Uridine diphospho-glucuronosyltransferases (UGTs) inactivate and facilitate the excretion of estrogens to glucuronides (-G), the most abundant circulating estrogen conjugates. The identity of the conjugated estrogens formed by all known overexpressed UGTs (n = 16) was analyzed by comparison with retention time and mass fragmentation of authentic standards by HPLC tandem mass spectrometry methods. Six UGTs, namely 1A1, 1A3, 1A8, 1A9, 1A10, and 2B7, were found to glucuronidate estradiol (E(2)) and estrone (E(1)), their hydroxyls (OH), and their methoxy derivatives (MeO). Addition of glucuronic acid was catalyzed by specific UGTs at positions 2, 3, and 4 of the estrogens, whereas only E(2) was conjugated at position 17 by UGT2B7. Kinetic parameters indicate that the conjugation of E(2) at position 3 was predominantly catalyzed by 1A1, 1A3, and 1A8 and by 1A8 for E(1). Conjugation of 2-OHE(1)/E(2) and 2- and 4-MeOE(1)/E(2) was selective at position 3, mostly catalyzed by 1A1 and 1A8. Of all UGTs, UGT2B7 demonstrated the highest catalytic activities for estrogens and at least 10- to 50-fold higher activity for the conjugation of genotoxic 4-hydroxycatecholestrogens at position 4, compared with the conjugation of E(2), E(1), and 2-hydroxycatecholestrogens. Its presence was further shown in the endometrium by RT-PCR and immunohistochemistry, localizing in the same cells expressing CYP1B1, involved locally in the formation of 4-hydroxycatecholestrogens. Data show that several UGT enzymes detected in the endometrium are involved in the glucuronidation of E(2) and its 2-OH, 4-OH, and 2-MeO metabolites that exert various biological effects in the tissue.

UDP-glucuronosyltransferase 1A1 (UGT1A1) is involved in a wide range of biological and pharmacological processes because of its critical role in the conjugation of a diverse array of endogenous and exogenous compounds. We now describe a new UGT1A1 isoform, referred to as isoform 2 (UGT1A1_i2), encoded by a 1495-bp complementary DNA isolated from human liver and generated by an alternative splicing event involving an additional exon found at the 3 end of the UGT1A locus. The N-terminal portion of the 45-kd UGT1A1_i2 protein is identical to UGT1A1 (55 kd, UGT1A1_i1); however, UGT1A1_i2 contains a unique 10-residue sequence instead of the 99 -amino acid C-terminal domain of UGT1A1_i1. RT-PCR and Western blot analyses with a specific antibody against UGT1A1 indicate that isoform 2 is differentially expressed in liver, kidney, colon, and small intestine at levels that reach or exceed, for some tissues, those of isoform 1. Western blots of different cell fractions and immunofluorescence experiments indicate that UGT1A1_i1 and UGT1A1_i2 colocalize in microsomes. Functional enzymatic data indicate that UGT1A1_i2, which lacks transferase activity when stably expressed alone in HEK293 cells, acts as a negative modulator of UGT1A1_i1, decreasing its activity by up to 78%. Coimmunoprecipitation of UGT1A1_i1 and UGT1A1_i2 suggests that this repression may occur via direct protein-protein interactions. Conclusion: Our results indicate that this newly discovered alternative splicing mechanism at the UGT1A locus amplifies the structural diversity of human UGT proteins and describes the identification of an additional posttranscriptional regulatory mechanism of the glucuronidation pathway. (HEPATOLOGY 2007;45:128-138.)

The oxidative metabolism of estrone (E 1 ) and estradiol (E 2 ) to form carcinogenic 4-hydroxy-catecholestrogens (4-OHCE) is associated with uterine and breast carcinogenesis. In this study, we conducted functional analyses of genetic variants in the UDP-glucuronosyltransferase UGT1A8, UGT1A9, and UGT2B7 enzymes primarily involved in the inactivation of 4-OHCEs. Compared with UGT2B7*2 (H 268 Y), UGT2B7*1 exhibited a 2-fold lower efficiency (intrinsic clearance) at conjugating 4-hydroxyestrone and 4-hydroxyestradiol at positions 3 and 4 caused by altered capacities (V max ) and affinities (K m ). The À79 G>A promoter variation, characterizing the UGT2B7*2g haplotype, leads to a 50% reduction of transcription (P < 0.001) in human endometrial carcinoma-1B cells. Furthermore, a >12-fold decreased intrinsic clearance of the *1 proteins was induced by selected amino acid substitutions in UGT1A8 (*3 C 277 Y) and UGT1A9 (*3 M 33 T). Frequencies of the low-activity alleles in Caucasians were 45% for UGT2B7*1, 5% for the À79A promoter variant, 1.2% for UGT1A8*3, and 2.2% for UGT1A9*3. Supporting a protective role in two organs sensitive to 4-OHCE-induced damages, the expression of UGT enzymes was shown by immunohistochemistry in normal breast and endometrial tissues and confirmed by Western blotting in a subset of samples. Altogether, findings suggest that specific polymorphisms in UGT genes may modulate the exposure to carcinogenic metabolites of E 2 and potentially lead to an altered risk of breast and endometrial cancers in women carrying the variant alleles. (Cancer Res 2006; 66(1): 125-33)

