Advertisement
Research ArticleArticle

The Glucuronidation of Δ4-3-Keto C19- and C21-Hydroxysteroids by Human Liver Microsomal and Recombinant UDP-glucuronosyltransferases (UGTs): 6α- and 21-Hydroxyprogesterone Are Selective Substrates for UGT2B7

K. Bowalgaha, D. J. Elliot, P. I. Mackenzie, K. M. Knights and J. O. Miners
Drug Metabolism and Disposition March 2007, 35 (3) 363-370; DOI: https://doi.org/10.1124/dmd.106.013052

Abstract

The stereo- and regioselective glucuronidation of 10 Δ4-3-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 6β-, 7α-, 11β- or 17α-positions, were not glucuronidated by HLMs. Of the remaining compounds, comparative kinetic and inhibition studies indicated that 6α- and 21-hydroxyprogesterone (OHP) were glucuronidated selectively by human liver microsomal UGT2B7. 6α-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 11α-OHP glucuronidation. In contrast, UGT2B15 and UGT2B17 were the major forms involved in human liver microsomal testosterone 17β-glucuronidation and the high-affinity component of 16α-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 16α- and 11α-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 6α- and 21-OHP may be used as selective “probes” for human liver microsomal UGT2B7 activity and, taken together, provide insights into the regio- and stereoselectivity of hydroxysteroid glucuronidation by human UGTs.

UDP-glucuronosyltransferase (UGT) enzymes catalyze the covalent linkage of glucuronic acid, derived from the cofactor UDP-glucuronic 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 stereo- and regioselective metabolism of steroids provides an alternative approach to the identification of enzyme-selective pathways.

Fig. 1.
Fig. 1.

Structures of the Δ4-3-keto hydroxysteroids.

The hydroxylation of C18-, C19- and 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 α-and β-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, regio- and stereoselective steroid hydroxylation may be used for identifying cytochrome P450 enzyme selective pathways. For example, it is well established that testosterone 6β-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), C19- and C21-steroids (Jin et al., 1997; Turgeon et al., 2001; Kuuranne et al., 2003; Lepine et al., 2004).

Δ4-3-Keto-hydroxylated C19- and C21-steroids provide a convenient series of compounds for investigating the regio- and stereoselectivity of hydroxysteroid glucuronidation. Numerous compounds are available commercially and the Δ4-3-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 6α-, 6β-, 7α-, 11α-, 11β-, 16α-, 17α-, 17β-, and 21-monohydroxylated derivatives of C19- and C21-steroids bearing the Δ4-3-keto function (Fig. 1). Whereas the stereo- and 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. 6α-Hydroxyprogesterone (6α-OHP), 6β-hydroxyprogesterone (6β-OHP), and 4-androsten-7α-ol-3, 17-dione (7α-OHAD) were purchased from Steraloids (Newport, RI). 4-Androsten-6β-ol-3,17-dione (6β-OHAD), testosterone (TST), 11α-hydroxyprogesterone (11α-OHP), 11β-hydroxyprogesterone (11β-OHP), 16α-hydroxyprogesterone (16α-OHP), 17α-hydroxyprogesterone (17α-OHP), 21-hydroxyprogesterone (21-OHP), 4-methylumbelliferone (4-MU), 4-methylumbelliferone-β-d-glucuronide (4-MUG), alamethicin (from Trichoderma viride), β-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 μg/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 below).

Before incubations, activities of recombinant UGT 1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, 2B17, and 2B28 were confirmed using the nonselective substrate 4-MU. For all enzymes, except UGT2B4 and UGT2B28, the conversion of 4-MU to 4-MUG was measured according to the fluorometric procedure of Miners et al. (1988), as modified by Uchaipichat et al. (2004). The concentration of 4-MU present in incubations corresponded to the known Km (or S50) for each enzyme (Sorich et al., 2002; Uchaipichat et al., 2004). Given the lower specific activities of UGT 2B4 and 2B28, 4-MU glucuronidation activity (at 1000 μM) was confirmed using a radiometric thin layer chromatographic procedure (Jin et al., 1997). The activity of UGT1A4 was demonstrated using lamotrigine as substrate, at the approximate Km for this substrate (1500 μM), according to the method of Rowland et al. (2006).

