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Background: High UGT2B17 is associated with poor prognosis in untreated chronic lymphocytic leukaemia (CLL) patients and its expression is induced in non-responders to fludarabine-containing regimens. We examined whether UGT2B17, the predominant lymphoid glucuronosyltransferase, affects leukaemic drug response and is involved in the metabolic inactivation of anti-leukaemic agents., Methods: Functional enzymatic assays and patients' plasma samples were analysed by mass-spectrometry to evaluate drug inactivation by UGT2B17. Cytotoxicity assays and RNA sequencing were used to assess drug response and transcriptome changes associated with high UGT2B17 levels., Results: High UGT2B17 in B-cell models led to reduced sensitivity to fludarabine, ibrutinib and idelalisib. UGT2B17 expression in leukaemic cells involved a non-canonical promoter and was induced by short-term treatment with these anti-leukaemics. Glucuronides of both fludarabine and ibrutinib were detected in CLL patients on respective treatment, however UGT2B17 conjugated fludarabine but not ibrutinib. AMP-activated protein kinase emerges as a pathway associated with high UGT2B17 in fludarabine-treated patients and drug-treated cell models. The expression changes linked to UGT2B17 exposed nuclear factor kappa B as a key regulatory hub., Conclusions: Data imply that UGT2B17 represents a mechanism altering drug response in CLL through direct inactivation but would also involve additional mechanisms for drugs not inactivated by UGT2B17.

Background: Perturbation of the major UGT2B17-dependent androgen catabolism pathway has the potential to affect prostate cancer (PCa) progression. The objective was to evaluate UGT2B17 protein expression in primary tumours in relation to hormone levels, disease characteristics and cancer evolution., Methods: We conducted an analysis of a high-density prostate tumour tissue microarray consisting of 239 localised PCa cases treated by radical prostatectomy (RP). Cox proportional hazard ratio analysis was used to evaluate biochemical recurrence (BCR), and a linear regression model evaluated variations in circulating hormone levels measured by mass spectrometry. The transcriptome of UGT2B17 in PCa was established by using RNA-sequencing data., Results: UGT2B17 expression in primary tumours was associated with node-positive disease at RP and linked to circulating levels of 3α-diol-17 glucuronide, a major circulating DHT metabolite produced by the UGT2B17 pathway. UGT2B17 was an independent prognostic factor linked to BCR after RP, and its overexpression was associated with development of metastasis. Finally, we demonstrated that distinctive alternative promoters dictate UGT2B17-dependent androgen catabolism in localised and metastatic PCa., Conclusions: The androgen-inactivating gene UGT2B17 is controlled by overlooked regulatory regions in PCa. UGT2B17 expression in primary tumours influences the steroidome, and is associated with relevant clinical outcomes, such as BCR and metastasis.

