Activation of cyclophosphamide in mouse limb bud cultures using a reconstituted cytochrome P-450 system

Purified phenobarbital-induced rat liver cytochrome P-450 was incorporated in a reconstituted system containing NADPH-cytochrome P-450 reductase, dilauroyl phosphatidyl choline and sodium cholate. This system was added to organ cultures of limb buds from mouse embryos on day 11 of gestation. Cyclophosphamide (100 micrograms per ml) was used as a "pre-teratogen" and activation was initiated by adding an NADPH-regenerating system. Due to extensive purification, toxicity of the enzyme preparations and residual solubilisation detergents could be greatly reduced. A reconstituted system containing 10-100 pmol cytochrome P-450 per ml without cyclophosphamide caused no noticeable interference with limb development. The same assay containing cyclophosphamide, however, resulted in a pronounced impairment of cartilage differentiation and in the formation of clearly abnormal structures, especially at the paw skeleton. The activity of the reconstituted system declined under the experimental conditions used, but some activating capacity towards cyclophosphamide was still demonstrable after about 2 h of incubation.