Glucuronidation of Hydroxysteroids by Human Liver Microsomes and Recombinant UGTs.Hydroxysteroid glucuronidation assays. Screening for hydroxysteroid glucuronidation by pooled HLMs (equal amounts of microsomal protein from the five livers used in kinetic studies) was performed at 37°C in a total incubation volume of 200 μl. Incubations contained microsomal protein (1 mg/ml), substrate (3 concentrations in the range of 5 to 250 μM), phosphate buffer (0.1 M, pH 7.4), MgCl2 (4 mM), and UDPGA (5 mM). Reaction mixtures were preincubated at 37°C for 5 min before initiation of the reaction by addition of cofactor (UDPGA). Reactions were carried out in air at 37°C (shaking water bath) for 120 min and then terminated by the addition of 2 μl of perchloric acid (11.6 M) and cooling on ice. Mixtures were vortex mixed and subsequently centrifuged (4000g for 10 min). A 30-μl aliquot of the supernatant fraction was analyzed by HPLC. Like experiments with HLMs, the ability of recombinant UGTs to glucuronidate hydroxysteroids was determined for three substrate concentrations in the range of 5 to 250 μM. Stock solutions of all hydroxysteroids were prepared in methanol; the final concentration of solvent in incubations was 1% v/v, which has a negligible effect on UGT enzyme activities (Uchaipichat et al., 2004). Activity-screening experiments with recombinant UGTs were performed with 1 mg/ml HEK cell lysate protein expressing the UGT of interest for 120 min.

Where activity was observed, kinetic studies were performed with microsomes from five separate human livers and the individual recombinant UGTs shown to glucuronidate each hydroxysteroid in activity-screening experiments. Kinetic experiments were performed in duplicate for 10 to 14 substrate concentrations spanning the Km (or S50). Linearity of product formation with respect to incubation time and protein concentration for HLMs and expressed UGT enzymes was established for all substrates investigated before kinetic analysis. Optimized incubation conditions for each hydroxysteroid investigated are given in Table 1.

View this table:
TABLE 1

Incubation conditions for the kinetic characterization of hydroxysteroid glucuronidation

Inhibition of hydroxysteroid glucuronide formation by Zidovudine. When hydroxysteroid glucuronidation appeared to involve UGT2B7, inhibition studies were performed with pooled HLMs (equal amounts of microsomal protein from five livers; see above) and UGT2B7 in the presence and absence of AZT, a known selective substrate for UGT2B7. Inhibition experiments were performed at an AZT concentration of 5 mM (which is ∼5-fold the Km for AZT glucuronidation by HLMs) (Boase and Miners 2002) and with the substrate concentration corresponding to the known Km (with HLMs as the enzyme source). When kinetics described by the two-enzyme Michaelis-Menten or Hill equations were observed, AZT inhibition experiments were carried out at a high and a low substrate concentration (see subsequent results) using the incubation conditions shown in Table 1.

Measurement of hydroxysteroid glucuronide formation. Hydroxysteroid glucuronidation was quantified by reversed phase HPLC. The HPLC system used (Agilent Technologies, Sydney, Australia) consisted of a gradient solvent delivery system, UV detector, and autosampler. The instrument was fitted with a Nova-Pak C18 (3.9 mm inside diameter) × 150 mm, 4 μm particle size) analytical column (Waters Corporation, Milford, MA). HPLC conditions for each hydroxysteroid are shown in Table 2. Analytes were eluted at a flow rate of 1 ml/min and detected by UV absorption at 241 nm. The Δ4-3-keto ring-A structure of hydroxysteroids provides a convenient chromophore for analysis of product formation by UV absorption at 241 nm. Chromatography was performed at 25°C.

View this table:
TABLE 2

Chromatographic conditions for the measurement of hydroxysteroid glucuronides

The identity of hydroxysteroid glucuronide formed in incubations with HLMs and expressed UGT enzymes was confirmed by hydrolysis with β-glucuronidase (from E. coli), acid hydrolysis, and comparison with blank incubations (which excluded UDPGA). Concentrations of TST glucuronide in incubations were determined by comparison of the peak areas with a standard curve constructed from an authentic TST glucuronide standard. For the remaining substrates, concentrations of hydroxysteroid glucuronides in incubations were determined by comparison of the peak areas with a standard curve constructed from known concentrations of each hydroxysteroid. Response factors of testosterone glucuronide and testosterone were shown to be equivalent. Nevertheless, Vmax values for other hydroxysteroid glucuronides should be considered “apparent.” Calibration standards were treated in the same manner as incubation samples. The lower limit of quantification for each hydroxysteroid assay, defined as five times background absorbance, was 0.04 μM hydroxysteroid glucuronide.

Within-day overall assay reproducibility for each hydroxysteroid assay was assessed by measuring the rate of glucuronide formation in eight separate incubations of pooled human liver microsomes. Assay reproducibility was assessed at two (low and high) substrate concentrations for each assay. For all hydroxysteroids tested, within-day coefficients of variation were <3.6% and <5.2% at the low and high concentrations, respectively.

Data Analysis. Kinetic constants for steroid glucuronidation were determined by fitting untransformed experimental data to equations for the single and two-enzyme Michaelis-Menten equations and the Hill equation (where indicated) using the nonlinear least-squares fitting program EnzFitter (version 2.0; Biosoft, Cambridge, UK). Goodness of fit was assessed from comparison of the parameter SE of fit, coefficient of determination (r2), and F-statistic.

