UGT2B7 Research Articles

Long-term stability study and evaluation of intact steroid conjugate ratios after the administration of endogenous steroids.

The most frequently detected substances prohibited by the World Anti-Doping Agency (WADA) belong to the anabolic steroids class. The most challenging compounds among this class are the endogenous anabolic steroids, which are detected by quantitative measurement of testosterone (T) and its metabolites with a so-called "steroid profiling" method. The current steroid profile is based on the concentrations and ratios of the sum of free and glucuronidated steroids. Recently, our group developed a steroid profiling method for the detection of three free steroids and 14 intact steroid conjugates, including both the glucuronic acid conjugated and sulfated fraction. The study aimed at evaluating the long-term stability of steroid conjugate concentrations and ratios, and the influence of different endogenous steroids on this extended steroid profile. A single dose of oral T undecanoate (TU), topical T gel, topical dihydrotestosterone (DHT) gel, and oral dehydroepiandrosterone (DHEA) was administered to six healthy male volunteers. One additional volunteer with a homozygote deletion of the UGT2B17 gene (del/del genotype) received a single topical dose of T gel. An intramuscular dose of TU was administered to another volunteer. To avoid fluctuation of steroid concentrations caused by variations in urinary flow rates, steroid ratios were calculated and evaluated as possible biomarkers for the detection of endogenous steroid abuse with low doses. Overall, sulfates do not have substantial additional value in prolonging detection times for the investigated endogenous steroids and administration doses. The already monitored glucuronides were overall the best markers and were sufficient to detect the administered steroids.

Novel mechanisms of testosterone detoxification in UGT2B17 gene deletion carriers and androgen activation by gut microbiome

Background and objectives Testosterone metabolism by the highly polymorphic UDP-glucuronosyltransferase 2B17 (UGT2B17) is widely recognized, but the metabolic fate of testosterone in the carriers of UGT2B17 gene deletion is not well understood. Similarly, testosterone glucuronide (TG) is prone to secretion into intestinal lumen (1); however, the role of microbiome in activation of testosterone is not studied. Therefore, the aims of this study were: i) to elucidate the alteration in the liver proteome and its effect on steroid metabolism pathways (i.e., metabolic switching) in the UGT2B17 gene deletion, and ii) to assess the TG deconjugation by gut β-glucuronidases. Methods Human liver S9 fraction with gene deletion and high expression (n=3) were used. Untargeted proteomics data was acquired using Easy nano-LC 1200 coupled with the high-resolution QE-HF Orbitrap. The proteomic data were analyzed, processed and pathway mapping was performed using MaxQuant, Perseus and STRING, respectively. In vitro studies in liver S9 fraction of UGT2B17 gene deletion were carried out to investigate testosterone metabolism by aldo-keto reductases (AKRs). The glucuronidation pathway of 5β-dihydrotestosterone (5β-DHT) and 5β-tetrahydrotestosterone (5β-THT) were identified using recombinant UGT2B isoforms. These data were verified using testosterone incubations in human hepatocyte and serum levels of 5β-THTG after oral testosterone dosing (800 mg) in human. Activation mechanism TG to testosterone in the human gut was investigated by incubating TG with recombinant human gut microbial β-glucuronidases and human fecal extracts. Results A total of 1002 liver proteins were quantified in the S9 fraction, of which 14 and 39 proteins were exclusively from the deletion and high-expressor groups, respectively, whereas 949 proteins were common. The following pathways were upregulated in UGT2B17 deletion group: oxidation-reduction, drug metabolic, steroid metabolic, and fatty acid metabolic process. Notable proteins that were upregulated in the gene deletion groups were: AKR1D1, AKR1C4, AKR7DA3, AKR1A1, DHCR7, ADH6, ADH1C, ALDH1A1, ALDH9A1, and ALDH5A1 A deeper molecular pathway analysis revealed that the AKR1D1 and AKR1C4 that metabolize testosterone to inactive 5β-DHT and 5β-THT, respectively (2) (Figure 1) were significantly upregulated in UGT2B17 deletion group. UGT2B7 is primarily responsible for glucuronidation of 5β-THT (Figure 1). Finally, incubation with purified bacterial β-glucuronidases and human fecal extracts confirmed facile but variable deconjugation of TG to testosterone in human gut. Conclusion Novel mechanisms of testosterone metabolic switching in UGT2B17 gene deletion and testosterone activation by gut microbiome have been identified. These findings have a potential to provide deeper insights into the mechanisms of testosterone associated pathophysiological conditions, such obesity, insulin sensitivity, and cancer. References Li, C. Y. et al. J Steroid Biochem Mol Biol (2019). Rižner et al. Steroids (2014).

