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Endocrinology Vol. 140, No. 12 5538-5548
Copyright © 1999 by The Endocrine Society

ARTICLES

UGT2B23, a Novel Uridine Diphosphate-Glucuronosyltransferase Enzyme Expressed in Steroid Target Tissues That Conjugates Androgen and Estrogen Metabolites

Olivier Barbier, Eric Lévesque, Alain Bélanger and Dean W. Hum

Laboratory of Molecular Endocrinology (O.B., E.L., D.W.H.), Medical Research Council Group in Molecular Endocrinology (A.B.), Centre Hospitalier de L’Université Laval Research Center, Laval University, Québec, Canada, G1V 4G2

Address all correspondence and requests for reprints to: Dr. Dean W. Hum or Dr. Alain Bélanger, Laboratory of Molecular Endocrinology, Centre Hospitalier de L’Université Laval Research Center, 2705 Laurier Boulevard, Québec G1V 4G2, Canada. E-mail: dean.hum@crchul.ulaval.ca; or alain.belanger{at}crchul.ulaval.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glucuronidation is widely accepted as a mechanism involved in the catabolism and elimination of steroid hormones from the body. However, relatively little is known about the enzymes involved, their specificity for the different steroids, and their site of expression and action. To characterize the pathway of steroid glucuronidation, a novel uridine diphosphate glucuronosyltransferase (UGT) enzyme was cloned and characterized. A 1768-bp complementary DNA, encoding UGT2B23 was isolated from a monkey liver library. Stable expression of UGT2B23 in human HK293 cells and Western blot analysis demonstrated the presence of a 51-kDa protein. The UGT2B23 transferase activity was tested with 62 potential endogenous substrates and was demonstrated to be active on 6 steroids and the bile acid, hyodeoxycholic acid. Kinetic analysis yielded apparent Michaelis constant (Km) values of 0.9, 13.5, 1.6, and 5.7 µM for the conjugation of androsterone (ADT), 3{alpha}-Diol, estriol, and 4-hydroxyestrone, respectively. RT-PCR analysis revealed that UGT2B23 transcript is expressed in several tissues, including the prostate, mammary gland, epididymis, testis, and ovary. Primary structure analysis shows that UGT2B23 is in the same family of enzymes as the previously characterized monkey isoforms UGT2B9 and UGT2B18, which are active on hydroxyandrogens. The characterization of UGT2B23 as a functional enzyme active on 3{alpha}-hydroxysteroids, and its expression in extrahepatic tissues, indicate that it may potentially play an important role in estrogen and androgen catabolism in peripheral steroid target tissues.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE LEVEL OF steroid hormones, which serve as specific ligands of nuclear receptors in steroid target tissues, is regulated by enzymes involved in steroid biosynthesis and catabolism (1, 2). The enzymes involved in steroid synthesis have been studied extensively; however, proteins required for catabolism are relatively less characterized. Uridine diphosphate (UDP)-glucuronosyltransferase (UGT) enzymes (EC 2.4.1.17) catalyze the transfer of the sugar group, from UDP-glucuronic acid (UDPGA) to small hydrophobic molecules (aglycons), which include hydroxylated steroid hormones (3, 4). Glucuronidated steroids are more polar and are generally eliminated from the body through the bile or urine.

In lower mammals, the testis is the exclusive source of androgens; whereas in humans, the adrenals secrete large amounts of the androgen precursors dehydroepiandrosterone (DHEA) and DHEA-sulfate. These precursor steroids are converted into potent androgens and estrogens in peripheral tissues (such as the prostate, breast, and skin) by steroidogenic enzymes, which include 3ß-hydroxysteroid dehydrogenase, 17ß-hydroxysteroid dehydrogenase, and 5{alpha}-reductase (5, 6). Moreover, it is now clear that extrahepatic tissues in humans also express steroid-conjugating UGT enzymes (1, 2, 7, 8). Based on the expression of UGT2B transcripts in most (if not all) steroid target tissues (9), and the significant concentration of glucuronidated steroids found in these tissues and in the circulation of humans, it has been proposed that UGT enzymes can glucuronidate steroids and contribute to modulate the steroid response in extrahepatic steroid target tissues (7, 9).

