Use of V79 cells with stably transfected cytochrome P450 cDNAs in studyingthe metabolism and effects of cytotoxic drugs

Purpose: Studying the metabolism of cytotoxic drugs has become increasingly necessary to predict clinically significant drug-drug interactions and to understand the basis of interindividual variations in the pharmacokinetics of anticancer agents. The aim of this study was to determine the feasibilit y of using V79 Chinese hamster fibroblasts, which are stably transfected wi th cytochrome P450 (CYP) cDNAs, to study the metabolism of cytotoxic drugs in vitro. Methods: The 3-[4.5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine cell survival after incubation wi th drugs. Gas chromatography/mass spectroscopy was used for the quantitatio n of metabolites of cyclophosphamide and ifosfamide in culture medium. The coculture technique was used to study the generation of cytotoxic metabolit es in culture medium. Results: After treatment with either cyclophosphamide or ifosfamide (100 mu M to 1 mM) cytotoxicity was demonstrated in only cyt ochrome CYP2B1- and cytochrome CYP3A4-expressing cells. Treatment of parent al nontransfected cells that were cocultured with CYP-expressing cells with cyclophosphamide resulted in increased sensitivity to this drug. All activ e and inactive metabolites of cyclophosphamide and ifosfamide were detected in the culture medium. Cyclophosphamide-induced cytotoxicity in CYP2B1- an d CYP3A4-expressing cells was abrogated by metyrapone and midazolam/trolean domycin, respectively. Paclitaxel showed greater cytotoxicity against paren tal V79 cells than against the CYP2B1-, 2E1-, or 3A4-expressing cells, whic h was also influenced by cotreatment with CYP inhibitors. Conclusions: Stab le expression of CYP cDNAs by V79 cells provided an in vitro system to stud y cytotoxic drug metabolism. Cell viability and metabolite assays were used to determine the differential metabolism and effects in different CYP-tran sfected cell lines treated with cytotoxic drugs. The potential use of this V79 cell expression system is in studying enzymes involved in the metabolis m of cytotoxic drugs, especially early in drug development. In addition, th is system may be used to determine drug interactions that may influence the outcome of therapy in patients with cancer.