UDP-glucuronosyltransferase 2B Research Articles

Naftopidil enantiomers suppress androgen accumulation and induce cell apoptosis via the UDP-glucuronosyltransferase 2B15 in benign prostate hyperplasia

Accumulation of androgens mediate alterations in prostate growth and has emerged as an essential factor in benign prostate hyperplasia (BPH). Dihydrotestosterone (DHT), the most potent natural androgen, binds to androgen receptors (AR) and regulates the prostate growth. Many inhibitors of DHT synthesis have been developed to reduce DHT levels and used in the treatment of prostate diseases. However, therapies targeting the elimination of the DHT remain limited. The DHT in prostate is metabolized by UDP-glucuronosyltransferase 2B (UGT2B) and transforms into inactive products. In this study, we analyzed and demonstrated that two enantiomers of naftopidil (NAF), an α1D/1A-adrenoceptor blocker, induced expression and activity of UGT2B in BPH rat prostate models as well as UGT2B15 in human prostate cells, BPH-1. The NAF enantiomers reduced intraprostatic and intracellular DHT levels, thus promoting cell apoptosis. Besides, assays with siRNA UGT2B15 transfection showed that UGT2B15 played an essential role in mediating the effects of the NAF enantiomers. The UGT2B15 mediated the inhibition of AR and PSA expression by NAF enantiomers. The data showed that the mechanism of upregulating UGT2B15 by the NAF enantiomers might differ from that of AR antagonists and 5α-reductase inhibitors. Together, our results demonstrated that NAF enantiomers could be potential and novel UGT2B15 regulators, which accelerated the DHT elimination and promoted apoptosis of BPH-1 cells. This study could help expand the clinical application of NAF and support the development of new therapeutic strategies targeting the elimination of androgens for the treatment of BPH and other androgen-sensitive diseases.

Species differences in phenobarbital-mediated UGT gene induction in rat and human liver microtissues.

Species differences in hepatic metabolism of thyroxine (T4) by uridine diphosphate glucuronosyl transferase (UGT) and susceptibility to thyroid hormone imbalance could underlie differences in thyroid carcinogenesis caused by hepatic enzyme inducers in rats and humans. To investigate this hypothesis we examined profiles of hepatic UGT induction by the prototypical CAR activator phenobarbital (PB) in rat and human liver 3D microtissues. The rationale for this approach was that 3D microtissues would generate data more relevant to humans. Rat and human liver 3D microtissues were exposed to PB over a range of concentrations (500 u M - 2000 u M) and times (24-96 hr). Microarray and proteomics analyses were performed on parallel samples to generate integrated differentially expressed gene (DEG) datasets. Bioinformatics analysis of DEG data, including CAR response element (CRE) sequence analysis of UGT promoters, was used to assess species differences in UGT induction relative to CAR-mediated transactivation potential. A higher proportion of human UGT promoters were found to contain consensus CREs compared to the rat homologs. UGTs 1a6, 2b17 and 2b37 were upregulated by PB in rat liver 3D microtissues, but unaltered in human liver 3D microtissues. By contrast, human UGTs 1A8, 1A10 and 2B10 showed higher levels of induction (RNA and /or protein) compared to the rat homologs. There was general concordance between the presence of CREs and the induction of UGT RNA. As UGT1A and 2B isoforms metabolise T4, these results suggest that differences in UGT induction could contribute to differential susceptibility to CAR-mediated thyroid carcinogenesis in rats and humans.

Perspective on the regulatory role of UGT2B28 as a conjugating enzyme in the progression of prostate cancer

The maintenance of steroid homeostasis in the prostate is critical, with perturbation of steroidogenesis contributing to the modulation of active ligands in the androgen pool. In this scenario, enzymes catalysing the biosynthesis, inactivation and conjugation of steroids are the key players, regulating active ligand levels and in so doing, the activation of the androgen receptor (AR). The glucuronidation of potent ligands renders them unable to bind the AR, allowing the secretion of conjugated steroids. Uridine diphosphate glucuronosyltransferase 2B type 28 (UGT2B28), one of the UGT enzymes catalyzing the glucuronidation of androgens, has recently been given a prominent role in the regulation of prostate steroidogenesis—one which stands in contrast to the accepted dogma that lower androgen levels resulting from increased conjugation are associated with decreased prostate cancer (PCa) risk and disease progression. Increased DHT and its precursors, T and androstenediol, were reported to be associated with increased UGT2B28 tumor expression levels, linked to lower PSA levels but higher Gleason scores and increased PCa risk. In addition, the complete deletion of UGT2B28, was associated with decreased T, DHT and glucuronide derivatives when compared to patients carrying both alleles. UGT2B28 is encoded by a single gene giving rise to UGT2B28 type I which catalyses androgen glucuronidation and, due to alternative splicing, also produces two distinct transcripts, UGT2B28 type II and III. Type II with its premature stop codon, is devoid of the cofactor binding domain while type III is devoid of the substrate binding domain, both catalytically inactive, truncated proteins. Increased UGT2B28 mRNA expression was reported in primary tumours, and while variable nuclear and strong cytoplasmic staining were distinctive of tumour cells, the expression levels and compartmentalization of the specific protein isoforms remain unknown. While increased expression of type I would contribute towards lowering androgen levels, increased expression of types II and III would not. The abundance of type III transcripts in multiple tissues may provide insight into a regulatory role with truncated isoforms possibly affecting androgen levels by regulating substrate and/or co-factor availability, dimerization or the formation of protein complexes with other UGTs, while protein-protein interaction may also impact cascade signaling pathways in PCa development and disease progression.

