RESUMEN
Identifying the molecular mechanisms by which genome-wide association study (GWAS) loci influence traits remains challenging. Chromatin accessibility quantitative trait loci (caQTLs) help identify GWAS loci that may alter GWAS traits by modulating chromatin structure, but caQTLs have been identified in a limited set of human tissues. Here we mapped caQTLs in human liver tissue in 20 liver samples and identified 3,123 caQTLs. The caQTL variants are enriched in liver tissue promoter and enhancer states and frequently disrupt binding motifs of transcription factors expressed in liver. We predicted target genes for 861 caQTL peaks using proximity, chromatin interactions, correlation with promoter accessibility or gene expression, and colocalization with expression QTLs. Using GWAS signals for 19 liver function and/or cardiometabolic traits, we identified 110 colocalized caQTLs and GWAS signals, 56 of which contained a predicted caPeak target gene. At the LITAF LDL-cholesterol GWAS locus, we validated that a caQTL variant showed allelic differences in protein binding and transcriptional activity. These caQTLs contribute to the epigenomic characterization of human liver and help identify molecular mechanisms and genes at GWAS loci.
Asunto(s)
Cromatina/metabolismo , Hígado/metabolismo , Sitios de Carácter Cuantitativo , Secuencias de Aminoácidos , Sitios de Unión , Ensamble y Desensamble de Cromatina , Elementos de Facilitación Genéticos , Variación Genética , Estudio de Asociación del Genoma Completo , Humanos , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción/química , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
Hepatic flavin-containing mono-oxygenase 3 (FMO3) metabolizes a broad array of nucleophilic heteroatom (e.g., N or S)-containing xenobiotics (e.g., amphetamine, sulindac, benzydamine, ranitidine, tamoxifen, nicotine, and ethionamide), as well as endogenous compounds (e.g., catecholamine and trimethylamine). To predict the effect of genetic and nongenetic factors on the hepatic metabolism of FMO3 substrates, we quantified FMO3 protein abundance in human liver microsomes (HLMs; n = 445) by liquid chromatography-tandem mass chromatography proteomics. Genotyping/gene resequencing, mRNA expression, and functional activity (with benzydamine as probe substrate) of FMO3 were also evaluated. FMO3 abundance increased 2.2-fold (13.0 ± 11.4 pmol/mg protein vs. 28.0 ± 11.8 pmol/mg protein) from neonates to adults. After 6 years of age, no significant difference in FMO3 abundance was found between children and adults. Female donors exhibited modestly higher mRNA fragments per kilobase per million reads values (139.9 ± 76.9 vs. 105.1 ± 73.1; P < 0.001) and protein FMO3 abundance (26.7 ± 12.0 pmol/mg protein vs. 24.1 ± 12.1 pmol/mg protein; P < 0.05) compared with males. Six single nucleotide polymorphisms (SNPs), including rs2064074, rs28363536, rs2266782 (E158K), rs909530 (N285N), rs2266780 (E308G), and rs909531, were associated with significantly decreased protein abundance. FMO3 abundance in individuals homozygous and heterozygous for haplotype 3 (H3), representing variant alleles for all these SNPs (except rs2066534), were 50.8% (P < 0.001) and 79.5% (P < 0.01), respectively, of those with the reference homozygous haplotype (H1, representing wild-type). In summary, FMO3 protein abundance is significantly associated with age, gender, and genotype. These data are important in predicting FMO3-mediated heteroatom-oxidation of xenobiotics and endogenous biomolecules in the human liver.
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Hígado/enzimología , Oxigenasas/genética , Oxigenasas/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/genética , Envejecimiento/metabolismo , Niño , Preescolar , Estudios de Cohortes , Femenino , Genotipo , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Caracteres Sexuales , Adulto JovenRESUMEN
A large haplotype on chromosome 19p13.11 tagged by rs10401969 in intron 8 of SURP and G patch domain containing 1 (SUGP1) is associated with coronary artery disease (CAD), plasma LDL cholesterol levels, and other energy metabolism phenotypes. Recent studies have suggested that TM6SF2 is the causal gene within the locus, but we postulated that this locus could harbor additional CAD risk genes, including the putative splicing factor SUGP1 Indeed, we found that rs10401969 regulates SUGP1 exon 8 skipping, causing non-sense-mediated mRNA decay. Hepatic Sugp1 overexpression in CD1 male mice increased plasma cholesterol levels 20-50%. In human hepatoma cell lines, SUGP1 knockdown stimulated 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) alternative splicing and decreased HMGCR transcript stability, thus reducing cholesterol synthesis and increasing LDL uptake. Our results strongly support a role for SUGP1 as a novel regulator of cholesterol metabolism and suggest that it contributes to the relationship between rs10401969 and plasma cholesterol.
