Role of acrolein in cyclophosphamide teratogenicity in rat embryos in vitro

To elucidate the role of acrolein in cyclophosphamide (CP) teratogenesis, we used the dechloro derivative of cyclophosphamide (D-CP). After activation, D-CP spontaneously breaks down to yield acrolein and dechlorophosphoramide mustard (D-PM), the nonalkylating derivative of phosphoramide mustard. At concentrations ranging from 6.25 to 50 micrograms/ml (33 to 262 microM), D-CP produced concentration-dependent cell death, growth retardation, and malformations in rat embryos cultured in vitro from Day 10 to 11 of gestation. D-PM, however, even at a concentration of 238 micrograms/ml (950 microM), had no effect on embryonic growth and development when added directly to standard culture medium containing Day 10 rat embryos. When embryos were exposed to acrolein (0.025 to 0.1 microgram/ml) directly in serum-free medium, this metabolite produced concentration-dependent decreases in growth parameters and abnormal flexion in some embryos. In no case, however, did acrolein-treated embryos resemble D-CP-treated embryos in terms of morphological malformations. Although we were able to show that D-CP was teratogenic in vitro, D-CP doses up to 50 mg/kg administered on Day 11 in vivo had no effect (with the exception of decreased growth at the highest dose) on growth or development at Day 20 of gestation. Our results suggest that acrolein plays a role in CP teratogenesis, but that the form in which it arrives at the teratogenically sensitive sites within the embryo is an important consideration in terms of the relative roles of acrolein and phosphoramide mustard in CP teratogenicity.