In humans, significant levels of steroid metabolites, in the form of glucuronide conjugates, are detected in circulation. It has been suggested that the plasma levels of 5{alpha}-reduced C19 steroid glucuronides, androsterone-glucuronide (ADT-G), and 5{alpha}-androstane-3{alpha},17ß-Diol-G (3{alpha}-Diol-G) reflect the peripheral tissue conversion of adrenal and gonadal precursor C19 steroids to active androgens (1). A recent study suggests that the serum levels of androgens or estrogens during aging are a poor indicator of total androgenic and estrogenic activities in men and women (10); whereas, the level of circulating conjugated androgen metabolites was shown to be correlated with the total androgen pool in men (10). In addition, the plasma levels of steroid glucuronides are increased in some hyperandrogenic pathologies, such as acne or hirsutism, which are related to the increased production of 5{alpha}-reduced C19 steroids (11). In women with hirsutism, the increased plasma concentration of 5{alpha}-reduced C19 steroid glucuronides may be a reflection of increased C19 steroid metabolism in peripheral tissues.

Based on the homology of primary structures, the mammalian UGT proteins have been categorized into two major families, UGT1 and UGT2; with the UGT2 family further divided into two groups, UGT2A and UGT2B (3). Enzymes of the UGT2B subfamily catalyze the glucuronidation of several endogenous compounds, including bile acids, steroids, fatty acids, and carboxylic acids (3). The characterizations of human UGT2B enzymes demonstrate an overlap of substrate specificities between the different proteins. However, each enzyme is active on specific classes of steroids, which are glucuronidated at their hydroxyl groups.

As found in humans, the monkey also has high plasma levels of androgen-glucuronides (12), which indicates that simians represent a relevant animal model for studying steroid glucuronidation. The isolation and characterization of simian UGT2B enzymes are important steps required to gain an understanding of the role of glucuronidation in steroid metabolism. In the present study, we report the isolation and characterization of a novel UGT enzyme, UGT2B23, which is expressed in several steroid target tissues, and which glucuronidates androgen (ADT, 3{alpha}-Diol) and estrogen metabolites (estriol, 4-hydroxyestrone).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
UDPGA and all aglycons were obtained from Sigma (St. Louis, MO) and ICN Pharmaceuticals, Inc. (Québec, Canada). Radiolabeled steroids (3H-ADT and 3H-3{alpha}-Diol) were purchased from NEN Life Science Products (Boston, MA). Radioinert steroids were purchased from Steraloids Inc. (Wilton, NH). [14C]UDPGA (285 mCi/mmol) was obtained from NEN Life Science Products, and {alpha}-[32P]-dCTP (3000 Ci/mmol) was from Amersham Pharmacia Biotech (Ontario, Canada). Geneticin (G418) and Lipofectin were obtained from Life Technologies (Ontario, Canada). Protein assay reagents were obtained from Bio-Rad Laboratories, Inc. (Richmond, CA). Restriction enzymes and other molecular biology reagents were from Pharmacia LKB Biotechnology Inc. (Milwaukee, WI), Life Technologies, Stratagene (La Jolla, CA) and Roche Molecular Biochemicals (Indianapolis, IN). AmpliTaq DNA polymerase was from Perkin-Elmer Cetus (Branchburg, NJ) and Roche Molecular Biochemicals. Human embryonic kidney 293 cells (HK293) were obtained from the American Type Culture Collection (Rockville, MD).

Monkey RNA isolation
Total RNA was isolated from monkey mammary gland, epididymis, prostate, kidney, testis, adrenal, bile duct, small intestine, brain, cerebellum, lung, colon, spleen, liver, ovary, seminal vesicle, thyroid, vagina, HK293 cells, and HK293 cells stably expressing UGT2B23, according to the Tri reagent acid phenol protocol as specified by the supplier (Molecular Research Center, Inc., Cincinnati, OH). The messenger RNAs (mRNAs) obtained from a monkey liver tissue was obtained by affinity chromatography through oligo (dT)-cellulose (Pharmacia LKB Biotechnology Inc.).

