RESUMO
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.
Assuntos
Perfilação da Expressão Gênica/métodos , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Preparações Farmacêuticas/metabolismo , Variantes Farmacogenômicos/genética , Transcriptoma/fisiologia , Humanos , Taxa de Depuração Metabólica/efeitos dos fármacos , Taxa de Depuração Metabólica/fisiologia , Preparações Farmacêuticas/administração & dosagem , Distribuição Tecidual/efeitos dos fármacos , Distribuição Tecidual/fisiologia , Transcriptoma/efeitos dos fármacosRESUMO
Previously, we observed strong positive associations between circulating concentrations of free testosterone and free dihydrotestosterone (DHT) in relation to Barrett's esophagus in a US male military population. To replicate these findings, we conducted a second study of sex steroid hormones and Barrett's esophagus in the Factors Influencing the Barrett/Adenocarcinoma Relationship (FINBAR) Study based in Northern Ireland and Ireland. We used mass spectrometry to quantitate EDTA plasma concentrations of nine sex steroid hormones and ELISA to quantitate sex hormone-binding globulin in 177 male Barrett's esophagus cases and 185 male general population controls within the FINBAR Study. Free testosterone, free DHT, and free estradiol were estimated using standard formulas. Multivariable logistic regression estimated odds ratios (OR) and 95% confidence intervals (95%CI) of associations between exposures and Barrett's esophagus. While plasma hormone and sex hormone-binding globulin concentrations were not associated with all cases of Barrett's esophagus, we did observe positive associations with estrogens in younger men (e.g. estrone + estradiol ORcontinuous per ½IQR = 2.92, 95%CI:1.08, 7.89), and free androgens in men with higher waist-to-hip ratios (e.g. free testosterone ORcontinuous per ½IQR = 2.71, 95%CI:1.06, 6.92). Stratification by body mass index, antireflux medications, and geographic location did not materially affect the results. This study found evidence for associations between circulating sex steroid hormones and Barrett's esophagus in younger men and men with higher waist-to-hip ratios. Further studies are necessary to elucidate whether sex steroid hormones are consistently associated with esophageal adenocarcinogenesis.
Assuntos
Esôfago de Barrett/sangue , Di-Hidrotestosterona/sangue , Estradiol/sangue , Globulina de Ligação a Hormônio Sexual/metabolismo , Testosterona/sangue , Fatores Etários , Idoso , Índice de Massa Corporal , Ensaio de Imunoadsorção Enzimática , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
Cyclic adenosine monophosphate (cAMP) plays a crucial role as a signaling molecule for capacitation, motility, and acrosome reaction in mammalian spermatozoa. It is well-known that cAMP degradation by phosphodiesterase (PDE) enzyme has a major impact on sperm functions. This study was undertaken to characterize cAMP-PDE activity in bovine spermatozoa. Total cAMP-PDE activity in cauda epididymal and ejaculated spermatozoa was 543.2 ± 49.5 and 1252.6 ± 86.5 fmoles/min/106 spermatozoa, respectively. Using different family-specific PDE inhibitors, we showed that in cauda epididymal and ejaculated spermatozoa, the major cAMP-PDE activity was papaverine-sensitive (44.5% and 57.5%, respectively, at 400 nm, papaverine is a specific inhibitor of the PDE10 family). These data are supporting the functional presence of PDE10 in bovine spermatozoa and were further confirmed by western blot to be PDE10A. Using immunocytochemistry, we showed immunoreactive signal for PDE10A present on the post-acrosomal region of the head and on the flagella of ejaculated spermatozoa. Using papaverine, we showed that it promotes tyrosine phosphorylation of sperm proteins, phosphorylation of Erk1 and Erk2, and Ca2+ release from Ca2+ store. These results suggest that PDE10 is functionally present in bovine spermatozoa and is affecting different molecular events involved in capacitation, most probably by cAMP local regulation.
