ACTIVATION OF THIO-TEPA CYTOTOXICITY TOWARD HUMAN BREAST-CANCER CELLSBY HEPATIC CYTOCHROME-P450

The anti-tumor alkylating agent thio-TEPA is metabolized by hepatic cy tochrome P450 to yield an oxo derivative. TEPA, plus one or more react ive metabolites. The contribution of this metabolism to the parent dru g's cytotoxicity was evaluated in cell culture using the human breast carcinoma cell line MCF-7 as a model. Incubation of thio-TEPA in the p resence of NADPH + an Aroclor 1254-induced rat liver 9000. x - superna tant fraction (S9 fraction) resulted in a dramatic increase in drug cy totoxicity, as measured using a clonogenic assay for cell survival. Th is increase in cytotoxicity was not evident when thio-TEPA was incubat ed with an uninduced rat liver homogenate, indicating that one or more Aroclor-inducible liver enzymes contribute to drug activation in this system. Metyrapone, a selective inhibitor of the Aroclor-inducible cy tochrome P450 2B1, completely blocked liver S9-dependent cytotoxicity, as did an inhibitory antibody directed against P450 2B1, demonstratin g that P450 2B1 is the S9 catalyst of thio-TEPA activation. Although T EPA is the major thio-TEPA metabolite formed by P450 2B 1, neither TEP A nor a TEPA metabolite is the cytotoxic species generated by the live r S9 fraction, as evidenced by the moderate cytotoxicity of TEPA in th is cellular system, by the failure of S9 to activate TEPA appreciably, and by the lack of synergism between TEPA and thio-TEPA. While glutat hione had little or no effect on the cytotoxicity of thio-TEPA or TEPA , low levels of extracellular glutathione (0.25-0.5 mM) fully blocked S9-dependent thio-TEPA activation, suggesting a cell surface site of a ction of the S9-generated cytotoxic metabolites. These metabolites may also act intracellulary, since depletion of intracellular glutathione by treatment with buthionine sulfoximine enhanced the cytotoxicity of thio-TEPA to a V79 cell transformant that stably expresses P450 2B1, but not to the parental V79 line or to MCF-7 cells. These studies esta blish a role for cytochrome P450 2B1 in the activation of thio-TEPA to cytotoxic metabolites that are distinct from TEPA, and furthermore su ggest a mechanism of action that includes the cell surface as a novel target of this cancer chemotherapeutic agent.