The Glucuronidation of -3-Keto C19- and C21-Hydroxysteroids by Human Liver Microsomal and Recombinant UDP-glucuronosyltransferases (UGTs): 6 - and 21- Hydroxyprogesterone Are Selective Substrates for UGT2B7

The stereoand regioselective glucuronidation of 10 -3-keto monohydroxylated androgens and pregnanes was investigated to identify UDP-glucuronosyltransferase (UGT) enzyme-selective substrates. Kinetic studies were performed using human liver microsomes (HLMs) and a panel of 12 recombinant human UGTs as the enzyme sources. Five of the steroids, which were hydroxylated in the 6 -, 7 -, 11 or 17 -positions, were not glucuronidated by HLMs. Of the remaining compounds, comparative kinetic and inhibition studies indicated that 6 and 21-hydroxyprogesterone (OHP) were glucuronidated selectively by human liver microsomal UGT2B7. 6 -OHP glucuronidation by HLMs and UGT2B7 followed Michaelis-Menten kinetics, whereas 21-OHP glucuronidation by these enzyme sources exhibited positive cooperativity. UGT2B7 was also identified as the enzyme responsible for the high-affinity component of human liver microsomal 11 -OHP glucuronidation. In contrast, UGT2B15 and UGT2B17 were the major forms involved in human liver microsomal testosterone 17 -glucuronidation and the high-affinity component of 16 -OHP glucuronidation. Activity of UGT1A subfamily enzymes toward the hepatically glucuronidated substrates was generally low, although UGT1A4 and UGT1A9 contribute to the low-affinity components of microsomal 16 and 11 -OHP glucuronidation, respectively. Interestingly, UGT1A10, which is expressed only in the gastrointestinal tract, exhibited activity toward most of the glucuronidated substrates. The results indicate that 6 and 21-OHP may be used as selective “probes” for human liver microsomal UGT2B7 activity and, taken together, provide insights into the regioand stereoselectivity of hydroxysteroid glucuronidation by human UGTs. UDP-glucuronosyltransferase (UGT) enzymes catalyze the covalent linkage of glucuronic acid, derived from the cofactor UDPglucuronic acid (UDPGA), to typically lipophilic substrates bearing a suitable acceptor group, most commonly hydroxyl, carboxylic acid, or amine. Given the ability of UGT to metabolize such commonly occurring chemical features, conjugation with glucuronic acid (“glucuronidation”) assumes importance for the elimination and detoxification of drugs, environmental chemicals, and endogenous compounds (Miners and Mackenzie, 1991). UGTs have been classified in two families, UGT1 and UGT2, based on the sequence identity of the encoded proteins (Mackenzie et al., 2005). Of the 19 human UGT proteins identified to date, 13 appear to exhibit significant activity toward drugs, environmental chemicals, and/or endogenous compounds: UGT 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, 2B17, and 2B28 (Miners et al., 2004). The individual UGTs possess distinct, albeit overlapping, substrate selectivities and differ in terms of regulation of expression. For example, age, diet, disease states, induction, and inhibition by coadministered chemicals, ethnicity, genetic polymorphism, and hormonal factors are all known to influence UGT activity (Miners and Mackenzie, 1991; Miners et al., 2001). Furthermore, differences occur in regulation of expression; whereas most UGTs are expressed in liver, UGT 1A7, 1A8, and 1A10 are localized in the gastrointestinal tract (Mojarrabi and Mackenzie, 1998; Tukey and Strassburg, 2000). Given these features of UGTs, recent attention has focused on identifying enzyme-selective probes, that is, compounds selectively glucuronidated by a single UGT. The availability of selective substrates is important for the reaction phenotyping of glucuronidation pathways and for evaluating the selectivity of drug-drug interactions and the functional significance of UGT genetic polymorphism (Court 2005; Miners et al., 2006). The majority of studies conducted to date have focused on the characterization of xenobiotic probe substrates for UGT enzymes. However, there is evidence with both cytochromes P450 and UGT to demonstrate that the stereoand regioselective This work was supported by a grant from the National Health and Medical Research Council of Australia. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.106.013052. ABBREVIATIONS: UGT, UDP-glucuronosyltransferase; UDPGA, UDP-glucuronic acid; HPLC, high performance liquid chromatography; 6 -OHP, 6 -hydroxyprogesterone; 6 -OHP, 6 -hydroxyprogesterone; 7 -OHAD, 4-androsten-7 -ol-3, 17-dione; 6 -OHAD, 4-androsten-6 -ol-3, 17dione; TST, testosterone; 16 -OHP, 16 -hydroxyprogesterone; 17 -OHP, 17 -hydroxyprogesterone; 21-OHP, 21-hydroxyprogesterone; 4-MU, 4-methylumbelliferone; 4-MUG, 4-methylumbelliferone-D-glucuronide; HLM, human liver microsome; HEK293, human embryo kidney 293 cell line; AZT, zidovudine; 11 -OHP, 11 -hydroxyprogesterone; 11 -OHP, 11 -hydroxyprogesterone. 