Assuntos
Papaverina/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Espermatozoides/enzimologia , Reação Acrossômica/efeitos dos fármacos , Animais , Cálcio/metabolismo , Bovinos , Epididimo/citologia , Epididimo/metabolismo , Masculino , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Espermatozoides/efeitos dos fármacosRESUMO
The second messenger cyclic adenosine monophosphate (cAMP) has a central role in sperm physiology. Extracellular cAMP can be sequentially degraded into 5'AMP and adenosine by ecto-phosphodiesterases (ecto-PDE) and ecto-nucleotidases, a phenomenon called extracellular cAMP-adenosine pathway. As cAMP-adenosine pathway is involved in sperm capacitation, we hypothesize that extracellular PDEs are functionally present in seminal plasma. Exclusively measuring cAMP-PDE activity, total activity in bovine seminal plasma was 10.1 ± 1.5 fmoles/min/µg. Using different family-specific PDE inhibitors, we showed that in seminal plasma, the major cAMP-PDE activity was papaverine sensitive (47.5%). These data support the presence of PDE10 in bovine seminal plasma and was further confirmed by western blot. In epididymal fluid, total cAMP-PDE activity was 48.2 ± 14.8 fmoles/min/µg and we showed that the major cAMP-PDE activity was 3-isobutyl-methylxanthine insensitive and thus ascribed to PDE8 family. PDE10A mRNAs were found in the testis, epididymis, and seminal vesicles. cAMP-PDE activity is present in bovine seminal plasma and epididymal fluid. The results suggest a role for ecto-PDEs present in those fluids in the signaling pathways involved in sperm functions.
Assuntos
AMP Cíclico/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Sêmen/metabolismo , Animais , Bovinos , Masculino , Inibidores de Fosfodiesterase/farmacologia , Sêmen/efeitos dos fármacos , Espermatozoides/metabolismoRESUMO
The antiretroviral protease inhibitor atazanavir inhibits hepatic uridine diphosphate glucuronosyltransferase (UGT) 1A1, thereby preventing the glucuronidation and elimination of bilirubin. Resultant indirect hyperbilirubinemia with jaundice can cause premature discontinuation of atazanavir. Risk for bilirubin-related discontinuation is highest among individuals who carry two UGT1A1 decreased function alleles (UGT1A1*28 or *37). We summarize published literature that supports this association and provide recommendations for atazanavir prescribing when UGT1A1 genotype is known (updates at www.pharmgkb.org).
Assuntos
Sulfato de Atazanavir/efeitos adversos , Glucuronosiltransferase/antagonistas & inibidores , Inibidores da Protease de HIV/efeitos adversos , Hiperbilirrubinemia/induzido quimicamente , Icterícia/induzido quimicamente , Fígado/efeitos dos fármacos , Farmacogenética/normas , Predisposição Genética para Doença , Genótipo , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Hiperbilirrubinemia/enzimologia , Hiperbilirrubinemia/genética , Icterícia/enzimologia , Icterícia/genética , Fígado/enzimologia , Fenótipo , Medição de Risco , Fatores de RiscoRESUMO
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.
Assuntos
Glucuronosiltransferase/genética , Desintoxicação Metabólica Fase II/genética , Transcriptoma , Mama/enzimologia , Neoplasias da Mama/enzimologia , Endométrio/enzimologia , Feminino , Glucuronosiltransferase/metabolismo , Humanos , Neoplasias Intestinais/enzimologia , Intestinos/enzimologia , Rim/enzimologia , Neoplasias Renais/enzimologia , Fígado/enzimologia , Neoplasias Hepáticas/enzimologia , Masculino , Especificidade de Órgãos , Próstata/enzimologia , Neoplasias da Próstata/enzimologia , RNA Mensageiro/metabolismo , Análise de Sequência de RNA/métodos , Neoplasias Uterinas/enzimologiaRESUMO
Acquisition of oocyte developmental competence needs to be understood to improve clinical outcomes of assisted reproduction. The stimulation of cumulus cell concentration of cyclic adenosine 3'5'-monophosphate (cAMP) by pharmacological agents during in vitro maturation (IVM) participates in improvement of oocyte quality. However, precise coordination and downstream targets of cAMP signaling in cumulus cells are largely unknown. We have previously demonstrated better embryo development after cAMP stimulation for first 6 h during IVM. Using this model, we investigated cAMP signaling in cumulus cells through in vitro culture of cumulus-oocyte complexes (COCs) in the presence of cAMP raising agents: forskolin, IBMX, and dipyridamole (here called FID treatment). Transcriptomic analysis of cumulus cells indicated that FID-induced differentially expressed transcripts were implicated in cumulus expansion, steroidogenesis, cell metabolism, and oocyte competence. Functional genomic analysis revealed that protein kinase-A (PKA), extracellular signal regulated kinases (ERK1/2), and calcium (Ca(2+)) pathways as key regulators of FID signaling. Inhibition of PKA (H89) in FID-supplemented COCs or substitution of FID with calcium ionophore (A23187) demonstrated that FID activated primarily the PKA pathway which inhibited ERK1/2 phosphorylation and was upstream of calcium signaling. Furthermore, inhibition of ERK1/2 phosphorylation by FID supported a regulation by dual specific phosphatase (DUSP1) via PKA. Our findings imply that cAMP (FID) regulates cell metabolism, steroidogenesis, intracellular signaling and cumulus expansion through PKA which modulates these functions through optimization of ERK1/2 phosphorylation and coordination of calcium signaling. These findings have implications for development of new strategies for improving oocyte in vitro maturation leading to better developmental competence.
