RESUMO
The mammalian erythrocyte micronucleus assay is frequently used to assess chemical-induced damage to the chromosomes or the mitotic apparatus of erythroblasts. Because quantitative analysis of micronuclei by microscopy is time consuming and laborious, several automatic scoring methodologies with image analysis have been reported. However, there have been cases in which it was difficult to examine the proportion of polychromatic erythrocytes (PCEs) among total erythrocytes as an index for bone marrow (BM) toxicity, and sample slide preparation has proven to be laborious with existing automatic methods. We developed an automatic scoring system with image analysis for the rodent erythrocyte micronucleus assay using 12-well plates employing high-content screening analyser. In our method, micronucleated PCEs (MNPCEs), PCEs and erythrocytes were identified from three kinds of images: bright field image, fluorescence image with Hoechst 33342, and fluorescence image with propidium iodide. The frequencies of MNPCEs and PCEs were subsequently calculated. A comparison of automatic and manual scoring was carried out using BM and peripheral blood (PB) obtained from mice treated with stepwise doses of mitomycin C. The scores obtained by automatic analysis corresponded to those obtained by manual scoring; the frequencies of MNPCEs in BM and PB obtained by automatic scoring were 132 and 113%, respectively, of those obtained by manual scoring, and the corresponding frequencies of PCEs were 95 and 120%, respectively. Furthermore, we performed five repeats of the examinations of mouse BM and PB treated with mitomycin C or vinblastine sulphate and showed that automatic scoring was equivalent to manual scoring in reproducibility. Meanwhile, the scoring data obtained by manual scoring tended to vary among observers. These results suggest that our automatic scoring system with image analysis is superior to manual microscopy scoring in terms of speed and objectivity, comparable in reproducibility and useful for the in vivo micronucleus assay.
RESUMO
The in vitro micronucleus (MN) test is an important component of genotoxicity screening and is used as an alternative to the in vitro chromosome aberration (CA) test. As the MN assay is more practical and simpler to use than the CA test, it is being applied as a high-throughput screening (HTS) assay. Therefore, we conducted a validation study of the MN test employing a confocal imaging plate reader, the IN Cell Analyzer 1000. We evaluated 30 chemicals, including clastogens and aneugens, using Chinese hamster lung cells (CHL/IU) seeded in 96-well microplates. The microplates were stained with Hoechst 33342 and CellMask Red for automated analysis, and MN were identified and counted automatically in fluorescence images. The MN test results for 30 chemicals obtained with this image analysis system, using the IN Cell Analyzer, were highly consistent with reference data for the in vitro MN test and CA test data obtained by microscopic analysis. In conclusion, this HTS assay for detecting MN offers high efficiency and accuracy in the early stages of chemical development.