0090-9556/07/3503-363–370$20.00 DRUG METABOLISM AND DISPOSITION Vol. 35, No. 3 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 13052/3178465 DMD 35:363–370, 2007 Printed in U.S.A. 363 at A PE T Jornals on A uust 7, 2017 dm d.aspurnals.org D ow nladed from metabolism of steroids provides an alternative approach to the identification of enzyme-selective pathways. The hydroxylation of C18-, C19and C21-steroids is known to occur at almost all nonbridgehead carbon atoms in humans, although certain positions may be favored, depending on the structure of the substrate (Setchell et al., 1976; Hobkirk, 1979). In addition, hydroxylation may be stereoselective, leading to the formation of -and -isomers. Many of these hydroxysteroids are excreted in urine as the glucuronide conjugates (Fotherby and James, 1972; Setchell et al., 1976; Musey et al., 1979). As noted above, regioand stereoselective steroid hydroxylation may be used for identifying cytochrome P450 enzyme selective pathways. For example, it is well established that testosterone 6 -hydroxylation is catalyzed selectively by CYP3A (Mei et al., 1999). Similarly, UGT1A and UGT2B enzymes appear to differentially contribute to the glucuronidation of hydroxylated derivatives of C18(estrogens), C19and C21-steroids (Jin et al., 1997; Turgeon et al., 2001; Kuuranne et al., 2003; Lepine et al., 2004). -3-Keto-hydroxylated C19and C21-steroids provide a convenient series of compounds for investigating the regioand stereoselectivity of hydroxysteroid glucuronidation. Numerous compounds are available commercially and the -3-keto-moiety provides a chromophore suitable for the UV detection of products by straightforward high-performance liquid chromatography (HPLC) methods. This study investigated the UGT enzyme selectivity of a series of 6 -, 6 -, 7 -, 11 -, 11 -, 16 -, 17 -, 17 -, and 21-monohydroxylated derivatives of C19and C21-steroids bearing the -3-keto function (Fig. 1). Whereas the stereoand regioselectivity of hydroxysteroid glucuronidation by recombinant UGTs and human liver microsomes was undertaken primarily to identify pathways that may be selective for hepatic UGT enzymes, the work provides further insights into the structural determinants of substrates that confer UGT enzyme selectivity. Materials and Methods Materials. 6 -Hydroxyprogesterone (6 -OHP), 6 -hydroxyprogesterone (6 -OHP), and 4-androsten-7 -ol-3, 17-dione (7 -OHAD) were purchased from Steraloids (Newport, RI). 4-Androsten-6 -ol-3,17-dione (6 -OHAD), testosterone (TST), 11 -hydroxyprogesterone (11 -OHP), 11 -hydroxyprogesterone (11 -OHP), 16 -hydroxyprogesterone (16 -OHP), 17 -hydroxyprogesterone (17 -OHP), 21-hydroxyprogesterone (21-OHP), 4-methylumbelliferone (4-MU), 4-methylumbelliferone-D-glucuronide (4-MUG), alamethicin (from Trichoderma viride), -glucuronidase (from Escherichia coli), and UDPGA (sodium salt) were purchased from Sigma-Aldrich (Sydney, Australia). Solvents and other reagents used were of analytical reagent grade. Methods. Human liver microsomes and recombinant UGTs. Microsomes were prepared from human liver tissue by differential centrifugation, as described by Bowalgaha et al. (2005). The five human livers used in this study (H7, H12, H13, H29, and H40) were obtained from the human liver “bank” of the Department of Clinical Pharmacology, Flinders Medical Centre. Approval for the use of human liver tissue for in vitro drug metabolism studies was granted by the Clinical Investigation Committee of Flinders Medical Centre. Human liver microsomes (HLMs) used to investigate hydroxysteroid glucuronidation were activated with alamethicin (50 g/mg microsomal protein), as described by Boase and Miners (2002). Human UGT 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, 2B17, and 2B28 cDNAs were stably expressed in a human embryonic kidney cell line (HEK293) according to Stone et al. (2003) and Uchaipichat et al. (2004). After growth to at least 80% confluence, cells were harvested and washed in phosphate-buffered saline. Cells were subsequently lysed by sonication using a Vibra Cell VCX 130 Ultrasonics Processor (Sonics and Materials, Newtown, CT). Cells expressing UGT1A enzymes were sonicated with four “bursts” each lasting 2 s, separated by 1 min with cooling on ice. Cells expressing UGT2B enzymes were treated using the same method, except sonication was limited to 1-s bursts. Lysates were centrifuged at 12,000g for 1 min at 4°C, and the supernatant fraction was separated and stored in phosphate buffer (0.1 M, pH 7.4) at 80°C until use. Expression of each UGT was demonstrated by immunoblotting with a commercial UGT1A antibody (BD Gentest, Woburn, MA) or a nonselective UGT antibody (raised against purified mouse Ugt) (Uchaipichat et al., 2004) and activity measurements (see