Complementary DNA (cDNA) isolation
Affinity-purified liver mRNA was used to construct a cDNA library in the {lambda} ZAP express vector, as specified by the manufacturer’s instructions (Stratagene). The library was not amplified for screening as previously described (8). The UGT2B23 cDNA clone was excised from the pBK-CMV vector using an helper phage (Stratagene). UGT2B23 cDNA clone was isolated and was sequenced in both directions, using specific UGT oligonucleotides (8).

Transcription/translation, in vitro, of the UGT2B23 cDNA
The entire UGT2B23 cDNA in the pBK-CMV vector was transcribed and translated using T3 RNA polymerase in the transcription/translation-coupled rabbit reticulocyte lysate system from Promega Corp. (Madison, WI) in the presence of [35S]-methionine. The protein product was separated on a 12% SDS/polyacrylamide gel and exposed on Hyperfilm-MP for 1 h.

Stable expression of UGT2B23
HK293 cells were grown in DMEM containing 4.5 g/l glucose, 10 mM HEPES, 110 µg/ml sodium pyruvate, 100 IU penicillin/ml, 100 µg/ml streptomycin, and 10% FBS in an humidified incubator, with an atmosphere of 5% CO2, at 37 C. Five micrograms of pBK-CMV-UGT2B23 was used to transfect HK293 cells using Lipofectin. A stable transfectant was selected in media containing 1 mg/ml G418, as previously reported (13).

Microsomal proteins isolation
Next, 8 x 106 transfected HK293 cells were homogenized in 5 ml homogenization buffer (14) and centrifuged at 12,000 x g, at 4 C, for 20 min. The supernatant was centrifuged at 105,000 x g for 1 h at 4 C. The microsome pellets was resuspended in 0.5 ml of homogenization buffer.

Endoglycosidase H digestion
Microsomal proteins from monkey liver (5 µg), ovary (25 µg), HK293 cells (25 µg), and HK293 cells stably expressing UGT2B23 (25 µg) were incubated with 20 mU endoglycosidase H, in the presence of 50 mM sodium acetate (pH 5.5) and 0.1% SDS, in a final vol of 20 µl for 16 h at 37 C. Proteins were separated on a 12% SDS-polyacrylamide gel, transferred onto nitrocellulose membrane, and probed with the EL-93 anti-UGT2B17 antisera (1:1500 dilution), as reported (15). An antirabbit IgG horse antibody conjugated with peroxidase (Amersham Pharmacia Biotech was used as the second antibody, and the resulting immunocomplexes were visualized using a chemiluminescence kit (ECL) (Renaissance, Québec, Canada) and exposed on hyperfilm for 1 min (Eastman Kodak Co., Rochester, NY).

Glucuronidation assay and Michaelis constant (Km) determination using cell homogenates and microsomal proteins
HK293 cells expressing exogenous UGT2B23 were suspended in Tris buffered saline containing 0.5 mM dithiothreitol and were homogenized using a Brinkmann Instruments, Inc. (Westbury, NY) polytron. For a first assessment of activity, enzyme assays were performed using 7.5 µM [14C]UDPGA, 92,5 µM unlabeled UDPGA, 200 µM of the various aglycons, and 150 µg protein from cell homogenates in 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 100 µg/ml phosphatidylcholine, and 8.5 mM saccharolactone in a final vol of 100 µl. Assays were performed for 16 h at 37 C and were terminated by adding 100 µl methanol. Chromatography analysis and formation of glucuronide were determined as previously described (8).

Compounds that demonstrated reactivity with UGT2B23 in the screening assay were subsequently reassayed with HK293-UGT2B23 microsomal proteins in 50 mM Tris-Hcl (pH 8), for 4 h at 37 C, in the presence of 7.5 µM [14C]UDPGA, 492.5 µM unlabeled UDPGA. Under these conditions, the enzyme reaction is linear for 6 h. The values represent the means of two independent experiments performed in triplicates. Kinetics analysis was realized, using microsomal proteins in the same conditions, except that final concentrations of estriol, 4-hydroxyestrone, and etiocholanolone were 1, 2, 4, 6, 8, 10, and 15 µM.