Assuntos
Células do Cúmulo/fisiologia , AMP Cíclico/metabolismo , Técnicas de Maturação in Vitro de Oócitos/métodos , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Cálcio/metabolismo , Bovinos , Células Cultivadas , Colforsina/farmacologia , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dipiridamol/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Understanding the mechanisms regulating oocyte developmental competence is essential to enhance the clinical efficiency of assisted reproduction. FSH orchestrates the acquisition of oocyte competence, both in vivo and in vitro. Multiple pathways are implicated in FSH signalling; however, their precise coordination remains unresolved. A robust system to investigate FSH signalling is oocyte in vitro maturation (IVM) and we have previously demonstrated better bovine embryo development after FSH addition for the first 6 h during IVM. Using this model, we investigated FSH signalling in cumulus through transcriptomic and pharmacological tools. We demonstrate modulation of cumulus transcriptome by FSH mainly through protein kinase A (PKA) and epidermal growth factor (EGF) pathways. Differentially expressed transcripts were implicated in cumulus expansion, steroidogenesis, cell metabolism and oocyte competence. FSH required rouse-sarcoma oncogene (SRC) for EGF receptor transactivation. PKA and EGF pathway crosstalk was investigated using extracellular signal-regulated kinases (ERK1/2) phosphorylation as the functional end-point. FSH enhanced ERK1/2 activation by the EGF pathway with a simultaneous diminution through PKA. More specifically, FSH increased dual specific phosphatase (DUSP1) transcripts via PKA although DUSP1 protein did not change since EGF was required to prevent degradation. Our findings implicate FSH in PKA and EGF pathway activation, which interact to maintain appropriate levels of ERK1/2 phosphorylation and eventually cumulus expansion, metabolism and steroidogenesis. Moreover, considering the implication of the EGF pathway in GDF9 and BMP15 actions, our findings suggest that FSH may have a role in modulation of the cumulus response to oocyte-secreted factors. This information has implications for improvement of IVM and hence oocyte developmental competence.
Assuntos
Células do Cúmulo/efeitos dos fármacos , Fármacos para a Fertilidade/farmacologia , Hormônio Foliculoestimulante/farmacologia , Oócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Bovinos , Biologia Computacional , Células do Cúmulo/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fosfatase 1 de Especificidade Dupla/metabolismo , Fator de Crescimento Epidérmico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Técnicas de Maturação in Vitro de Oócitos , Oócitos/metabolismo , Fosforilação , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Fatores de Tempo , TranscriptomaRESUMO
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.
Assuntos
Camptotecina/análogos & derivados , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Glucuronosiltransferase/genética , Neutropenia/induzido quimicamente , Neutropenia/genética , Antineoplásicos Fitogênicos/efeitos adversos , Antineoplásicos Fitogênicos/uso terapêutico , Biomarcadores Tumorais/genética , Camptotecina/efeitos adversos , Camptotecina/uso terapêutico , Canadá , Feminino , Testes Genéticos/métodos , Haplótipos/genética , Humanos , Irinotecano , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Glucuronidation by uridine diphospho-glucuronosyltransferase enzymes (UGTs) is a major phase II biotransformation pathway and, complementary to phase I metabolism and membrane transport, one of the most important cellular defense mechanisms responsible for the inactivation of therapeutic drugs, other xenobiotics, and endogenous molecules. Interindividual variability in UGT pathways is significant and may have profound pharmacological and toxicological implications. Several genetic and genomic processes underlie this variability and are discussed in relation to drug metabolism and diseases such as cancer.