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LDL-Colesterol/genética , Colesterol/genética , Enfermedad de la Arteria Coronaria/genética , Metabolismo de los Lípidos/genética , Factores de Empalme de ARN/genética , Empalme Alternativo/genética , Animales , Colesterol/sangre , LDL-Colesterol/sangre , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/patología , Exones/genética , Regulación de la Expresión Génica , Haplotipos , Células Hep G2 , Humanos , Masculino , Ratones , Polimorfismo de Nucleótido Simple , Factores de Empalme de ARN/biosíntesis , Estabilidad del ARNRESUMEN
The cytochrome P450 (P450) enzymes are the predominant enzyme system involved in human drug metabolism. Alterations in the expression and/or activity of these enzymes result in changes in pharmacokinetics (and consequently the pharmacodynamics) of drugs that are metabolized by this set of enzymes. Apart from changes in activity as a result of drug-drug interactions (by P450 induction or inhibition), the P450 enzymes can exhibit substantial interindividual variation in basal expression and/or activity, leading to differences in the rates of drug elimination and response. This interindividual variation can result from a myriad of factors, including genetic variation in the promoter or coding regions, variation in transcriptional regulators, alterations in microRNA that affect P450 expression, and ontogenic changes due to exposure to xenobiotics during the developmental and early postnatal periods. Other than administering a probe drug or cocktail of drugs to obtain the phenotype or conducting a genetic analysis to determine genotype, methods to determine interindividual variation are limited. Phenotyping via a probe drug requires exposure to a xenobiotic, and genotyping is not always well correlated with phenotype, making both methodologies less than ideal. This article describes recent work evaluating the effect of some of these factors on interindividual variation in human P450-mediated metabolism and the potential utility of endogenous probe compounds to assess rates of drug metabolism among individuals.
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Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Variación Genética/genética , Inactivación Metabólica/genética , Xenobióticos/metabolismo , Animales , Interacciones Farmacológicas/genética , Humanos , FenotipoRESUMEN
CYP2C19 rs12769205 alters an intron 2 branch point adenine leading to an alternative mRNA in human liver with complete inclusion of intron 2 (exon 2B). rs12769205 changes the mRNA reading frame, introduces 87 amino acids, and leads to a premature stop codon. The 1000 Genomes project (http://browser.1000genomes.org/index.html) indicated rs12769205 is in linkage disequilibrium with rs4244285 on CYP2C19*2, but found alone on CYP2C19*35 in Blacks. Minigenes containing rs12769205 transfected into HepG2 cells demonstrated this single nucleotide polymorphism (SNP) alone leads to exon 2B and decreases CYP2C19 canonical mRNA. A residual amount of CYP2C19 protein was detectable by quantitative proteomics with tandem mass spectrometry in CYP2C19*2/*2 and *1/*35 liver microsomes with an exon 2 probe. However, an exon 4 probe, downstream from rs12769205, but upstream of rs4244285, failed to detect CYP2C19 protein in livers homozygous for rs12769205, demonstrating rs12769205 alone can lead to complete loss of CYP2C19 protein. CYP2C19 genotypes and mephenytoin phenotype were compared in 104 Ethiopians. Poor metabolism of mephenytoin was seen in persons homozygous for both rs12769205 and rs4244285 (CYP2C19*2/*2), but with little effect on mephenytoin disposition of CYP2C19*1/*2, CYP2C19*1/*3, or CYP2C19*1/*35 heterozygous alleles. Extended haplotype homozygosity tests of the HapMap Yorubans (YRI) showed both haplotypes carrying rs12769205 (CYP2C19*35 and CYP2C19*2) are under significant natural selection, with CYP2C19*35 having a higher relative extended haplotype homozygosity score. The phylogenetic tree of the YRI CYP2C19 haplotypes revealed rs12769205 arose first on CYP2C19*35 and that rs4244285 was added later, creating CYP2C19*2. In conclusion, rs12769205 is the ancestral polymorphism leading to aberrant splicing of CYP2C19*35 and CYP2C19*2 alleles in liver.