Re-evaluation of combined ((ES/EG)/(TS/TG)) ratio as a marker of testosterone intake in men.

To detect doping with pseudo-endogenous anabolic steroids in sports, a urinary steroid profile with glucuronidated plus unconjugated androgens is used. In addition to analyze androgen glucuronide metabolites, it can be of interest to also include sulfate metabolites in the urinary steroid profile. The combined ratios of epitestosterone sulfate/epitestosterone glucuronide to the ratios of testosterone sulfate/testosterone glucuronide ((ES/EG)/(TS/TG)) have previously been investigated as a complementary biomarker for testosterone doping. In this restudy, the aim was to evaluate this biomarker in a larger study sample population. A single dose of 500-mg testosterone enanthate was administered to 54 healthy male volunteers. Urine was collected prior to (Day 0) administration and throughout 15 days and analyzed for the sulfate and glucuronide conjugates of testosterone and epitestosterone. The results show that the combined ratio increased to a larger extent than the traditional T/E ratio in all subjects. This increase was independent on UGT2B17 gene polymorphism. Moreover, a delayed peak of the combined ratio was observed in ~60% of the participants. The results confirm that complementary analyses of the sulfate metabolites may be a useful approach to detect testosterone doping in men.

1H, 13C and 15N chemical shift assignments of the C-terminal domain of human UDP-Glucuronosyltransferase 2B7 (UGT2B7-C).

The human UDP-glucuronosyltransferase (UGT) family of enzymes catalyze the covalent addition of glucuronic acid to a wide range of compounds, generally rendering them inactive. Although important for clearance of environmental toxins and metabolites, UGT activation can lead to inappropriate glucuronidation of therapeutics underlying drug resistance. Indeed, 50% of medications are glucuronidated. To better understand this mode of resistance, we studied the UGT2B7 enzyme associated with glucuronidation of cancer drugs such as Tamoxifen and Sorafenib. We report 1H,13C and 15N backbone (> 90%) and side-chain assignments (~ 78% completeness according to CYANA) for the C-terminal domain of UGT2B7 (UGT2B7-C). Given the biomedical importance of this family of enzymes, our assignments will provide a key tool for improving understanding of the biochemical basis for substrate selectivity and other aspects of enzyme activity. This in turn will inform on drug design to overcome UGT-related drug resistance.

Pharmacokinetics and Pharmacogenetics of Dabigatran

Dabigatran etexilate is a prodrug of dabigatran, a oral direct inhibitor of thrombin. Pharmacokinetics of dabigatran etexilate doesn’t have the disadvantages of vitamin K antagonists. However, pharmacokinetics and pharmacogenetics of dabigatran are variable. This can affect both effectiveness and safety of anticoagulant therapy. It is considered that CES1 enzyme and P-glycoprotein (CES1 and ABCB1 genes accordingly) play important role in pharmacokinetics of dabigatran etexilate. UDP-glucuronosyltransferase enzymes UGT2B15, UGT1A9, UGT2B7 (UGT2B15, UGT1A9, UGT2B7 genes accordingly) take part in the metabolism of active dabigatran. Presence of these gene’s single-nucleotide variants (SNV) can affect effectiveness and safety of dabigatran etexilate usage. The goal of this review is analysis of associated researches of SNV genes CES1 and ABCB1 and search for new candidate genes that reveal effectiveness and safety of dabigatran etexilate usage.Materials and methods. The search for full-text publications in Russian and English languages containing key words “dabigatran etexilate”, “dabigatran”, “pharmacokinetics”, “effectiveness”, “safety” was carried out amongst literature of the past twenty years with the use of eLibrary, PubMed, Web of Science, OMIM data bases. Pharmacokinetics and pharmacogenetics of dabigatran etexilate are considered in this review. The hypothesis about UDP-glucuronosyltransferase enzymes influence on dabigatran metabolism was examined. Nowadays more than 2000 SNV CES1 and ABCB1 genes are identified, but their potential influence on pharmacokinetics of dabigatran etexilate and its active metabolite (dabigatran) in clinical practice needs to be further researched. Role of SNV UDP-glucuronosyltransferase genes (UGT2B15, UGT1A9, UGT2B7) in dabigatran’s effectiveness and safety is not explored enough. However, UGT2B15 gene can be a potential candidate gene for research on safety of this drug.

