Chemically induced growth inhibition and cell cycle perturbations in cultures of differentiating rodent embryonic cells

Abstract

Ethylnitrosourea (ENU) is a proven animal teratogen, although the mechanism of its developmental toxicity is unknown. The micromass rat embryo midbrain (CNS) and limb bud (LB) cultures were used in an effort to determine potential mechanisms by which ENU may exert its teratogenic effect. When cultured at high cell densities, both cell types undergo several rounds of replication while differentiating into discrete foci of neuronal cells and chondrocytes, respectively. Differentiation was monitored after 5 days by staining with hematoxylin (CNS) and alcian blue (LB). Our objectives were to (1) apply flow cytometry technology to the micromass cultures and (2) determine how ENU disrupts the normal growth, differentiation, and cell cycling of these cultures. Dose-dependent decreases in cell attachment and viability were observed in the first 24 hr after ENU exposure. Exposed cultures also exhibited dose-dependent growth inhibition over 5 days in culture as determined by cell counts. Flow cytometric cell cycle analysis of treated cultures revealed a dose-related accumulation of CNS cells in late G 1/early S. Treated LB cells also displayed dose-related cell cycle changes with cells accumulating throughout the S phase. The concentration-dependent changes in both the CNS and the LB cell cycle profiles were observed in the attached cell populations which had >94 ± 3% viability at all ENU concentrations tested. This suggests that flow cytometric analysis allowed description of cellular alterations that would have been overlooked if only cell viability had been examined. Our examinations suggest that the effects of ENU on cell differentiation are related to its early effects on cell attachment, cell cycling, and cell proliferation.