Results

Hydroxysteroid Glucuronidation by Human Liver Microsomes and Recombinant UGTs. The hydroxysteroids shown in Fig. 1 were screened for glucuronide formation by pooled HLMs. Only TST, 6α-OHP, 11α-OHP, 16α-OHP, and 21-OHP were glucuronidated at a rate >0.5 pmol/min/mg by HLMs (data not shown). For example, glucuronidation activities ranged from 66 pmol/min/mg for 16α-OHP to 1290 pmol/min/mg for 11α-OHP at a substrate concentration of 50 μM. Thus, the glucuronidation of TST, 6α-OHP, 11α-OHP, 16α-OHP, and 21-OHP by both HLMs and recombinant UGTs was investigated further. Kinetic parameters for hydroxysteroid glucuronidation by HLMs and UGTs are summarized in Table 3. Eadie-Hofstee plots for hydroxysteroid glucuronidation by microsomes from a representative liver (H13) are shown in Fig. 2.

View this table:
TABLE 3

Kinetic parameters for hydroxysteroid glucuronidation by HLMs and expressed UGT enzymes

As indicated under Materials and Methods, each hydroxysteroid was screened for glucuronidation activity by 12 recombinant human UGTs. It was found that UGT 1A3, 1A4, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15, and 2B17 variably contributed to the glucuronidation of the hydroxysteroids investigated (see subsequent results). Representative Eadie-Hofstee plots for hydroxysteroid glucuronidation by recombinant human UGT 2B7 and 2B17 are shown in Fig. 3. UGT 1A1, 1A6, 2B4, and 2B28 did not glucuronidate any of the substrates investigated here, at least to the limit of detection of the assays used. It should be noted that where activity was observed with UGT 1A7, 1A8, and 1A10, kinetic studies were not performed because these enzymes are not expressed in liver.

TST Glucuronidation. TST glucuronidation exhibited Michaelis-Menten kinetics in all five livers investigated (Fig. 2A; Table 3), with respective mean ± S.D. derived Km and Vmax values of 5.6 ± 0.96 μM and 196 ± 113 pmol/min/mg. Seven recombinant enzymes (UGT 1A3, 1A4, 1A8, 1A10, 2B7, 2B15, and 2B17) glucuronidated TST. Rates of TST glucuronidation (at 100 μM) by recombinant UGT 1A10, 2B15, and 2B17 were in the range of 5.0 to 475 pmol/min/mg, whereas those of UGT 1A3, 1A4, 1A8, and 2B7 were <2.5 pmol/min/mg. TST glucuronidation by UGT 2B15 and 2B17 (Fig. 3A) followed Michaelis-Menten kinetics, with respective Km values of 5.7 and 3.8 μM (Table 3).

Fig. 2.
Fig. 2.

Representative Eadie-Hofstee plots for TST (A), 6α-OHP (B), 11α-OHP (C), 16α-OHP (D), and 21-OHP (E) glucuronidation by HLMs (liver H13). Points show experimentally determined values, whereas the curves are the computer-generated curves of best fit.

6α-OHP Glucuronidation. 6α-OHP glucuronidation similarly exhibited Michaelis-Menten kinetics in all five livers investigated (Fig. 2B; Table 3), with derived mean Km and Vmax values of 94 ± 22 μM and 1828 ± 743 pmol/min/mg, respectively. Recombinant UGT 1A3, 1A10, and 2B7 glucuronidated 6α-OHP. The rate of 6α-OHP glucuronidation by UGT2B7 at a substrate concentration of 250 μM was 1210 pmol/min/mg, whereas UGT 1A3 and 1A10 exhibited rates <2.5 pmol/min/mg. The glucuronidation of 6α-OHP by UGT2B7 followed Michaelis-Menten kinetics (Fig. 3B), with respective Km and Vmax values of 55 μM and 269 pmol/min/mg. AZT inhibition experiments were conducted at a 6α-OHP concentration of 100 μM (the mean approximate Km for the HLM-catalyzed reaction). AZT (5 mM) inhibited human liver microsomal 6α-OHP glucuronidation by 74% and UGT2B7 catalyzed 6α-OHP glucuronidation by 83%.

Fig. 3.
Fig. 3.

Eadie-Hofstee plots for TST (A), 6α-OHP (B), 11α-OHP (C), 16α-OHP (D), and 21-OHP (E) glucuronidation by the principal recombinant human UGT enzyme involved in the glucuronidation of each hydroxysteroid (typically UGT 2B7 and 2B17). Points show experimentally determined values and the computer-generated curves of best fit.

