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Biotransformation of N,N',N''-Triethylenethiophosphoramide: Oxidative Desulfuration to Yield N,N',N''-Triethylenephosphoramide Associated with Suicide Inactivation of a Phenobarbital-inducible Hepatic P-450 Monooxygenase¹

Department of Biological Chemistry and Molecular Pharmacology and Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115

Oxidative metabolism of the polyfunctional alkylating agent N,N',N''-triethylenethiophosphoramide (thio-TEPA) was studied in isolated rat liver microsomes and purified, reconstituted cytochrome P-450 (P-450) enzyme systems in order to elucidate the pathways of drug oxidation and to identify the possible contributions of individual P-450 enzymes to the bioactivation of this chemotherapeutic agent. Rat liver microsomes were found to catalyze conversion of thio-TEPA to its oxo metabolite, N,N',N''-triethylenephosphoramide (TEPA), in a P-450-dependent reaction that was markedly stimulated by prior in vivo treatment with drug inducers of hepatic P-450 subfamily IIB (phenobarbital), but not by pretreatment with inducers of P-450 subfamilies IA (ß-naphthoflavone) or IIE (isoniazid). Thio-TEPA depletion and TEPA formation catalyzed by phenobarbital-induced liver microsomes were both inhibited by >90% by antibodies selectively reactive with P-450 PB-4 (gene product IIB1), the major phenobarbital-inducible rat liver microsomal P-450 form, but not by antibodies inhibitory toward 7 other rat hepatic P-450s. Oxidation of thio-TEPA to TEPA was also catalyzed by purified P-450 PB-4 (K_m (app) 19 µM; V_max (app) = 11 mol thio-TEPA metabolized/min/mol P-450 PB-4) following reconstitution of the cytochrome with NADPH P-450 reductase in a lipid environment. Metabolism of thio-TEPA by P-450 PB-4 was associated with a suicide inactivation of the cytochrome characterized by k_inactivation = 0.096 min^-1, K_I = 24 µM, and a partition ratio of 136 ± 28 (SD) mol thio-TEPA metabolized/mol P-450 inactivated. The thio-TEPA metabolite TEPA, however, did not inactivate the cytochrome, nor was it subject to further detectable metabolism. In microsomal incubations, metabolism of thio-TEPA led to the inactivation of P-450 PB-4 (steroid 16ß-hydroxylase) as well as P-450 IIIA-related enzymes (steroid 6ß-hydroxylase) and the P-450-independent enzyme steroid 17ß-hydroxysteroid:NADP⁺ 17-oxidoreductase, as demonstrated by use of the P-450 form-selective steroidal substrate androst-4-ene-3,17-dione. In contrast, little or no inactivation of microsomal P-450 IIA-related enzymes (steroid 7-hydroxylase) or microsomal NADPH P-450 reductase was observed. Substantial protection of steroid 6ß-hydroxylase and steroid 17ß-hydroxysteroid:NADP⁺ 17-oxidoreductase but not steroid 16ß-hydroxylase (P-450 PB-4) was afforded by the sulfhydrylcontaining nucleophiles cysteine and glutathione; this suggests that inactivation of these microsomal enzymes is mediated by diffusible, reactive metabolite(s) of thio-TEPA, but that in the case of P-450 PB-4, inactivation occurs before the metabolite(s) depart from the active site. These findings demonstrate that P-450 PB-4 can oxidize thio-TEPA to chemically reactive metabolite(s) that may potentially contribute to drug cytotoxicity.

¹ Supported in part by Grant BC-462 from the American Cancer Society (D. J. W.).

² To whom requests for reprints should be addressed, at Dana-Farber Cancer Institute, JF-525, 44 Binney Street, Boston, MA 02115.

Received 8/11/89. Revised 10/27/89. Accepted 10/31/89.

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