Metabolic activation and cytotoxicity of cyclophosphamide in primary cultures of postnatal rat hepatocytes

We have developed a model of primary cultures of postnatal rat hepatocytes to characterize the metabolic activation of xenobiotics to toxic intermediates and to study the mechanism(s) by which these chemicals produce cellular injury. This model was employed to investigate the cytochrome P-450 mediated biotransformation of cyclophosphamide (CP) to cytotoxic metabolites that nonspecifically alkylate DNA and cellular proteins. The parenchymal cells were isolated by an in situ collagenase perfusion technique and cultured for 24 hr prior to drug treatment. The cultures were then exposed to CP concentrations ranging from 1 × 10^?4 M to 1 × 10^?3 M for 24 hr. Initial studies indicated minimal toxicity to non-replicating parenchymal hepatocytes maintained in ornithine-supplemented, arginine-deficient medium. The addition of arginine permitted the overgrowth of fibroblasts in the same culture system. These fibroblasts then became the target of alkylating CP metabolites produced by the par-enchymal cells. By day 3 after CP administration, cell number and total protein per dish decreased by over 40 percent. The morphology of the cultures changed dramatically because of fibroblast destruction. The cytotoxicity to dividing fibroblasts was eliminated by administering 2-diethylaminoethyl-2, 2-diphenylvalerate hydrochloride (SKF 525-A), an inhibitor of the cytochrome P-450 monooxygenase system, to the co-cultures treated with CP. The alkylating metabolites of CP produced by the parenchymal cells and released into the culture medium were quantitated by reacting aliquots of medium from CP-treated cells with 4-(p-nitrobenzyl)pyridine. These results provide both direct and indirect evidence of drug metabolism in cultured cells and suggest that this co-culture system can be utilized to evaluate the metabolic activation of xenobiotics.