The Grapefruit Juice Effect Is Not Limited to Cytochrome P450 (P450) 3A4: Evidence for Bergamottin-Dependent Inactivation, Heme Destruction, and Covalent Binding to Protein in P450s 2B6 and 3A5

Abstract

Bergamottin (BG), a component of grapefruit juice, is a mechanism-based inactivator of cytochromes P450 (P450) 2B6 and 3A5 in the reconstituted system. The inactivation of both P450s was NADPH-dependent and irreversible. The kinetic constants for the inactivation of the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of P450 2B6 were: K I , 5 M; k inact , 0.09 min Ϫ1 ; and t 1/2 , 8 min. The kinetic constants obtained for the inactivation of the testosterone 6␤-hydroxylation activity of P450 3A5 were: K I , 20 M; k inact , 0.045 min Ϫ1 ; and t 1/2 , 15 min. Incubations of P450s 2B6 and 3A5 with 20 M BG at 37°C for 20 min resulted in an ϳ60% loss in the catalytic activity that was accompanied by a significant loss in intact heme and a similar decrease in the reduced CO difference spectrum. The extrapolated partition ratios for BG with P450s 2B6 and 3A5 were ϳ2 and ϳ20, respectively. Liquid chromatography-mass spectroscopy analysis of the BG-inactivated samples showed that the mass of the inactivated apoprotein had increased by approximately 388 Da for both P450 2B6 and P450 3A5. SDSpolyacrylamide gel electrophoresis analysis demonstrated that [ 14 C]BG was irreversibly bound to the apoprotein in the BGinactivated samples. The stoichiometry of binding was ϳ0.5 mol BG metabolite/mol of each P450 inactivated. High-pressure liquid chromatography analysis of the metabolites of BG showed that P450 2B6 generated two major metabolites, whereas P450 3A5 generated three additional metabolites. Two of metabolites were identified as 6Ј,7Ј-dihydroxybergamottin and bergaptol.

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

“…In general, we and others have found that mass assignments using LC-MS analysis of adducted human P450s has been difficult and often exhibit unacceptably large errors. Perhaps the adducted enzyme has a greater tendency to aggregate and is poorly ionized.The results with the 17EE-adducted P450 2B6 are similar to our previous studies where a bergamottin-adducted P450 2B6 could be detected, but due to the significant error in the mass assignment it was not possible to accurately determine the number of oxygen atoms attached to the reactive intermediate (32). Others have also observed that inactivated apo-P450 3A4 was difficult to analyze by a variety of mass spectrometry techniques (33).…”

Identification of the Residue in Human CYP3A4 That Is Covalently Modified by Bergamottin and the Reactive Intermediate That Contributes to the Grapefruit Juice Effect

“…14 C]BG to the CYP3A4 apoprotein by SDS-polyacrylamide gel electrophoresis (PAGE) (Lin et al, 2005). However, in those studies, the mass increase of the adducted apoprotein (apo)-3A4 and the mass of the reactive intermediate species were not characterized.…”

“…14 C]BG as described previously (He et al, 1998;Lin et al, 2005). The [ 14 C]BG-labeled apo-3A4 was separated from the reductase by SDS-PAGE and recovered by electroblotting to a nitrocellulose (NC) membrane or to a polyvinylidene difluoride (PVDF) membrane for digestion by lysyl endopeptidase (Lys C) or cyanogen bromide (CNBr), respectively (Roberts et al, 1993).…”

Inactivation of Cytochrome P450 (P450) 3A4 but not P450 3A5 by OSI-930, a Thiophene-Containing Anticancer Drug

“…Cytochrome b 5 was purified from liver microsomes of phenobarbital-treated Long-Evans rats. The purification procedures for all the P450s, cytochrome b 5 , and NADPH-cytochrome P450 reductase (reductase) were described previously (Lin et al, 2005).…”

“…The plasmids for P450 3A4, 3A5, 2B6, and 2D6 were expressed as His-tagged proteins in Escherichia coli TOPP3 cells (Domanski et al, 2001;Lin et al, 2005). Cytochrome b 5 was purified from liver microsomes of phenobarbital-treated Long-Evans rats.…”