Hepatocyte activation of indirect mutagens during the first days in primary culture

Changes in activation capacity of hepatocytes during the first days after hepatocyte isolation were investigated by measuring the cytogenetic effects of the indirect mutagens cyclophosphamide (CP), dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1). On the day of hepatocyte isolation and on the subsequent 4 days, hepatocytes were co-cultured with V79 cells, and SCE- and chromosome aberration-tests were performed. CP- and DMN-induced SCE frequencies declined with increasing hepatocyte age. Such a decline was also found for AFB1-induced aberration frequencies, whereas the SCE-inducing effect of AFB1 remained stable up to 4 days after hepatocyte isolation. This indicates that SCE-inducing and clastogenic AFB1 metabolites are generated by different enzymatic pathways. The clastogenic metabolites of AFB1--as well as the SCE-inducing CP- and DMN-metabolites--are generated by cytochrome P450-dependent monooxygenases whose activity is obviously lost during the first days of primary hepatocyte culture. Activation of AFB1 to SCE-inducing metabolites may be performed by more stable monooxygenases. Our results show that the ability of hepatocytes to activate indirect mutagens decreases during the first few days after hepatocyte isolation. Furthermore, the results with AFB1 indicate that routine culture methods are not suitable to maintain the balance of the different enzymatic functions which are involved in metabolism of indirect mutagens.