Abiraterone (Abi) acetate (AA) is a prodrug of Abi, a CYP17A1 inhibitor used to treat patients with advanced prostate cancer. Abi is a selective steroidal inhibitor that blocks the biosynthesis of androgens. It undergoes extensive biotransformation by steroid pathways, leading to the formation of pharmacologically active Δ 4 -Abi (D4A) and 5 α -Abi. This study aimed to characterize the glucuronidation pathway of Abi and its two active metabolites. We show that Abi, its metabolites, and another steroidal inhibitor galeterone (Gal) undergo secondary metabolism to form glucuronides (G) in human liver microsomes with minor formation by intestine and kidney microsomal preparations. The potential clinical relevance of this pathway is supported by the detection by liquid chromatography-tandem mass spectrometry of Abi-G, D4A-G, and 5 α -Abi-G in patients under AA therapy. A screening of UGT enzymes reveals that UGT1A4 is the main enzyme involved. This is supported by inhibition experiments using a selective UGT1A4 inhibitor hecogenin. A number of common and rare nonsynonymous variants significantly abrogate the UGT1A4-mediated formation of Abi-G, D4A-G, and 5 α -Abi-G in vitro. We also identify Gal, Abi, and its metabolites as highly potent inhibitors of steroid inactivation by the UGT pathway with submicromolar inhibitor constant values. They reduce the glucuronidation of both the adrenal precursors and potent androgens in human liver, prostate cancer cells, and by recombinant UGTs involved in their inactivation. In conclusion, tested CYP17A1 inhibitors are metabolized through UGT1A4, and germline variations affecting this metabolic pathway may also influence drug metabolism. SIGNIFICANCE STATEMENT: The antiandrogen abiraterone (Abi) is a selective steroidal inhibitor of the cytochrome P450 17α-hydroxy/17,20-lyase, an enzyme involved in the biosynthesis of androgens. Abi is metabolized to pharmacologically active metabolites by steroidogenic enzymes. We demonstrate that Abi and its metabolites are glucuronidated in the liver and that their glucuronide derivatives are detected at variable levels in circulation of treated prostate cancer patients. UDP-glucuronosyltransferase (UGT)1A4 is the primary enzyme involved, and nonsynonymous germline variations affect this metabolic pathway in vitro, suggesting a potential influence of drug metabolism and action in patients. Their inhibitory effect on drug and steroid glucuronidation raises the possibility that these pharmacological compounds might affect the UGT-associated drug-metabolizing system and pre-receptor control of androgen metabolism in patients., Competing Interests: No conflict of interests was disclosed., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Accurate quantification of the metabolic enzyme uridine diphospho-glucuronosyltransferase (UGT) UGT2B17 has been hampered by the high sequence identity with other UGT2B enzymes (as high as 94%) and by the lack of a specific antibody. Knowing the significance of the UGT2B17 pathway in drug and hormone metabolism and cancer, we developed a specific monoclonal antibody (EL-2B17mAb), initially validated by the lack of detection in liver microsomes of an individual carrying no UGT2B17 gene copy and in supersomes expressing UGT2B enzymes. Immunohistochemical detection in livers revealed strong labeling of bile ducts and variable labeling of hepatocytes. Expression levels assessed by immunoblotting were highly correlated to mass spectrometry-based quantification ( r = 0.93), and three major expression patterns (absent, low, or high) were evidenced. Livers with very low expression were carriers of the functional rs59678213 G variant, located in the binding site for the transcription factor forkhead box A1 (FOXA1) of the UGT2B17 promoter. The highest level of expression was observed for individuals carrying at least one rs59678213 A allele. Multiple regression analysis indicated that the number of gene copies explained only 8% of UGT2B17 protein expression, 49% when adding rs59678213, reaching 54% when including sex. The novel EL-2B17mAb antibody allowed specific UGT2B17 quantification and exposed different patterns of hepatic expression. It further suggests that FOXA1 is a key driver of UGT2B17 expression in the liver. The availability of this molecular tool will help characterize the UGT2B17 level in various disease states and establish more precisely the contribution of the UGT2B17 enzyme to drug and hormone metabolism., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

The detoxification enzyme UDP-glucuronosyltransferase UGT2B10 is specialized in the N-linked glucuronidation of many drugs and xenobiotics. Preferred substrates possess tertiary aliphatic amines and heterocyclic amines, such as tobacco carcinogens and several antidepressants and antipsychotics. We hypothesized that alternative splicing (AS) constitutes a means to regulate steady-state levels of UGT2B10 and enzyme activity. We established the transcriptome of UGT2B10 in normal and tumoral tissues of multiple individuals. The highest expression was in the liver, where 10 AS transcripts represented 50% of the UGT2B10 transcriptome in 50 normal livers and 44 hepatocellular carcinomas. One abundant class of transcripts involves a novel exonic sequence and leads to two alternative (alt.) variants with novel in-frame C termini of 10 or 65 amino acids. Their hepatic expression was highly variable among individuals, correlated with canonical transcript levels, and was 3.5-fold higher in tumors. Evidence for their translation in liver tissues was acquired by mass spectrometry. In cell models, they colocalized with the enzyme and influenced the conjugation of amitriptyline and levomedetomidine by repressing or activating the enzyme (40%-70%; P < 0.01) in a cell context-specific manner. A high turnover rate for the alt. proteins, regulated by the proteasome, was observed in contrast to the more stable UGT2B10 enzyme. Moreover, a drug-induced remodeling of UGT2B10 splicing was demonstrated in the HepaRG hepatic cell model, which favored alt. variants expression over the canonical transcript. Our findings support a significant contribution of AS in the regulation of UGT2B10 expression in the liver with an impact on enzyme activity., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Alternative splicing as a mean to control gene expression and diversify function is suspected to considerably influence drug response and clearance. We report the quantitative expression profiles of the human UGT genes including alternatively spliced variants not previously annotated established by deep RNA-sequencing in tissues of pharmacological importance. We reveal a comprehensive quantification of the alternative UGT transcriptome that differ across tissues and among individuals. Alternative transcripts that comprise novel in-frame sequences associated or not with truncations of the 5'- and/or 3'- termini, significantly contribute to the total expression levels of each UGT1 and UGT2 gene averaging 21% in normal tissues, with expression of UGT2 variants surpassing those of UGT1. Quantitative data expose preferential tissue expression patterns and remodeling in favor of alternative variants upon tumorigenesis. These complex alternative splicing programs have the strong potential to contribute to interindividual variability in drug metabolism in addition to diversify the UGT proteome.

