FLOW CYTOMETRIC ANALYSIS OF THE CELL-CYCLE DISTRIBUTION OF SPLEEN LYMPHOCYTES ISOLATED FROM FISCHER-344 RATS EXPOSED TO ETHYL NITROSOUREA

Current studies in our laboratory are designed to determine the freque ncy of genotoxic responses induced in lymphocytes isolated from Fische r 344 rats. To evaluate the effect of a model compound, N-ethyl-N-nitr osourea (ENU), on the cell-cycle distribution of spleen lymphocytes, 8 -week old, female Fischer 344 rats were injected ip. with ENU and sacr ificed 1, 2, 4, and 6 weeks after exposure. Four replicate cultures pe r dose per exposure period were established and cells were cultured fo r 66 hr. Colcemid, an agent which blocks cells in mitosis and induces an accumulation of cells in the G2 + M peak, was added to two of the f our cultures as a positive control. After a 3 hr incubation, the cells were harvested, the nuclei stained with propidium iodide, and the DNA content of the individual nuclei was quantified by flow cytometry. As expected, exposure to Colcemid resulted in an accumulation of cells i n the G2 + M phase of the cell cycle, which was accompanied by a decre ase in the G0 + G1 population. The increase in the G2 + M population w as significant (p < 0.05) in cultures of lymphocytes assayed at 4 and 6 weeks after exposure. The effect of increasing ENU concentration was an increase in the percentage of S-phase cells (p = 0.05) and a decre ase (p < 0.02) in the percentage of G0 + G1 cells. This finding was ob served only in those lymphocytes isolated 1 week after exposure. These findings indicate that flow cytometric analysis of the distribution o f cells within the cell-cycle may provide insight into the effects of toxicant exposure on mammalian cells.