Comparative mouse micronucleus evaluation in bone marrow and spleen using immunofluorescence and Wright's Giemsa

Bone marrow and spleen toxicity, clastogenicity and aneugenicity were analyzed in the CD1 mouse using an antikinetochore antibody (AKA) procedure (Krishna et al., Mutation Res., 282, 159-169, 1992). Further, to verify the fluorescence micronucleus (MN) analysis, additional slides were stained with Wright's Giemsa and results were compared. 5 mice per sex were treated with cyclophosphamide (CP) (40 mg/kg) or vincristine (VC) (0.1 or 0.2 mg/kg). Slides were prepared 24 h postdose using a column fractionation procedure. Per animal, 400 total erythrocytes (TEs) for toxicity and 2000 polychromatic erythrocytes (PCEs) for MN per tissue were analyzed. In the fluorescent method, the clastogen, CP, produced MNPCEs predominantly devoid of kinetochores (K) and the aneugen, VC, produced mostly MNPCEs containing K. The MNPCE frequency did not differ significantly between tissues; however, it differed statistically between sexes. On an overall basis, spleen had significantly lower PCE to TE ratios compared to bone marrow. In general, CP and VC caused a small, but statistically significant decrease in PCE frequencies compared to controls, suggesting possible toxicity to these tissues at the given doses. The data on Wright's stain indicated that the proportion of PCEs and MNPCEs in general, were comparable to those using fluorescent stain. This study further confirms the usefulness of an AKA-staining technique in a multiple genetic endpoint evaluation under a single set of microscopic conditions.