A critical evaluation of centromeric labeling to distinguish micronuclei induced by chromosomal loss and breakage in vitro.

The in vitro micronucleus assay in conjunction with CREST-staining and fluorescence in situ hybridization (FISH) with centromere-specific DNA probes is being increasingly utilized for the detection of clastogenic and aneuploidy-inducing agents. Although potentially powerful techniques, both methods have unique characteristics that can influence sample processing and the interpretation of results. In this article, the use of the CREST and the FISH modifications of the in vitro micronucleus assay have been used to characterize the origin of the micronuclei induced by cyclophosphamide, 4,4'-methylene-bis(2-chloroaniline), 4-nitroquinoline N-oxide and ionizing radiation in metabolically competent MCL-5 cells or a derived cell line lacking metabolic activation. Using these results and our previous experiences with these techniques, a detailed comparison including the strengths and limitations of each technique as well as potential problems in performing each assay and in analyzing the data is discussed. In spite of their limitations, our results to date indicate that CREST-staining as well as FISH with centromere-specific DNA probes can be used to accurately distinguish micronuclei formed from chromosome loss from those originating from chromosome breakage and that these techniques can be valuable complements to the in vitro micronucleus assay.