In vitro metabolism of cyclophosphamide in limb bud culture

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Abstract
The presence of a drug‐metabolizing system in any in vitro bioassay for teratogenicity is necessary because many chemical agents require metabolism before their activity can be expressed, and limb bud cells lack the enzymes necessary to activate these chemicals. We have taken three approaches to add a drug‐metabolizing system to limb bud culture; addition of a 9,000 × g supernatant (S9) or a purified microsomal fraction, both from mouse liver, and co‐incubation of limb buds with hamster embryo cells (HEC). The liver preparations were able to convert the teratogen cyclophosphamide to five separate metabolites with alkylating activity in limb bud culture but the cytotoxicity of these preparations limited their usefulness as metabolizing systems in limb bud culture.HEC did not exert a toxic effect on limb buds and they were able to continue metabolism of cyclophosphamide for three days. Analysis of the metabolites indicated that HEC did not convert cyclophosphamide to the same products as the liver preparations. The metabolic products were capable of inducing abnormal limb development in vitro. Addition of 14C‐cyclophosphamide in the presence of an HEC activating system led to uptake of radioactivity into limb buds. In the absence of HEC, little radioactivity was detected in the target tissue. These results suggest that culturing intact cells capable of drug metabolism with limb buds may be the most likely method to follow in achieving in vitro activation of chemicals.