CYTOTOXICITY AND APOPTOSIS PRODUCED BY ARACHIDONIC-ACID IN HEP G2 CELLS OVEREXPRESSING HUMAN CYTOCHROME P4502E1

The goal of the current study was to evaluate the effects of arachidon ic acid, as a representative polyunsaturated fatty acid, on the viabil ity 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 C YP2E1, but little or no toxicity was found with control Hep G2-MV-5 ce lls, 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 involv e a lipid peroxidation type of mechanism since toxicity was enhanced a fter depletion of cellular glutathione; formation of malondialdehyde a nd 4-hydroxy-2-nonenal was markedly elevated in the cells expressing C YP2E1, and toxicity was prevented by antioxidants such as alpha-tocoph erol 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 enhance d the arachidonic acid toxicity, whereas transfection with plasmid con taining CYP2E1 in the antisense orientation decreased toxicity. The CY P2E1-dependent arachidonic acid toxicity appeared to involve apoptosis , as demonstrated by terminal deoxynucleotidyltransferase-mediated dUT P nick end labeling and DNA laddering experiments. Trolox, which preve nted toxicity of arachidonic acid, also prevented the apoptosis. Trans fection with a plasmid containing bcl-2 resulted in complete protectio n against the CYP2E1-dependent arachidonic acid toxicity. It is propos ed that elevated production of reactive oxygen intermediates by cells expressing CYP2E1 can cause lipid peroxidation, which subsequently pro motes apoptosis and cell toxicity when the cells are enriched with pol yunsaturated fatty acids such as arachidonic acid. The Hep G2-MV2E1-9 cells appear to be a valuable model to study interaction between CYP2E 1, polyunsaturated fatty acids, reactive radicals, and the consequence of these interactions on cell viability and to reproduce several of t he key features associated with ethanol hepatotoxicity in the intragas tric infusion model of ethanol treatment.