Km determination in intact HK293 cells
Km determinations were performed by incubating intact HK293 cells with radiolabeled steroid substrates, as previously described (13). Cells were incubated for 6 h at 37 C with 0.25, 0.5, 0.75, 1, 2.5, and 5 µM radioinert ADT or 3{alpha}-Diol and 20 nM of corresponding radiolabeled substrate. The medium was removed and analyzed for glucuronide conjugates by organic extraction and scintillation counting, as previously described (16). The data obtained were normalized by DNA content quantitated by fluorometric assay with 3,5-diaminobenzoic acid (17).

RT-PCR analysis
The tissue distribution of UGT2B23 was achieved using a RT-PCR technique, as previously reported (18). Five micrograms of total RNA from cynomolgus monkey tissues HK293 cells and HK293 cells stably expressing UGT2B23 were used. Reverse transcriptase reactions were performed using 500 pmol of the UGT2B23-specific antisense primer 5'-GAAAAGAAATCCTCCACAATGCTTTTCAAAAACA3' and 2 pmol of the control antisense GAPDH primer, 5'-CCCAGCGTCAAAGGTGG-3', in the presence of 200 U SuperScript II reverse transcriptase, according to the manufacturer’s instructions (Life Technologies). The PCR reaction was carried out with one fourth of the complementary RNA product and 100 pmol of the specific sense primer 5'-CCTGAGTTTGAGAATATAGTCACGCAAGAGA3' using ampli Taq DNA polymerase (Perkin-Elmer Cetus). The PCR was performed for 30 cycles (1 min at 94 C, 1 min at 67 C, 1 min at 72 C), after which one fifth of the PCR product was electrophoresed on an ethidium bromide-stained 1% agarose gel. The sense and antisense primers start at positions 250 and 567, respectively, in relation to the adenine of the initiator codon, which is designated nucleotide 1 (Fig. 1Go). All PCR reactions were controlled using the sense primer for GAPDH, 5'-TGGGTGTGAACCATGAG-3'. The identity of all PCR products was verified by direct sequencing (19).



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  		Figure 1. Nucleotide and amino acid sequence of UGT2B23 and alignment with UGT2B9 and UGT2B18. Nucleotide sequence: the initial and stop codons are in bold, and the arrows indicate the position of the two specific primers used for RT-PCR analysis. Amino acid sequence: the putative membrane insertion signal is denoted by the overline, and the sequence of the putative membrane-anchoring domain is underlined. Potential asparagine-linked [NX(S/T)] glycosylation sites are indicated by the boxes, and the dashed box indicates the consensus signature sequence found in UGT enzymes.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation of the UGT2B23 cDNA
To isolate simian UGT2B cDNA clones, a library was constructed with liver RNA isolated from the cynomolgus monkey and was screened with a combination of probes synthesized from the full-length human cDNAs of UGT2B7, UGT2B10, and UGT2B15 (14). Twenty positive cDNAs were isolated, and nucleotide sequence analysis revealed five novel clones (UGT2B9, UGT2B18, UGT2B19, UGT2B20, and UGT2B23). The cDNA encoding UGT2B23 is 1768 bp in length and contains an open reading frame of 1587 bp, a 5' untranslated region of 27 bp, and a 3' untranslated region of 151 bp (Fig. 1Go). Two putative polyadenylation signals are present at nucleotides 1724 and 1735, with a poly(A+) tail starting at position 1750. The nucleotides proximal to the first AUG codon is consistent with it being the initiator methionine, according to the findings of Kozak (20). The protein primary structure of 529 amino acids, which is deduced from the nucleotide sequence, reveals the presence of several conserved sequences found in most of the UGT2B enzymes (Fig. 1Go) (3). The UGT2B23 protein contains a characteristic hydrophobic signal peptide from residue 1–23, a hydrophobic transmembrane region between amino acids 494 and 510, and 2 potential asparagine glycosylation sites [NX(S/T)] present at positions 68 and 69 (Fig. 1Go).

Amino acid sequence alignments show that UGT2B23 is 94% identical to the previously characterized monkey UGT2B9 and UGT2B18 isoenzymes (Fig. 2Go). Of the 32 amino acid differences found between UGT2B23 and UGT2B18, 10 are found in the carboxylterminal domain between residues 291 and 530, which was proposed to bind the cofactor UDPGA (Fig. 1Go). Comparisons with UGT2B9 reveal 13 different residues in the carboxylterminal domain, whereas 18 variant residues are found in the aminoterminal half of the proteins. UGT2B23 is 82, 87, 77, and 76% identical to the human UGT2B4, UGT2B7, UGT2B15, and UGT2B17 isoenzymes, respectively (Fig. 2Go).