11α-OHP Glucuronidation. Non-Michaelis-Menten kinetics were observed for 11α-OHP glucuronidation by microsomes from four of the five livers investigated (Table 3). Data for microsomes from H12, H13, H29, and H40 were well described by the two-enzyme Michaelis-Menten equation (Fig. 2C), with apparent Km values of 3.8 ± 1.8 and 125 ± 172 μM for the high- and low-affinity components, respectively. In contrast, data for microsomes from liver H7 were consistent single-enzyme Michaelis-Menten kinetics (Km 11 μM) (Table 3). Recombinant UGT 1A3, 1A4, 1A7, 1A9, 1A10, and 2B7 glucuronidated 11α-OHP. The kinetics of 11α-OHP glucuronidation by recombinant UGT 1A9 and 2B7 were characterized on the basis of activities >2.5 pmol/min/mg at a substrate concentration of 250 μM. Conversion of 11α-OHP to its glucuronide by UGT1A9 (Km 93 μM) and UGT2B7 (Km 6.9 μM) (Fig. 3C) followed single-enzyme Michaelis-Menten kinetics. AZT inhibition experiments were performed at 11α-OHP concentrations of 4 and 100 μM. (Substitution of the mean Km and Vmax values for the high- and low-affinity components of 11α-OHP glucuronidation by HLMs (Table 3) in the two-enzyme Michaelis-Menten equation indicates that the high-affinity enzyme is responsible for 89% of activity at a substrate concentration of 4 μM and 52% of activity at a substrate concentration of 100 μM.) AZT (5 mM) inhibited human liver microsomal 11α-OHP glucuronidation by 75% at the lower substrate concentration and by 29% at the higher substrate concentration.

16α-OHP Glucuronidation. 16α-OHP glucuronidation by HLMs exhibited biphasic kinetics for all five livers investigated (Fig. 2D; Table 3). Fitting of experimental data to the two-enzyme Michaelis-Menten equation gave mean ± S.D. apparent Km values for the high- and low-affinity components of 4.0 ± 1.5 and 130 ± 103 μM, respectively, and Vmax values of 49 ± 29 and 113 ± 46 pmol/min/mg, respectively. UGT 1A4, 1A10, 2B7, 2B15, and 2B17 all glucuronidated 16α-OHP. Kinetic data for 16α-OHP glucuronidation by UGT1A4 (Km 73 μM), UGT2B7 (Km 211 μM), 2B15 (Km 3.0 μM), and 2B17 (Km 3.0 μM) (Fig. 3D) all followed hyperbolic kinetics. AZT inhibition experiments were conducted at 16α-OHP concentrations of 20 and 100 μM. Substitution of the mean Km and Vmax values for the high- and low-affinity components of 16α-OHP glucuronidation by HLMs (Table 3) in the two-enzyme Michaelis-Menten equation indicates that low-affinity enzyme is responsible for 29 and 51% of activity at substrate concentrations of 20 and 100 μM, respectively. AZT (5 mM) inhibited human liver microsomal 16α-OHP glucuronidation by 30 and 40% at the low- and high-substrate concentrations, respectively.

Fig. 4.
Fig. 4.

Proposed binding modes (A–D) of hydroxypregnanes in the UGT2B7 active site, showing sites of glucuronidation associated with each binding mode.

21-OHP Glucuronidation. 21-OHP glucuronidation by HLMs was best described by the Hill equation with positive cooperativity (autoactivation) in four of the five livers studied, with a mean S50 value of 34 ± 12 μM, Hill coefficient (n) of 1.22 ± 0.14, and Vmax of 217 ± 54 pmol/min/mg (Table 3; Fig. 2E). In contrast, data for microsomes from liver H29 were best described by the single-enzyme Michaelis-Menten equation (Table 3). Of the recombinant UGTs screened, UGT 1A10 and 2B7 glucuronidated 21-OHP. Glucuronidation of this compound by UGT2B7 exhibited sigmoidal kinetics, which were modeled using the Hill equation (Fig. 3E). Derived S50 and n values were 19 μM and 1.16, respectively. AZT inhibition experiments were conducted at 21-OHP concentration of 20 μM (the approximate S50). AZT inhibited human liver microsomal and UGT2B7 catalyzed 21-OHP glucuronide formation by 86 and 100%, respectively.

Discussion

The liver is generally considered to be the main organ responsible for the glucuronidation of circulating hydroxysteroids, although extrahepatic UGTs are presumably of importance as a “local” detoxification mechanism (Hum et al., 1999). In this work we sought primarily to identify substrate probes for human hepatic UGT enzymes using a series of Δ4-3-keto hydroxysteroids. Of the compounds screened, TST and 6α-, 11α-, 16α-, and 21-OHP were glucuronidated by HLMs, whereas 6β- and 7α-OHAD and 6β-, 11β-, and 17α-OHP lacked measurable glucuronidation activity. Comparative kinetic studies with a panel of recombinant enzymes indicated that, of the hepatically expressed enzymes, UGT 2B7, 2B15, and 2B17 were the major contributors to the glucuronidation of the compounds metabolized by HLMs. UGT 1A3, 1A4, 1A7, 1A8, 1A9, and 1A10 variably glucuronidated the substrates metabolized by HLMs. However, with some exceptions, activity of the UGT1A subfamily enzymes was generally low (<2.5 pmol/min/mg). UGT1A10, which is expressed solely in the gastrointestinal tract, significantly glucuronidated TST and 16α- and 21-OHP. UGT1A9 appeared to be the enzyme responsible for the low-affinity component of human liver microsomal 11α-OHP glucuronidation, and a contribution of UGT1A4 to the low-affinity component of 16α-OHP glucuronidation cannot be discounted.

