A proposal for a simple way to distinguish aneugens from clastogens in the in vitro micronucleus test

In our previous in vitro micronucleus (MN) study, we showed that aneugens, in addition to inducing micronuclei, induce a higher frequency of polynuclear (PN) and mitotic (M) cells than clastogens. We hypothesized that the frequency of PN and M cells induced can distinguish aneugens from clastogens. To test the hypothesis, we conducted the micronucleus tests with mitomycin C (MMC), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), vincristine (VINC) and diazepam in a Chinese hamster cell line (CHL) and VINC, benzo[a]pyrene (BP) and 7,12-dimethylbenz[a]anthracene (DMBA) in a subclone of V79 cells (V79-MZ). All chemicals increased the frequency of M cells with statistical significance. All chemicals except diazepam increased the frequency of PN cells with statistical significance. Three of the aneugens (VINC, BP and DMBA) induced >/=200 PN cells/1000 cells while the clastogens (MNNG and MMC) induced 100 PN cells at most. All the aneugens but no clastogens significantly increased the frequency of M cells. We propose that micronucleus test-positive chemicals that induce >/=200 PN cells/1000 cells and significantly increase the frequency of M cells are aneugens and those that induce at most 100 PN cells/1000 cells and do not significantly increase the frequency of M cells in our MN test protocol are clastogens. Diazepam, however, did not induce PN cells, although it increased the frequency of M cells dose dependently. We explain this fact in relation to diazepam's mode of action. Our proposal suggests a quick, easy and practical way to distinguish aneugens from clastogens for screening purposes.