UGT concentrations in human rectal tissue after multidose,oral curcumin.

In vitro studies have demonstrated that curcumin is a substrate for uridine diphosphate glucuronosyltransferase (UGTs), with a putative ability to both induce expression and inhibit function, highlighting the potential for interaction with some drugs. Therefore, we sought to evaluate the effect of oral curcumin on intestinal UGT expression. Healthy volunteers, ages 40–80 years, who had received recent screening colonoscopy were recruited. Participants did not have any gastrointestinal or bleeding disorders, lab abnormalities, or recent antibiotic use. All participants received daily curcuminoid extract, 4 g, for 30 days. Untreated, rectal mucosal pinch biopsies were obtained at baseline and at 30 days. Microsomes were prepared from biopsy samples, using sequential centrifugation. Quantification of 14 UGT 2As and 2Bs was performed by LC‐MS/MS(MS, mass spectrometry), using quantitative‐ targeted absolute proteomics. Lowest LODs were ~0.1 pmol/mg protein. Comparisons were performed using Wilcoxon signed‐rank test. Paired baseline and 30 days biopsy samples were available for 38 participants. UGTs 1A10 and 2B17 were detected in 35 and 33 paired samples, respectively, while all other UGTs were below the limit of quantification (BLOQ). Median baseline UGT1A10 concentration was 0.60 pmol/mg (95% CI:0.32–0.92), and 0.60 pmol/mg (95% CI:0.43–1.00) after 30 days (P = 0.23). For UGT2B17, median baseline concentration was 0.83 pmol/mg (95% CI:0.32–1.62), and 1.18 pmol/mg (95% CI:0.39–1.77) after 30 days (P = 0.24). We found no differences in rectal mucosal UGT concentrations before and after 30 days of oral curcumin administration, indicating that daily curcumin use is unlikely to alter colonic UGT expression. Distal gut biopsies may not accurately reflect the proximal gut environment where UGT expression and curcumin concentrations may be higher.

Abstract A01: ADRB2 regulates phase II steroid metabolism and determines development of castration-resistant prostate cancer

Abstract The underlying mechanisms responsible for the development of castration-resistant prostate cancer (CRPC) are not fully understood. The β2-adrenergic receptor (ADRB2) is a key regulator of a wide range of metabolic processes in the body, and it has been implicated in androgen receptor signaling and development of CRPC. We have unpublished data which shows that low expression of ADRB2 predicts a more rapid development of CRPC. Based on this finding, we wanted to investigate whether the ADRB2 level/activity impacts cellular features to help explain how and why the receptor has prognostic value in prostate cancer. We stably transfected androgen-dependent LNCaP cells with shRNAs to mimic the clinical situation where patients have differential levels of ADRB2 in their prostate epithelial carcinomas. Gene expression profiling revealed changes in expression of several metabolic genes. Among the most regulated were two androgen-glucuronidating UDP-glucuronosyltransferase 2B (UGT2B) enzymes, UGT2B15 and UGT2B17. Both enzymes are critical in the phase-II metabolic pathway responsible for elimination of androgens by glucuronidation in the prostate, and they were highly down-regulated at the mRNA level in two LNCaP cell sublines expressing low levels of ADRB2 (shADRB2). By mixing androgen substrates with protein lysates from the cell and measuring glucuronide formation by LC-MS/MS, we found that glucuronide formation mirrored the UGT2B15/2B17 expression levels. To further complement these findings, we measured androgen levels in the cells by LC-MS, and found higher levels of bioactive testosterone. As this theoretically should invoke a change in androgen receptor activity of the cells, we measured androgen regulated luciferase activities as well as prostate-specific antigen (PSA/KLK3)-expression and secretion upon stimulation with the glucuronidable androgen dihydrotestosterone. The experiments revealed a noticeable increase in androgen responsiveness in cells with low levels of ADRB2 compared to cells with normal levels of ADRB2. Upon supplementation with the synthetic, non-glucuronidable androgen R1881, no induction in androgen responsiveness was observed in the shADRB2 cells compared to control cells. Dihydrotestosterone responsiveness was inhibited upon supplementation of diclofenac sodium, an inhibitor of UDP-glucuronosyltransferase actvity, and also upon rescue of ADRB2 expression level. Finally, using immunohistochemistry with an anti-UGT2B17 antibody, we found that patients showing strong cytoplasmic UGT2B17 immunostaining intensity progressed more rapidly to CRPC. Altering metabolic pathways, such as the steroid catabolic pathways, may be a powerful adaptive tool for cells to increase survival in an androgen-deprived micromilieu, and thus also a potential drug target. As LNCaP cells with low levels of ADRB2 display lowered glucuronidation activity, higher androgen responsiveness, and higher testosterone levels, we propose that this may be a novel metabolic mechanism by which ADRB2 affects the survival of androgen-dependent cells during androgen-deprivation therapy, and thereby development of CRPC. Citation Format: Peder Rustøen Braadland, Helene Hartvedt Grytli, Håkon Ramberg, Betina Katz, Lois Gauthier-Landry, Ralf Kellmann, Kurt Allen Krobert, Wanzhong Wang, Aud Svindland, Finn Olav Levy, Viktor Berge, Gunnar Mellgren, Olivier Barbier, Kristin Austlid Taskén. ADRB2 regulates phase II steroid metabolism and determines development of castration-resistant prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A01.