Assuntos
Variação Genética , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Farmacogenética , Interações Medicamentosas , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/enzimologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/genética , Genótipo , Glucuronídeos/metabolismo , Humanos , Isoenzimas , Desintoxicação Metabólica Fase II/genética , Farmacocinética , Fenótipo , Medicina de Precisão , Medição de Risco , Fatores de RiscoRESUMO
The UDP-glucuronosyltransferase UGT1A gene is a major biotransformation gene involved in the metabolism of a vast array of molecules. Recently, we uncovered a new series of alternative spliced isoforms referred to as isoforms 2 or UGT1As_i2 that use an alternative exon 5 (5b). The function of such mRNAs and the corresponding 45 kDa proteins still remains unclear. Although devoid of glucuronosyltransferase activity, UGT1As_i2 are widely co-expressed with the enzymatically active and classical UGT1A isoforms (UGT1As_i1). In this study, we observed abundant signal in human colon tissue samples, predominantly along intestinal crypts. In human cells, UGT1A_i2 proteins are expressed in similar subcellular compartments as UGT1As_i1. Cellular properties of i2-spliced forms were then studied using synthetic small-interfering RNA (siRNA) in two human colon cancer cell lines that show a significant amount of exon 5a- and exon 5b-containing mRNAs and that display enzymatic activities for UGT1As substrates. We observed that siRNA-mediated knockdown of endogenous i2 upregulates cellular glucuronidation activities by 120-170% (P<0.01) for all substrates tested. Functional data support a dominant-negative function for endogenous exon 5b-spliced forms of UGT1A, hence potentially affecting in vivo glucuronidation capacity. This new regulatory strategy may ensure an additional mean to modulate cellular response to endo/xeno stimulus.
Assuntos
Processamento Alternativo , Glucuronídeos/biossíntese , Glucuronosiltransferase/genética , Western Blotting , Linhagem Celular Tumoral , Colo/enzimologia , Colo/patologia , Neoplasias do Colo/enzimologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Humanos , Imuno-Histoquímica , Desintoxicação Metabólica Fase II , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade por SubstratoRESUMO
Previous studies have shown that using nonspecific phosphodiesterase (PDE) inhibitors such as caffeine improved milk production, supporting the premise that modulation of intracellular concentration of cyclic nucleotides (cyclic AMP, cyclic guanosine 3'-5'-monophosphate) is involved. Intracellular cyclic nucleotides are degraded by the PDE enzyme family. The contribution of type IV PDE (PDE4) in the secretion of casein has been reported in rat mammary gland. The objective of this study was to demonstrate the functional presence of the PDE4 family in the bovine mammary gland. To understand the enzymatic expression pattern in the mammary gland, tissue samples were taken randomly from udders obtained from a local slaughterhouse. Reverse transcription PCR revealed that the PDE4D transcript was amplified, and the expected size fragment was obtained in a 1% agarose gel. Sequence analysis of the amplicon resulted in 99% homology to PDE4D. Moreover, Western blotting using a specific PDE4D antibody has confirmed that the protein of the isoenzyme PDE4D1 is present. A clear immunoreactive signal was also observed within the acini where epithelial cells are located. Assaying cyclic AMP PDE activity reported a total activity of 38.71 +/- 3.22 fmol/min per microg of total protein. Rolipram, a specific PDE4 inhibitor, showed a sensitive activity of 8.48 +/- 1.28 fmol/min per microg of total protein, indicating that PDE4 is responsible for one-fifth of the total enzymatic activity of PDE in the mammary gland. To further validate the presence of PDE4D in the bovine mammary epithelial cells, protein extracts from bovine mammary epithelial cells were separated on SDS-PAGE gels, and PDE4D protein was detected. The PDE assays reported a total activity of 30.16 +/- 4.82 fmol/min per microg of total protein. Rolipram showed a sensible activity of 11.91 +/- 5.93 fmol/min per microg of total protein. In conclusion, these results not only demonstrate the presence of PDE4D transcript and protein, but also show an active enzyme, suggesting a functional role of PDE4D in bovine mammary gland.