Evidence for Enzymatic Activation and Oxygen Involvement in Cytotoxicity and Antitumor Activity of N,N′,N″-Triethylenethiophosphoramide

Abstract

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.