The UGT2B28 Sex-steroid Inactivation Pathway Is a Regulator of Steroidogenesis and Modifies the Risk of Prostate Cancer Progression

BackgroundAndrogen inactivation occurs mainly through the glucuronidation conjugative reaction mediated by UDP-glucuronosyltransferases (UGTs). This metabolic process is involved in the control of systemic and local androgen bioavailability. ObjectiveTo examine the relationship among expression of the androgen-inactivating UGT2B28 enzyme, circulating steroid hormone levels, and clinical phenotype in prostate cancer (PCa). Design, setting, and participantsWe conducted an analysis of a high-density prostate tumor tissue microarray consisting of 239 localized PCa cases. The study of 51 additional PCa patients with no copies of UDP glucuronosyltransferase 2B subfamily, polypeptide B28 (UGT2B28) in their genomes was performed to confirm the importance of the enzyme on circulating hormone levels. Outcome measurements and statistical analysisSteroid hormones were measured by mass spectrometry. Multivariate Cox proportional hazard models assessed the influence of UGT2B28 on progression, and general linear model regression evaluated variations in hormone levels. Results and limitationsTumor overexpression of UGT2B28 was associated with lower prostate-specific antigen levels at diagnosis, higher Gleason scores, margin and nodal invasion status, and it was shown to be an independent prognostic factor associated with progression. Enzyme overexpression correlated with 30% higher circulating levels of testosterone (T) and dihydrotestosterone (DHT). Patients with no copies of UGT2B28 in their genomes have lower levels of T (19%), DHT (17%), its glucuronide metabolites (18–38%), and enhanced levels of the adrenal precursor androstenedione (36%). ConclusionsThe UGT2B28 steroid-inactivating pathway modifies circulating T and DHT levels, and UGT2B28 overexpression is associated with high-grade PCa. Our work has uncovered the role of UGT2B28 as a regulator of steroidogenesis and underscores the interconnectivity among the steroid-inactivation capacity of cancer cells, hormone levels, disease characteristics, and the risk of cancer progression. Patient summaryThe androgen-inactivating UGT2B28 enzyme influences hormone levels, clinical and pathologic factors, and the risk of cancer progression.

Abstract P3-06-43: A genetic deletion in the uridine diphosphate glucuronosyltransferase 2B17 affects the pharmacokinetics of exemestane

