Cell-cycle alterations underlie cyclophosphamide-induced teratogenesis in the chick embryo

Cyclophosphamide (CP) embryotoxicity was documented in several studies on different experimental models. We investigate quantitatively the relationship between the embryotoxic effect of CP and the disturbance of the cell-cycle using flow cytometry.

Chick embryos on Days 2-4 were treated with 0.5, 1, 2, 4, 8, and 16 microg doses of pure substance of CP by intraamniotic or subgerminal administration routes. Cell-cycle analysis was carried out in the brain, limb buds, hearts, and facial outgrowths dissected from the embryos 6 hr after administration. Samples of nuclear suspensions were obtained by enzymatic and mechanical disintegration of solid tissues in collagenase-dispase, followed by detergent and RNA-ase mediated cytolysis. Nuclei were stained by ethidium bromide.

A dose-dependent increase of S-phase cells followed by decrement of G2M cell compartment was observed. The significant block of S-phase cells, however, was not always associated with malformations. The degree of cell-cycle disturbance was expressed more readily by the ratio G2M/S that demonstrated consistently the threshold character of both teratogenic and lethal effects.

CP-induced cytotoxicity manifested by dose-dependent disturbance of cell-cycle resulted in an overall depression of proliferation activity clearly associated with the occurrence of malformations and embryonic death. Although a non-significant depression of mitotic activity appeared sufficient to produce malformations on Day 2, remarkably deeper disturbance was needed to interfere with the development of the embryos in more advanced stages. Changes in proliferation rate appear to be a primary and most important event in teratogenesis induced by general toxic agents.