The UDP glucuronosyltransferase (UGT) superfamily comprises glycoproteins that reside in the endoplasmic reticulum membranes and that undergo post-translational modifications (PTMs). UGT2B7 is of particular interest because of its action on a wide variety of drugs. Most studies currently survey common variants and examine only a small fraction of the genetic diversity; however, rare variants (frequency <1%) might have a significant effect because they are predicted to greatly outnumber common variants in the human genome. We discovered a rare single nucleotide UGT2B7 variant of potential pharmacogenetic relevance that encodes a nonconservative amino acid substitution at codon 121. This low-frequency variation, found in two individuals of a population of 305 healthy volunteers, leads to the translation of an asparagine instead of an aspartic acid (UGT2B7 p.D 121 N). This amino acid change was predicted to create a putative N-glycosylation motif NX(S/T) subsequently validated upon endoglycosidase H treatment of microsomal fractions and inhibition of N-glycosylation of endogenously produced UGT2B7 with tunicamycin in human embryonic kidney (HEK293) cells. The presence of an additional N-linked glycan on the UGT2B7 enzyme, likely affecting proper protein folding, resulted in a significant decrease of 49% and 40% in the formation of zidovudine and mycophenolic acid glucuronides, respectively. A systematic survey of the Short Genetic Variations database uncovered 32 rare, naturally occurring missense variations predicted to create or disrupt N-glycosylation sequence motifs in the other UGT2B enzymes. Collectively, these variants have the potential to increase the proportion of variance explained in the UGT pathway resulting from changes in PTMs, such as N-linked glycosylation with consequences on drug metabolism., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Maintenance of cellular homeostasis and xenobiotic detoxification is mediated by 19 human UDP-glucuronosyltransferase enzymes (UGTs) encoded by ten genes that comprise the glucuronidation pathway. Deep RNA sequencing of major metabolic organs exposes a substantial expansion of the UGT transcriptome by alternative splicing, with variants representing 20% to 60% of canonical transcript expression. Nearly a fifth of expressed variants comprise in-frame sequences that may create distinct structural and functional features. Follow-up cell-based assays reveal biological functions for these alternative UGT proteins. Some isoforms were found to inhibit or induce inactivation of drugs and steroids in addition to perturbing global cell metabolism (energy, amino acids, nucleotides), cell adhesion, and proliferation. This work highlights the biological relevance of alternative UGT expression, which we propose increases protein diversity through the evolution of metabolic regulators from specific enzymes., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Androgen 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., Objective: To 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 Participants: We 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 Analysis: Steroid 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 Limitations: Tumor 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%)., Conclusions: The 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 Summary: The androgen-inactivating UGT2B28 enzyme influences hormone levels, clinical and pathologic factors, and the risk of cancer progression., (Copyright © 2015 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

A comprehensive view of the human UDP-glucuronosyltransferase (UGT) transcriptome is a prerequisite to the establishment of an individual's UGT metabolic glucuronidation signature. Here, we uncover the transcriptome landscape of the 10 human UGT gene loci in normal and tumoral metabolic tissues by targeted RNA next-generation sequencing. Alignment on the human hg19 reference genome identifies 234 novel exon-exon junctions. We recover all previously known UGT1 and UGT2 enzyme-coding transcripts and identify over 130 structurally and functionally diverse novel UGT variants. We further expose a revised genomic structure of UGT loci and provide a comprehensive repertoire of transcripts for each UGT gene. Data also uncover a remodelling of the UGT transcriptome occurring in a tissue- and tumor-specific manner. The complex alternative splicing program regulating UGT expression and protein functions is likely critical in determining detoxification capacity of an organ and stress-related responses, with significant impact on drug responses and diseases.

The risk of severe irinotecan-induced neutropenia has been shown to be related to the UGT1 variant UGT1A1*28, which increases exposure to the potent metabolite SN-38. Our goal was to identify a novel UGT1 marker(s) using 28 haplotype-tagged single nucleotide polymorphisms genotyped by mass spectrometry. By characterizing the UGT1 sequence from a cohort of 167 Canadian metastatic colorectal cancer (mCRC) patients and a validation cohort of 250 Italian mCRC patients, we found rs11563250G, located in the intergenic region downstream of UGT1, to be significantly associated with reduced risk of severe neutropenia (odds ratio (OR)=0.21; P=0.043 and OR=0.27; P=0.036, respectively, and OR=0.31 when combined; P=0.001), which remained significant upon correction for multiple testing in the combined cohort (P=0.041). For the two-marker haplotype rs11563250G and UGT1A1*1 (rs8175347 TA6), the OR was of 0.17 (P=0.0004). Genetic testing of this marker may identify patients who might benefit from increased irinotecan dosing.

