Analysis of micronucleated cells by flow cytometry. 4. Kinetic analysis of cytogenetic damage in blood

Micronucleated cells (MN cells) generated spontaneously or by clastogen action accumulate in the peripheral blood of the mouse, and their presence reflects the level of chromosome damage. Traditionally, micronucleated cells have been scored by visual inspection. With the development of flow cytometry based scoring procedures, vast numbers of cells can be analyzed, making it possible to determine the change in the number of MN cells in the total peripheral blood pool. This report describes experiments whereby initial blood samples were obtained before dosing, providing mouse-specific controls for measuring subsequent changes in MN cells. Mice were then dosed with saline (solvent control), methyl methanesulfonate, cyclophosphamide or colchicine every 48 h and bled every 96 h for 12 days. For each blood sample, one million fixed erythrocytes (RBCs) were interrogated for the presence of micronuclei, and regression analysis was used to determine the rate of MN cell influx per day for each animal or sets of animals. To evaluate the effect of treatment on MN induction, the mean slopes of solvent and chemically treated animals were compared using t-tests. The results of these experiments indicate that the kinetics of MN induction continues near the background frequency for saline dosed mice, whereas clastogenic agents or spindle poisons cause a significant influx of MN events into the blood. The results suggest that some studies may benefit from a flow cytometry based analysis of multiple blood samples, especially when the number of mice is limited, or when a weak clastogen is being investigated.