Cytochrome P450-mediated Oxidation of Glucuronide Derivatives: Example of Estradiol-17 -glucuronide Oxidation to 2-hydroxy- Estradiol-17 -glucuronide by Cyp 2c8

In the classical metabolic oxidation scheme, hydrophobic endogenous or xenobiotic compounds undergo phase I oxidation, generally catalyzed in the liver by cytochromes P450, followed by phase II conjugation reactions, in a way that allows much more polar metabolites to be expelled from the cell through active transport mechanisms. Cytochrome P450-mediated oxidation of steroid sulfate has been described, suggesting that oxidation of polar metabolites such as glucuronide derivatives of endogenous compounds can occur. As an example, we report here that hydroxyestradiol-17 -glucuronide can be directly formed through oxidation of estradiol-17 -glucuronide on the aromatic C2 position. This reaction is specifically catalyzed by CYP 2C8, which is more active in female than in male human liver microsomes. A thorough docking of the molecule within the CYP 2C8 crystal structure shows that the active site is large enough to handle a glucuronide conjugate. Moreover, the most energetically favored position of the bound ligand is fully consistent with the recently published structural determinants of substrate specificity of the CYP 2C8 active site. This is the first demonstration of cytochrome P450-mediated oxidation of a steroid glucuro-conjugate. Such oxidation of a glucuronide should be a general process since, in addition to estradiol and testosterone glucuronide, it has been observed for xenobiotic compounds, e.g., diclofenac or naproxen glucuronide. Cytochrome P450 enzymes (P450s) have long been of interest because of their role in the metabolism of xenobiotic or endogenous compounds (Guengerich, 1995; Lewis, 2001). Steroid metabolism has been extensively studied in the context of the formation of metabolites with hormonal activities or those involved in cancer proliferation. Steroids are primarily metabolized in gonads, and hepatic metabolism is also observed for many of them, leading to regioor stereoselective oxidations (Aoyama et al., 1990; Lee et al., 2003). In this regard, hepatic metabolism of steroids is still an interesting tool to investigate the specific involvement of a given P450. It is generally admitted that P450-mediated reaction, also called phase I metabolism, converts hydrophobic compounds into more polar metabolites, some of them having toxicological effects. Phase II metabolism involves an adduct formation via a conjugation reaction, leading to a decrease of hydrophobicity or to a charge modification of the given metabolite. Several conjugation pathways are known, such as methylation, sulfation, and glucuronidation (Raftogianis et al., 2000). Metabolites are generally recovered in urine or feces after active transport out of the cells via ATP-binding cassette transporters (Goh et al., 2002; Zelcer et al., 2003). In addition to these classical metabolic pathways, it has been demonstrated that the sulfate derivatives of different classes of steroids, such as testosterone and estrogens, can be oxidized by cytochromes P450. Oxidation usually takes place at the opposite moiety as compared with the sulfate group position (Ingelman-Sundberg et al., 1975; Milewich et al., 1986). The main P450 form involved in dehydroepiandrosterone (DHEA) sulfate oxidation has been identified as CYP 3A7, an isoform present in human liver fetus (Kitada et al., 1987; Ohmori et al., 1998). Oxidation of 5 -androstane-3 ,17 -diol3,17-disulfate has been observed in rat microsomes and attributed to the female isoform P450i (Ryan et al., 1984). This isoform is classified as CYP 2C11 in rat (Guengerich, 1995), and its sequence is very similar to that of human P450 2C (http://p450.abc.hu). In the present study, we demonstrate the formation of 2-hydroxy-estradiol-17 glucuronide by direct CYP 2C8 oxidation of estradiol-17 -glucuronide. This metabolic pathway is observed in human liver microsomal preparations as well as in heterologously expressed cytochrome P450 isoforms. Materials and Methods Chemicals. Estradiol, 2-hydroxy-, 4-hydroxy-, and 6 -hydroxy-estradiol, estriol, estradiol-3-glucuronide, estradiol-17 -glucuronide, estriol-17 -glucuronide, NADPH, NADP, glucose 6-phosphate (G6P), glucose-6-phosphate dehydrogenase (G6PDH), Helix pomatia or Escherichia coli glucuronidases, Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.104.002097. ABBREVIATIONS: P450, cytochrome P450; G6P, glucose 6-phosphate; G6PDH, glucose-6-phosphate dehydrogenase; DEX, dexamethasone; PB, phenobarbitone; UDPGT, UDP-glucuronosyltransferase; HPLC, high-performance liquid chromatography; MS, mass spectrometry; MS-MS, tandem mass spectrometry; XOH E2-glu, oxidized metabolite of estradiol-17 -glucuronide; E2-glu, estradiol-17 -glucuronide; XOH E2, oxidized metabolite of estradiol; 2OH E2, 2-hydroxy-estradiol; 4OH E2, 4-hydroxy-estradiol; amu, atomic mass unit(s); DHEA, dehydroepiandrosterone. 0090-9556/05/3303-466–473$20.00 DRUG METABOLISM AND DISPOSITION Vol. 33, No. 3 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 2097/1196250 DMD 33:466–473, 2005 Printed in U.S.A. 466 at A PE T Jornals on M ay 9, 2017 dm d.aspurnals.org D ow nladed from dexamethasone (DEX), sodium phenobarbitone salt (PB), furafylline, ketoconazole, quinidine, sulfaphenazole, bromocriptine, and paclitaxel were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France). 6 -Hydroxyl-estradiol was obtained from Steraloids (Newport, RI), and tritiated estradiol-17 -glucuronide [estradiol-6,7-H (N)] was obtained from PerkinElmer Life and Analytical Sciences (Paris, France). All other chemicals used were of the highest quality commercially available. Preparation of Microsomes. Male rats were housed and treated according to French legislation, in a facility authorized by the Ministry of Agriculture. Male Sprague-Dawley rats (200–220 g) (Iffa Credo, l’Arbresle, France) were treated with either PB (80 mg/kg i.p. in saline for 3 days) or DEX (100 mg/kg i.p. in corn oil for 3 days). Control rats [untreated (UT)] received only corn oil (0.5 ml/day for 3 days). Rats were sacrificed 1 day after the last treatment. Microsomes were prepared from a pool of four to six livers as previously described (Kremers et al., 1981), frozen in liquid nitrogen, and stored at 80°C until use. Human liver samples were kindly supplied by the Franche Comté Hospital surgery department of Université de Besançon, France and prepared as previously described (Kremers et al., 1981). Monkey, mouse, and dog male or female human liver microsomes were obtained from XenoTech LLC, Tebu-Bio (Le Perray en Yvelines, France). Yeast-expressed human P450s were prepared as already described and produced by SPI-BIO (Massy, France) (Peyronneau et al., 1992). The mutated CYP 2C8 isoform R241A expressed in yeast was kindly provided by A. Melet (Unité Mixte Recherche 8601, Centre National de la Recherche Scientifique, Paris, France). E. coliexpressed P450 forms were obtained from Cypex Ltd., Tebu-Bio. Bactosomeexpressed human CYP 3A7 and UDPGT2B7 were obtained from BD Gentest (Le Pont de Claix, France). Protein content in microsomal suspensions was determined by the method of Lowry et al. (1951) using bovine serum albumin as standard. P450 concentration was measured as described by Omura and Sato (1964). Human specific isoform activities in human liver microsomes were determined by XenoTech LLC, Tebu-Bio. Metabolism. Metabolism of estradiol derivatives was studied at 37°C in 0.1 M potassium phosphate buffer (pH 7.4) with 1 M P450 from rat liver or human liver microsomes, using 50 or 100 M substrate and an NADPHgenerating system (1 mM NADP , 10 mM G6P, and 1 IU G6PDH) for a 0.5-ml incubation. Radioactive incubations were performed using 50 M estradiol-17 -glucuronide and 0.5 Ci [H]estradiol-17 -glucuronide. The incubations were stopped at the indicated times by addition of the same volume of acetonitrile and a 10% volume of 1 M acetic acid. The mixture was centrifuged at 10,000 rpm for 5 min and then frozen until analysis, before which it was remixed. Incubations of E. coli or Bactosomes Containing Human P450. Metabolism of 50 M estradiol or estradiol glucuronides was studied at 37°C in 200l final volume phosphate buffer (0.1 M, pH 7.4), using 0.5 M P450, and in the presence of an NADPH-generating system (1 mM NADP , 10 mM G6P, and