Cytokinesis-block micronucleus assay in primary human liver fibroblasts exposed to griseofulvin and mitomycin C

Primary liver fibroblasts were applied in a cytokinesis-block micronucleus assay in combination with fluorescence in situ hybridization (FISH) using t wo protocols. In protocol A (Prot. A), cytochalasin B (Cyt B) was added at the end of the treatment time directly to the medium containing the standar d compounds, whereas in protocol B (Prot, B) the chemical-containing medium was removed and fresh medium with Cyt B was added. The study was performed using the aneugen griseofulvin (GF) and the clastogen mitomycin C (MMC) as standard compounds. With both protocols GF induced a significant increase in MN frequency over controls in a dose-related manner at the lower concent rations tested (7.5 and 15 mu g/ml). At the highest dose (30 mu g/ml) the a neugen effect was substantially reduced. MN induction obtained with Prot. A was significantly higher (similar to 3-fold) than with Prot. B at the most effective concentration. The aneugen effect induced by GF did not change w hen different cell densities were used, but again with Prot. A we obtained the highest effect. MN induced by MMC showed a dose- and time-dependent inc rease in both protocols. In contrast to GF, the greater clastogenic respons e induced by MMC in human liver fibroblasts was obtained with Prot. B, simi lar to 3-fold higher than Prot. A at the most effective concentration and s imilar to 2-fold with 24 h treatment at 0.17 mu g/ml MMC, With GF, the FISH data in human liver fibroblasts (80% C + MN) were fairly consistent with t hose obtained in the rodent cell lines, In human whole blood cultures, the same dose used in our experiment produced a relatively higher percentage of C + MN. FISH analysis showed that MMC induced mainly MN containing acentri c fragments rather than whole chromosomes. In conclusion we have demonstrat ed that chemically induced genetic effects are strongly dependent on the ce ll culture employed, treatment schedule and intra- and post-treatment exper imental conditions.