Molecular modeling study of the testosterone metabolizing enzyme UDP-glucuronosyltransferase 2B17

The dominant sex hormone testosterone is mainly metabolized by liver enzymes belonging to the uridine-diphospho (UDP) glucuronosyltransferase (UGT) family. These enzymes are the main phase II enzymes, and they have an important role in the detoxification of endogenous and exogenous compounds in humans. The aim of the present study was to improve the understanding of the binding properties of UGT2B17. A homology modelling procedure was used to generate models of the UGT2B17 enzyme based on templates with known crystal structures. Molecular docking of inhibitors was performed to gain further insights in the interactions between ligand and binding site, and to determine which of the models had the best accuracy. ROC curves were made to evaluate the ability of the models to differentiate between binders (inhibitors) and non-binders (decoys). When comparing the four models, which were based on four different crystal structures, the model based on the 4AMG crystal structure was the most accurate in distinguishing between true binders and non-binders. Investigating pharmacological UGT2B17 inhibition may provide novel treatment for patients with low testosterone levels. Such treatment may elevate endogenous testosterone levels and provide a more predictable increase in serum concentrations rather than un-physiological elevation of serum levels through direct treatment with testosterone, and this could be favorable both for giving a predictable treatment regime with reduced chances of serious adverse effects. The present study may serve as a tool in the search for novel drugs aiming for increasing testosterone levels.

Pharmacokinetics of the CYP3A4 and CYP2B6 Inducer Carbamazepine and Its Drug-Drug Interaction Potential: A Physiologically Based Pharmacokinetic Modeling Approach.

The anticonvulsant carbamazepine is frequently used in the long-term therapy of epilepsy and is a known substrate and inducer of cytochrome P450 (CYP) 3A4 and CYP2B6. Carbamazepine induces the metabolism of various drugs (including its own); on the other hand, its metabolism can be affected by various CYP inhibitors and inducers. The aim of this work was to develop a physiologically based pharmacokinetic (PBPK) parent−metabolite model of carbamazepine and its metabolite carbamazepine-10,11-epoxide, including carbamazepine autoinduction, to be applied for drug–drug interaction (DDI) prediction. The model was developed in PK-Sim, using a total of 92 plasma concentration−time profiles (dosing range 50–800 mg), as well as fractions excreted unchanged in urine measurements. The carbamazepine model applies metabolism by CYP3A4 and CYP2C8 to produce carbamazepine-10,11-epoxide, metabolism by CYP2B6 and UDP-glucuronosyltransferase (UGT) 2B7 and glomerular filtration. The carbamazepine-10,11-epoxide model applies metabolism by epoxide hydroxylase 1 (EPHX1) and glomerular filtration. Good DDI performance was demonstrated by the prediction of carbamazepine DDIs with alprazolam, bupropion, erythromycin, efavirenz and simvastatin, where 14/15 DDI AUClast ratios and 11/15 DDI Cmax ratios were within the prediction success limits proposed by Guest et al. The thoroughly evaluated model will be freely available in the Open Systems Pharmacology model repository.

UGT1A6 and UGT2B7 Gene Polymorphism and its Effect in Pediatric Epileptic Patients on Sodium Valproate Monotherapy

To evaluate the pattern of UGT1A6 and UGT2B7 gene polymorphism in pediatric epileptic patients and to compare the sodium valproate concentration in different patterns of UGT gene polymorphism. In this cross-sectional study, 99 pediatric epileptic patients aged 2-18 y receiving Sodium valproate monotherapy for the past one month were included from JusticeK S Hegde Charitable hospital, Mangalore after obtaining informed consent. Genetic polymorphism patterns were evaluated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Trough level serum valproate concentration was estimated by high-performance liquid chromatography (HPLC). Sodium valproate concentration in different UGT genotypes was compared by Analysis of Variance (ANOVA). P value <0.05 was considered significant. In the present study population, the predominant mutant allele pattern was observed in UGT1A6 (T19G, A541G, A552C) gene. In UGT2B7 (A268G, C161T) showed predominant mutant allele pattern while (G211T) showed predominant wild type. Mean steady-state sodium valproate concentration was 105.40 ± 49.9μg/ml and adjusted sodium valproate concentration was 5.5± 3.2mg/kg/L. It was found that there was no statistical difference in sodium valproate concentration in different UGT1A6 and UGT2B7 gene polymorphism. The present study concluded that though there was a difference in pattern of gene polymorphism with concerning UGT1A6 and UGT2B7, however, it has not contributed to variation in serum concentration of sodium valproate in the present study population.

