FLOW CYTOMETRIC ANALYSIS OF IN-VITRO MICRONUCLEUS INDUCTION IN HEPATOCYTES TREATED WITH CARCINOGENS

The micronucleus test is frequently used in established cell lines to detect genotoxic chemicals. In contrast, there is only very limited experience concerning the applicability of this test in primary cultures of hepatocytes. The induction of micronuclei (MN) by methyl methanesulphonate (2 mm) and by the indirect carcinogens cyclophosphamide (CP, 0.4-4 mM) and diethylnitrosamine (DEN, 1-10 mm) has therefore been studied in mt fiver cells in vitro. Analysis and quantification of MN, as well as determination of the proliferative activity of the hepatocytes, was performed by flow cytometric techniques. All three chemicals increased the frequency of MN at incubation times of more than 48 hr. The relative increase in MN compared with that in untreated or solvent-treated cultures, however, was at the most only three-fold, since the frequency of MN increased markedly in the control cultures also. There was a marked decrease in proliferative activity of the hepatocytes, as shown by the decrease in frequency of cells in the second G1-phase at the highest concentration of CP and at all concentrations of DEN. In conclusion, flow cytometric analysis of MN enables a fast and reliable determination of cytogenetic effects in hepatocyte cultures treated with chemicals. However, the large number of MN in untreated hepatocytes, which is possibly a consequence of DNA damage induced by the isolation procedure, may limit the sensitivity of the method.