Abstract Background Exemestane (EXE) is an aromatase inactivator used in the prevention and treatment of breast cancer (BC). The majority of EXE and its active metabolite 17-dihydroEXE are excreted as glucuronide conjugates by uridine diphosphate glucuronosyltransferase (UGT). The UGT2B17 enzyme is the most expressed in human liver. A deletion spanning the entire UGT2B17 gene (*2) decreased the EXE glucuronidation process by 14-fold in human liver microsomes (HLM) from UGT2B17(*2/*2) genotype subjects compared to wild-type UGT2B17(*1/*1) HLMs. Aim The aim of this study was to investigate whether the UGT2B17 deletion is associated with increased serum levels of EXE and 17-dihydroEXE and to assess if this deletion predicts the anti-proliferative effect of EXE in BC tissue as measured by Ki67 changes 6 weeks apart. Methods In a phase II pre-surgical trial, 50 postmenopausal women with histologically-confirmed ER positive BC; stage T1-2, N0-1, M0 were assigned to EXE 25 mg/d for 6 weeks before surgery. Morning fasting blood was collected at baseline (B) and after 6 weeks and stored at -80° C until assayed. Time of last drug intake was collected at blood draw. DNA was extracted from whole blood (Qiagen, Italy). We used Taqman copy number variation assay (Life Technologies, Monza, Italy) for the UGT2B17 genotyping. EXE and 17-dihydroEXE concentrations were determined by mass spectrometry (MS) using Waters® Xevo™ TQ MS in electrospray positive ionization mode (Waters, Manchester, UK), optimized by multiple reaction monitoring mode. We used Zorbax Eclipse Plus C18 columns and mobile phase MeOH/H2O with 0.1% formic acid. EXE pure substance was provided by Pfizer Inc.,17β-hydroxy EXE and EXE-19-d3 purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). Wilcoxon Signed Rank Test was used to assess differences in serum concentrations of EXE, its metabolites, and post-pretreatment tissue Ki67 changes according to UGT2B17 genotypes. Results Median age and BMI were 62 years and 26 kg/m2, respectively. The UGT2B17 genotype (n=50) was 24 homozygote wt (*1/*1), 20 heterozygote (*1/*2) and 6 homozygote variant (*2/*2). Minor allele frequency = 0.32. Hardy-Weinberg Equilibrium (P=0.57) was respected. Median and interquartile ranges (IQRs) of EXE and 17-dihydroEXE at 6 weeks, by time elapsed since last drug intakeTime since last drugEXE (nM)17-dihydroEXE (nM)≤24 hours (n=32)10.25 (5.55, 17.25)2.75 (1.85, 3.6)>24 hours (n=15)2.10 (1.20, 2.20)*1.50 (1.20, 2.00)*** P=.0001 vs ≤24h; **P=.0003 vs ≤24h ; Median and IQRs of EXE, 17-dihydroEXE and Ki67 change from B by UGT2B17 genotypesGenotypeEXE (nM)17-dihydroEXE (nM)Ki67 changeBMIUGT2B17 *1/*1 (n=22)5.3 (2.2, 11.4)1.85 (1.4, 3.06)-9 (-16, -5)28 (24, 32)UGT2B17 *1/*2, *2/*2 (n=25)9.3 (2.6, 17.6)*2.5 (2.0, 3.6)**-11 (-19, -3)♦26 (23, 30)♦♦P-values between genotype groups:*P=.28; **P=.04; ♦P=.82; ♦♦P=.41 Conclusions Our study demonstrates a significant association of the UGT2B17 deletion with increased serum concentrations of 17-dihydroEXE, irrespective of time since last drug intake. Neither 17-dihydroEXE, nor genotype explained the significant anti-proliferative effect of EXE on BC tissue. Larger studies should determine the clinical implications of this gene on EXE efficacy. Citation Format: Harriet Johansson, Valentina Aristarco, Jennifer Gjerde, Sara Gandini, Aliana Guerrieri-Gonzaga, Matteo Lazzeroni, Serena Mora, Debora Macis, Davide Serrano, Antonio Toesca, Luca Bottiglieri, Gunnar Mellgren, Giuseppe Viale, Andrea DeCensi, Bernardo Bonanni. A genetic deletion in the uridine diphosphate glucuronosyltransferase 2B17 affects the pharmacokinetics of exemestane [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-43.

Tissue distributions of 4-(hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone glucuronide, a stable form of reactive intermediate produced from 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, in the rat

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), induces lung tumors in rodents and has been suggested as a causative factor in human lung cancer. NNK is activated by α-hydroxylation at either the methyl or methylene carbon adjacent to the N-nitroso group to yield unstable intermediates that spontaneously decompose to produce alkylating agents. 4-(Hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (HO-methyl NNK) glucuronide, a glucuronide of the reactive intermediate of NNK has been identified. However, there are no available data concerning HO-methyl NNK glucuronide. In the present study, we investigated the tissue distribution of HO-methyl NNK glucuronide in control and phenobarbital (PB)-treated rats after intraperitoneal administration of NNK. In PB-treated rats, HO-methyl NNK glucuronide was detected in plasma, kidney, liver, lung, and pancreas. On the contrary, in the control rats, HO-methyl NNK glucuronide was detected only in plasma, kidney and liver at low concentrations compared with PB-treated rats. The results of cumulative urinary excretion of HO-methyl NNK glucuronide in Wistar and Gunn rats suggested that PB-inducible UDP-glucuronosyltransferase 2B isoforms mainly contribute to the formation of HO-methyl NNK glucuronide.

