Mechanism of clofibrate hepatotoxicity: mitochondrial damage and oxidative stress in hepatocytes

Abstract

Peroxisome proliferators have been found to induce hepatocarcinogenesis in rodents, and may cause mitochondrial damage. Consistent with this, clofibrate increased hepatic mitochondrial oxidative DNA and protein damage in mice. The present investigation aimed to study the mechanism by which this might occur by examining the effect of clofibrate on freshly isolated mouse liver mitochondria and a cultured hepatocyte cell line, AML-12. Mitochondrial membrane potential (ΔΨ_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 ΔΨ_m both in liver cells and in isolated mitochondria. The loss of ΔΨ_m occurred prior to cell death and appeared to involve the mitochondrial permeability transition (MPT), as revealed by calcein fluorescence studies and the protective effect of cyclosporin A (CsA) on the decrease in ΔΨ_m. Levels of reactive oxygen species (ROS) were measured with the fluorescent probes 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate (DCFDA) and dihydrorhodamine 123 (DHR123). Treatment of the hepatocytes with clofibrate caused a significant increase in intracellular and mitochondrial ROS. Antioxidants such as vitamin C, deferoxamine, and catalase were able 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 formation of ROS, which eventually contribute to cell death.

Introduction

Peroxisome proliferators, such as clofibrate, are a structurally diverse group of compounds that contain a number of rodent nongenotoxic hepatocarcinogens [1], [2], [3], [4], [5]. These chemicals can increase the size and the number of peroxisomes and induce the expression of genes encoding enzymes of the peroxisomal β-oxidation pathway, resulting in a dramatic elevation of the activity of peroxisomal acyl-CoA oxidase [3], [4], [5]. Increased production of H₂O₂ by this pathway is thought to play a key role in oncogenesis [5], [6], [7], although this view has been challenged [8], [9], [10]. Peroxisome proliferators can affect not only peroxisomes but also mitochondria. They increase the expression of several genes in mitochondrial DNA [11], [12], enhance the activities of some mitochondrial enzymes [13], [14], and may inhibit mitochondrial oxidative phosphorylation [15], [16]. These effects on mitochondria have been suggested to be early steps in the mechanism by which clofibrate and other peroxisome proliferators cause hepatic damage [15], [17], [18].

Recently, we have found that the levels of oxidative damage to mtDNA and proteins are elevated in livers of mice treated with clofibrate [18], suggesting that mitochondria may be a major target of cell injury caused by clofibrate-induced ROS. The present study aimed to investigate the mechanism by which this might occur, using a hepatocyte cell line and isolated mitochondrial fractions.