Analysis of DES-induced micronuclei in binucleated rat fibroblasts: comparison between FISH with a rat satellite I probe and immunocytochemical staining with CREST serum

Abstract

The usefulness of fluorescence in situ hybridization (FISH) with rat satellite I DNA was compared with immunocytochemical staining with CREST serum for the analysis of the content of micronuclei from primary rat fibroblasts. We analyzed micronuclei induced in vitro by the aneugenic compound diethylstilbestrol (DES) or the clastogenic compound mitomycin C (MMC). Since a centromeric probe was not available for the rat, we isolated rat satellite I DNA by PCR with primers designed on the basis of the known rat satellite I DNA sequence. The PCR products obtained as well as the cloned PCR products showed hybridization to the centromeric regions of a large number of chromosomes, but not of chromosome 1, 19, 20, X and Y. Clone 18-5 was further analyzed and was shown to contain at least 4 repeats of the rat satellite I family. This probe, which hybridizes in the centromeric region of 34 of the 42 chromosomes, was used throughout the study as a probe for the FISH analysis of the micronuclei. For the immunocytochemical staining, the commonly used commercial anti-centromeric antibodies could not be used because of the weakness of the fluorescent signals given. Consequently, CREST serum of a single patient was used, which showed bright and distinct signals on the kinetochores of each chromosome. After treatment of the cells with the aneugen DES an increase in centromere (FISH) and kinetochore (CREST) positive micronuclei was found, whereas after treatment with the clastogen MMC, the percentage of centromere-positive micronuclei was similar to that observed in controls. Analysis of a large number of DES-induced micronuclei showed that the immunocytochemical method is equally as or slightly less sensitive for the detection of chromosomes in micronuclei and we therefore recommend FISH with probe 18-5 for the detection of chromosome loss in rat cells.