Cell-cycle alterations within chick embryonic anlagen after cyclophosphamide treatment

With the aim of explaining a mechanism underlying the dose-dependent change in prevalence of different malformation types (shift in malformation spectra) in a population of chick embryos treated with general cytotoxic agents, we have investigated the effect of cyclophosphamide (CP) on the cell cycle in different organ rudiments. CP was administered intra-amniotically in doses of 2, 4, 8, and 16 micrograms to chick embryos on day 4. Six hours later, the embryos were removed, and limb buds, facial region, brain, and heart rudiments were dissected and treated for isolation of nuclei. The tissues were dissociated mechanically and enzymatically with collagenase-dispase, and suspensions of nuclei were prepared by a detergent and RNAase-mediated cytolysis. Ethidium bromide added to the solution allowed DNA analysis by flow cytometry, which, within the embryotoxic range of doses (4-16 micrograms), revealed a dose-dependent block of cells in the S phase, followed by a decrease of cell numbers in the G2-M phase. The effect of CP on the cell cycle was associated with the degree of damage to the embryo, and dysmorphogenesis appeared proportionate to the magnitude of mitotic inhibition. The results are consistent with the idea that the dose-dependent shift in malformation spectra is causally associated with the dose-dependent and organ-specific depression of mitotic activity.