RESUMEN
Vascular aging affects multiple organ systems, including the brain, where it can lead to vascular dementia. However, a concrete understanding of how aging specifically affects the brain vasculature, along with molecular readouts, remains vastly incomplete. Here, we demonstrate that aging is associated with a marked decline in Notch3 signaling in both murine and human brain vessels. To clarify the consequences of Notch3 loss in the brain vasculature, we used single-cell transcriptomics and found that Notch3 inactivation alters regulation of calcium and contractile function and promotes a notable increase in extracellular matrix. These alterations adversely impact vascular reactivity, manifesting as dilation, tortuosity, microaneurysms, and decreased cerebral blood flow, as observed by MRI. Combined, these vascular impairments hinder glymphatic flow and result in buildup of glycosaminoglycans within the brain parenchyma. Remarkably, this phenomenon mirrors a key pathological feature found in brains of patients with CADASIL, a hereditary vascular dementia associated with NOTCH3 missense mutations. Additionally, single-cell RNA sequencing of the neuronal compartment in aging Notch3-null mice unveiled patterns reminiscent of those observed in neurodegenerative diseases. These findings offer direct evidence that age-related NOTCH3 deficiencies trigger a progressive decline in vascular function, subsequently affecting glymphatic flow and culminating in neurodegeneration.
Asunto(s)
Encéfalo , Demencia Vascular , Receptor Notch3 , Animales , Humanos , Ratones , Encéfalo/metabolismo , CADASIL/genética , CADASIL/patología , Demencia Vascular/metabolismo , Ratones Noqueados , Mutación , Receptor Notch3/genéticaRESUMEN
Metastases largely rely on hematogenous dissemination of tumor cells via the vascular system and significantly limit prognosis of patients with solid tumors. To colonize distant sites, circulating tumor cells must destabilize the endothelial barrier and transmigrate across the vessel wall. Here we performed a high-content screen to identify drugs that block tumor cell extravasation by testing 3,520 compounds on a transendothelial invasion coculture assay. Hits were further characterized and validated using a series of in vitro assays, a zebrafish model enabling three-dimensional (3D) visualization of tumor cell extravasation, and mouse models of lung metastasis. The initial screen advanced 38 compounds as potential hits, of which, four compounds enhanced endothelial barrier stability while concurrently suppressing tumor cell motility. Two compounds niclosamide and forskolin significantly reduced tumor cell extravasation in zebrafish, and niclosamide drastically impaired metastasis in mice. Because niclosamide had not previously been linked with effects on barrier function, single-cell RNA sequencing uncovered mechanistic effects of the drug on both tumor and endothelial cells. Importantly, niclosamide affected homotypic and heterotypic signaling critical to intercellular junctions, cell-matrix interactions, and cytoskeletal regulation. Proteomic analysis indicated that niclosamide-treated mice also showed reduced levels of kininogen, the precursor to the permeability mediator bradykinin. Our findings designate niclosamide as an effective drug that restricts tumor cell extravasation through modulation of signaling pathways, chemokines, and tumor-endothelial cell interactions. SIGNIFICANCE: A high-content screen identified niclosamide as an effective drug that restricts tumor cell extravasation by enhancing endothelial barrier stability through modulation of molecular signaling, chemokines, and tumor-endothelial cell interactions. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/3/619/F1.large.jpg.
Asunto(s)
Colforsina/farmacología , Endotelio Vascular , Neoplasias Pulmonares/patología , Células Neoplásicas Circulantes , Niclosamida/farmacología , Migración Transendotelial y Transepitelial/efectos de los fármacos , Animales , Comunicación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular , Ensayos de Selección de Medicamentos Antitumorales/métodos , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Quininógenos/análisis , Masculino , Metabolómica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Invasividad Neoplásica , Proteómica , Pez CebraRESUMEN
PURPOSE: Gliomas with isocitrate dehydrogenase 1 mutations (IDH1mut) are less aggressive than IDH1 wild-type (IDH1wt) gliomas and have global genomic hypermethylation. Yet it is unclear how specific hypermethylation events contribute to the IDH1mut phenotype. Previously, we showed that the gene encoding the procoagulant tissue factor (TF), F3, is among the most hypermethylated and downregulated genes in IDH1mut gliomas, correlating with greatly reduced thrombosis in patients with IDH1mut glioma. Because TF also increases the aggressiveness of many cancers, the current study explored the contribution of TF suppression to the reduced malignancy of IDH1mut gliomas.Experimental Design: TF expression was manipulated in patient-derived IDH1mut and IDH1wt glioma cells, followed by evaluation of in vitro and in vivo behavior and analyses of cell signaling pathways. RESULTS: A demethylating agent, decitabine, increased F3 transcription and TF-dependent coagulative activity in IDH1mut cells, but not in IDH1wt cells. TF induction enhanced the proliferation, invasion, and colony formation of IDH1mut cells, and increased the intracranial engraftment of IDH1mut GBM164 from 0% to 100% (P = 0.0001). Conversely, TF knockdown doubled the median survival of mice engrafted with IDH1wt/EGFRvIIIamp GBM6, and caused complete regression of IDH1wt/EGFRamp GBM12 (P = 0.001). In vitro and in vivo effects were linked to activation of receptor tyrosine kinases (RTK) by TF through a Src-dependent intracellular pathway, even when extracellular RTK stimulation was blocked. TF stimulated invasion predominately through upregulation of ß-catenin. CONCLUSIONS: These data show that TF suppression is a component of IDH1mut glioma behavior, and that it may therefore be an attractive target against IDH1wt gliomas.
