A kinetic analysis of rat embryo response to cyclophosphamide exposure in vitro

Day 10 rat embryos were exposed in vitro to a teratogenic dose of cyclophosphamide (25 micrograms/ml) in the presence of a metabolic system (activated CP) for varying lengths of time and then were recultured in drug-free medium. Development was assessed after a total of 24-26 hours of culture. Exposure periods of 2.5 hours or less had no significant effects on growth, incidence of malformed embryos, mitotic index, or necrotic index when compared to control cultures from which cofactors for metabolic activation were omitted. Embryos exposed to activated CP for 5 hours and then recultured in drug-free medium for 19-21 hours were indistinguishable from embryos exposed to activated CP continuously over a 24-26-hour period. These embryos are characterized by retarded growth as well as abnormalities of the prosencephalon, mandibular arches, limbs, and tail. Histological examination of embryos exposed to activated CP for 5 hours and immediately fixed revealed a decrease in the mitotic index and an increase in the necrotic index of the neuroepithelium and the surrounding mesenchyme of the prosencephalon. Our findings indicate that a 5-hour exposure to activated CP during day 10 of rat gestation is sufficient to produce the molecular lesions necessary to elicit the teratogenic effects of cyclophosphamide.