Novel associations of UDP-glucuronosyltransferase 2B gene variants with prostate cancer risk in a multiethnic study

BackgroundWe have previously shown that a functional polymorphism of the UGT2B15 gene (rs1902023) was associated with increased risk of prostate cancer (PC). Novel functional polymorphisms of the UGT2B17 and UGT2B15 genes have been recently characterized by in vitro assays but have not been evaluated in epidemiologic studies.MethodsFifteen functional SNPs of the UGT2B17 and UGT2B15 genes, including cis-acting UGT2B gene SNPs, were genotyped in African American and Caucasian men (233 PC cases and 342 controls). Regression models were used to analyze the association between SNPs and PC risk.ResultsAfter adjusting for race, age and BMI, we found that six UGT2B15 SNPs (rs4148269, rs3100, rs9994887, rs13112099, rs7686914 and rs7696472) were associated with an increased risk of PC in log-additive models (p < 0.05). A SNP cis-acting on UGT2B17 and UGT2B15 expression (rs17147338) was also associated with increased risk of prostate cancer (OR = 1.65, 95% CI = 1.00-2.70); while a stronger association among men with high Gleason sum was observed for SNPs rs4148269 and rs3100.ConclusionsAlthough small sample size limits inference, we report novel associations between UGT2B15 and UGT2B17 variants and PC risk. These associations with PC risk in men with high Gleason sum, more frequently found in African American men, support the relevance of genetic differences in the androgen metabolism pathway, which could explain, in part, the high incidence of PC among African American men. Larger studies are required.

Association of Uridine Diphosphate-Glucuronosyltransferase 2B Gene Variants with Serum Glucuronide Levels and Prostate Cancer Risk

Uridine diphosphate-glucuronosyltransferase 2B (UGT2B) enzymes conjugate testosterone metabolites to enable their excretion in humans. The functional significance of the UGT2B genetic variants has never been described in humans. We evaluated UGT2B variants in relation to plasma androstane-3α,17β-diol-glucuronide (AAG) levels and the prostate cancer risk. AAG levels were measured in sera from 150 controls and compared to the polymorphisms of UGT2B17, UGT2B15, and UGT2B7. Genomic DNA from controls (301) and cases (148) was genotyped for the polymorphisms, and odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated using unconditional logistic regression analyses. Having two copies of UGT2B17 was associated with higher AAG levels in controls among Whites (p=0.02), but not Blacks (p=0.82). Logistic regression models adjusting for age and race revealed that homozygosity for the G allele of the UGT2B15(D85Y) polymorphism was directly associated with the prostate cancer risk (OR=2.70, 95% CI=1.28, 5.55). While the small sample size limits inference, our findings suggest that an association between the UGT2B17 copy number variant (CNV) and serum AAG levels in Whites, but unexpectedly not in Blacks. This novel observation suggests that genetic determinants of AAG levels in Blacks are unrelated to the UGT2B17 CNV. This study replicates the results that show an association of UGT215(D85Y) with an increased prostate cancer risk.

Proteome profiling of tolbutamide‐treated rat primary hepatocytes using nano LC‐MS/MS and label‐free protein quantitation

Tolbutamide is used as a first line oral antihyperglycemic drug for type 2 diabetes. One side effect of this drug, hepatotoxicity, is well recognized; however, the precise mechanisms underlying tolbutamide-induced hepatotoxicity remain unclear. In this respect, proteomics techniques were used to gain further insight into the mechanistic processes of the hepatotoxicity induced by this drug. In this study, we aimed to identify molecular pathways based on proteins responding to cellular toxicity in tolbutamide-treated primary hepatocytes, using nano UPLC-MS/MS analysis. Rat primary hepatocytes were treated with an IC(20) concentration for 24 h to study the hepatotoxic effects of tolbutamide. For high-throughput label-free quantitation, tryptic-digested peptides of proteins from cell lysates were analyzed using LC-MS/MS and quantitated using the IDEAL-Q software, in which several parameters, such as assisted sequence, elution time, and mass-to-charge ratio were included. We quantified a total of 330 distinct proteins from the tolbutamide-treated hepatocytes and identified 55 upregulated and 82 downregulated proteins with expression changes. Among these differentially expressed proteins, we focused mainly on the 18 upregulated proteins belonging to xenobiotic cytochrome P450 (CYP), drug metabolism/detoxification, oxidative stress/antioxidant response, and cell damage pathway. CYP2D1, CYP2C11, UDP-glucuronosyltransferase 2B (UGT2B), superoxide dismutase 2 (SOD2), 60 kDa heat shock protein (HSPD1), heat shock protein 90 (HSP90), and catalase (CAT) were confirmed by Western blot analysis. In addition, various xenobiotic CYP proteins upregulated in the tolbutamide-treated group, CYP2D1, CYP2C13, and CYP2C11 were confirmed by reverse transcriptase-PCR analysis. Our results offer important new insights into the molecular mechanisms of tolbutamide-induced hepatotoxicity.