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Citocromo P-450 CYP2C19/genética , Hígado/enzimología , Acetilación , Adulto , Alelos , Citocromo P-450 CYP2C19/metabolismo , ADN Complementario/biosíntesis , ADN Complementario/genética , Etiopía/epidemiología , Exones , Femenino , Frecuencia de los Genes , Haplotipos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Mefenitoína/farmacocinética , Fenotipo , Filogenia , Polimorfismo de Nucleótido Simple , Empalme del ARN , Espectrometría de Masas en TándemRESUMEN
25-Hydroxyvitamin D3 (25OHD3) is used as a clinical biomarker for assessment of vitamin D status. Blood levels of 25OHD3 represent a balance between its formation rate and clearance by several oxidative and conjugative processes. In the present study, the identity of human uridine 5'-diphosphoglucuronyltransferases (UGTs) capable of catalyzing the 25OHD3 glucuronidation reaction was investigated. Two isozymes, UGT1A4 and UGT1A3, were identified as the principal catalysts of 25OHD3 glucuronidation in human liver. Three 25OHD3 monoglucuronides (25OHD3-25-glucuronide, 25OHD3-3-glucuronide, and 5,6-trans-25OHD3-25-glucuronide) were generated by recombinant UGT1A4/UGT1A3, human liver microsomes, and human hepatocytes. The kinetics of 25OHD3 glucuronide formation in all systems tested conformed to the Michaelis-Menten model. An association between the UGT1A4*3 (Leu48Val) gene polymorphism with the rates of glucuronide formation was also investigated using human liver microsomes isolated from 80 genotyped livers. A variant allele dose effect was observed: the homozygous UGT1A4*3 livers (GG) had the highest glucuronidation activity, whereas the wild type (TT) had the lowest activity. Induction of UGT1A4 and UGT1A3 gene expression was also determined in human hepatocytes treated with pregnane X receptor/constitutive androstane receptor agonists, such as rifampin, carbamazepine, and phenobarbital. Although UGT mRNA levels were increased significantly by all of the known pregnane X receptor/constitutive androstane receptor agonists tested, rifampin, the most potent of the inducers, significantly induced total 25OHD3 glucuronide formation activity in human hepatocytes measured after 2, but not 4 and 24 hours, of incubation. Finally, the presence of 25OHD3-3-glucuronide in both human plasma and bile was confirmed, suggesting that the glucuronidation pathway might be physiologically relevant and contribute to vitamin D homeostasis in humans.
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Calcifediol/metabolismo , Glucuronosiltransferasa/metabolismo , Humanos , Técnicas In Vitro , Cinética , Microsomas Hepáticos/metabolismoRESUMEN
When investigating the potential for xanthine oxidase (XO)-mediated metabolism of a new chemical entity in vitro, selective chemical inhibition experiments are typically used. Most commonly, these inhibition experiments are performed using the inhibitor allopurinol (AP) and commercially prepared human liver cytosol (HLC) as the enzyme source. For reasons detailed herein, it is also a common practice to perfuse livers with solutions containing AP prior to liver harvest. The exposure to AP in HLC preparations could obviously pose a problem for measuring in vitro XO activity. To investigate this potential problem, an HPLC-MS/MS assay was developed to determine whether AP and its primary metabolite, oxypurinol, are retained within the cytosol for livers that were treated with AP during liver harvest. Differences in enzymatic activity for XO and aldehyde oxidase (AO) in human cytosol that can be ascribed to AP exposure were also evaluated. The results confirmed the presence of residual AP (some) and oxypurinol (all) human liver cytosol preparations that had been perfused with an AP-containing solution. In every case where oxypurinol was detected, XO activity was not observed. In contrast, the presence of AP and oxypurinol did not appear to have an impact on AO activity. Pooled HLC that was purchased from a commercial source also contained residual oxypurinol and did not show any XO activity. In the future, it is recommended that each HLC batch is screened for oxypurinol and/or XO activity prior to testing for XO-mediated metabolism of a new chemical entity.