Importantly, comparison of kinetic and AZT inhibition data using HLMs and recombinant UGTs as the enzyme sources indicated that 6α- and 21-OHP were glucuronidated selectively by UGT2B7. In addition, UGT2B7 appeared to be the high- and low-affinity enzyme responsible for human liver microsomal 11α-OHP and 16α-OHP glucuronidation, respectively. Thus, 6α- and 21-OHP may serve as substrate probes for human hepatic UGT2B7 activity in vitro. Of these, 6α-OHP has two advantages; higher activity (∼10-fold higher Vmax with HLMs) and 6α-OHP glucuronidation exhibits Michaelis-Menten kinetics (as opposed to the atypical kinetics observed for 21-OHP glucuronidation). The Vmax value for 6α-OHP glucuronidation by HLMs is comparable with those for zidovudine and morphine glucuronidation, commonly used xenobiotic probes for hepatic UGT2B7 activity (Court 2005; Miners et al., 2006). Of the human UGTs, UGT2B7 is arguably the most important in terms of drug metabolism. For example, UGT2B7 contributes to the glucuronidation of opioids (Coffman et al., 1998; Court et al., 2003; Stone et al., 2003), nonsteroidal anti-inflammatory agents (Jin et al., 1993), anticancer agents (Miners et al., 1997; Innocenti et al., 2001), lamotrigine (Rowland et al., 2006), and zidovudine (Barbier et al., 2000; Court et al., 2003).

The data reported here provide further insights into the stereo- and regioselectivity of hydroxysteroid glucuronidation by human UGTs. Of the compounds not glucuronidated by HLMs, the hydroxyl groups of 6β- and 7α-OHAD and 6β- and 11β-OHP are axially oriented in rings B/C, whereas the hydroxyl group of 17α-OHP is hindered by the pregnane 17β side chain. Thus, glucuronidation in these positions is presumably not favored because of steric hindrance.

Consistent with a previous report (Turgeon et al., 2001), UGT 2B15 and 2B17 selectively glucuronidated TST. Km values were comparable with both enzymes, although the Vmax for the UGT2B17-catalyzed reaction was two orders of magnitude higher. However, because it is not possible to quantify the relative expression of these enzymes (in either HLMs or HEK293 cells) comparison of intrinsic clearances (Vmax/Km) is not meaningful. UGT 2B15 and UGT 2B17 also exhibited the lowest Km values (3 μM) with 16α-OHP. A comparison of the glucuronidation of 13 anabolic androgenic steroids by recombinant human UGTs (Kuuranne et al., 2003) and data from activity-screening studies (Green et al., 1994; Beaulieu et al., 1996) suggests that UGT 2B15 and 2B17 preferentially glucuronidate 16α- and 17β-hydroxyl groups in ring D of androgens or pregnanes.

Also consistent with data presented here, UGT2B7 has previously been shown in this and other laboratories to glucuronidate numerous hydroxylated C18-, C19-, and C21-steroids (Ritter et al., 1990; Jin et al., 1993, 1997; Coffman et al., 1998; Turgeon et al., 2001; Kuuranne et al., 2003; Lepine et al., 2004). Apart from the substrates identified in the present study, UGT2B7 efficiently glucuronidates 3α-hydroxyandrogens, 3α-hydroxypregnanes and 3,4-catecholestrogens. Taken together, these data permit the development of structure-function relationships. It may be hypothesized that electrostatic interaction via the 20-keto function orientates the OH group of 3α-hydroxypregnanes for glucuronidation within the UGT2B7 active site (Fig. 4A; “normal” mode 1). (The presence of 16α- and 17β-hydroxy (and 17-keto) groups similarly orientate C18- and C19–3-hydroxysteroids within the UGT2B7 active site for glucuronidation at this position (Jin et al., 1997).) The preferential glucuronidation of 16α- and 21-OHP may be explained by binding to the UGT2B7 active site in the “reverse” mode (i.e., rotation through 180 along the x-axis), whereby an electrostatic interaction via the 3-keto function orientates these functional groups in a catalytically favorable orientation (Fig. 4B). Similar considerations apply to the glucuronidation of ring B/C hydroxypregnanes (Fig. 4, C and D). Electrostatic interactions involving the 3- and 20-keto groups orientate 6α-OHP and 11α-OHP for glucuronidation in the normal and reverse modes, respectively. (Alternatively, 6α- and 11α-OHP may be overlaid by rotation through 180 through the z-axis.). The normal and reverse binding modes illustrated in Fig. 4, C and D, suggest that the equatorially oriented groups of 6β- and 12β-OHP might also be glucuronidated by UGT2B7, but these compounds were not available for investigation. Relationships between the two normal binding modes (1 and 2) cannot be inferred from the present data.