Effect of UGT2B7 Gene Polymorphism with Clinical Pediatric Epileptic patients on Sodium Valproate Monotherapy

Background: Pediatric epilepsy comprises of a chronic neurological disorders characterized by recurrent seizure attack. Sodium valproate is one of the common anti-epileptic drugs used in the treatment. Glucuronide conjugation is the major metabolic pathway of sodium valproate, carried out by the enzyme Uridine 5’-diphospho (UDP) glucuronosyl transferase (UGT) whose genetic polymorphisms may alter clinical outcome. Aim: To find the association between UGT2B7 genetic polymorphism and clinical outcome in terms of efficacy and tolerability in pediatric epileptic patients on sodium valproate monotherapy. Methods and Materials: In this cohort study, 75 pediatric epileptic patients aged 2-18 years receiving sodium valproate monotherapy for past one month were included from Justice K S Hegde Charitable Hospital, Mangalore, India after obtaining informed consent. Genetic polymorphism patterns of UGT2B7 (C161T, A268G, G211T) was evaluated by PCR-RFLP. Clinical outcome was measured in terms of responders and non-responders based on seizure control during the 6 month observation period. Tolerability was measured by estimating the hepatic, renal and other lab parameters. Clinical outcome in different UGT genotypes was compared by Chi square test. P value <0.05 was considered as significant. Results: Out of 75 patients, CC (41.3%), CT (38.7%), TT (20%) pattern was observed in UGT2B7 (C161T) gene, AA(14.7%), AG(42.7%), GG(42.7%) in (A268G) gene and GG(80%), GT(18.7%), TT(1.3%) in (G211T) gene. It was found that there was no statistical difference in clinical outcome with different UGT2B7 genetic polymorphism patterns. Conclusion: We conclude from our study that genetic polymorphism of UGT2B7 doesn’t have any role on the clinical outcome of epilepsy.

A Study on the Relationship between UGT2B7 Gene Polymorphism and Clinical Prognosis

Breast cancer (BC) is the most common cancer diagnosis in women, often referred to as the "pink killer" [1], and its incidence ranks first among female malignancies, accounting for 25% of all cancer cases and 15% of all cancer deaths in the world [2]. With the improvement of medical level, the treatment methods of breast cancer have become relatively mature, including chemical therapy, adjuvant chemotherapy, neoadjuvant chemotherapy (mainly anthracycline and zisanchun drugs), endocrine therapy (tamoxifen, etc.), anti-estrogen drugs, aromatase inhibitors, etc. [3].In recent years, the role of UGT2B7 in drug metabolism and prognosis has been gradually discovered. To summarize the important role of UGT2B7 in prognosis diagnosis in recent years. In this paper, we search for qualified research by searching Pubmed, Uniprot and Chinese knowledge network. The data analysis and conclusion are helpful to the glucuronidation of many therapeutic drugs, including opioid drugs (such as codeine, morphine, naloxone), anticancer drugs (such as antitumor drugs: epirubicin, diclofenac, naproxen, etc.). In each treatment, the prognostic effect of UGT2B7 is gradually reflected, which is expected to be an important prognostic index.

Promising areas of research on the pharmacogenetics of dabigatran etexilate

Dabigatran etexilate is a prodrug of dabigatran, a direct inhibitor of thrombin. Pharmacokinetics of dabigatran etexilate doesn’t have the disadvantages of vitamin K antagonists. It is considered that CES1 enzyme and P-glycoprotein (CES1 and ABCB1 genes accordingly) play important role in pharmacokinetics of dabigatran etexilate. UDP-glucuronosyltransferase enzymes UGT2B15, UGT1A9, UGT2B7 (UGT2B15, UGT1A9, UGT2B7 genes accordingly) take part in the metabolism of active dabigatran. Presence of these gene’s single-nucleotide variants (SNV) can affect effectiveness and safety of dabigatran etexilate usage. The goal of this review is analysis of promising areas of associated researches of SNV of genes CES1 and ABCB1 and search for new candidate genes that reveal effectiveness and safety of dabigatran etexilate usage.

