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 monooxygenase

Abstract

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 (beta-naphthoflavone) or IIE (isoniazid). Thio-TEPA depletion and TEPA formation catalyzed by phenobarbital-induced liver microsomes were both inhibited by greater than 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 (Km (app) 19 microM; Vmax (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 kinactivation = 0.096 min-1, KI = 24 microM, 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 16 beta-hydroxylase) as well as P-450 IIIA-related enzymes (steroid 6 beta-hydroxylase) and the P-450-independent enzyme steroid 17 beta-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 alpha-hydroxylase) or microsomal NADPH P-450 reductase was observed.(ABSTRACT TRUNCATED AT 400 WORDS)