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Evidence for Enzymatic Activation and Oxygen Involvement in Cytotoxicity and Antitumor Activity of N,N',N''-Triethylenethiophosphoramide¹

Division of Cancer Pharmacology, Dana-Farber Cancer Institute [B. A. T., D. J. W., S. A. H., Y. W., L. C., E. A. S., S. M. J., E. F.], and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School [D. J. W., L. C.], Boston, Massachusetts 02115

The cytotoxicity of N,N',N''-triethylenethiophosphoramide (thiotepa) was studied in vitro in the MCF-7 human breast carcinoma cell line and in vivo using the EMT6 mouse mammary tumor model, under various conditions of oxygenation and in the presence and absence of Aroclor 1254-induced liver preparations. The cytotoxicity of thiotepa toward exponentially growing MCF-7 cells was markedly dependent on the presence of oxygen during the period of drug exposure, with 3 log greater cell kill at 500 µM thiotepa being observed when the cells were normally oxygenated compared with hypoxic cells. Incubation of thiotepa with an Aroclor 1254-induced rat liver S-9 homogenate, in the presence of a NADPH-regenerating system, resulted in an 8-fold increase in cytotoxicity towards the MCF-7 cells over a wide range of drug concentrations. Thiotepa was shown to be metabolized under these conditions in a NADPH- and O₂-dependent reaction that was catalyzed by one or more microsomal cytochrome P-450 enzymes that were present in the S-9 fraction. The thiotepa metabolite triethylene phosphoramide, which hydrolyzes significantly faster than thiotepa, was significantly less cytotoxic toward the MCF-7 cells than was thiotepa itself, suggesting that it is unlikely to be the S-9 metabolite responsible for the observed increase in drug cytotoxicity. Moreover, triethylene phosphoramide cytotoxicity was only partially O₂ dependent and was largely unaffected by incubation in the presence of the S-9 preparation, indicating a mechanism of action distinct from that of thiotepa. Tumor cell survival experiments with the EMT6 mouse mammary carcinoma system revealed that a 3.6-fold increase in thiotepa cytotoxicity was obtained by prior administration of the liver inducer Aroclor 1254 to the tumor-bearing animals, 5 days before drug treatment. Finally, the therapeutic effectiveness of thiotepa was significantly enhanced (3- to 5.8-fold increase in tumor growth delay) when an increase in oxygenation was achieved, by carbogen breathing, in animals given the perfluorochemical emulsion Fluosol-DA. These findings establish that the cytotoxic effects of thiotepa are oxygen dependent and may involve, at least in part, metabolic processes catalyzed by cytochrome P-450 enzymes.

¹ This work was supported by grants from Lederle Laboratories, Pearl River, NY (B. A. T.); by National Cancer Institute Grant 1PO1-CA-38493 (E. F.); and by American Cancer Society Grant BC-462 (D. J. W.).

² To whom requests for reprints should be addressed.

Received 4/12/89. Revised 6/ 9/89. Accepted 6/15/89.

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