Analysis of micronuclei induced by 2-chlorobenzylidene malonitrile (CS) using fluorescence in situ hybridization with telomeric and centromeric DNA probes, and flow cytometry

Abstract

Micronuclei (MN) induced in NIH 3T3 cells by the tear gas 2-chlorobenzylidene malonitrile (CS) were studied in detail using fluorescence in situ hybridization (FISH). The chromosomal composition of CS-induced MN was analysed by simultaneous use of DNA probes for the telomeric hexamer repeat (TTAGGG) and for mouse major satellite DNA. The majority of CS-induced MN, 63-73% of all CS-induced MN at doses from 10 to 30 microM CS, revealed centromeric signals and several telomeric signals suggesting their origin from whole chromosomes. Almost 50% of all CS-induced MN showed one centromeric signal and were assumed to contain one single chromosome. Only 4.5% of all MN did not show any signal and 23-28% showed telomeric signals only, thus containing acentric fragments. Based on the experimental data from FISH the distribution of the DNA content of CS-induced MN was calculated assuming random breakage of chromosomes, and random combination of chromosomes and chromosome fragments. Good agreement between calculated MN distributions and distributions measured by flow cytometry was obtained. By sorting MN with distinct DNA content and hybridization of the sorted MN with the centromeric probe, regions in the MN distribution containing mainly MN with single whole chromosomes could be demonstrated.