In vitro test to determine the effect of cytostatic drugs on co-cultured rat hepatocytes and hepatoma cells.

Abstract

Using uptake of the fluorescent bile acid derivative cholylglycylamido-fluorescein (FITC-GC) as a measurement of liver cell population size and function, the antiproliferative and toxic effects of the well known cytostatic drug, cisplatin was evaluated on rapidly growing rat hepatoma McA-RH7777 cells and rat hepatocytes in primary culture under non-proliferating conditions. Co-culture set up to mimic the in vivo situation of tumour and extratumoural liver tissue exposed to cytostatic chemotherapy does not markedly affect the survival or the growth dynamics of both cell types. FITC-GC uptake as corrected for DNA and protein contents in the dish was significantly lower in hepatoma cells than in rat hepatocytes throughout the experimental period (96 h). Effect of 0.1-100 microM cisplatin exposure from 24 to 96 h of culture on cell population size, as measured by protein and DNA contents in the culture dishes, were consistent with changes observed in total FITC-GC uptake. Cisplatin concentrations lower than 50 microM did not affect FITC-GC uptake by rat hepatocytes. By contrast, a progressively increasing effect on hepatoma cells as from 2 microM cisplatin was observed. Two phases in the decay of FITC-GC uptake versus cisplatin concentrations were found in co-cultures exposed to this drug. The first segment, between 2 microM and 50 microM, was characterized by a slow decay that matched the response of hepatoma cells to cisplatin exposure. This was considered to be due to the antiproliferative effect of cisplatin. The second segment, with a steeper decay, matched the effect of cisplatin on hepatocytes. This was interpreted as being due to non-specific toxicity. These results suggest that FITC-GC uptake by co-culture of hepatocytes and tumour cells provides a useful experimental model to explore the mechanism of action and the size of beneficial effect window for new drugs in vitro.