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Alopurinol/farmacología , Citosol/enzimología , Inhibidores Enzimáticos/farmacología , Hígado/enzimología , Oxipurinol/farmacología , Xantina Oxidasa/metabolismo , Aldehído Oxidasa/metabolismo , Alopurinol/análisis , Alopurinol/metabolismo , Cromatografía Líquida de Alta Presión , Citosol/efectos de los fármacos , Inhibidores Enzimáticos/análisis , Inhibidores Enzimáticos/metabolismo , Femenino , Humanos , Límite de Detección , Hígado/efectos de los fármacos , Masculino , Oxipurinol/análisis , Oxipurinol/metabolismo , Perfusión , Espectrometría de Masas en Tándem , Técnicas de Cultivo de Tejidos/métodos , Xantina Oxidasa/antagonistas & inhibidoresRESUMEN
Human liver gene regulatory (Bayesian) network analysis was previously used to identify a cytochrome P450 (P450) gene subnetwork with Aldo-keto reductase 1D1 (AKR1D1) as a key regulatory driver of this subnetwork. This study assessed the biologic importance of AKR1D1 [a key enzyme in the synthesis of bile acids, ligand activators of farnesoid X receptor (FXR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR), known transcriptional regulators of P450s] to hepatic P450 expression. Overexpression of AKR1D1 in primary human hepatocytes led to increased expression of CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2B6. Conversely, AKR1D1 knockdown decreased expression of these P450s. We resequenced AKR1D1 from 98 donor livers and identified a 3'-untranslated region (UTR) (rs1872930) single nucleotide polymorphism (SNP) significantly associated with higher AKR1D1 mRNA expression. AKR1D1 3'-UTR-luciferase reporter studies showed that the variant allele resulted in higher luciferase activity, suggesting that the SNP increases AKR1D1 mRNA stability and/or translation efficiency. Consistent with AKR1D1's putative role as a driver of the P450 subnetwork, the AKR1D1 3'-UTR SNP was significantly associated with increased hepatic mRNA expression of multiple P450s (CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2B6) and CYP3A4, CYP2C8, CYP2C19, and CYP2B6 activities. After adjusting for multiple testing, the association remained significant for AKR1D1, CYP2C9, and CYP2C8 mRNA expression and CYP2C8 activity. These results provide new insights into the variation in expression and activity of P450s that can account for interindividual differences in drug metabolism/efficacy and adverse drug events. In conclusion, we provide the first experimental evidence supporting a role for AKR1D1 as a key genetic regulator of the P450 network.
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Sistema Enzimático del Citocromo P-450/genética , Regulación Enzimológica de la Expresión Génica , Oxidorreductasas/genética , Empalme Alternativo , Variación Genética , Células Hep G2 , Hepatocitos/enzimología , Humanos , Hígado/enzimología , Oxidorreductasas/fisiología , Polimorfismo de Nucleótido Simple , ARN Mensajero/análisis , ARN Mensajero/químicaRESUMEN
PURPOSE: To investigate the role of ontogeny in sorafenib metabolism to the equipotent active metabolite sorafenib N-oxide. EXPERIMENTAL DESIGN: Steady-state pharmacokinetic studies of sorafenib and metabolites were conducted in 30 children and young adults (17 males; median age, 9.5 years) receiving sorafenib 150 mg/m(2) or 200 mg/m(2) twice daily. Sorafenib metabolism was evaluated in vitro at 10 µmol/L using a panel of purified human cytochrome P450 (CYP) enzymes. Sorafenib metabolism and CYP3A4 expression was evaluated in 52 human liver samples from donors of ≤20 years old. The drug-drug interaction potential between sorafenib and azole antifungal agents was evaluated in vitro and in vivo. RESULTS: No age-related differences in sorafenib apparent oral clearance were observed. Mean sorafenib N-oxide metabolite ratio was 0.27 ± 0.14. In children of ≤10 years of age, boys had approximately 2-fold higher N-oxide ratios than girls (0.40 ± 0.15 vs. 0.22 ± 0.12, P = 0.026). Of the CYPs evaluated, sorafenib was exclusively metabolized to sorafenib N-oxide by CYP3A4. A trend for increased N-oxide formation in boys was observed in liver samples, which correlated with CYP3A4 mRNA expression. Posaconazole and voriconazole potently inhibited sorafenib N-oxide formation in vitro, and reduced sorafenib N-oxide formation in 3 children given sorafenib concurrent with azoles. CONCLUSION: We have identified several factors affecting interpatient variability in sorafenib metabolism to the active N-oxide metabolite including age, sex, and concurrent treatment with azole antifungals. This knowledge may provide important considerations for the clinical use of sorafenib in children and possibly other kinase inhibitors undergoing CYP3A4-mediated metabolism.