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Glândulas Mamárias Animais/enzimologia , Animais , Bovinos , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glândulas Mamárias Animais/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Glucuronosiltransferase/genética , Transplante de Rim/imunologia , Ácido Micofenólico/análogos & derivados , Polimorfismo de Nucleotídeo Único , Códon/genética , Diarreia/induzido quimicamente , Doenças Hematológicas/induzido quimicamente , Humanos , Infecções/epidemiologia , Ácido Micofenólico/efeitos adversos , Estudos RetrospectivosRESUMO
We studied whether polymorphisms in the UGT1A8, UGT1A9, and UGT2B7 genes, the enzymes producing the phenolic (MPAG) and acyl (AcMPAG) glucuronides of mycophenolic acid (MPA), could contribute to the interindividual variation observed in mycophenolate mofetil (MMF) pharmacokinetics (PKs). This study enrolled 17 healthy volunteers with no polymorphisms (controls) and 17 carriers of UGT1A9 -275/-2152 selected among 305 individuals genetically screened for UDP-glucuronosyltransferase (UGT) polymorphisms. Additional investigative groups included carriers of UGT1A8*2 (A173G) (n=9), UGT1A8*3 (C277Y) (n=4), and UGT1A9*3 (M33T) (n=5). Genetic analysis also included UGT2B7 to detect UGT2B7*2 (His268Tyr) and the promoter haplotype -1248A>G, -1241T>C, -1054T>C, -842G>A, -268A>G, -102T>C. Kinetics were measured in plasma and urine after a single 1.5 g oral dose of MMF, by high-performance liquid chromatography coupled with tandem mass spectrometry, over 12 h after drug intake. Compared to controls, MPA exposure was significantly lower for UGT1A9 -275/-2152 carriers, with no significant changes in MPAG. The estimates of enterohepatic (re)cycling (area under the concentration-time curve (AUC6-12 h/AUC0-12 h)) were significantly lower for MPA, MPAG, and AcMPAG in UGT1A9 -275/-2152 subjects. Compared with controls, UGT1A9*3 carriers had higher MPA and AcMPAG exposure, whereas homozygosity for the UGT1A8*2 allele and heterozygosity for UGT1A8*3 allele had no impact on MPA PKs. Compared with UGT2B7*1/*1 individuals (n=10), UGT2B7*2/*2 subjects (n=17) presented significantly higher free MPA C(max) values and elevated free and total MPA. Results indicate that after a single oral dose of MMF in healthy volunteers, specific UGT genotypes significantly alter MPA PKs and this clearly warrants additional studies with complete and detailed genetic profiling of UGT1A8, UGT1A9, and UGT2B7 genes.
Assuntos
Antibióticos Antineoplásicos/farmacocinética , Glucuronosiltransferase/genética , Ácido Micofenólico/farmacocinética , Polimorfismo Genético/fisiologia , Adolescente , Adulto , Área Sob a Curva , Disponibilidade Biológica , Biotransformação , Estudos de Coortes , Feminino , Frequência do Gene , Haplótipos , Heterozigoto , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , UDP-Glucuronosiltransferase 1ARESUMO
UDP-glucuronosyltransferase (UGT) enzymes comprise a superfamily of key proteins that catalyze the glucuronidation reaction on a wide range of structurally diverse endogenous and exogenous chemicals. Glucuronidation is one of the major phase II drug-metabolizing reactions that contributes to drug biotransformation. This biochemical process is also involved in the protection against environmental toxicants, carcinogens, dietary toxins and participates in the homeostasis of numerous endogenous molecules, including bilirubin, steroid hormones and biliary acids. Over the years, significant progress was made in the field of glucuronidation, especially with regard to the identification of human UGTs, study of their tissue distribution and substrate specificities. More recently, the degree of allelic diversity has also been revealed for several human UGT genes. Some polymorphic UGTs have demonstrated a significant pharmacological impact in addition to being relevant to drug-induced adverse reactions and cancer susceptibility. This review focuses on human UGTs, the description of the nature of polymorphic variations and their functional impact. The pharmacogenomic implication of polymorphic UGTs is presented, more specifically the role of UGT polymorphisms in modifying cancer risk and their impact on individual risk to drug-induced toxicities.
