Effect of 17-α-Ethynylestradiol on Activities of Cytochrome P450 2B (P450 2B) Enzymes: Characterization of Inactivation of P450s 2B1 and 2B6 and Identification of Metabolites

Abstract

17-␣-Ethynylestradiol (17EE) inactivated purified, reconstituted rat hepatic cytochrome P450 (P450) 2B1 and human P450 2B6 in a mechanism-based manner. Little or no inactivation was observed when P450s 2B2 or 2B4 were incubated with 17EE. The inactivation of P450s 2B1 and 2B6 was entirely dependent on both NADPH and 17EE and followed pseudo-first order kinetics. The maximal rate constants for the inactivation of P450s 2B1 and 2B6 at 30°C were 0.2 and 0.03 min Ϫ1 , respectively. For P450s 2B1 and 2B6 the apparent K I was 11 and 0.8 M, respectively. Incubation of P450 2B1 with 17EE and NADPH for 20 min resulted in a 75% loss in enzymatic activity and a concurrent 20 to 25% loss of the enzyme's ability to form a reduced CO complex. With P450 2B6, an 83% loss in enzymatic activity and a 5 to 10% loss in the CO reduced spectrum were observed. The extrapolated partition ratios for 17EE with P450 2B1 and 2B6 were 21 and 13, respectively. Simultaneous incubation of an alternate substrate together with 17EE protected both enzymes from inactivation. A 1.3:1 stoichiometry of labeling for binding of the radiolabeled 17EE to P450 2B1 and 2B6 was seen. These results indicate that 17EE inactivates P450s 2B1 and 2B6 in a mechanism-based manner, primarily by the binding of a reactive intermediate of 17EE to the apoprotein. Analysis of the 17EE metabolites showed that 2B enzymes that become inactivated differ primarily by their ability to generate two metabolites that were not produced by P450s 2B2 or 2B4.

Identification of 17-α-Ethynylestradiol-Modified Active Site Peptides and Glutathione Conjugates Formed during Metabolism and Inactivation of P450s 2B1 and 2B6

“…The metabolism of 17EE by human P450 enzymes such as P450s 3A4 and 2B6 results in the production of reactive intermediates that are able to alkylate the P450 heme or modify the apoprotein (16)(17)(18). This observation not only raises a potential concern about drug interactions with other therapeutics that are also metabolized by these enzymes but the ensuing reactive 17EE intermediates may also lead to toxic or carcinogenic effects (3).…”

“…The resultant intermediate then undergoes a rearrangement involving ring expansion to an aldehyde that could potentially be further oxidized to the corresponding carboxylic acid (36). A metabolite corresponding to this 17-formyl-D-homosteroid has been observed in incubation mixtures from P450 2B and 3A enzymes that were inactivated by 17EE (17,18). Alternatively such a reactive intermediate could also react with either the heme or the apoprotein.…”

“…This observation not only raises a potential concern about drug interactions with other therapeutics that are also metabolized by these enzymes but the ensuing reactive 17EE intermediates may also lead to toxic or carcinogenic effects (3). A previous study demonstrated that in the presence of 17EE and NADPH, a loss of approximately 70% of the enzymatic activity of P450s 2B1 and 2B6 occurred (17). Because the same samples did not show a comparable loss in the ability of the 2B P450s to bind CO, it was proposed that this loss in enzymatic activity was primarily due to the formation of an adduct with the apo-P450 by a reactive intermediate.…”

“…We have previously reported that P450 2B2 was not inactivated and P450 2B4 was not significantly inactivated by 17EE (17). Therefore, we have compared the amino acid sequences for P450s 2B1, 2B6, 2B2 and 2B4 between residues 347 and 376 (Table 2).…”

“…Previously, 17EE was shown to be a mechanism-based inactivator for P450s 3A4, 2B1, and 2B6 (16)(17)(18). Interestingly, the activity of P450s 2B2 and 2B4, which share a greater than 70% sequence identity with P450 2B1, was only minimally affected by incubations with 17EE in the presence of NADPH (17).…”