Phase II metabolism is prominently governed by UDP-glucuronosyltransferases (UGTs) in humans. These enzymes regulate the bioactivity of many drugs and endogenous small molecules in many organs, including the liver, a major site of regulation by the glucuronidation pathway. This study determined the expression of hepatic UGTs by targeted proteomics in 48 liver samples and by measuring the glucuronidation activity using probe substrates. It demonstrates the sensitivity and accuracy of nano-ultra-performance liquid chromatography with tandem mass spectrometry to establish the complex expression profiles of 14 hepatic UGTs in a single analysis. UGT2B7 is the most abundant UGT in our collection of livers, expressed at 69 pmol/mg microsomal proteins, whereas UGT1A1, UGT1A4, UGT2B4, and UGT2B15 are similarly abundant, averaging 30-34 pmol/mg proteins. The average relative abundance of these five UGTs represents 81% of the measured hepatic UGTs. Our data further highlight the strong relationships in the expression of several UGTs. Most notably, UGT1A4 correlates with most measured UGTs, and the expression levels of UGT2B4/UGT2B7 displayed the strongest correlation. However, significant interindividual variability is observed for all UGTs, both at the level of enzyme concentrations and activity (coefficient of variation: 45%-184%). The reliability of targeted proteomics quantification is supported by the high correlation between UGT concentration and activity. Collectively, these findings expand our understanding of hepatic UGT profiles by establishing absolute hepatic concentrations of 14 UGTs and further suggest coregulated expression between most abundant hepatic UGTs. Data support the value of multiplexed targeted quantitative proteomics to accurately assess specific UGT concentrations in liver samples and hepatic glucuronidation potential., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Renal metabolism by UDP-glucuronosyltransferase (UGT) enzymes is central to the clearance of many drugs. However, significant discrepancies about the relative abundance and activity of individual UGT enzymes in the normal kidney prevail among reports, whereas glucuronidation in tumoral kidney has not been examined. In this study, we performed an extensive profiling of glucuronidation metabolism in normal (n = 12) and tumor (n = 14) kidneys using targeted mass spectrometry quantification of human UGTs. We then correlated UGT protein concentrations with mRNA levels assessed by quantitative polymerase chain reaction and with conjugation activity for the major renal UGTs. Beyond the wide interindividual variability in expression levels observed among kidney samples, UGT1A9, UGT2B7, and UGT1A6 are the most abundant renal UGTs in both normal and tumoral tissues based on protein quantification. In normal kidney tissues, only UGT1A9 protein levels correlated with mRNA levels, whereas UGT1A6, UGT1A9, and UGT2B7 quantification correlated significantly with their mRNA levels in tumor kidneys. Data support that posttranscriptional regulation of UGT2B7 and UGT1A6 expression is modulating glucuronidation in the kidney. Importantly, our study reveals a significant decreased glucuronidation capacity of neoplastic kidneys versus normal kidneys that is paralleled by drastically reduced UGT1A9 and UGT2B7 mRNA and protein expression. UGT2B7 activity is the most repressed in tumors relative to normal tissues, with a 96-fold decrease in zidovudine metabolism, whereas propofol and sorafenib glucuronidation is decreased by 7.6- and 5.2-fold, respectively. Findings demonstrate that renal drug metabolism is predominantly mediated by UGT1A9 and UGT2B7 and is greatly reduced in kidney tumors., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Alternative splicing (AS) is one of the most significant components of the functional complexity of human UDP-glucuronosyltransferase enzymes (UGTs), particularly for the UGT1A gene, which represents one of the best examples of a drug-metabolizing gene regulated by AS. Shorter UGT1A isoforms [isoform 2 (i2)] are deficient in glucuronic acid transferase activity but function as negative regulators of enzyme activity through protein-protein interaction. Their abundance, relative to active UGT1A enzymes, is expected to be a determinant of the global transferase activity of cells and tissues. Here we tested whether i2-mediated inhibition increases with greater abundance of the i2 protein relative to the isoform 1 (i1) enzyme, using the extrahepatic UGT1A7 as a model and a series of 23 human embryonic kidney 293 clonal cell lines expressing variable contents of i1 and i2 proteins. Upon normalization for i1, a significant reduction of 7-ethyl-10-hydroxycamptothecin glucuronide formation was observed for i1+i2 clones (mean of 53%) compared with the reference i1 cell line. In these clones, the i2 protein content varied greatly (38-263% relative to i1) and revealed two groups: 17 clones with i2 < i1 (60% ± 3%) and 6 clones with i2 ≥ i1 (153% ± 24%). The inhibition induced by i2 was more substantial for clones displaying i2 ≥ i1 (74.5%; P = 0.001) compared with those with i2 < i1 (45.5%). Coimmunoprecipitation supports a more substantial i1-i2 complex formation when i2 exceeds i1. We conclude that the relative abundance of regulatory i2 proteins has the potential to drastically alter the local drug metabolism in the cells, particularly when i2 surpasses the protein content of i1.

