Practical threshold for micronucleated reticulocyte induction observed for low doses of mitomycin C, Ara-C and colchicine

Micronucleus induction was studied for the DNA target clastogens mitomycin C (MMC) and 1-β-D-arabinofuranosylcytosine (Ara-C), and also the non-DNA target aneugen colchicine (COL) in order to evaluate the dose-response relationship at very low dose levels. The acridine orange (AO) supravital staining method was used for microscopy and the anti-CD71-FITC based method was used for flow cytometric analysis. In the AO method, 2000 reticulocytes were analysed as commonly advised, but in the flow cytometric method, 2000, 20000, 200000 and 1000000 reticulocytes were analysed for each sample to increase the detecting power (i.e. sensitivity) of the assay. The present data show that increasing the number of cells scored increases the statistical power of the assay when the cell was considered as a statistical unit. Even so, statistically significant differences from respective vehicle controls were not observed at the lowest dose level for MMC and Ara-C, or the lower four dose levels for COL, even after one million cells were analysed. When the animal was considered as a statistical unit, only the top dose group for each chemical showed significant increase of micronucleated reticulocytes frequency. As non-linear dose-response curves were obtained for each of the three chemicals studied, these observations provide evidence for the existence of a practical threshold for the DNA target clastogens as well as the non-DNA target aneugen studied.