246 CI = confidence interval; COMT = catechol-O-methyltransferase; CYP = cytochrome P450; E 1 = estrone; E 1 S = estrone sulfate; E 2 = estradiol; ER = estrogen receptor; OHCE = hydroxy-catecholestrogen; OR = odds ratio; UGT = UDP-glucuronosyltransferase. Breast Cancer ResearchVol 6 No 6 Guillemette et al. IntroductionEstrogens are essential for development of the reproductive system in women, in whom they exert beneficial effects in a large number of tissues, including breast, bone, brain, and cardiovascular system. In contrast, the proliferation and genetic instability induced by estrogens in breast and uterus have been considered to increase further the likelihood that normal cells will transform into a malignant type. Over the past 30 years a large number of case-control and cohort studies have been conducted that examined circulating levels of estrogens and/or their urinary excretion to detect any differences in estrogen concentrations that may contribute to cancer. Based on their findings it has been suggested that increased estrogen exposure for lengthy periods of time may induce breast cancer [1-3].Several observations have also associated in situ production of hydroxylated estrogen metabolites, namely catecholestrogens, with the development of estrogensensitive cancers. Oxidative reactions are also important because 2-and 4-hydroxylated metabolites possess distinctive biologic properties as compared with estradiol (E 2 ), the 4-hydroxylated metabolites being particularly important to the carcinogenic effects of estrogen [4][5][6]. It is also important to emphasize that these metabolic reactions not only take place in the liver but also in estrogen target tissues such as breast, ovary and uterus, AbstractThe breast tissue is the site of major metabolic conversions of estradiol (E 2 ) mediated by specific cytochromes P450 hydroxylations and methylation by catechol-O-methyltransferase. In addition to E 2 itself, recent findings highlight the significance of 4-hydroxylated estrogen metabolites as chemical mediators and their link to breast cancer development and progression, whereas, in opposition, 2-methoxylated estrogens appear to be protective. Recent data also indicate that breast tissue possesses enzymatic machinery to inactivate and eliminate E 2 and its oxidized and methoxylated metabolites through conjugation catalyzed by UDP-glucuronosyltransferases (UGTs), which involves the covalent addition of glucuronic acid. In opposition to other metabolic pathways of estrogen, the UGT-mediated process leads to the formation of glucuronides that are devoid of biologic activity and are readily excreted from the tissue into the circulation. This review addresses the most recent findings on the identification of UGT enzymes that are responsible for the glucuronidation of E 2 and its metabolites, and evidence regarding their potential role in breast cancer.

Circulating levels of steroids were associated with increased risk of EC. Estrogens may represent novel biomarkers predictive of clinical characteristics, including evidence for an increased risk of relapse.

We concluded that the higher levels of circulating estrogens in women with endometrial cancer are likely associated with an imbalance of multiple biotransformation pathways in endometrial tumor tissues.

UDP-glucuronosyltransferase (UGT) 1A1 is involved in the inactivation of estradiol (E 2 ) and its oxidized metabolites. These metabolites have been shown to contribute to the development of endometrial cancer in animal studies. Thus UGT1A1 represents a candidate gene in endometrial carcinogenesis. In this study, we established the substrate specificity of UGT1A1 for E 2 and its 2-and 4-hydroxylated metabolites. Intrinsic clearances indicated that UGT1A1 had a preference for the glucuronidation of 2-hydroxyestradiol, a metabolite associated with antiproliferative activity. Expression analysis demonstrated that UGT1A1 is present in the nonmalignant endometrium. Subsequently, we sought to determine whether the common UGT1A1 promoter allele, UGT1A1*28 [A(TA) 7

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