Androgens and estrogens play major roles in cell differentiation, cell growth, and peptide secretion in steroid target tissues. In addition to the binding of these hormones to their receptors, formation and metabolism are important in the action of steroids. Metabolism of the potent steroid hormones includes glucuronidation, a major pathway of steroid elimination in liver and several steroid target tissues. Glucuronidation is catalyzed by UDP-glucuronosyltransferases (UGTs), which transfer the polar moiety from UDP-glucuronic acid to a wide variety of endogenous compounds, including steroid hormones. The UGT superfamily of enzymes is subdivided into two families, UGT1 and UGT2, on the basis of sequence homology. To date, six UGT2B proteins have been isolated, namely UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, and UGT2B17, all of which have been demonstrated to be active on steroid molecules, except for UGT2B10 and UGT2B11, for which no substrate was found. The relative activity of these enzymes on steroidal compounds remains unknown due to variable levels of UGT2B expression in different in vitro cell line models and various conditions of the enzymatic assays. Comparison of the glucuronidation rates of these enzymes requires a unique system for UGT2B protein expression, protein normalization, and enzymatic assays. In this study we have stably expressed UGT2B4, UGT2B7, UGT2B15, and UGT2B17 in the HK293 cell line, which is devoid of steroid UGT activity; characterized their kinetic properties relative to UGT protein expression; determined their transcript and protein stabilities; and established extensively their tissular distributions. UGT2B7 was demonstrated to glucuronidate estrogens, catechol estrogens, and androstane-3alpha,17beta-diol more efficiently than any other human UGTB isoform. UGT2B15 and UGT2B17 showed similar glucuronidation activity for androstane-3alpha,17beta-diol (30% lower than that of UGT2B7), whereas UGT2B17 demonstrated the highest activity for androsterone, testosterone, and dihydrotestosterone. UGT2B4 demonstrates reactivity toward 5alpha-reduced androgens and catechol estrogens, but at a significantly lower level than UGT2B7, 2B15, and 2B17. Cycloheximide treatment of stably transfected HK293 cells demonstrated that the UGT2B17 protein is more labile than the other enzymes; the protein levels decrease after 1 h of treatment, whereas other UGT2B proteins were stable for at least 12 h. Treatment of stable cells with actinomycin D reveals that UGT2B transcripts are stable for 12 h, except for the UGT2B4 transcript, which was decreased by 50% after the 12-h incubation period. Tissue distribution of the UGT2B enzymes demonstrated that UGT2B isoforms are expressed in the liver as well as in several extrahepatic steroid target tissues, namely, kidney, breast, lung, and prostate. This study clearly demonstrates the relative activities and the major substrates of human steroid-metabolizing UGT2B enzymes, which are expressed in a wide variety of steroid target tissues.

UDP-glucuronosyltransferase (UGT) 2B17 is one of the most important conjugating enzymes in androgen metabolism and shares more than 95% homology with UGT2B15. Although UGT2B15 has been fully characterized for its ability to conjugate drugs, environmental pollutants, and dietary components, UGT2B17 received less attention for its capacity to glucuronidate xenobiotics. In the present study, more than 55 exogenous compounds belonging to several categories of compounds were analyzed as potential substrates for UGT2B17. Glucuronidation activity was observed with several coumarins, anthraquinones, and flavonoids. The higher glucuronidation activity was measured with alizarin (125 pmol x min(-1) x mg protein(-1)), whereas UGT2B17 conjugated eugenol, scopoletin, and galangin with glucuronidation rates of 102.5, 102, and 58 pmol x min(-1) x mg protein(-1), respectively. The characterization of UGT2B17 as a xenobiotics-conjugating enzyme demonstrates that its role is not limited to androgen metabolism and that its specificity for exogenous substrates is different from other UGT2B isoforms. Taken together, these data suggest a role of UGT2B17 for the hepatic detoxification.

UDP-glucuronosyltransferase (UGT) enzymes belonging to the UGT2B subfamily catalyze the transfer of glucuronic acid to a large number of endogenous compounds, particularly steroids, to facilitate their elimination from target cells. A novel human UGT2B cDNA of 1666 bp was isolated and encodes a 529-amino acid protein named UGT2B28 type I. Glucuronidation assays demonstrated that UGT2B28 type I catalyzes the conjugation of endogenous and exogenous compounds. The tissue distribution of UGT2B28 revealed the expression of the type I transcript in the liver, breast, and LNCaP cells. Two other UGT2B cDNAs were isolated, and sequence analysis led to the identification of two truncated UGT2B28 species. UGT2B28 type II differs from type I by a deletion of 308 bp in the cofactor binding domain, whereas UGT2B28 type III lacks 351 bp in the putative substrate binding domain. All UGT2B28 isoforms are encoded by a single UGT2B28 gene which has a genomic organization similar to that of the other UGT2B genes characterized thus far. Although no substrates could be identified for the shorter isoforms, the three subtypes were shown to be located in the endoplasmic reticulum and the perinuclear membrane, demonstrating that the missing domains are not required for the subcellular localization of these UGT2B proteins. However, all the domains remain necessary for observing glucuronidation activity. The expression of UGT2B28 type I in the breast and liver suggests a role of this enzyme in the androgen and estrogen metabolism in these tissues.

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