Uridine diphosphate-glucuronosyltransferases (UGTs) are a family of phase II-metabolizing enzymes involved in glucuronic acid conjugation of sex steroid hormones. UGT1A1 and UGT2B7 are expressed in the uterus and involved in the conjugation and elimination of estrogens. Chronic exposure to estrogens is associated with endometrial cancer. Functional polymorphisms have been identified in UGT1A1 and UGT2B7. We hypothesized that these variants may be associated with endometrial cancer risk. We conducted a case-control study nested within the Nurses' Health Study and the Women's Health Study to investigate the associations between five polymorphisms and endometrial cancer risk using 593 invasive endometrial cancer cases and 1545 controls. We did observe the suggestion of an inverse association with homozygote variant carriers of UGT1A1*28 and endometrial cancer risk. We did not observe significant associations between individual single nucleotide polymorphisms and UGT1A1 haplotypes and endometrial cancer risk. Our data suggest that these UGT polymorphisms do not contribute significantly to endometrial cancer risk.

Tissue iron overload constitutes a major health problem for people who require regular blood transfusions, such as those with beta-thalassemia major. Deferiprone is a hydroxypyridinone iron chelator used therapeutically to remove this excess iron and prevent tissue damage. Deferiprone is metabolized by UDP-glucuronosyltransferases (UGTs) into deferiprone 3-O-glucuronide (DG), but a systematic evaluation of the contribution of individual human UGTs and the impact of genetic variations of UGTs have not been conducted. Sixteen human UGT1A and UGT2B were studied for deferiprone glucuronidation, and their clearances were compared in human tissue samples. DG was measured by liquid chromatography coupled with mass spectrometry. DG was primarily produced in vitro by UGT1A6, and a second glucuronide metabolite was discovered. UGT1A6, as well as liver and kidney human microsomes, had similar kinetic profiles and clearance (Cl(int) = 1.4-3.0 mul/min/mg), but clearance by intestinal microsomes was much lower (0.04 mul/min/mg). Binding of deferiprone to microsomal preparations was not significant. Genetic variants of UGT1A6 had K(m) values similar to the reference protein (UGT1A6*1), but their V(max) values were reduced by 25 to 70%. The UGT1A6 splice variant isoform 2, detected in the liver and kidney, had no transferase activity for deferiprone. When UGT1A6&#95;i2 was coexpressed with the classic UGT1A6&#95;i1 isoform, velocity was reduced for deferiprone but remained similar for 4-nitrophenol or serotonin glucuronidation. In conclusion, deferiprone glucuronidation seems to depend almost totally on UGT1A6, especially in the liver. Genetic variations and differences in the expression of splice variants represent a potential source of variation in deferiprone metabolism.

Glucuronidation by the UDP-glucuronosyltransferase enzymes (UGTs) is one of the primary detoxification pathways of dietary heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons (PAHs). In a population-based case-control study of 537 cases and 866 controls, we investigated whether colon cancer was associated with genetic variations in UGT1A1 and UGT1A9 genes and we determined if those variations modify the association between colon cancer and dietary HCA and PAH exposure. We measured functional UGT1A1 polymorphisms at positions -53 (28; A(TA)6TAA to A(TA)7TAA), -3156 (G>A), -3279 (T>G) and the UGT1A9-275(T>A) polymorphism, and found no association with colon cancer overall. However, when stratified by race, the UGT1A1-3279 GG/TG intermediate/low activity genotypes were associated with an increased risk of colon cancer (odds ratio (OR)=1.5, 95% confidence interval (CI)=1.1-2.0) in Caucasians. This finding is also supported by haplotype analyses where the UGT1A1-3279G-allele-bearing haplotype is overrepresented in case group. Overall, UGT1A1-53 and -3156 genotypes modified the association between dietary benzo(a)pyrene (BaP) and colon cancer (P for interaction=0.02 and 0.03, respectively). The strongest association was observed for those with <7.7 ng/day BaP exposure and the low activity genotypes, for both UGT1A1 28/28 (OR=1.8, 95% CI=1.1-2.9) and -3156AA (OR=1.7, 95% CI=1.0-3.0), compared to >or=7.7 ng/day and combined high/intermediate genotypes. These data support a hypothesis that UGTs modify the association between meat-derived PAH exposure and colon cancer by their role in the elimination of dietary carcinogens.