UGT1A enzymes exhibit greater selectivity toward C19- and C21-hydroxysteroids. As noted above, UGT 1A3, 1A4, 1A7, 1A8, 1A9, and 1A10 variably glucuronidated the compounds investigated here but, of the hepatically expressed enzymes, appreciable activity was observed only with the UGT1A4-catalyzed glucuronidation of 16α-OHP and the UGT1A9-catalyzed glucuronidation of 11α-OHP. Previous activity screening studies have reported that UGT 1A3, 1A4, 1A8, 1A9, and 1A10 have the capacity to glucuronidate hydroxylated C19- and C21-steroids (Green and Tephly 1996; Cheng et al., 1999; Kuuranne et al., 2003), although structure-function relationships with these enzymes were generally not investigated. Of interest was our observation that UGT1A10 glucuronidated most hydroxysteroids screened for activity. Together with the activities identified for UGT 1A7 and 1A8, this indicates that many hydroxysteroids may potentially be glucuronidated by enzymes present in the gastrointestinal tract.

In summary, comparative kinetic and inhibitor studies with HLMs and a panel of recombinant human UGTs demonstrated that 6α- and 21-OHP may serve as substrate probes for human hepatic UGT2B7 activity in vitro. Of these compounds, 6α-OHP has advantages of higher activity and Michaelis-Menten kinetics (compared with the atypical kinetics observed for 21-OHP glucuronidation). UGT2B7 was also identified as the enzyme responsible for the high-affinity component of human liver microsomal 11α-OHP glucuronidation, whereas UGT2B15 and UGT2B17 were the major forms involved in human liver microsomal TST 17β-glucuronidation and the high-affinity component of 16α-OHP glucuronidation. Activity of UGT1A subfamily enzymes toward the hepatically glucuronidated substrates was generally low. These data confirm the apparent preference of UGT2B subfamily enzymes in the glucuronidation of hydroxylated C19- and C21-steroids. Interestingly, however, UGT1A10, which is expressed only in the gastrointestinal tract, exhibited activity toward most of the glucuronidated substrates, suggesting possible prehepatic first-pass extraction of dietary hydroxysteroids.

Footnotes

  • 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; 6α-OHP, 6α-hydroxyprogesterone; 6β-OHP, 6β-hydroxyprogesterone; 7α-OHAD, 4-androsten-7α-ol-3, 17-dione; 6β-OHAD, 4-androsten-6β-ol-3, 17-dione; TST, testosterone; 16α-OHP, 16α-hydroxyprogesterone; 17α-OHP, 17α-hydroxyprogesterone; 21-OHP, 21-hydroxyprogesterone; 4-MU, 4-methylumbelliferone; 4-MUG, 4-methylumbelliferone-β-d-glucuronide; HLM, human liver microsome; HEK293, human embryo kidney 293 cell line; AZT, zidovudine; 11α-OHP, 11α-hydroxyprogesterone; 11β-OHP, 11β-hydroxyprogesterone.

    • Received September 20, 2006.
    • Accepted December 1, 2006.

