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

In order to standardize automated scoring for the in vivo micronucleus(MN) test a flow cytometric method which recognized micronucleatedpolychromatic erythrocytes (MPCE) and micronucleated normochromaticerythrocytes (MNCE) in mouse peripheral blood developed by Grawéet al. (1992) has been modified and applied. Blood samples werepurified with 35% percoll solution and stained with the RNA-specificdye thiazole orange (TO) and with the DNA-specific dye Hoechst33342 (HO) for dual laser flow cytometry. The TO fluorescentsignals permitted the discrimination between polychromatic andnormochromatic erythrocytes (PCE and NCE). Cytotoxic effectscould be assessed by the reduction of PCE counts. The blue fluorescentsignals of HO permitted the scoring of MN. The MPCE and MNCEwere 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 industrialchemical acrylamide were tested at 24 h intervals after singleintraperitoneal injection up to 72 h after treatment of male(l02/ElC3H/EI)F₁ mice. All three chemicals showed a dose-relatedmaximum of the MPCE frequencies at 48 h while the MNCE frequenciesstayed within the control range up to 72 h. The data obtainedwith the flow cytometric method were in good agreement withpublished results. The flow cytometric technique presented hereis a fast, accurate and automated method for quantifying MPCEand MNCE in peripheral blood as an indicator of cytogeneticdamage induced hi the bone marrow and scored in peripheral bloodsamples. With minor modifications the technique will also beapplicable to bone marrow samples.