Abstract P3-12-07: Polymorphisms in the UDP-Glucuronosyltransferase 2B Gene Family and Risk of Breast Cancer in Women of African Descent

Abstract Background: The UDP-glucuronosyltransferase 2B (UGT2B) enzymes catalyze glucuronidation, which is an important clearance pathway of steroid hormones, and medications, and potential carcinogens. As steroid hormones play a central role in breast cancer development, genetic polymorphisms in the UGT2B gene family may affect breast cancer susceptibility. Methods: We evaluate two missense mutations in UGT2B15 (rs1902023, D85Y and rs4148269, K523T), one missense mutation in UGT2B7 (rs7439366, H268Y), and one single nucleotide polymorphism (SNP) 497 bp upstream to UGT2B4 (rs13129471) using pooled case-control studies of breast cancer, which included 1,287 cases and 851 controls. Cases and controls were enrolled in Nigeria, Barbados, and the US, and all are women of African ancestry. Results: All four SNPs followed Hardy-Weinberg equilibrium in each ethnicity group. We found the rs13129471 SNP was significantly associated with breast cancer risk after adjusting for ethnicity (P=0.003). Compared with homozygous genotype for the A-rs13129471 allele, the odds ratio for heterozygous genotype and homozygous genotype for the G-rs13129471 allele was 1.02 (95% CI: 0.81-1.28) and 1.50 (1.15-1.95), respectively. The association was stronger in Nigerian women (P=4x10- 5) and the corresponding odds ratio was 1.13 (95% CI: 0.85-1.49) and 2.39 (1.61-3.55). No significant association was found for the other three non-synonymous SNPs. Conclusion: Our results suggest that polymorphism in the UGT2B4 gene may be associated with breast cancer risk in women of African ancestry and the association may follow a recessive model. The finding needs to be confirmed in other populations. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-12-07.

Potentially harmful advantage to athletes: a putative connection between UGT2B17 gene deletion polymorphism and renal disorders with prolonged use of anabolic androgenic steroids

Background and objectiveWith prolonged use of anabolic androgenic steroids (AAS), occasional incidents of renal disorders have been observed. Independently, it has also been established that there are considerable inter-individual and inter-ethnic differences, in particular with reference to the uridine diphosphate-glucuronosyltransferase 2B17 (UGT2B17) gene, in metabolising these compounds. This report postulates the association of deletion polymorphism in the UGT2B17 gene with the occurrence of renal disorders on chronic exposure to AAS.Presentation of the hypothesisThe major deactivation and elimination pathway of AASs is through glucuronide conjugation, chiefly catalyzed by the UGT2B17 enzyme, followed by excretion in urine. Excretion of steroids is affected in individuals with a deletion mutation in the UGT2B17 gene. We hypothesize that UGT2B17 deficient individuals are more vulnerable to developing renal disorders with prolonged use of AAS owing to increases in body mass index and possible direct toxic effects of steroids on the kidneys. Elevated serum levels of biologically active steroids due to inadequate elimination can lead to prolonged muscle build up. An increase in body mass index may cause renal injuries due to sustained elevated glomerular pressure and flow rate.Testing the hypothesisIn the absence of controlled clinical trials in humans, observational studies can be carried out. Real time PCR with allelic discrimination should be employed to examine the prevalence of different UGT2B17 genotypes in patients with impaired renal function and AAS abuse. In individuals with the UGT2B17 deletion polymorphism, blood tests, biofluid analyses, urinalysis, and hair analyses following the administration of an anabolic steroid can be used to determine the fate of the substance once in the body.Implications of the hypothesisIf the hypothesis is upheld, anabolic steroid users with a deletion mutation in the UGT2B17 gene may be exposed to an increased risk of developing renal disorders. In the current detecting - sanctioning anti-doping system, athletes motivated by the potential to evade detection owing to their unique genetic make-up could subject themselves to a serious health consequence. More research on AAS metabolism in the presence of UGT2B17 gene deletion is required. Benefit - harm evaluations in therapeutic use of anabolic steroids should also consider this potential link between UGT2B17 gene deletion polymorphism and renal disorders.

Molecular Cloning of the Baboon UDP-Glucuronosyltransferase 2B Gene Family and Their Activity in Conjugating Morphine