References

  1. ↵
    Barbier O, Turgeon D, Girard C, Green MD, Tephly TR, Hum DW, and Belanger A (2000) 3′-Azido-3′-deoxythymidine (AZT) is glucuronidated by human liver UDP-glucuronosyltransferase 2B7 (UGT2B7). Drug Metab Dispos 28: 497–502.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    Beaulieu M, Levesque E, Hum DW, and Belanger A (1996) Isolation and characterization of a novel cDNA encoding a human UDP-glucuronosyltransferase active on C19 steroids. J Biol Chem 271: 22855–22862.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    Boase S and Miners JO (2002) In vitro–in vivo correlations for drugs eliminated by glucuronidation: investigations with the model substrate zidovudine. Br J Clin Pharmacol 54: 493–503.
    OpenUrlCrossRefPubMed
  4. ↵
    Bowalgaha K, Elliot DJ, Mackenzie PI, Knights KM, Swedmark S, and Miners JO (2005) Naproxen and desmethylnaproxen glucuronidation by human liver microsomes and recombinant human UDP-glucuronosyltransferases (UGT): Role of UGT2B7 in the elimination of naproxen Br J Clin Pharmacol 60: 423–433.
    OpenUrlCrossRefPubMed
  5. ↵
    Cheng Z, Radominska-Pandya A, and Tephly TM (1999) Studies on the substrate specificity of human intestinal UDP-glucuronosyltransferases 1A8 and 1A10. Drug Metab Dispos 27: 1165–1170.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Coffman BL, King CD, Rios GS, and Tephly TR (1998) The glucuronidation of opioids, other xenobiotics, and androgens by human UGT2B7Y(268) and UGT2B7H(268). Drug Metab Dispos 26: 73–77.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Court MH, Krishnaswamy S, Hao Q, Duan SX, Patten CJ, von Moltke LL, and Greenblatt DJ (2003) Evaluation of 3′-azido-3′-deoxythymidine, morphine and codeine as probe substrates for UDP-glucuronosyltransferase 2B7 (UGT2B7) in human liver microsomes: specificity and influence of the UGT2B7*2 polymorphism. Drug Metab Dispos 31: 1125–1133.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Court MH (2005) Isoform-selective probe substrates for in vitro studies of human UDP-glucuronosyltransferases. Methods Enzymol 400: 104–116.
    OpenUrlPubMed
  9. ↵
    Fotherby K and James F (1972) Metabolism of synthetic steroids, in Advances in Steroid Biochemistry and Pharmacology (Briggs MH and Christie GA eds) vol 3, pp 67–165, Academic Press, London.
    OpenUrl
  10. ↵
    Green MD, Oturu EM, and Tephly TM (1994) Stable expression of a human liver UDP-glucuronosyltransferase (UGT2B15) with activity toward steroid and xenobiotic substrates. Drug Metab Dispos 22: 799–805.
    OpenUrlAbstract
  11. ↵
    Green MD and Tephly TR (1996) Glucuronidation of amines and hydroxylated xenobiotics and endobiotics catalyzed by expressed human UGT1.4 protein. Drug Metab Dispos 24: 356–363.
    OpenUrlAbstract
  12. ↵
    Hobkirk R (1979) Hydroxylated C18 and C19 steroids: their significance and factors related to their biosynthesis, in Steroid Biochemistry (Hobkirk R ed) vol 2, pp 133–179, CRC Press, Boca Raton, FL.
    OpenUrl
  13. ↵
    Hum DW, Belanger A, Levesque E, Barbier O, Beaulieu M, Albert C, Vallee M, Guillemette C, Techernof A, Turgeon G, et al. (1999) Characterization of UDP-glucuronosyltransferases active on steroid hormones. J Steroid Chem Mol Biol 69: 413–423.
    OpenUrlCrossRef
  14. ↵
    Innocenti F, Iyer L, Ramirez J, Green MD, and Ratain MJ (2001) Epirubicin glucuronidation is catalyzed by human UDP-glucuronosyltransferase 2B7. Drug Metab Dispos 29: 686–692.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Jin C-J, Mackenzie PI, and Miners JO (1997) The regio- and stereo-selectivity of C19 and C21 hydroxysteroid glucuronidation by UGT2B7 and UGT2B11. Arch Biochem Biophys 341: 207–211.
    OpenUrlCrossRefPubMed
  16. ↵
    Jin C-J, Miners JO, Lillywhite KJ, and Mackenzie PI (1993) Complementary deoxyribonucleic acid cloning and expression of a human liver uridine diphosphate glucuronosyltransferase glucuronidating carboxylic acid containing drugs. J Pharmacol Exp Ther 264: 475–479.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Kuuranne T, Kurkela M, Thevis M, Schanzer W, Finel M, and Kostianen R (2003) Glucuronidation of anabolic androgenic steroids by recombinant human UDP-glucuronosyltransferases. Drug Metab Dispos 31: 1117–1124.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Lepine J, Bernard O, Plante M, Tetu B, Pelletier G, Labrie F, Belanger A, and Guillemette C (2004) Specificity and regioselectivity of the conjugation of estradiol, estrone, and their catecholestrogen and methoxyestrogen metabolites by human uridine diphosphoglucuronosyltransferases expressed in endometrium. J Clin Endocrinol Metab 89: 5222–5232.
    OpenUrlCrossRefPubMed
  19. ↵
    Mackenzie PI, Bock KW, Burchell B, Guillemette C, Ikushiro S, Iyanagi T, Miners JO, Owens IS, and Nebert DW (2005) Nomenclature update for the mammalian UDP glycosyltransferase (UGT) superfamily. Pharmacogenet Genomics 15: 677–685.