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  		Figure 2. Dendrogram of the UGT2B enzymes isolated from rat, rabbit (rab), human (hum), and monkey (mon), and homology between the deduced amino acid sequence of UGT2B23 and other UGT2B isoenzymes. The protein sequences were obtained from the GenBank database. The sequence identity of the amino-terminal domain from residues 1–290, the carboxy-terminal domain from residues 291–530, and the entire protein is as indicated. The steroid specificity of each enzyme is indicated. ADT, androsterone; E3, estriol; Etio, etiocholanolone; 4-OH-E1, 4-hydroxyestrone; 3{alpha}-Diol, 5{alpha}-androstane-3{alpha},17ß-diol; Testo, testosterone; 4-OH-E2, 4-hydroxyestradiol.

 
Stable expression and characterization of the UGT2B23 protein
The predicted coding region of the UGT2B23 cDNA was confirmed by in vitro transcription and translation, which produced a protein of 51 kDa (Fig. 3AGo). To characterize the UGT2B23 protein, the cDNA was stably transfected into HK293 cells, and the derived cell line was demonstrated to express a 51-kDa protein, by Western blot analysis of microsomal proteins (Fig. 3BGo). Microsomes from monkey liver and ovary also contain proteins of similar apparent molecular mass, which are recognized by the anti-UGT2B17 antibody. In contrast, untranfected control HK293 cells did not express immunoreactive proteins. To determine whether the UGT2B23 protein was glycosylated, microsomal protein from HK293 cells stably expressing UGT2B23 was treated with endoglycosidase H. Immunoreactive protein from the liver and ovary displayed faster migration after endoglycosidase H treatment, indicative of glycosylated UGT2B enzyme. However, the migration of UGT2B23 remained the same, which suggests that this protein is not glycosylated (Fig. 3BGo). When separated on the same gel by SDS-PAGE, the proteins UGT2B9, UGT2B18, and UGT2B23 all migrated at the same rate but faster than the UGT2B20 protein (data not shown), which was previously found to be glycosylated (13). Considering that all four proteins are comprised of 528–530 residues, this result is consistent with the possibility that UGT2B9 and UGT2B18, which have two potential overlapping glycosylation sites identical to UGT2B23, are not glycosylated; whereas UGT2B20, which has four different potential glycosylation sites, is glycosylated.



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  		Figure 3. In vitro transcription/translation of UGT2B23 (A), and immunoblot analysis of HK293 cells stably expressing UGT2B23 (B). A, The in vitro transcription/translation product of the UGT2B23 cDNA was separated by 12% SDS-PAGE. B, Microsomal proteins isolated from monkey liver (5 µg), ovary (25 µg), HK293 cells (25 µg), and transfected HK293 cells expressing UGT2B23 (25 µg) were digested by endoglycosidase H. Proteins were separated on a 12% SDS-polyacrylamide gel. The proteins were transferred, and the membrane was probed with the anti-UGT2B17 polyclonal antibody, demonstrating the presence of immunoreactive UGT2B protein. The top arrow on the right indicates the immunoreactive protein(s) seen in liver and ovary extracts. The lower arrow indicates the faster migrating immunoreactive protein(s) seen in liver and ovary microsome after endoglycosidase H treatment. The migration of UGT2B23 is not altered after endoglycosidase H treatment and comigrates with the deglycosylated proteins seen in liver and ovary microsomes. Immunoreactive protein is not seen in control microsome preparation from HK293 cells.

 
To determine the substrate specificity of UGT2B23, HK293 cell homogenates containing stably expressed UGT2B23 were incubated with potential substrates, and conjugation was assessed by TLC (Fig. 4Go). Of the 62 different endogenous compounds tested, only 7 were conjugated by UGT2B23 (Table 1Go). Glucuronidation of these compounds was not detected using control HK293 cell homogenates. The main substrates of UGT2B23 are 3{alpha}-hydroxyandrogens (etiocholanolone and 3{alpha}-Diol), 4-hydroxyestrone, estriol, and the bile acid HDCA (hyodeoxycholic acid); however, UGT2B23 does not glucuronidate neuromodulators and C21 steroids. As found with most other UGT2B enzymes, UGT2B23 is active on planar phenols, including 4-propyl-phenol, 1-naphthol, and eugenol (data not shown).



