IMMORTALIZED HEPATOCYTES AS IN-VITRO MODEL SYSTEMS FOR TOXICITY TESTING - THE COMPARATIVE TOXICITY OF MENADIONE IN IMMORTALIZED CELLS, PRIMARY CULTURES OF HEPATOCYTES AND HTC HEPATOMA-CELLS

Immortalized differentiated liver cell lines capable of continuous pro liferation, and expressing stable liver-specific functions, would be v aluable for in vitro toxicity testing in the pharmaceutical, chemical, food and cosmetics industries. Immortalized rat hepatocyte cell lines have been produced by transfection of SV40 DNA by electroporation or calcium phosphate precipitation. Their utility has been assessed by st udying the toxicity of a model compound, menadione, and by measuring t he activities of DT-diaphorase and NADPH cytochrome c reductase. Enzym e activities and toxicity were compared in freshly isolated hepatocyte s, the immortalized cell lines and dedifferentiated HTC hepatoma cells . In HTC cells DT-diaphorase activity was 100-fold elevated compared w ith freshly isolated hepatocytes. In only one cell line, C2. 1.2. (pro duced by calcium phosphate precipitation), was DT-diaphorase activity increased (twofold) compared with freshly isolated hepatocytes. Menadi one caused loss of viability at similar concentrations (40-80 mu M) in the immortalized cell lines and 24-hr primary cultures of hepatocytes , whereas HTC cells showed loss of viability only with menadione conce ntrations above 200 mu M. The immortalized lines therefore appear to h ave potential for predicting toxicity and for menadione this can be co rrelated with the expression of DT-diaphorase. (C) 1997 Elsevier Scien ce Ltd.