Predicting Opioid Therapy Safety In Pancreatic Cancer Patients

Background — Obligatory use of strong opioids for treating chronic pain syndrome in patients with pancreatic cancer provides the implementation of opioid-associated adverse reactions. Genetic and non-genetic risk factors are predictive of the opioid therapy safety. Contemporary methods of information analysis allow using prognostic risk models for practical application. Objective — Identification of significant risk factors for the development of opioid-associated adverse drug reactions in patients with chronic pain syndrome against the background of pancreatic cancer. Material and Methods — The study included 90 patients with chronic pain against the background of pancreatic cancer, randomized at a ratio of 1: 1. Group 1 received morphine sulfate (MS), group 2 received fentanyl transdermal therapeutic system (FTTS) with standard adjuvant therapy (ketoprofen, diazepam, amitriptyline). To assess pain level, the 10-point Digital Rating Scale, the Visual Analogue Scale and the pain questionnaires were used. The assessment of the treatment safety was conducted by the Naranjo Scale. Assessment of prognostic genetic and non-genetic factors was carried out using ROC analysis with calculation of AUC (the area under the ROC-curve). Results — Prognostic models of good quality were determined with the optimal ratio of sensitivity and specificity for the influence of genetic and non-genetic risk factors on the development of opioid-associated adverse drug reactions (OA-ADRs) in comparison groups. Various prognostic factors, complementing each other, were identified in the comparison groups. Conclusion — The following OA-ADRs predicting factors were identified: for FTTS-associated nausea and vomiting – age and carriage of rs7438135 AG genotype of UGT2B7 gene; for local reactions – the sum of points on the ESAS scale and carriage of rs7438135 AA genotype of UGT2B7 gene; for difficulty urinating – the level of glomerular filtration rate; for neurotoxicity – the level of AST and bilirubin, and the carriage of rs1128503 GG genotype of ABCB1 gene; for pruritus – carriage of rs1045642642 AA genotype of ABCB1 gene. The prognostic factors for the implementation of MS-associated neurotoxicity were age and comorbidity; for dry mouth was predicted best from the sum of points on the MMCE scale; weakness was predicted by the carriage of rs7668258 TT genotype of UGT2B7 gene.

Exposure of Slovenian children and adolescents to bisphenols, parabens and triclosan: Urinary levels, exposure patterns, determinants of exposure and susceptibility

Chemicals such as bisphenols, parabens and triclosan are endocrine disrupting chemicals. They are used in a wide variety of consumer products, making human exposure to those chemicals widespread.In the present study, levels of three bisphenols (bisphenol A, F and S), 7 parabens (methyl-, ethyl-, isopropyl-, propyl-, isobutyl-, butyl-, benzyl paraben) and triclosan were measured in first morning void from 246 Slovenian children and adolescents, aged 6–9 and 11–15 years and living in a rural region of Slovenia. Median levels of specific-gravity corrected levels for bisphenol A, bisphenol F, methyl paraben and ethyl paraben were 1.9, 0.085, 5.4 and 2.5 µg/L for children and 1.6, 0.11, 7.2 and 6.0 µg/L for adolescents, respectively. Median levels for all other endocrine disrupting chemicals were < LOQ. The levels are comparable with the levels reported in studies across the world. Exposure was age, sex, and location specific. Higher levels of bisphenol F and ethyl paraben were found in the samples of adolescents, while higher levels of methyl paraben were found in samples from girls. Furthermore, individuals living in one of the sampling locations, Goričko, were exposed to higher levels of bisphenol F and ethyl paraben than those in the remaining two sampling locations. Information about participants’ dietary habits, use of food packaging and personal care products was obtained through questionnaires, and used to investigate associations between urinary levels of the biomarkers and potential exposure sources. High fat foods were associated with bisphenol A exposure, and cosmetics items such as lipstick and perfume with methyl paraben exposure. Significant correlation between methyl- and propyl paraben was observed in children’s samples, suggesting similar exposure sources, while other compounds were not largely correlated, indicating independent sources. Furthermore, association between a single nucleotide polymorphism (SNP) in UGT2B15 gene and urinary levels of methyl and ethyl paraben was observed, showing the role of UGT2B15 isoform in methyl and ethyl paraben metabolism as well as indicating the SNP rs1902023 as a potential biomarker of susceptibility to adverse effects caused by the exposure.The present study reports exposure of children and adolescents in Slovenia to a wide range of different endocrine disrupting chemicals for the first time, connecting it to exposure patterns and exposure sources. The study is to the authors’ knowledge the first that investigates direct connection between levels of urinary endocrine disrupting chemical biomarkers and genetic polymorphism in UGT2B15.