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  		Figure 4. Glucuronidation of C18 and C19 steroids by UGT2B23, analyzed by TLC (A). The glucuronidation of etiocholanolone, 3{alpha}-Diol, ADT (A, B), 4-hydroxyestrone, and estriol (A, C) was assessed by incubation of UGT2B23 cell homogenates with 7.5 µM [14C ]UDPGA and 492.5 µM unlabeled UDPGA. Testosterone, DHT, estrone, estradiol, and 2-hydroxyestrone (A, D) were not glucuronidated. Testo, Testosterone; Etio, etiocholanolone: 3{alpha}-Diol, 5{alpha}-androstane-3{alpha},17ß-diol; 3ß-Diol, 5{alpha}-androstane-3ß,17ß-diol; 4-OH-Estrone, 4-hydroxyestrone.

 

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  		Table 1. Reactivity of endogenous compounds with UGT2B23 expressed in HK293 cells

 
Kinetic analysis for UGT2B23 conjugation of 3{alpha}-Diol, and ADT, was performed using intact HK293 cells stably expressing UGT2B23 (Fig. 5BGo). The apparent Km value for conjugation of 3{alpha}-Diol (13.5 µM) is more than 10-fold higher than for ADT (0.9 µM), as determined by double reciprocal plots (Lineweaver-Burk). The apparent maximal velocity (Vmax) values obtained for these two substrates were similar, thus the Vmax/Km ratio demonstrates a 10-fold higher efficiency for the glucuronidation of ADT. Similar experiments performed using microsomal proteins demonstrate Km values of 3.5, 1.6, and 5.7 µM for etiocholanolone, estriol, and 4-hydroxyestrone, respectively (Fig. 5AGo). The Vmax/Km ratio is similar for estriol and etiocholanolone, indicating the same efficiency of glucuronidation, whereas 4-hydroxyestrone presents a 5-fold lower efficiency of conjugation by UGT2B23.



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  		Figure 5. Lineweaver-Burk plots for the conjugation of estriol, 4-hydroxyestrone, and etiocholanolone (A); androsterone and 3{alpha}-Diol (B). Experiments were performed using microsome preparations from HK293 cells stably expressing UGT2B23 (A) and intact cells (B), as described in Materials and Methods. Values represent the mean ± SD of two experiments, each performed in triplicate. ADT, Androsterone; Etio, etiocholanolone; 3{alpha}-Diol, 5{alpha}-androstane-3{alpha},17ß-diol.

 
Tissue distribution of UGT2B23 transcript
To ascertain the tissue distribution of UGT2B23 transcript, RT-PCR analysis was performed with total RNA isolated from monkey tissues. RT was performed using a specific UGT2B23 primer, and the oligonucleotides used for PCR were specific for UGT2B23 and designed to not amplify the previously characterized monkey transcripts UGT2B9, UGT2B18, UGT2B19, and UGT2B20. UGT2B23 transcript is expressed in the mammary gland, epididymis, prostate, testis, bile duct, small intestine, brain, cerebellum, colon, liver, ovary, and vagina. However, the kidney, adrenal, lung, spleen, seminal vesicle, and thyroid do not express detectable levels of this mRNA (Fig. 6Go). Identity of the 318-bp UGT2B23 RT-PCR product was confirmed by direct sequencing. The integrity of each RNA sample was verified by amplification of the GAPDH transcript, using specific oligonucleotides.