Asunto(s)
Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Glioma/genética , Glioma/patología , Isocitrato Deshidrogenasa/genética , Mutación , Tromboplastina/genética , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Expresión Génica Ectópica , Epigénesis Genética , Técnicas de Silenciamiento del Gen , Glioma/metabolismo , Humanos , Isocitrato Deshidrogenasa/metabolismo , Ratones , Clasificación del Tumor , Unión Proteica , Proteómica/métodos , Tromboplastina/metabolismo , Activación Transcripcional , Ensayo de Tumor de Célula MadreRESUMEN
Background: Among diffusely infiltrative gliomas in adults, 20%-30% contain a point mutation in isocitrate dehydrogenase 1 (IDH1mut), which increases production of D-2-hydroxyglutarate (D2HG). This is so efficient that D2HG often reaches 30 mM within IDH1mut gliomas. Yet, while up to 100 µM D2HG can be detected in the circulating cerebrospinal fluid of IDH1mut glioma patients, the exposure of nonneoplastic cells within and surrounding an IDH1mut glioma to D2HG is unknown and difficult to measure directly. Methods: Conditioned medium from patient-derived wild type IDH1 (IDH1wt) and IDH1mut glioma cells was analyzed for D2HG by liquid chromatography-mass spectrometry (LC-MS). Mathematical models of D2HG release and diffusion around an IDH1mut glioma were independently generated based on fluid dynamics within the brain and on previously reported intratumoral and cerebrospinal D2HG concentrations. Results: LC-MS analysis indicates that patient-derived IDH1mut glioma cells release 3.7-97.0 pg D2HG per cell per week. Extrapolating this to an average-sized tumor (30 mL glioma volume and 1 × 108 cells/mL tumor), the rate of D2HG release by an IDH1mut glioma (SA) is estimated at 3.2-83.0 × 10-12 mol/mL/sec. Mathematical models estimate an SA of 2.9-12.9 × 10-12 mol/mL/sec, within the range of the in vitro LC-MS data. In even the most conservative of these models, the extracellular concentration of D2HG exceeds 3 mM within a 2 cm radius from the center of an IDH1mut glioma. Conclusions: The microenvironment of an IDH1mut glioma is likely being exposed to high concentrations of D2HG, in the low millimolar range. This has implications for understanding how D2HG affects nonneoplastic cells in an IDH1mut glioma.
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Sistema Nervioso Central/patología , Glioma/patología , Glutaratos/metabolismo , Isocitrato Deshidrogenasa/genética , Modelos Teóricos , Mutación , Sistema Nervioso Central/metabolismo , Difusión , Glioma/genética , Glioma/metabolismo , Humanos , Células Tumorales Cultivadas , Microambiente TumoralRESUMEN
1. There is limited knowledge regarding the metabolism of megestrol acetate (MA), as it was approved by FDA in 1971, prior to the availability of modern tools for identifying specific drug-metabolizing enzymes. We determined the cytochrome P450s (P450s) and UDP-glucuronosyltransferases (UGTs) that metabolize MA, identified oxidative metabolites and determined pharmacologic activity at the progesterone, androgen and glucocorticoid receptors (PR, AR and GR, respectively). 2. Oxidative metabolites were produced using human liver microsomes (HLMs), and isolated for mass spectral (MS) and nuclear magnetic resonance (NMR) analyses. We screened recombinant P450s using MA at 62 µM (HLM Km for metabolite 1; M1) and 28 µM (HLM Km for metabolite 2; M2). UGT isoforms were simultaneously incubated with UDPGA, nicotinamide adenine dinucleotide phosphate (NADPH), CYP3A4 and MA. Metabolites were evaluated for pharmacologic activity on the PR, AR and GR. CYP3A4 and CYP3A5 are responsible for oxidative metabolism of 62 µM MA. 3. At 28 µM substrate concentration, CYP3A4 was the only contributing enzyme. Mass spectral and NMR data suggest metabolism of MA to two alcohols. After oxidation, MA is converted into two secondary glucuronides by UGT2B17 among other UGTs. MA, M1 and M2 had significant pharmacologic activity on the PR while only MA showed activity on the AR and GR.