Assuntos
Glucuronosiltransferase/genética , Farmacogenética/métodos , Animais , Glucuronosiltransferase/metabolismo , HumanosRESUMO
BACKGROUND: UDP-glucuronosyltransferase 1A7 (UGT1A7) detoxifies several tobacco carcinogens. We determined whether UGT1A7 expression is observed in normal orolaryngeal tissue and whether UGT1A7 allelic variations are associated with the risk for orolaryngeal cancer. METHODS: UGT1A7 expression in normal orolaryngeal tissue was determined by semiquantitative reverse transcription-polymerase chain reaction (PCR). Buccal cell DNA isolated from 194 case subjects with orolaryngeal cancer and from 388 control subjects who were matched by sex, age, and race was subjected to UGT1A7 genotyping with the use of combined PCR-restriction fragment length polymorphism and allelic discrimination analysis. All statistical tests were two-sided. RESULTS: UGT1A7 messenger RNA was expressed at similar levels in the esophagus, tongue, tonsil, floor of the mouth, and larynx. Genotyping revealed the presence of three variant reduced-activity UGT1A7 alleles in both Caucasians and African-Americans. Individuals with any of the predicted low-activity UGT1A7 genotypes had an increased risk of orolaryngeal cancer (odds ratio [OR] = 3.7; 95% confidence interval [CI] = 1.7 to 8.7) relative to subjects with the wild-type genotype. Both Caucasians and African-Americans with the low-activity genotypes had statistically significantly increased orolaryngeal cancer risk compared with Caucasians and African-Americans with the wild-type genotype (OR = 2.8 [95% CI = 1.1 to 7.6] and OR = 6.2 [95% CI = 1.2 to 31], respectively). For subjects with the predicted low-activity genotypes, the risks of oral cavity cancer (OR = 4.2; 95% CI = 1.7 to 10) and laryngeal cancer (OR = 3.7; 95% CI = 0.99 to 14) were similar. There was no association between UGT1A7 genotype and orolaryngeal cancer risk in never smokers, whereas subjects with predicted low-activity UGT1A7 genotypes who were light smokers (OR = 3.7; 95% CI = 1.1 to 12) or heavy smokers (OR = 6.1; 95% CI = 1.5 to 25) had an increased risk. CONCLUSIONS: The tissue expression of UGT1A7 is consistent with the possibility of a physiologic role in orolaryngeal cancer. Variations in the UGT1A7 gene that reduce UGT1A7 activity may affect the risk of smoking-related orolaryngeal cancer.
Assuntos
Carcinógenos/farmacocinética , Carcinoma de Células Escamosas/enzimologia , Glucuronosiltransferase/fisiologia , Neoplasias Laríngeas/enzimologia , Neoplasias Bucais/enzimologia , Nicotiana , Fumaça/análise , Alelos , Substituição de Aminoácidos , População Negra/genética , Carcinoma de Células Escamosas/epidemiologia , Carcinoma de Células Escamosas/etiologia , Códon , Estudos de Coortes , Esôfago/enzimologia , Feminino , Predisposição Genética para Doença , Genótipo , Glucuronosiltransferase/análise , Glucuronosiltransferase/genética , Humanos , Inativação Metabólica/genética , Neoplasias Laríngeas/epidemiologia , Neoplasias Laríngeas/etiologia , Laringe/enzimologia , Estilo de Vida , Fígado/enzimologia , Masculino , Pessoa de Meia-Idade , Boca/enzimologia , Neoplasias Bucais/epidemiologia , Neoplasias Bucais/etiologia , New York/epidemiologia , Especificidade de Órgãos , Tonsila Palatina/enzimologia , Philadelphia/epidemiologia , Polimorfismo Genético , Risco , Fumar/efeitos adversos , Inquéritos e Questionários , Língua/enzimologia , População Branca/genéticaRESUMO
UDP-glucuronosyltransferases (UGTs) catalyze the detoxification and the elimination of a large number of endogenous and exogenous compounds in the liver and extrahepatic tissues. One of the UGT1A family members, UGT1A1, is involved in estradiol metabolism and, therefore, represents a candidate gene in breast carcinogenesis. A common insertion/deletion polymorphism in the TATA-box of the promoter region of UGT1A1 results in decreased initiation of transcription. In a previous study, we found a positive association between the UGT1A1 low-transcriptional alleles and premenopausal breast cancer risk in an African-American population. In the present study, we sought to determine whether the low-transcription UGT1A1 promoter allele, UGT1A1*28 [A(TA)(7)TAA], was associated with increased breast cancer risk among primarily Caucasian women in a nested case-control study within the Nurses' Health Study cohort. No significant association between the UGT1A1*28 [A(TA)(7)TAA] allele and breast cancer was observed. Compared with women homozygous for the UGT1A1*1 [A(TA)(6)TAA] allele, the relative risk was 0.80 (confidence interval, 0.49-1.29) for women homozygous for the UGT1A1*28 allele. The effect of the UGT1A1 genotype on plasma hormone levels in postmenopausal women not using hormone replacement was also evaluated, and overall, no significant differences in hormone levels by genotypes were observed. When restricted to women who had at least one UGT1A1*28 allele and a body mass index at blood draw of >27 kg/m(2), particularly in combination with the cytochrome p450c17alpha genotype, estrone and estradiol levels tended to vary by UGT1A1 genotypes. The results presented do not support a strong association between the UGT1A1 promoter polymorphism and the risk of breast cancer.