    OpenUrlCrossRefPubMed
  20. ↵
    Mei Q, Tang C, Assang C, Lin Y, Slaughter D, Rodrigues AD, Baillie TA, Rushmore TH, and Shou M (1999) Role of potent inhibitory monoclonal antibody to cytochrome P450 3A4 in assessment of human drug metabolism. J Pharmacol Exp Ther 291: 749–759.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Miners JO, Knights KM, Houston JB, and Mackenzie PI (2006) In vitro–in vivo correlation for drugs and other compounds eliminated by glucuronidation in humans: Pitfalls and promises. Biochem Pharmacol 71: 1531–1539.
    OpenUrlCrossRefPubMed
  22. ↵
    Miners JO, Lillywhite KJ, Matthews AP, Jones ME, and Birkett DJ (1988) Kinetic and inhibitor studies of 4-methylumbelliferone and 1-naphthol glucuronidation in human liver microsomes. Biochem Pharmacol 37: 665–671.
    OpenUrlCrossRefPubMed
  23. ↵
    Miners JO and Mackenzie PI (1991) Drug glucuronidation in humans. Pharmacol Ther 51: 347–369.
    OpenUrlCrossRefPubMed
  24. ↵
    Miners JO, McKinnon RA, and Mackenzie PI (2001) Genetic polymorphisms of UDP-glucuronosyltransferases and their functional significance. Toxicology 181: 453–456.
    OpenUrlCrossRef
  25. ↵
    Miners JO, Smith PA, Sorich MJ, McKinnon RA, and Mackenzie PI (2004) Predicting human drug glucuronidation parameters: application of in vitro and in silico modeling approaches. Annu Rev Pharmacol Toxicol 44: 1–25.
    OpenUrlCrossRefPubMed
  26. ↵
    Miners JO, Valente L, Lillywhite KJ, Mackenzie PI, Burchell B, Baguley BC, and Kestell P (1997) Preclinical prediction of factors influencing the elimination of 5,6-dimethylxanthenone-4-acetic acid, anew anticancer drug. Cancer Res 57: 284–289.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    Mojarrabi B and Mackenzie PI (1998) Characterization of two UDP glucuronosyltransferases that are predominantly expressed in human colon. Biochem Biophys Res Commun 247: 704–709.
    OpenUrlCrossRefPubMed
  28. ↵
    Musey PI, Wright K, Preedy JRK, and Collins DC (1979) Formation and metabolism of steroid conjugates: Effect of conjugation on excretion and tissue distribution, in Steroid Biochemistry (Hobkirk R ed) vol 2, pp 81–131, CRC Press, Boca Raton, FL.
    OpenUrl
  29. ↵
    Ritter JK, Sheen YY, and Owens IS (1990) Cloning and expression of human liver UDP-glucuronosyltransferase in COS-1 cells. J Biol Chem 265: 7900–7906.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Rowland A, Elliot DJ, Williams JA, Mackenzie PI, Dickinson RG, and Miners JO (2006) In vitro characterization of lamotrigine N2-glucuronidation and lamotrigine-valproic acid interaction Drug Metab Dispos 34: 1055–10626.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Setchell KD, Alme B, Axelson M, and Sjovall J (1976) The multicomponent analysis of conjugates of neutral steroids in urine by lipophilic ion exchange chromatography and computerized gas chromatography–mass spectrometry. J Steroid Biochem 20: 615–629.
    OpenUrl
  32. ↵
    Sorich MJ, Smith PA, McKinnon RA, and Miners JO (2002) Pharmacophore and quantitative structure relationship modeling of UDP-glucuronosyltransferase 1A1 (UGT1A1) substrates. Pharmacogenetics 12: 635–645.
    OpenUrlCrossRefPubMed
  33. ↵
    Stone AN, Mackenzie PI, Galetin A, Houston JB, and Miners JO (2003) Isoform selectivity and kinetics of morphine 3- and 6-glucuronidation by human UDP-glucuronosyltransferases: evidence for atypical glucuronidation kinetics by UGT2B7. Drug Metab Dispos 31: 1086–1089.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Tukey RH and Strassburg CP (2000) Human UDP-glucuronosyltransferase: metabolism, expression and disease. Annu Rev Pharmacol Toxicol 40: 581–616.
    OpenUrlCrossRefPubMed
  35. ↵
    Turgeon D, Carrier J-S, Levesque E, Hum DW, and Belanger A (2001) Relative enzymatic activity, protein stability, and tissue distribution of human steroid-metabolizing UGT2B subfamily members. Endocrinology 142: 778–787.
    OpenUrlCrossRefPubMed
  36. ↵
    Uchaipichat V, Mackenzie PI, Guo X-H, Gardner-Stephen D, Galetin A, Houston JB, and Miners JO (2004) Human UDP-glucuronosyltransferases: Isoform selectivity and kinetics of 4-methylumbelliferone and 1-naphthol glucuronidation, effects of organic solvents, and inhibition by diclofenac and probenecid. Drug Metab Dispos 32: 413–442.
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Download PDF
Article Alerts
Email Article
Citation Tools
Research ArticleArticle

The Glucuronidation of Δ4-3-Keto C19- and C21-Hydroxysteroids by Human Liver Microsomal and Recombinant UDP-glucuronosyltransferases (UGTs): 6α- and 21-Hydroxyprogesterone Are Selective Substrates for UGT2B7

K. Bowalgaha, D. J. Elliot, P. I. Mackenzie, K. M. Knights and J. O. Miners
Drug Metabolism and Disposition March 1, 2007, 35 (3) 363-370; DOI: https://doi.org/10.1124/dmd.106.013052
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Jump to section