IDENTIFICATION AND CHARACTERIZATION OF ANEUPLOIDY-INDUCING AGENTS BY DNA-PROTEIN FLOW-CYTOMETRY IN V79 CHINESE-HAMSTER CELLS

The reproducibility and usefulness of DNA-protein flow analysis as an in vitro test for aneuploidy in V79 Chinese hamster lung fibroblasts was demonstrated by making repeated measurements in untreated cultures and by testing three established aneugenic compounds: carbendazim, nocodazole, and estriol. Studies were completed by microscopic examination of the cells and results compared with data obtained from ketotifen, a chemical with a cell cycle-inhibiting activity only. Nocodazole and carbendazim had similar affects on cellular DNA and protein content. After the 6-h exposure, a dose-dependent pronounced decrease in the relative number of cells in the G0/G1 phase of the cell cycle and an increase in G2 phase cells were observed. This increase was associated with an increase in the relative number of metaphase cells, cells with malformed nuclei, and in the DNA dispersion in G0/G1 phase cells (coefficient of variation, CV1). Carbendazim increased the relative number of S phase cells whereas nocodazole caused a slight but not significant increase within the 6-h exposure period. Eighteen hours after the compounds were removed, the cells had almost completely recovered. Estriol, however, induced no increase in S phase cells after the 6-h exposure period but a larger increase in CV1 values. This suggests a different mode of aneugenic activity. The arrest occurs at a later stage in the G2/M phase without feedback on S phase cells. In contrast, ketotifen affected neither the relative number of cells with malformed nuclei nor the CV1 values. An increase in cellular protein content was found after the 6-h exposure to estriol and ketotifen. This is interpreted as an adaptive protective response of the cells. It seems that a concentration-dependent pattern of alterations in cell cycle compartments together with an increase in CV1 values at low, subtoxic exposure levels and at a narrow effective exposure range is a reliable indicator that a chemical has aneugenic activity. Thus, this flow analysis method allows aneugenic chemicals to be identified and their different modes of action to be differentiated.