Development of a modified micronucleus assay in vitro for detection of aneugenic effects

A simple and rapid micronucleus (MN) assay in vitro has been developed with V79 cells enabling preferential detection of aneugen-induced MN. V79 cells were treated with test compounds for 3 hr, the mitoses were shaken off and transferred into new flasks, and after a 3.5-hr recovery period interphase cells were analysed for MN. Using this time schedule only cells that were treated during mitosis or G2-phase plus mitosis were analysed. Four suspected aneuploidy-inducing agents (aneugens) were tested: griseofulvin, methyl 2-benzimidazole carbamate, diazepam and thiobendazole. All four compounds induced MN in the modified V79 assay. For further characterization of the modified V79/MN assay the clastogen mitomycin C (MMC) was investigated with respect to the time-dependency of MN induction. MMC did not induce MN in our modified V79 assay, but was positive after 14 hr of exposure. Induced MN were characterized by CREST staining and by measuring their size. The frequencies of CREST-positive and large MN were increased by the four suspected aneugens analysed. Furthermore, the use of the external metabolization systems, S-9 mix, and hepatocytes, was examined.