This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ng, S.-f. Articles by Waxman, D. J. Search for Related Content PubMed PubMed Citation Articles by Ng, S.-f. Articles by Waxman, D. J.

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.

This article has been cited by other articles:

				E. Harleton, M. Webster, N. N. Bumpus, U. M. Kent, J. M. Rae, and P. F. Hollenberg Metabolism of N,N',N''-Triethylenethiophosphoramide by CYP2B1 and CYP2B6 Results in the Inactivation of Both Isoforms by Two Distinct Mechanisms J. Pharmacol. Exp. Ther., September 1, 2004; 310(3): 1011 - 1019. [Abstract] [Full Text] [PDF]

				J. M. Rae, N. V. Soukhova, D. A. Flockhart, and Z. Desta Triethylenethiophosphoramide Is a Specific Inhibitor of Cytochrome P450 2B6: Implications for Cyclophosphamide Metabolism Drug Metab. Dispos., May 1, 2002; 30(5): 525 - 530. [Abstract] [Full Text] [PDF]

				M. J. van Maanen, I. M. Tijhof, J. M. A. Damen, C. Versluis, J. J. K. van den Bosch, A. J. R. Heck, S. Rodenhuis, and J. H. Beijnen A Search for New Metabolites of N,N',N''-Triethylenethiophosphoramide Cancer Res., September 1, 1999; 59(18): 4720 - 4724. [Abstract] [Full Text] [PDF]