Assuntos
Neoplasias da Mama/genética , Estradiol/metabolismo , Glucuronosiltransferase/genética , Polimorfismo Genético , Adulto , Neoplasias da Mama/patologia , Estudos de Casos e Controles , Estradiol/sangue , Feminino , Genótipo , Humanos , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Fatores de Risco , TATA Box/genética , Ativação TranscricionalRESUMO
One of the most important mechanisms involved in host defense against xenobiotic chemicals and endogenous toxins is the glucuronidation catalysed by UDP-glucuronosyltransferase enzymes (UGT). The role of genetic factors in determining variable rates of glucuronidation is not well understood, but phenotypic evidence in support of such variation has been reported. In the present study, six single nucleotide polymorphisms were discovered in the first exon of the UGT1A7 gene, which codes for the putative substrate-binding domain, revealing a high structural heterogeneity at the UGT1 gene locus. The new UGT1A7 proteins differ in their primary structure at amino acid positions 129, 131 and 208, creating four distinct UGT1A7 allelic variants in the human population: UGT1A7*1 (N129 R131 W208), *2 (K129 K131 W208), *3 (K129 K131 R208), and *4 (N129 R131 R208). In functional studies, HEK cells stably transfected to express the four allelic UGT1A7 variants exhibited significant differences in catalytic activity towards 3-, 7-, and 9-hydroxy-benzo(a)pyrene. UGT1A7*3 exhibited a 5.8-fold lower relative Vmax compared to wild-type *1, whereas *2 and *4 had a 2.6- and 2.8-fold lower relative Vmax than *1, respectively, suggesting that these mutations confer slow glucuronidation phenotype. Kinetic characterization suggested that these differences were primarily attributable to altered Vmax. Additionally, it suggested that each amino acid substitutions can independently affect the UGT1A7 catalytic activity, and that their effects are additive. The expression pattern of UGT1A7 studied herein and its catalytic activity profile suggest a possible role of UGT1A7 in the detoxification and elimination of carcinogenic products in lung. A population study demonstrated that a considerable proportion of the population (15.3%) was found homozygous for the low activity allele containing all three missense mutations, UGT1A7*3. These findings suggest that further studies are needed to investigate the impact of the low UGT1A7 conjugator genotype on individual susceptibility to chemical-induced diseases and responses to therapeutic drugs.
Assuntos
Glucuronosiltransferase/genética , Mutação de Sentido Incorreto , Sequência de Bases , Linhagem Celular Transformada , Primers do DNA , Feminino , Genótipo , Glucuronosiltransferase/metabolismo , Humanos , Cinética , Masculino , Linhagem , Reação em Cadeia da PolimeraseRESUMO
We have implemented a method for multiplexed detection of polymorphic sites and direct determination of haplotypes in 10-kilobase-size DNA fragments using single-walled carbon nanotube (SWNT) atomic force microscopy (AFM) probes. Labeled oligonucleotides are hybridized specifically to complementary target sequences in template DNA, and the positions of the tagged sequences are detected by direct SWNT tip imaging. We demonstrated this concept by detecting streptavidin and IRD800 labels at two different sequences in M13mp18. Our approach also permits haplotype determination from simple visual inspection of AFM images of individual DNA molecules, which we have done on UGT1A7, a gene under study as a cancer risk factor. The haplotypes of individuals heterozygous at two critical loci, which together influence cancer risk, can be easily and directly distinguished from AFM images. The application of this technique to haplotyping in population-based genetic disease studies and other genomic screening problems is discussed.