Glucuronidation by UDP-glucuronyltransferase 2B enzymes (UGT2Bs) is a major pathway for the elimination of endobiotics and xenobiotics, including therapeutic drugs. Morphine, a probe drug for UGT2B7, is metabolized to morphine-3-beta-glucuronide (M3G) and morphine-6-beta-glucuronide (M6G) in humans. Morphine has been used in a series of experiments in the baboon to characterize developmental changes in fetal glucuronidation. This study identifies the baboon UGT2B family of enzymes, compares them with that of the human and the monkey (Macaca fascicularis), and measures the activity of the individual baboon UGT2Bs toward morphine. UGT2B cDNAs were cloned from the liver of adult and newborn baboons and expressed in human embryonic kidney 293 cells. The UGT activity toward morphine was assessed by the rate of formation of M3G and M6G by high-performance liquid chromatography. Eight baboon UGT2Bs were cloned and identified: UGT2B41 and UGT2B42, which are 90% homologous to human UGT2B4; UGT2B43, which is 93% homologous to human UGT2B15; and UGT2B39, UGT2B40, UGT2B44, UGT2B45, and UGT2B46, which are 89 to 91% homologous to human UGT2B7. Homology between baboon and monkey UGT2B ranged from 92.6 to 99.1%, with the primary protein structure of UGT2B43 being 99.1% identical to monkey UGT2B20, including a unique R96I substitution. Gene conversion interfered with the phylogenetic signal in the baboon UGT2B7-like and the monkey UGT2B4-like groups and led to concerted evolution of these enzymes. All of the baboon UGT2Bs metabolized morphine to both M3G and M6G. This study lays the foundation for investigating the regulation of UGT2B enzymes during fetal and neonatal development in the baboon.

Copy-number variations (CNVs) of the human sex steroid metabolizing genesUGT2B17andUGT2B28and their associations with aUGT2B15functional polymorphism

UGT2B17 and UGT2B28 are among the most commonly deleted genes in humans and encode members of the uridine diphosphate (UDP)-glucuronosyltransferase 2B (UGT2B) subfamily. They are involved, along with UGT2B15, in the catabolism of sex-steroid hormones. Despite the recent biomedical interest in UGT2B17 and UGT2B28 copy-number variations (CNVs) within human populations, the impact of their gene dosage has been hampered by the lack of precise molecular identification of the common deletion breakpoints within high homology sequence regions on chromosome 4. We have characterized these common deletions and report their coexistence in Caucasians, along with the p.D85Y (rs1902023:G>T) functional polymorphism of UGT2B15. Segmental duplications of 4.9 kb for UGT2B17 and 6.8 kb for UGT2B28 comprise purine-rich recombination sites located 117 kb and 108 kb apart on both ends of the deletions. CNVs of UGT2B17 and UGT2B28 occur in Caucasians at 27% and 13.5%, respectively. While only 43% have two copies of both genes, 57% harbor at least one deletion. Their co-occurrence on 5% of chromosomes creates a 225-kb genomic gap. CNVs of both UGT2B17 and UGT2B28, with the co-occurrence of UGT2B15:p.D85Y, generate seven distinct haplotypes. Restricting the analyses to CNV of the UGT2B17 gene without evaluating UGT2B28 CNV, along with the genotype of UGT2B15, may over- or underestimate the impact of each gene under physiological conditions or disease states.

Association Between Polymorphisms in HSD3B1 and UGT2B17 and Prostate Cancer Risk

Androgens, especially dihydrotestosterone, have been postulated to modify the risk of prostate cancer. 3-Beta-hydroxysteroid dehydrogenase1 (HSD3B1) and uridine diphosphate-glucuronosyltransferase 2B17 (UGT2B17) are enzymes that inactivate dihydrotestosterone in the prostate and may affect dihydrotestosterone concentration in prostatic tissue. The purpose of this study was to determine whether polymorphisms in HSD3B1 and UGT2B17 increase the risk of prostate cancer. In a case-control study of 356 patients with incident primary prostate cancer and 363 age-matched controls, the frequencies of HSD3B1 N367T and UGT2B17 null polymorphisms in genomic DNA were compared between the patients and controls. No evidence was found for a main effect of the HSD3B1 codon 367 polymorphism on prostate cancer risk. However, among white men with family history of prostate cancer, the HSD3B1 367Thr allele was positively associated with prostate cancer (odds ratio 3.0, 95% confidence interval 1.0 to 9.2). A significant association was observed between the UGT2B17 null polymorphism and prostate cancer risk (odds ratio 1.7, 95% confidence interval 1.03 to 2.9). An association with the UGT2B17 null polymorphism was further elevated (odds ratio 2.7, 95% confidence interval 1.1 to 6.5) among individuals with HSD3B1 Asn/Asn genotype. These results suggest that the HSD3B1 N367T and UGT2B17 null polymorphisms may modify the risk of prostate cancer, particularly among men with a family history of the disease.

Pharmacogenomics of Deferiprone Metabolism.