Mycophenolate mofetil (MMF) is an immunosuppressive prodrug approved for use in transplantation. Its active metabolite, mycophenolic acid, is mainly metabolized by UDP-glucuronosyltransferase (UGT) enzymes. In this study, we retrospectively analyzed 74 kidney transplant patients who had been prescribed MMF as part of their immunosuppression regimen. Polymorphisms in UGT1A8 (-999C > T, codon 255A > G, codon 277G > A) were correlated with the occurrence of side effects, such as diarrhea, blood disorders, and infections. The infectious episodes were more frequently observed among individuals receiving MMF (2 g/d) who carryied the variant UGT1A8 codon 277A (P = .031), the haplotype UGT1A8H5 (-999C/codon 55A/codon 277A; P = .02), and the diplotype UGT1A8H2/H5 (-999CC/codon 255AA/codon 277GA; P = .015). The molecular data from this study suggest that UGT polymorphisms may be a factor influencing clinical outcomes among patients receiving MMF for transplant therapy; however, larger studies are warranted.

Polymorphisms in UGT1A9 were associated with reduced toxicity and increased response to irinotecan in cancer patients. UDP-glucuronosyltransferase (UGT) protein expression, glucuronidation activities for 7-ethyl-10-hydroxycamptothecin (SN-38), and probe substrates of the UGT1A9 and UGT1A1 were measured in 48 human livers to clarify the role of UGT1A9 variants on the in vitro glucuronidation of SN-38. Genotypes were assessed for UGT1A9 (-2152C>T, -275T>A, and -118T(9>10)), three novel UGT1A9 variants (-5366G>T, -4549T>C, and I399C>T), and UGT1A1 (-53TA(6>7), -3156G>A, and -3279T>G). Of all the variants, the UGT1A9 I399C>T was associated with the most dramatic change in SN-38-glucuronide (SN-38G) (2.64-fold; p = 0.0007). Compared with UGT1A9 I399C/C, homozygous I399T/T presented elevated UGT1A1 and UGT1A9 proteins and higher glucuronidation of UGT1A9 and UGT1A1 substrates (p < 0.05). The very low linkage disequilibrium (r(2) < 0.19) between UGT1A9 I399 and all the other UGT1A1 and UGT1A9 variants suggests a direct effect or linkage to unknown functional variant(s) relevant to SN-38 glucuronidation. The UGT1A9 -118T(9/10) was also linked to alteration of SN-38 glucuronidation profiles in the liver (p < 0.05) and was associated with higher UGT1A1 protein (p = 0.03). However, UGT1A9 -118T(10) appears to have low functional impact as a result of the lack of correlation with UGT1A9 protein levels and a modest 1.4-fold higher reporter gene expression associated with the -118T(10) allele in HepG2 cells (p = 0.004). In contrast, the UGT1A9 -5366T, -4549C, -2152T, and -275A, associated with higher UGT1A9 protein (2-fold; p < 0.05), have no influence on SN-38G. Despite limitations resulting from sample size, results indicate that UGT1A9 I399 and -118T(9/10) may represent additional candidates in combination with UGT1A1 promoter haplotypes for the prediction of SN-38 glucuronidation profile in vivo.