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  		Figure 6. Tissue distribution of UGT2B23 transcript. Total RNA isolated from cynomolgus monkey tissues, HK293 cells, and HK293 cells stably expressing UGT2B23 were analyzed by specific RT-PCR analysis. One fifth of each RT-PCR product was separated on a 1% agarose gel. The 318-bp PCR product represents amplification of the UGT2B23 transcript, as confirmed by direct sequencing.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In a recent study, which measured the circulating levels of 5{alpha}-reduced C19 steroid glucuronides among nine mammalian species, it was found that humans and simians are unique in having significant levels of ADT-G and 3{alpha}-Diol-G (12). Moreover, several of the extrahepatic tissues in these two species were shown to express steroid-conjugating UGT2B transcripts (14). In addition, the cloning and characterization of other steroidogenic enzymes (such as 5{alpha}-reductase from the rhesus monkey) demonstrated high identity in primary structure and very similar biochemical properties as the human protein (21). Thus, taken together, these data suggest that the monkey is an appropriate animal model in which to study steroidogenesis and the role of steroid glucuronidation in extrahepatic steroid target tissues. Similar to several of the human UGT2B enzymes, the ability of UGT2B23 to conjugate androgens and estrogens is consistent with its potential role in steroid metabolism.

Of the 13 different androgens tested as potential substrates in this study, glucuronidation by UGT2B23 was detected only on C19 steroids, which are hydroxylated at the 3{alpha}-position (etiocholanolone, ADT, and 3{alpha}-Diol) (Table 1Go and Fig. 4Go). Interestingly, the glucuronidation of 3ß-hydroxylated androgens was not detected, which suggests a differential specificity of UGT2B23 for different stereoisomers of the C19 steroid molecule. UGT2B23 conjugates 5{alpha}- and 5ß-reduced androgens and is in contrast to the monkey UGT2B19 enzyme (18), which is more active on 5ß-reduced compounds (Table 2Go).


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  		Table 2. Steroid substrate specificity of monkey UGT2B9 ([14], UGT2B18 [22], UGT2B19 [18], UGT12B20 [13], and UGT2B23

 
The primary structure of UGT2B23 is highly homologous to the other simian UGT2B proteins (Fig. 2Go), including UGT2B9 (94% identity) and UGT2B18 (94% identity). In contrast to UGT2B23, which is specific for 3{alpha}-hydroxylated steroids, UGT2B9 is capable of glucuronidating steroids at both the 3{alpha}-hydroxy and 17ß-hydroxy positions [ADT, testosterone, and dihydrotestosterone (DHT)] (14), whereas UGT2B18 is specific for 3{alpha}-hydroxyandrogens and does not conjugate C18 steroids (22) (Table 2Go). These differences in substrate specificity are most likely conferred by differences in primary structure at the aminoterminal halves of the proteins, which have been proposed to contain the aglycon binding site. More specifically, the region between residues 90 to 229 contains variant amino acids at the identical position of the UGT2B9, UGT2B18, and UGT2B23 proteins (Fig. 1Go).

In humans, UGT2B4 (23, 24), UGT2B7 (23), UGT2B15 (25), and UGT2B17 (8) are steroid-conjugating enzymes. However, despite the high homology between UGT2B proteins from the two species, it is interesting that there exist differences in their steroid specificity. For example, UGT2B7, which is the human protein most homologous to UGT2B23, is active on 3{alpha}-hydroxyandrogens, estrogen metabolites (estriol, 2-hydroxyestrone, 4-hydroxyestrone), and C21 steroids (23). It is clear that the steroid specificity of UGT2B enzymes between two species, or even within the same species, cannot be predicted based on sequence homology. In addition, the rat UGT2B2 enzyme has the same androgen specificity as the monkey UGT2B23, in that they can both conjugate ADT (3{alpha}-OH) and not DHT nor testosterone (17ß-OH) (26). Thus, based on steroid substrate specificity, it is possible that a human orthologue of UGT2B23 remains to be isolated and characterized.