Influence of Polymorphisms on Mycophenolate Mofetil - Induced Diarrhoea in Renal Transplanted Children

Purpose: The objective of this study was to investigate the factors involved in mycophenolate mofetil (MMF) disposition on the risk of diarrhoea in renal transplanted children. Methods: Patients’ characteristics, immunosuppression and polymorphisms of UGT1A8, UGT1A9, UGT2B7, ABCC2, IMPDH1, and IMPDH2 genes were explored. Statistical analyses were performed using logistic regression. Results: Eighty three renal transplanted patients were included and 28/83 (33%) developed diarrhoea requiring MMF discontinuation or switched to the enteric-coated formulation EC-MPS during follow-up. In the multivariate analysis, the risk of diarrhoea was significantly higher in ABCC2 -24CC wild-type patients carrying IMPDH2 IVS7 + 10 T>C variant. Conclusion: IMPDH2 (IVS7+10T>C) and ABCC2 (c.-24C>T) are biomarkers associated with diarrhoea in children treated with MMF.

Variation in the UGT2B17 genotype, exemestane metabolism and menopause-related toxicities in the CCTG MAP.3 trial.

To examine associations between the UGT2B17 gene deletion and exemestane metabolites, and commonly reported side effects (fatigue, hot flashes, and joint pain) among postmenopausal women participating in the MAP.3 chemoprevention trial. The analytical samples for the UGT2B17 analysis comprised 1752 women on exemestane and 1721 women on placebo; the exemestane metabolite analysis included 1360 women on exemestane with one-year serum samples. Both the UGT2B17 gene deletion and metabolites were measured in blood. The metabolites were conceptualized as a ratio (17-DHE-Gluc:17-DHE). Symptoms were assessed using the CTCAE v4.0 at approximately 1-year intervals. Log-binomial regression was used to examine the associations between UGT2B17 deletion, exemestane metabolites and each side effect at 1 and up to 5-year follow-up, adjusting for potential confounders. Among individuals on exemestane with the UGT2B17 gene deletion (i.e., lower detoxification), a higher risk of severe fatigue (RR = 2.59 95% CI: 1.14-5.89) was observed at up to 5-year follow-up. Among individuals on placebo, those with the UGT2B17 gene deletion had a higher risk of any fatigue (RR = 1.39, 95% CI: 1.02-1.89) at year 1. A lower metabolite ratio (poor detoxification) was associated with a higher risk of any fatigue, hot flashes and joint pain at year 1 (fatigue: RR = 1.89, 95% CI: 1.16-3.09; hot flashes: RR = 1.77, 95% CI: 1.40-2.24; joint pain: RR = 2.05, 95% CI: 1.35-3.12); similar associations were observed at 5-year follow-up. Variation in the metabolism of exemestane through the UGT2B17-mediated pathway is associated with subsequent risk of commonly reported symptoms in MAP.3.

Expression of UDP Glucuronosyltransferases 2B15 and 2B17 is associated with methylation status in prostate cancer cells

ABSTRACT Studies have suggested that abrogated expression of detoxification enzymes, UGT2B15 and UGT2B17, are associated with prostate tumour risk and progression. We investigated the role of EGF on the expression of these enzymes since it interacts with signalling pathways to also affect prostate tumour progression and is additionally associated with decreased DNA methylation. The expression of UGT2B15, UGT2B17, de novo methyltransferases, DNMT3A and DNMT3B was assessed in prostate cancer cells (LNCaP) treated with EGF, an EGFR inhibitor PD16893, and the methyltransferase inhibitor, 5-azacytidine, respectively. The results showed that EGF treatment decreased levels of expression of all four genes and that their expression was reversed by PD16893. Treatment with 5-azacytidine, markedly decreased expression of UGT2B15 and UGT2B17 over 85% as well as significantly decreased expression of DNMT3B, but not the expression of DNMT3A. DNMT3B siRNA treated LNCaP cells had decreased expression of UGT2B15 and UGT2B17, while DNMT3A siRNA treated cells had only moderately decreased UGT2B15 expression. Treatment with DNMT methyltransferase inhibitor, RG108, significantly decreased UGT2B17 expression. Additionally, methylation differences between prostate cancer samples and benign prostate samples from an Illumina 450K Methylation Array study were assessed. The results taken together suggest that hypomethylation of the UGT2B15 and UGT2B17 genes contributes to increased risk of prostate cancer and may provide a putative biomarker or epigenetic target for chemotherapeutics. Mechanistic studies are warranted to determine the role of the methylation marks in prostate cancer.

