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Activation and Inactivation of Cancer Chemotherapeutic Agents by Rat Hepatocytes Cocultured with Human Tumor Cell Lines¹

Departments of Urology [M. C. A., M. M. L.] and Oncology [G. P., P. L. A., K. L. K.], Mayo Clinic and Foundation, Rochester, Minnesota 55905

While colony formation assays provide sensitive indices of tumor cell proliferation and growth inhibition imposed by many chemotherapeutic agents, drugs which require metabolic activation lack activity in such assays. In the present study, we have utilized freshly isolated rat hepatocytes for the activation of drugs which are metabolized by hepatic microsomal as well as extramicrosomal enzymes. Hepatocytes in fluid medium are placed over soft-agarose matrix containing tumor-derived cells (e.g., A204, A549) within 35-mm culture dishes; drug and/or drug vehicle is added directly to the hepatocyte layer, and cultures are incubated for 24 hr prior to removal of the hepatocyte layer. Tumor cell colony formation is assessed following 7 to 10 days of incubation. Cyclophosphamide was used as a prototype agent to assess utility of the coculture methodology. In vivo treatment of rats with phenobarbital prior to hepatocyte isolation enhances cyclophosphamide toxicity in vitro, whereas pretreatment with carbon tetrachloride markedly reduced subsequent in vitro cyclophosphamide cytotoxicity. Hepatocyte:tumor cell cocultures provide an efficient means to detect metabolic activation and inactivation of several selected cancer chemotherapeutic agents as well. In the presence of hepatocytes, the 50% growth-inhibitory concentrations for cyclophosphamide, indicine N-oxide, and procarbazine are markedly decreased, whereas the 50% growth-inhibitory concentrations for [2,5-bis(1-aziridinyl)-3,6-diazo-1,4-cyclohexadiene-1,4-diyl]bis)carbamic acid)diethyl ester, 1,3-bis-chloro(2-chloroethyl)-1-nitrosourea, dacarbazine, 5-fluorouracil, ftorafur, 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea, and vincristine are significantly increased. By contrast, the 50% growth-inhibitory concentrations for actinomycin D, mitomycin C, 6-mercaptopurine, and other agents are unaffected by hepatocyte presence. Cryopreserved hepatocytes exhibit detectable levels of drug activation, although inadequate for routine use. Results suggest that hepatocyte:tumor cell cocultures may be well-suited for assessing the degree to which hepatic metabolism may activate or inactivate new anticancer drugs.

¹ This work was supported in part by a grant from the Fraternal Order of the Eagles' Cancer Research Fund and by NIH Grant CA 36512.

² To whom requests for reprints should be addressed, at Laboratory of Developmental Oncology Research, Department of Oncology, Mayo Clinic and Foundation, Rochester, MN 55905.

Received 7/13/83. Accepted 11/ 1/83.

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