The relationship between DNA content and centromere content in micronucleated mouse bone marrow erythrocytes analysed by flow cytometry and fluorescent in situ hybridization.

A fixation method for mouse bone marrow erythrocytes was developed which allows both flow cytometric enumeration of micronucleated polychromatic erythrocytes (MPCEs) and detection of centromeres using fluorescent in situ hybridization (FISH) with a mouse gamma satellite probe on flow-sorted MPCEs. Male CBA mice were treated with clastogens or spindle poisons: X-irradiation (XR; 0.5 Gy), cyclophosphamide (CPA; 80 mg/kg), vincristine sulphate (VCR; 0.125 mg/kg) and colchicine (COL; 1 mg/kg). At 30 and 50 h after treatment bone marrow suspensions were prepared and subsequently analysed by flow cytometry to enumerate and determine the DNA content of induced MPCEs. The mean DNA content in MPCEs was found to be higher after treatments with the two aneugens, VCR and COL, than with the two clastogens, CPA and XR. The mean DNA content of MPCEs was positively correlated with the mean proportion of micronuclei (MN) containing centromeres, indicating that determination of the mean DNA content alone can give information about the mechanism of MN induction. When the MPCEs induced with VCR were outsorted according to four classes of increasing DNA content and the presence or absence of centromeres was determined with FISH, it was found that only the class with the lowest DNA content (0.8-1.7% of the diploid DNA content) had a low proportion (< 20%) of centromere-containing micronuclei while in the three classes with higher DNA content (1.7-10.2% of the diploid DNA content) > 80% of the MN had centromeres. This was in contrast to the results from treatments with CPA where the proportion of centromere-containing MN did not increase with increasing DNA content.