The function of uterine UDP-glucuronosyltransferase 1A8 (UGT1A8) and UDP-glucuronosyltransferase 2B7 (UGT2B7) is involved in endometrial cancer based on estrogen metabolism regulation.

The progression of endometrial cancer (EC) is closely related to estrogen levels. UDP-glucuronosyltransferases (UGTs) are an essential class of phase II metabolizing enzymes that play a pivotal role in detoxifying steroid hormone. In this study, we aimed to uncover the role of UGTs in estrogen metabolism and the pathogenesis of EC. A total of 100 unrelated EC patients (mean age 52.15 ± 10.04y) and 100 healthy subjects (mean age 50.26 ± 8.80y) were recruited for analysis of the UGT gene polymorphism and estrogen level. In six cases of EC, EC-adjacent tissues and cancer tissues were collected for detection of UGT expression. Our results showed that the estrogen homeostasis profile was disturbed in EC patients, with carcinogenic catechol estrogens (4-OHE1, 2-OHE1, 2-OHE2) significantly accumulated in the serum of these patients. Also, levels of estrogen-glucuronides were decreased significantly, and the expression of UGT1A8 and UGT2B7 in uterine tissues was downregulated in EC patients. Consistent with this, we observed that the distribution of genotypes and allele frequencies in UGT1A8 rs1042597 and UGT2B7 rs7439366 was significantly different between EC patients and healthy volunteers. These results indicated that UGT1A8 and UGT2B7 may contribute to the estrogen signaling pathway and the pathogenesis of EC.

Metabolism and Toxicity of Emodin: Genome-Wide Association Studies Reveal Hepatocyte Nuclear Factor 4α Regulates UGT2B7 and Emodin Glucuronidation.

Emodin is the main toxic component in Chinese medicinal herbs such as rhubarb. Our previous studies demonstrated that genetic polymorphisms of UDP-glucuronosyltransferase 2B7 (UGT2B7) had an effect on the glucuronidation and detoxification of emodin. This study aimed to reveal the transcriptional regulation mechanism of UGT2B7 on emodin glucuronidation and its effect on toxicity. Emodin glucuronic activity and genome and transcriptome data were obtained from 36 clinical human kidney tissues. The genome-wide association studies (GWAS) identified that four single nucleotide polymorphisms (SNPs) (rs6093966, rs2868094, rs2071197, and rs6073433), which were located on the hepatocyte nuclear factor 4α (HNF4A) gene, were significantly associated with the emodin glucuronidation (p < 0.05). Notably, rs2071197 was significantly associated with the gene expression of HNF4A and UGT2B7 and the glucuronidation of emodin. The gene expression of HNF4A showed a high correlation with UGT2B7 (R2 = 0.721, p = 5.83 × 10-11). The luciferase activity was increased 7.68-fold in 293T cells and 2.03-fold in HepG2 cells, confirming a significant transcriptional activation of UGT2B7 promoter by HNF4A. The knockdown of HNF4A in HepG2 cells (36.6%) led to a significant decrease of UGT2B7 (19.8%) and higher cytotoxicity (p < 0.05). The overexpression of HNF4A in HepG2 cells (31.2%) led to a significant increase of UGT2B7 (24.4%) and improved cell viability (p < 0.05). Besides, HNF4A and UGT2B7 were both decreased in HepG2 cells and rats after treatment with emodin. In conclusion, emodin used long term or in high doses could inhibit the expression of HNF4A, thereby reducing the expression of UGT2B7 and causing hepatotoxicity.

