A. Matsuoka

Evaluation of the micronucleus test using a Chinese hamster cell line as an alternative to the conventional in vitro chromosomal aberration test

The in vitro micronucleus (MN) test was carried out simultaneously with the conventional chromosomal aberration (CA) test on 11 clastogenic chemicals or spindle poisons with different modes of action using a Chinese hamster cell line (CHL). The method of slide preparation for the MN test was the same as that for the conventional metaphase analysis, except that 1% acetic acid in methanol was used as the cell suspension medium for air-drying (to preserve the cytoplasm around the nucleus). All chemicals tested induced micronuclei reproducibly and dose-dependently in good agreement with the results of metaphase analysis (r = 0.99). Since the MN test methodology is simple and the observation of MN is less subjective than that of CA, we conclude that the in vitro MN test would be a good alternative to the conventional CA test for screening the genotoxicity of chemicals.

Micronucleated reticulocyte induction by ethylating agents in mice

Six model ethylating agents were tested for clastogenic potency by means of a new technique of the micronucleus assay with mouse peripheral blood cells using acridine orange (AO)-coated slides, to evaluate the test. The alkylating agents were: N-ethyl-N-nitro-N-nitrosoguanidine (ENNG), N-ethyl-N-nitrosourea (ENU), diethylsulfate (DES), ethyl methanesulfonate (EMS), epichlorohydrin (ECH) and ethylene dibromide (EDB). The animals were given a single intraperitoneal injection of the following doses of the chemicals: ENNG and ENU, 25, 50 and 100 mg/kg; EMS and DES, 100, 200 and 400 mg/kg body weight. For EDB and ECH, the doses were 50, 100 and 200 mg/kg, given twice, 24 h apart. Before and after the injection, blood samples were taken from the tails at 24-h intervals up to 72 h and preparations were made on AO-coated slides. For each dose group, 4 animals were used and 1000 reticulocytes were examined per slide for the presence of micronuclei. At the optimum induction time of 48 h, ENU induced micronucleated reticulocytes (MNRETs) at all 3 doses. ENNG and EMS induced MNRETs significantly at 2 dose levels each and DES only at the highest dose. ECH and EDB failed to induce MNRETs. On the basis of the dose of chemical needed to double the spontaneous frequency, the order of clastogenic potency was ENU greater than ENNG greater than EMS greater than DES. The results obtained compared favorably with those from other in vivo methods. The present technique proves to be simple, flexible and relatively sensitive. Shifts in the optimum induction peak in individual animals and by some chemicals can be picked up easily which is important when testing weak mutagens and chemicals with an unknown mechanism of action.