Cytotoxicity and Apoptosis Produced by Arachidonic Acid in Hep G2 Cells Overexpressing Human Cytochrome P4502E1

Abstract

The goal of the current study was to evaluate the effects of arachidonic acid, as a representative polyunsaturated fatty acid, on the viability of a Hep G2 cell line, which has been transduced to express human cytochrome P4502E1 (CYP2E1). Arachidonic acid produced a concentration- and time-dependent toxicity to Hep G2-MV2E1-9 cells, which express CYP2E1, but little or no toxicity was found with control Hep G2-MV-5 cells, which were infected with retrovirus lacking human CYP2E1 cDNA. In contrast to arachidonic acid, oleic acid was not toxic to the Hep G2-MV2E1-9 cells. The cytotoxicity of arachidonic acid appeared to involve a lipid peroxidation type of mechanism since toxicity was enhanced after depletion of cellular glutathione; formation of malondialdehyde and 4-hydroxy-2-nonenal was markedly elevated in the cells expressing CYP2E1, and toxicity was prevented by antioxidants such as alpha-tocopherol phosphate, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), propylgallate, ascorbate, and diphenylphenylenediamine, and the iron chelator desferrioxamine. Transfection of the Hep G2-MV2E1-9 cells with plasmid containing CYP2E1 in the sense orientation enhanced the arachidonic acid toxicity, whereas transfection with plasmid containing CYP2E1 in the antisense orientation decreased toxicity. The CYP2E1-dependent arachidonic acid toxicity appeared to involve apoptosis, as demonstrated by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and DNA laddering experiments. Trolox, which prevented toxicity of arachidonic acid, also prevented the apoptosis. Transfection with a plasmid containing bcl-2 resulted in complete protection against the CYP2E1-dependent arachidonic acid toxicity. It is proposed that elevated production of reactive oxygen intermediates by cells expressing CYP2E1 can cause lipid peroxidation, which subsequently promotes apoptosis and cell toxicity when the cells are enriched with polyunsaturated fatty acids such as arachidonic acid. The Hep G2-MV2E1-9 cells appear to be a valuable model to study interaction between CYP2E1, polyunsaturated fatty acids, reactive radicals, and the consequence of these interactions on cell viability and to reproduce several of the key features associated with ethanol hepatotoxicity in the intragastric infusion model of ethanol treatment.