Impact of UGT2B7 gene deletion genotype on human liver proteome

Background and objectivesUDP‐Glucuronosyltransferase 2B17 (UGT2B17) is a highly variable enzyme, which plays an important role in metabolism of endogenous steroids (e.g., testosterone), drugs or active metabolites (e.g., vorinostat, 17‐dihydroexemestane), and environmental chemicals (e.g., nicotine nitrosamine ketone, NNK). UGT2B17 is the most commonly deleted gene and we have previously demonstrated that age, sex, copy number variation, and certain single nucleotide polymorphisms can affect its abundance and activity (1). The aim of this study was to identify steroid and drug metabolism pathways that are impacted by UGT2B17 deletion using label‐free untargeted proteomics approach.MethodsThe untargeted proteomics study was performed on the liver S9 samples isolated from liver tissue of subjects with UGT2B17 gene deletion (n=3) and UGT2B17 high expression (n=3) (1). The proteins were reduced, alkylated, and digested with trypsin using an optimized protocol (1). Proteomics data were acquired in high resolution mode in QE‐HF (Thermo Scientific) coupled with Easy nano‐LC 1200. The tryptic digest was injected onto a nano‐column for peptide separation and the peptides were detected using data‐dependent acquisition (DDA) method. The survey scans of peptide precursors were performed from 375 to 1500 m/z at 120 K resolution with a 3e6 ion count target and a maximum injection time of 50 ms. The untargeted proteomic data was analyzed using MaxQuant with 1% FDR followed by data mining using Perseus and STRING analysis for pathway mapping.Results and conclusionA total of 1002 liver proteins were quantified based on the signature peptides. Out of 1002 proteins, 14 and 39 proteins were exclusively detected and quantified in UGT2B17 deletion and high expression groups, respectively (Fig. 1A), whereas 949 proteins were quantified in both the groups. The following pathways were upregulated in UGT2B17 deletion group: oxidation reduction process, drug metabolic process, steroid and fatty acid metabolic process. Notable proteins that were differentially expressed in the gene deletion groups were: a) AKR1D1, AKR1C4, AKR7DA3, AKR1A1, DHCR7, ADH6, ADH1C, ALDH1A1, ALDH9A1, and ALDH5A1 (upregulated) and b) CYP3A5, CYP1A2, LDHA, NNMT, Kynureninase, and ACACB (downregulated). A deeper molecular pathway analysis reveals that the enzymes associated with steroidal and bile acid homeostasis (e.g., aldo‐keto reductases (AKRs) and aldehyde dehydrogenases) were significantly elevated in the UGT2B17 deletion group. Interestingly, AKR1D1 acts as the sole Δ4‐3‐ketosteroid‐5β‐reductase, that metabolizes testosterone to inactive 5β‐dihydrotestosterone (2). In contrast, this pathway is a minor contributor to testosterone metabolism in the UGT2B17 high expressers. Similarly, AKR1C4 and UGT2B7, which are responsible for sequential conversion of 5β‐ dihydrotestosterone to 3α‐5β‐tetrahydrotestosterone (THT) to THT‐glucuronide (Fig. 1B), are upregulated in the UGT2B17 gene deletion groups. These novel findings of testosterone homeostasis in the UGT2B17 gene individuals can provide deeper insights into the mechanisms of UGT2B17 gene deletion‐associated pathophysiological conditions (osteoporosis, obesity, insulin sensitivity, and cancers).A) Number of hepatic proteins detected in UGT2B17 gene deletion (red) and high expressers (blue) individuals. B) Proposed compensatory mechanism of testosterone hepatic metabolism in UGT2B17 gene deletion individuals.Figure 1

Coordinated Regulation of UGT2B15 Expression by Long Noncoding RNA LINC00574 and hsa-miR-129-5p in HepaRG Cells.

Recent studies have shown that microRNAs and long noncoding RNAs (lncRNAs) regulate the expression of drug metabolizing enzymes (DMEs) in human hepatic cells and that a set of DMEs, including UDP glucuronosyltransferase (UGT) 2B15, is down-regulated dramatically in liver cells by toxic acetaminophen (APAP) concentrations. In this study we analyzed mRNA, microRNA, and lncRNA expression profiles in APAP-treated HepaRG cells to explore noncoding RNA-dependent regulation of DME expression. The expression of UGT2B15 and lncRNA LINC00574 was decreased in APAP-treated HepaRG cells. UGT2B15 levels were diminished by LINC00574 suppression using antisense oligonucleotides or small interfering RNA. Furthermore, we found that hsa-miR-129-5p suppressed LINC00574 and decreased UGT2B15 expression via LINC00574 in HepaRG cells. In conclusion, our results indicate that LINC00574 acts as an important regulator of UGT2B15 expression in human hepatic cells, providing experimental evidence and new clues to understand the role of cross-talk between noncoding RNAs. SIGNIFICANCE STATEMENT: We showed a molecular network that displays the cross-talk and consequences among mRNA, micro RNA, long noncoding RNA, and proteins in acetaminophen (APAP)-treated HepaRG cells. APAP treatment increased the level of hsa-miR-129-5p and decreased that of LINC00574, ultimately decreasing the production of UDP glucuronosyltransferase (UGT) 2B15. The proposed regulatory network suppresses UGT2B15 expression through interaction of hsa-miR-129-5p and LINC00574, which may be achieved potentially by recruiting RNA binding proteins.