Individuals with transfusion-dependent β-thalassemia can be affected by iron accumulation in the heart and liver, resulting in liver fibrosis, cardiomyopathy and eventual death. As a result, iron chelation treatment is a necessary concurrent therapy for many patients. Deferiprone (1,2-dimethyl-3-hydroxypyrid-4-one) is currently the only orally active iron chelator approved outside the U.S. for the treatment of iron overload. Despite the efficacy of deferiprone in reducing serum ferritin levels and total body iron burden, not all patients respond to the drug, and serious adverse events can require discontinuation of therapy. The variability in drug response, combined with a high rate of side effects, indicate that there may be genetic factors that influence how the drug is metabolized. We have identified the uridine diphosphate glucuronosyltransferase (UGT) enzyme that metabolizes deferiprone. Utilizing human liver microsomes and insect cell supersomes, we have screened UGT 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17 enzymes for deferiprone glucuronidation. Deferiprone glucuronide was detected solely with UGT1A6. Previous reports of linkage disequilibrium between the UGT1A1*28 and UGT1A6*2 alleles (Kohle et. al., 2003; Peters et. al., 2003), combined with the association of the UGT1A1*28 allele with an increased risk of hyperbilirubinemia and gallstone formation in the hemoglobinopathies (Haverfield et. al., 2005), substantiate the importance of elucidating the pharmacogenomics of deferiprone metabolism. From this preliminary data, it will be possible to examine UGT1A6 genetic variation that may contribute to differences in drug efficacy and toxicity, and further delineate any additional enzymes involved in deferiprone metabolism.

Characterization of a common deletion polymorphism of the UGT2B17 gene linked to UGT2B15

Members of the human UDP-glucuronosyltransferase 2B family are located in a cluster on chromosome 4q13 and code for enzymes whose gene products are responsible for the normal catabolism of steroid hormones. Two members of this family, UGT2B15 and UGT2B17, share over 95% sequence identity. However, UGT2B17 exhibits broader substrate specificity due to a single amino acid difference. Using gene-specific primers to explore the genomic organization of these two genes, it was determined that UGT2B17 is absent in some human DNA samples. The gene-specific primers demonstrated the presence or absence of a 150 kb genomic interval spanning the entire UGT2B17 gene, revealing that UGT2B17 is present in the human genome as a deletion polymorphism linked to UGT2B15. Furthermore, it is shown that the UGT2B17 deletion polymorphism shows Mendelian segregation and allele frequencies that differ between African Americans and Caucasians.

Uridine diphosphate sugar-selective conjugation of an aldose reductase inhibitor (AS-3201) by UDP-glucuronosyltransferase 2B subfamily in human liver microsomes

N-Glucosidation is known as a major metabolic reaction for barbiturates in humans. However, the enzyme(s) involved in this N-glucosidation has not been clarified yet. Thus, to clarify the enzyme(s) involved in the N-glucosidation in human liver microsomes, we investigated the N-glucosyltransferase activity in recombinant UDP-glucuronosyltransferases (UGTs) using AS-3201, an aldose reductase inhibitor, as a substrate. AS-3201 was found to be biotransformed to both N-glucoside and N-glucuronide in human liver microsomes. The N-glucosyltransferase activities were detectable with multiple UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT2B4, UGT2B7, and UGT2B15). In contrast, the N-glucuronyltransferase activities for the same substrate were seen with UGT1A (UGT1A1, UGT1A3, UGT1A4, and UGT1A9) but not UGT2B isoforms. We then determined the relative activity factor of each recombinant UGT and estimated the contribution of each UGT isoform to the N-glucosidation in human liver microsomes. The results showed that UGT2B isoforms mainly contribute to AS-3201 N-glucosidation in human liver microsomes. In addition, the activity of AS-3201 N-glucosyltransferase significantly correlated with that of amobarbital N-glucosyltransferase in microsomes from sixteen human livers ( r=0.964, P<0.01), indicating that UGT2B isoforms were also involved in the barbiturate N-glucosidation in humans. The findings of this study clearly show that UGT2B specifically utilizes UDP-glucose but not UDP-glucuronic acid as a sugar donor for the conjugation of AS-3201 in human liver microsomes.

CDNA Cloning and Expression of Two New Members of the Human Liver UDP-Glucuronosyltransferase 2B Subfamily

Two new UDP-glucuronosyltransferase cDNAs, designated UGT2B10 and UGT2B11, encoding 528 amino acid proteins were isolated from a human liver cDNA library. The deduced amino acid sequences of UGTs 2B10 and 2B11 share > 76% sequence similarity with other known human liver UGT2B subfamily isoforms and < 48% sequence similarity with UGT1 family proteins. COS-7 cells transfected with UGT 2B10 and 2B11 synthesized proteins with respective molecular masses of 49kDa and 51 kDa. UGT2B11 expressed in COS-7 cells glucuronidated a number of polyhydroxylated estrogens (estriol, 4-hydroxyestrone and 2-hydroxyestriol) and xenobiotics (4-methylumbelliferone, 1-naphthol, 4-nitrophenol, 2-aminophenol, 4-hydroxybiphenyl and menthol). Despite the screening of more than forty potential substrates, glucuronidation activity was not observed for expressed UGT2B10.