Primary culture of rat hepatocytes in 96-well plates: Effects of extracellular matrix configuration on cytochrome P450 enzyme activity and inducibility, and its application in in vitro cytotoxicity screening

Abstract

Basal level enzyme activities and enzyme inducibility were compared for rat hepatocytes that were cultured in 96-well plates with three different extracellular matrix configurations: single layer (SL) collagen type I, SL Matrigel, and collagen/Matrigel (C/M) sandwich. Overall, C/M sandwich and SL Matrigel plates were both superior to SL collagen type I plates in maintaining enzyme activities and inducibility and C/M sandwich plates had higher induced activity for CYP3A enzymes than SL Matrigel plates did. Cytotoxicity of nine reference compounds to rat hepatocytes (C/M sandwich configuration), rat hepatoma H4IIE and mouse fibroblast Balb/c 3T3 (3T3) cells was evaluated in 96-well plates using neutral red uptake (for 3T3) and tetrazolium salt MTS assays (for H4IIE and rat hepatocytes). For compounds chlorpromazine, quinidine, trichlorfon, thiopental, and antipyrine, the absolute differences in cytotoxicity Log IC 50 values obtained from different cell types were relatively small and without an obvious trend. The Δ Log IC 50 values between cultured hepatocytes and the cell lines were much larger for acetaminophen and cyclophosphamide (1.35 ⩽ ∣Δ Log IC 50∣ ⩽ 3.40), and for clofibrate and thioacetamide (not cytotoxic in hepatocytes at their highest dose levels). These large differences were likely the result of metabolism of these compounds in rat hepatocytes. The relationship between in vitro cytotoxicity Log IC 50 values and in vivo mouse or rat oral acute Log LD 50 values showed that compared to the cell lines, cultured rat hepatocytes improved correlation for acetaminophen and cyclophosphamide. The potential benefit of conducting in vitro cytotoxicity screening using a combination of permanent cell lines and cultured hepatocytes would allow us to obtain mechanistic insight on bioactivation, as well as improve the predictability of metabolism-mediated toxicity.