Flow cytometric detection of micronuclei induced by chemicals in poly- and normochromatic erythrocytes of mouse peripheral blood.

Abstract

In order to standardize automated scoring for the in vivo micronucleus (MN) test a flow cytometric method which recognized micronucleated polychromatic erythrocytes (MPCE) and micronucleated normochromatic erythrocytes (MNCE) in mouse peripheral blood developed by Grawé et al. (1992) has been modified and applied. Blood samples were purified with 35% percoll solution and stained with the RNA-specific dye thiazole orange (TO) and with the DNA-specific dye Hoechst 33342 (HO) for dual laser flow cytometry. The TO fluorescent signals permitted the discrimination between polychromatic and normochromatic erythrocytes (PCE and NCE). Cytotoxic effects could be assessed by the reduction of PCE counts. The blue fluorescent signals of HO permitted the scoring of MN. The MPCE and MNCE were flow sorted for microscopic analysis and showed that 95% of the sorted cells actually contained MN. Three model chemicals, the clastogen mitomycin C, the aneugen colchicine and the industrial chemical acrylamide were tested at 24 h intervals after single intraperitoneal injection up to 72 h after treatment of male (102/ElxC3H/El)F1 mice. All three chemicals showed a dose-related maximum of the MPCE frequencies at 48 h while the MNCE frequencies stayed within the control range up to 72 h. The data obtained with the flow cytometric method were in good agreement with published results. The flow cytometric technique presented here is a fast, accurate and automated method for quantifying MPCE and MNCE in peripheral blood as an indicator of cytogenetic damage induced in the bone marrow and scored in peripheral blood samples. With minor modifications the technique will also be applicable to bone marrow samples.