Purpose: Irinotecan is used in the first-line treatment of metastatic colorectal cancer. The UGT1A1-metabolizing enzyme, expressed in liver and colon, is primarily involved in the inactivation of its active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38). Herein, we explored the role of DNA methylation in the silencing of UGT1A1 gene expression in colon cancer and its influence on cellular SN-38 detoxification., Experimental Design and Results: UGT1A1 mRNA was repressed in most primary tumors (41 of 50; 82%) and in three colon cancer cell lines (HCT-116, HCT-15, and COLO-320DM). Bisulfite sequencing of the UGT1A1 gene revealed the aberrant methylation of specific CpG islands in UGT1A1-negative cells. Conversely, hypomethylation was observed in HT-29, HT-115, and LOVO cells that overexpress UGT1A1. Direct methylation of the UGT1A1 promoter resulted in the complete repression of transcriptional activity. Treatment with demethylating and histone deacetylase inhibitor agents had the capacity to reverse aberrant hypermethylation and to restore UGT1A1 expression in hypermethylated UGT1A1-negative cells but not in hypomethylated cells. Loss of UGT1A1 methylation was further associated with an increase in UGT1A1 protein content and with an enhanced inactivation of SN-38 by 300% in HCT-116 cells., Conclusions: We conclude that DNA methylation represses UGT1A1 expression in colon cancer and that this process may contribute to the level of tumoral inactivation of the anticancer agent SN-38 and potentially influence clinical response.

PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-f]pyridine), the most abundant heterocyclic amine in diet, is involved in the etiology of cancer. PhIP and its carcinogenic metabolite N-hydroxy-PhIP (N-OH-PhIP) are extensively conjugated by UDP-glucuronosyltransferase (UGTs) with wide variability. This study aimed to determine the genetic influence of UGTs on the hepatic detoxification of this carcinogen. The formation of N-OH-PhIP glucuronides was studied in 48 human liver samples by mass spectrometry. Liver samples were genotyped for common polymorphisms and correlated with UGT protein levels and N-OH-PhIP glucuronidation activities. The formation of four different N-OH-PhIP glucuronide metabolites was observed in all livers. The major metabolite was N-OH-PhIP-N(2)-glucuronide (N(2)G), which is the primary metabolite found in human urine, and showed a high interindividual variability (up to 28-fold). Using an heterologous expression system, the bilirubin-conjugating UGT1A1 enzyme was identified among all known UGTs (n = 16) as the predominant enzyme involved. The significant correlation between UGT1A1 protein content and formation of N(2)G (Rs = 0.87; P < .0001) suggests a critical role for UGT1A1 in the hepatic metabolism of this carcinogen. UGT1A1 expression was strongly determined by the presence of the common promoter polymorphisms, UGT1A1*28 (TATA box polymorphism) (P = .0031), -3156G/A (P = .0006) and -3279G/T (P = .0017), and rates of N(2)G were indeed correlated with these polymorphisms (P < .05), whether analyzed individually or in combination (haplotypes). In conclusion, UGT1A1 polymorphisms modulate the hepatic metabolism of the carcinogenic intermediate of PhIP and may determine the level of its exposure and potentially influence the risk of cancer through dietary exposure to HCAs.

UDP-glucuronosyltransferase (UGT) enzymes catalyze the glucuronidation reaction, which is a major pathway in the catabolism and elimination of numerous endo- and xenobiotics. Among the UGT enzyme family members, the UGT1A7, UGT1A8, UGT1A9, and UGT1A10 isoforms are issued from a single gene through differential splicing. However, these enzymes display distinct tissue-specific expression patterns. Indeed, UGT1A7, UGT1A8, and UGT1A10 are exclusively expressed in extrahepatic tissues, whereas UGT1A9 transcripts are found at high concentrations in liver. In the present study, we report that the liver-enriched hepatocyte nuclear factor 4 (HNF4)-alpha controls the hepatic expression of the UGT1A9 enzyme. Liver-specific disruption of the HNF4alpha gene in mice drastically decreases liver UGT1A9 mRNA levels. Furthermore, an HNF4alpha response element (HNF4alpha RE) was identified in the promoter of human UGT1A9 at position -372 to -360 base pairs by transient transfection, electrophoretic mobility shift assays, and chromatin immunoprecipitation experiments. It is interesting that this response element is absent in the proximal UGT1A7, UGT1A8, and UGT1A10 gene promoters. In conclusion, the present study identifies HNF4alpha as a major factor for the control of UGT1A9 hepatic expression and suggests that the absence of UGT1A7, UGT1A8, and UGT1A10 expression in the liver is caused by, at least in part, a few base pair changes in their promoter sequences in the region corresponding to the HNF4alpha RE of the UGT1A9 gene.

