Differential effects of 4-hydroperoxycyclophosphamide on limb development in vitro

Cyclophosphamide must be metabolically activated to produce malformations in limbs developing in culture; 4-hydroperoxycyclophosphamide is an analog of the active metabolite of cyclophosphamide, 4-hydroxycyclophosphamide, that breaks down spontaneously in solution to form 4-hydroxycyclophosphamide. To study the mechanism by which metabolites of cyclophosphamide produce limb malformations in vitro we determined the effects of exposure of cultured limb buds to 4-hydroperoxycyclophosphamide. Fore- and hindlimbs were excised from ICR mice on day 12 of gestation and cultured in roller bottles for 6 days. Limbs were exposed to 4-hydroperoxycyclophosphamide for the first 20 hours of the culture period. Addition of 10 micrograms/ml of 4-hydroperoxycyclophosphamide to forelimb or to hindlimb buds in culture produced limb reduction malformations. A dramatic decrease in total limb bone area in fore- and hindlimbs was observed with 10 micrograms/ml of 4-hydroperoxycyclophosphamide. In forelimbs, the long bone area decreased and the paw area remained constant so that the relative contribution of the long bone area to total limb bone area was decreased. In hindlimbs treated with 10 micrograms/ml of 4-hydroperoxycyclophosphamide, no paw skeleton was observed. The DNA, RNA, and protein contents of the limbs were not affected by exposure to 1 microgram/ml of 4-hydroperoxycyclophosphamide, but were decreased by exposure to 10 micrograms/ml of this compound. Exposure to the higher concentration of 4-hydroperoxycyclophosphamide also decreased alkaline phosphatase activity, a marker for osteogenesis, in both fore- and hindlimbs; in contrast, neither concentration of 4-hydroperoxycyclophosphamide had an effect on creatine phosphokinase activity, a marker for myogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)