A mixture of antioxidants and fatty acids improves the viability of cultured rat hepatocytes untreated or treated with doxorubicin

The objective of this study was to determine whether medium supplementation by antioxidants and fatty acids would improve the viability of cultured rat hepatocytes and protect them against doxorubicin toxicity. We examined the effects of three agents: vitamin E, sodium pyruvate and egg yolk (the combination of vitamin E, sodium pyruvate and fatty acids is a proprietary, patented technology of Warner Lambert called CRT) 0.3% (v/v) as a source of fatty acids, on cell viability measured by the dehydrogenase-dependent bioreduction of a tetrazolium salt (MTS). Untreated hepatocytes and hepatocytes treated with carbon tetrachloride (CCl 4, EC 50 5.7 m m) or doxorubicin (1 and 30 μ m) were exposed to different amounts of a mixture of antioxidants and fatty acids. The mixture, identified as 1X, provided a final concentration of 5 units of vitamin E, 0.1% egg yolk and 10 m m sodium pyruvate while the 3X and 5X mixtures contained proportionately higher concentrations of these components. The mixtures were added 18 hr prior to, simultaneously with or following treatment with doxorubicin and just simultaneously with CCl 4. Neither vitamin E, sodium pyruvate nor egg yolk alone improved viability. However, the viability of untreated hepatocytes improved significantly when the 3X mixture was added after 18 hr as indicated by determination of MTS reduction activity 24 hr later. The viability of doxorubicin treated cultures (1 and 30 μ m) increased significantly when exposed either to the 3X or 5X mixtures simultaneously. A significant increase in viability was also seen when cells were exposed to the 3X mixture following doxorubicin (1 μ m). The mixtures did not protect against toxicity caused by CCl 4, perhaps due to the overwhelming level of damage at its EC 50 concentration. It is proposed that the antioxidant properties of vitamin E and sodium pyruvate protect the cells from low levels of reactive oxygen species generated spontaneously in culture and by doxorubicin metabolism while the fatty acids help to maintain the integrity of hepatocyte membranes, resulting in greater viability of the hepatocytes.