The effects of isolation technique, substrata and pyrazole on cytochrome P450 enzymes in cultured male rat hepatocytes.

The cytochrome P450 enzymes are of particular interest in the study of drug metabolism since they play a key role in the transformation of xenobiotics. The recent explosion of in vitro techniques has facilitated detailed examination of drug metabolism in isolated systems such as primary hepatocyte culture. However, the expression of cytochrome P450 enzymes declines under culture conditions, limiting the use of this method. These studies examined the effects of combinations of isolation technique and substrata on the activity and amount of immunodetectable protein of several cytochrome P450 enzymes in male rat hepatocytes cultured for up to 96 hours. Two techniques, collagenase and EDTA, were used to isolate hepatocytes subsequently cultured on Matrigel®, Vitrogen®, fibronectin or uncoated plastic. Collagenase digestion was a significantly superior method of isolating hepatocytes compared to EDTA, in terms of cell yield and viability. The low cell yield and viability following EDTA isolation suggest that this technique is unsuitable for routine use. The data generated show that both substrata and, to a greater degree, isolation technique significantly affected the activity and immunodetectable amounts of several cytochrome P450 enzymes in rat hepatocytes throughout the culture period examined. The rates of decline of these enzymes, and the effects of isolation technique and substrata, were variable. No combination of isolation technique and substratum was found to halt the decline in cytochrome P450 enzymes and it is likely that a number of factors involved in their expression are lacking in the culture system The expression of CYP2E1 in cultured rat hepatocytes was also examined following exposure to the CYP2E1 inducing agent pyrazole. Increases in both CYP2E1 activity and amount of immunodetectable protein show that the hepatocytes have retained the ability to respond to pyrazole, demonstrating that the decline in cytochrome P450 expression is not irreversible