured hepatocyte cell line, AML-12. Mitochondrial membrane potential (Delta Psi (m)) was determined by using the fluorescent dye 5,5',6,6'-tetrachloro-1,1', 3,3'-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) and tetramethylrhodamine methyl ester (TMRM). Application of clofibrate at concentrations greater than 0.3 mM rapidly collapsed the Delt a Psi (m) both in liver cells and in isolated mitochondria. The loss of Del ta Psi (m) occurred prior to cell death and appeared to involve the mitocho ndrial permeability transition (MPT), as revealed by calcein fluorescence s tudies and the protective effect of cyclosporin A (CsA) on the decrease in Delta Psi (m). Levels of reactive oxygen species (ROS) were measured with t he fluorescent probes 5-(and-6)carboxy-2',7'-dichlorofluoreseein diacetate (DCFDA) and dihydrorhodamine 123 (DHR123). Treatment of the hepatocytes wit h clofibrate caused a significant increase in intracellular and mitochondri al ROS. Antioxidants such as vitamin C, deferoxamine, and catalase were abl e to protect the cells against the clofibrate-induced loss of viability, as was CsA, but to a lesser extent. These results suggest that one action of clofibrate might be to impair mitochondrial function, so stimulating format ion of ROS, which eventually contribute to cell death. (C) 2001 Elsevier Sc ience Inc.