Objectives: Polymorphisms in UDP-glucuronosyltransferases (UGTs) can influence detoxifying capacities and have considerable therapeutic implications in addition to influence various (patho)physiological processes. UGT1A9 plays a central role in the metabolism of various classes of therapeutic drugs in addition to carcinogens and steroids. The great interindividual variability of UGT1A9-mediated glucuronidation remains poorly explained, while evidence for its genetic origin exists., Methods: The proximal UGT1A9 promoter was screened for polymorphisms by sequencing and, the contribution of single nucleotide polymorphisms (SNPs) to the variability of UGT1A9 protein levels and activity was evaluated., Results: We confirmed the presence of the -109 to -98 T10 polymorphism and found ten novel SNPs that generated a diversity of haplotypes in two independent populations. In a panel of 48 human liver microsomes, the UGT1A9 expression varied by 17-fold and was significantly correlated with SNPs -275, -331/-440, -665 and -2152. The base insertion T10 reported to increase reporter gene expression in HepG2 cells [] was not linked to -275 and -2152 SNPs and was not associated with changes in UGT1A9 protein levels. Compared to wild-type individuals, there were statistically significant higher glucuronidating activities in livers with the -275 and -2152 using mycophenolic acid and propofol as UGT1A9 substrates, indicating an extensive glucuronidator phenotype associated with these variants., Conclusions: This is the first study to demonstrate that naturally occurring sequence variations in the UGT1A9 promoter are informative in predicting the levels of protein and glucuronidating activity, providing a potential mechanism for interindividual variation in UGT1A9-mediated metabolism.

In vitro metabolic studies revealed that along with UDP-glucuronosyltransferase (UGT) 1A1, the hepatic UGT1A9 and the extrahepatic UGT1A7 are involved in the biotransformation of the active and toxic metabolite of irinotecan, 7-ethyl-10-hydroxycamptothecin (SN-38). Variant UGT1A1 and UGT1A7 alleles have been reported but the polymorphic nature of the UGT1A9 gene has not been revealed yet. To further clarify the molecular determinants of irinotecan-induced toxicity, we have identified and characterized the functionality of novel UGT1A9 polymorphisms and determined whether additional missense polymorphisms exist in UGT1A7. Using direct DNA sequencing, four single nucleotide polymorphisms (SNPs) were identified in the first exons of UGT1A7 and UGT1A9. One of the two amino acid substitutions found in the UGT1A9 gene, UGT1A9*3 (M33T), results in a dramatic decrease in SN-38 glucuronide formation, with 3.8% of the activity of the UGT1A9*1 allele. In turn, the glucuronidation of flavopiridol, an anticancer drug biotransformed predominantly by UGT1A9, remains unaffected, indicating a substrate-dependent impact of this variant. UGT1A9*3 is detected only in Caucasians and 4.4% of the population tested was found heterozygous (*1/*3). Two additional UGT1A7 SNPs were found exclusively in African-American subjects and generate five alleles (UGT1A7*5 to *9) when combined to the four known SNPs present in UGT1A7*2, *3, and *4. Upon functional analysis with SN-38, five out of nine UGT1A7 allozymes exhibited much lower SN-38 glucuronidation activities compared with UGT1A7*1, all having in common the mutational changes at codons 115 or 208. Results suggest that these low SN-38 glucuronidating alleles may represent additional molecular determinants of irinotecan-induced toxicity and warrant further investigations.

Peroxisome proliferator-activated receptor (PPAR) alpha and gamma are ligand-activated transcription factors belonging to the nuclear receptor family. PPAR alpha mediates the hypolipidemic action of the fibrates, whereas PPAR gamma is a receptor for the antidiabetic glitazones. In the present study, the UDP-glucuronosyltransferase (UGT) 1A9 enzyme is identified as a PPAR alpha and PPAR gamma target gene. UGTs catalyze the glucuronidation reaction, which is a major pathway in the catabolism and elimination of numerous endo- and xenobiotics. Among the UGT1A family enzymes, UGT1A9 metabolizes endogenous compounds, including catecholestrogens, and xenobiotics, such as fibrates and to a lesser extent troglitazone. Treatment of human hepatocytes and macrophages and murine adipocytes with activators of PPAR alpha or PPAR gamma resulted in an enhanced UGT1A9 expression and activity. In addition, disruption of the PPAR alpha gene in mice completely abolished the PPAR alpha agonist-induced UGT1A9 mRNA and activity levels. A PPAR response element was identified in the promoter of UGT1A9 at positions -719 to -706 bp by transient transfection and electromobility shift assays. Considering the role of UGT1A9 in catecholestrogen metabolism, PPAR alpha and PPAR gamma activation may contribute to the protection against genotoxic catecholestrogens by stimulating their inactivation in glucuronide derivatives. Furthermore, since UGT1A9 is involved in the catabolism of fibrates, these results suggest that PPAR alpha and PPAR gamma may control the intracellular level of active fibrates.