Androgens have important physiological functions, including the regulation of sex organ development in the embryonic stage and acquisition of male secondary sexual characteristics at puberty. In adult life, androgens are involved in maintaining homeostasis; however, in certain pathologies (such as prostate cancer) C19 steroids can have deleterious effects and lead to increased cancer cell proliferation. In peripheral steroid target tissues [such as the breast, skin and prostate, which express UGT2B transcripts (8, 25)], it is clear that the level of steroids is regulated by anabolic and catabolic enzymes. In these tissues, it has been proposed that glucuronidation of steroids abolishes their specific interaction with nuclear receptor and leads to their excretion from the tissue (1). As determined by RT-PCR, UGT2B23 transcript is expressed in steroid target tissues, which is consistent with this enzyme being involved in steroid catabolism at these sites. UGT2B23 transcript was not detected in the seminal vesicle, which is a known androgen-responsive tissue. However, considering that the family of UGT2B enzymes has overlapping (but distinct) patterns of substrate specificity, it is possible that other androgen-conjugating UGT2B proteins are expressed in this tissue. UGT2B23 can glucuronidate ADT with a 10-fold higher efficiency (ratio Vmax/Km) than 3{alpha}-Diol, which correlates with the 10:1 ratio of ADT-G/3{alpha}-Diol-G found in the circulation (12). This 10:1 ratio is also observed in human plasma, but the human steroid-conjugating UGT2B enzymes characterized to date do not exhibit a 10-fold higher efficiency of conjugation for ADT over 3{alpha}-Diol.

Similar to androgens, estrogens exert many important functions via interaction with their receptors (27). The catabolism of estrogens include oxidative metabolism and conjugative metabolism, by glucuronidation, sulfonation, and O-methylation, to decrease the effect of the parent hormone (28). The activity of UGT2B23 on estriol, with an apparent Km value of 1.6 µM, and its expression in extrahepatic estrogen-sensitive tissues (such as the breast, ovary, and prostate) is consistent with this enzyme playing a role in steroid metabolism. Conjugation of estrogens by UGT2B23 in these tissues may be required to promote steroid elimination to maintain homeostasis or to increase elimination required after increased estrogen synthesis in response to physiological conditions.

The high activity of UGT2B23 on 4-hydroxyestrone, with an apparent Km value in the micromolar range, suggests a physiological role of this enzyme on the catecholestrogen. Although 2-hydroxylation of estradiol and estrone is the dominant pathway for catecholestrogen formation in liver microsomes, recent data indicate that formation of 4-hydroxylated catecholestrogens is a dominant pathway in several extrahepatic steroid target tissue, such as the breast and uterus (29, 30). Glucuronidation of the catecholestrogen 4-hydroxyestrone is potentially an important catabolic pathway required to eliminate these genotoxic steroid metabolites from a given tissue and to prevent cell damage (31, 32, 33). Catecholestrogens can undergo metabolic redox cycling catalyzed by P450 enzymes. The hydroperoxide-dependent oxidation of catecholestrogens to quinones, and the NADPH-dependent reduction of the quinones back to hydroquinones, yield semiquinone free radical intermediates and superoxide radicals (34). The continuous generation of the free radicals by the redox cycle have been postulated to mediate DNA damage (such as single-strand breaks, 8-hydroxylation of guanine bases, and depurination of adenine-guanine adducts) leading to tumor development (31, 34, 35). This potential problem of catecholestrogens is particularly relevant in estrogen sensitive-tissues (such as the breast, ovary, and uterus) that express steroidogenic enzymes (including aromatase required for estrogen synthesis) and the enzymes (such as cytochrome P4501B1) that yield catecholestrogens (30, 36, 37, 38). It is interesting that several steroid-specific UGT enzymes (including UGT2B23) are also expressed in these tissues; thus, it will be important to determine the role that each of these proteins may have in metabolizing estrogens and, subsequently, influencing their effects.

In summary, the present study demonstrates that the simian UGT2B23 enzyme is capable of conjugating specific 3{alpha}-hydroxylated C18 and C19 steroids. The glucuronidation of steroids by UGT2B23 with apparent Km values in the low micromolar range and the expression of its transcript in the breast, ovary, testis, and prostate suggest that this enzyme plays a role in steroid metabolism in extrahepatic steroid target tissues. The high homology between the simian and human proteins, and the ability of these enzymes to conjugate steroids, correlates well with the finding that these two species have similar mechanisms of steroid glucuronidation and are thus far unique in containing significant amounts of glucuronidated androgen metabolites in the plasma. Characterization of the simian UGT2B enzymes establishes the monkey as a relevant animal model for further study, to understand the role and significance of steroid glucuronidation in steroid target tissues.


   Acknowledgments
 
We gratefully thank Dr. Pei Min Rong and Lina Berthiaume for their excellent technical assistance in DNA sequencing and Western blot analysis. GenBank accession number for UGT2B23: AF112113.

Received April 8, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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