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Citocromo P-450 CYP3A/metabolismo , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Inhibidores de Proteínas Quinasas , Adolescente , Factores de Edad , Azoles/farmacología , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Hígado/citología , Hígado/efectos de los fármacos , Masculino , Niacinamida/administración & dosificación , Niacinamida/sangre , Niacinamida/farmacocinética , Compuestos de Fenilurea/administración & dosificación , Compuestos de Fenilurea/sangre , Compuestos de Fenilurea/farmacocinética , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacocinética , Pirimidinas/farmacología , Factores Sexuales , Sorafenib , Triazoles/farmacología , Voriconazol , Adulto JovenRESUMEN
UNLABELLED: The role of organic anion transporting polypeptides (OATPs), particularly the members of OATP1B subfamily, in hepatocellular handling of endogenous and exogenous compounds is an important and emerging area of research. Using a mouse model lacking Slco1b2, the murine ortholog of the OATP1B subfamily, we have demonstrated previously that genetic ablation causes reduced hepatic clearance capacity for substrates. In this study, we focused on the physiological function of the hepatic OATP1B transporters. First, we studied the influence of the Oatp1b2 deletion on bile acid (BA) metabolism, showing that lack of the transporter results in a significantly reduced expression of Cyp7a1, the key enzyme of BA synthesis, resulting in elevated cholesterol levels after high dietary fat challenge. Furthermore, Slco1b2-/- mice exhibited delayed clearance after oral glucose challenge resulting from reduced hepatic glucose uptake. In addition to increased hepatic glycogen content, Slco1b2-/- mice exhibited reduced glucose output after pyruvate challenge. This is in accordance with reduced hepatic expression of phosphoenolpyruvate carboxykinase (PEPCK) in knockout mice. We show that this phenotype is due to the loss of liver-specific Oatp1b2-mediated hepatocellular thyroid hormone entry, which then leads to reduced transcriptional activation of target genes of hepatic thyroid hormone receptor (TR), including Cyp7a1 and Pepck but also Dio1 and Glut2. Importantly, we assessed human relevance using a cohort of archived human livers in which OATP1B1 expression was noted to be highly associated with TR target genes, especially for glucose facilitating transporter 2 (GLUT2). Furthermore, GLUT2 expression was significantly decreased in livers harboring a common genetic polymorphism in SLCO1B1. CONCLUSION: Our findings reveal that OATP1B-mediated hepatic thyroid hormone entry is a key determinant of cholesterol and glucose homeostasis.
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Colesterol/fisiología , Glucosa/fisiología , Homeostasis , Transportadores de Anión Orgánico/fisiología , Receptores de Hormona Tiroidea/fisiología , Animales , Humanos , RatonesRESUMEN
The commonly prescribed antiepileptic drug phenytoin has a narrow therapeutic range and wide interindividual variability in clearance explained in part by CYP2C9 and CYP2C19 coding variants. After finding a paradoxically low urinary phenytoin metabolite (S)/(R) ratio in subjects receiving phenytoin maintenance therapy with a CYP2C9*1/*1 and CYP2C19*1/*2 genotype, we hypothesized that CYP2C9 regulatory polymorphisms (rPMs), G-3089A and -2663delTG, in linkage disequilibrium with CYP2C19*2 were responsible. These rPMs explained as much as 10% of the variation in phenytoin maintenance dose in epileptic patients, but were not correlated with other patients' warfarin dose requirements or with phenytoin metabolite ratio in human liver microsomes. We hypothesized the rPMs affected CYP2C9 induction by phenytoin, a pregnane X receptor (PXR), and constitutive androstane receptor (CAR) activator. Transfection studies showed that CYP2C9 reporters with wild-type versus variant alleles had similar basal activity but significantly greater phenytoin induction by cotransfected PXR, CAR, and Nrf2 and less Yin Yang 1 transcription factor repression. Phenytoin induction of CYP2C9 was greater in human hepatocytes with the CYP2C9 wild type versus variant haplotype. Therefore, CYP2C9 rPMs affect phenytoin-dependent induction of CYP2C9 and phenytoin metabolism in humans, with an effect size comparable with that for CYP2C9*2 and 2C9*3. These findings may also be relevant to the clinical use of other PXR, CAR, and Nrf2 activators.