Micronuclei induced by 2-chlorobenzylidene malonitrile contain single chromosomes as demonstrated by the combined use of flow cytometry and immunofluorescent staining with anti-kinetochore antibodies.

The effects of the tear gas 2-chlorobenzylidene malonitrile (CS) on micronucleus induction and cell cycle kinetics were studied in Chinese hamster and Ehrlich ascites tumour cells using flow cytometric analysis of micronuclei and nuclei in suspension, and indirect immunofluorescent staining of kinetochores in micronuclei. In both cell lines CS induced a concentration-dependent inhibition of the fraction of cells in mitosis as observed by simultaneous flow cytometric measurements of DNA content and side scatter intensities of cell nuclei. Micronucleus frequency increased during the delayed division of cells accumulated by CS in mitosis and reached a plateau when most of these cells have divided. The height of this plateau depended on the CS concentration. Results obtained by flow cytometric analysis of the frequency of CS-induced micronuclei did not agree quantitatively with results obtained by microscopic analysis due to cells showing CS-induced fragmented nuclei. Nearly all CS-induced micronuclei exhibited kinetochores, the majority of which (60-70%) showed one kinetochore per micronucleus implying the presence of a single metaphase chromosome in these micronuclei. DNA distributions of micronuclei measured by flow cytometry showed pronounced peaks corresponding to the DNA distribution of chromosomes measured by flow karyotyping. Even micronuclei containing two of the large chromosomes could be observed as distinct peaks in the distributions. The combined results of flow cytometric analysis of micronucleus distributions and immunofluorescence staining of kinetochores in micronuclei suggest that CS induces micronuclei mainly by damaging the spindle fibres of single chromosomes during mitosis, thus possibly leading to aneuploidy and polyploidy.