Asunto(s)
Acetato de Megestrol/metabolismo , Metaboloma , Línea Celular Tumoral , Citocromo P-450 CYP3A/metabolismo , Inhibidores del Citocromo P-450 CYP3A/farmacología , Glucurónidos/metabolismo , Humanos , Cetoconazol/farmacología , Cinética , Acetato de Megestrol/química , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Oxidación-Reducción , Antígeno Prostático Específico/metabolismo , Espectroscopía de Protones por Resonancia Magnética , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato/efectos de los fármacos , Troleandomicina/farmacologíaRESUMEN
UDP-glucuronosyltransferases (UGTs) are important phase II drug metabolism enzymes. The aim of this study was to explore the relationship between age and changes in mRNA expression and activity of major human hepatic UGTs, as well as to understand the potential regulatory mechanism underlying this relationship. Using previously generated data, we investigated age-dependent mRNA expression levels of 11 hepatic UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B10, UGT2B15, and UGT2B17) and 16 transcription factors (AHR, AR, CAR, ESR2, FXR, GCCR, HNF1a, HNF3a, HNF3b, HNF4a, PPARA, PPARG, PPARGC, PXR, SP1, and STAT3) in liver tissue of donors (n = 38) ranging from 0 to 25 years of age. We also examined the correlation between age and microsomal activities using 14 known UGT drug substrates in the liver samples (n = 19) of children donors. We found a statistically significant increase (nominal p < 0.05) in the expression of UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT2B7, and UGT2B17, as well as glucuronidation activities of serotonin, testosterone, and vorinostat during the first 25 years of life. Expression of estrogen receptor 1 and pregnane X receptor, two strong UGT transcriptional regulators, were significantly correlated with both age and UGT mRNA expression (p ≤ 0.05). These results suggest that both UGT expression and activity increase during childhood and adolescence, possibly driven in part by hormonal signaling. Our findings may help explain inter-patient variability in response to medications among children.
RESUMEN
1. Aprepitant, an oral antiemetic, commonly used in the prevention of chemotherapy-induced nausea and vomiting, is primarily metabolized by CYP3A4. Aprepitant glucuronidation has yet to be evaluated in humans. The contribution of human UDP-glucuronosyltransferase (UGT) isoforms to the metabolism of aprepitant was investigated by performing kinetic studies, inhibition studies and correlation analyses. In addition, aprepitant was evaluated as an inhibitor of UGTs. 2. Glucuronidation of aprepitant was catalyzed by UGT1A4 (82%), UGT1A3 (12%) and UGT1A8 (6%) and Kms were 161.6 ± 15.6, 69.4 ± 1.9 and 197.1 ± 28.2 µM, respectively. Aprepitant glucuronidation was significantly correlated with both UGT1A4 substrates anastrazole and imipramine (rs = 0.77, p < 0.0001 for both substrates; n = 44), and with the UGT1A3 substrate thyroxine (rs = 0.58, p < 0.0001; n = 44). 3. We found aprepitant to be a moderate inhibitor of UGT2B7 with a Ki of â¼10 µM for 4-MU, morphine and zidovudine. Our results suggest that aprepitant can alter clearance of drugs primarily eliminated by UGT2B7. Given the likelihood for first-pass metabolism by intestinal UGT2B7, this is of particular concern for oral aprepitant co-administered with oral substrates of UGT2B7, such as zidovudine and morphine.
Asunto(s)
Inhibidores Enzimáticos/química , Glucuronosiltransferasa/antagonistas & inhibidores , Glucuronosiltransferasa/química , Morfolinas/química , Aprepitant , Citocromo P-450 CYP3A/química , HumanosRESUMEN
OTS167 is a potent maternal embryonic leucine zipper kinase inhibitor undergoing clinical testing as antineoplastic agent. We aimed to identify the UDP-glucuronosyltransferases (UGTs) involved in OTS167 metabolism, study the relationship between UGT genetic polymorphisms and hepatic OTS167 glucuronidation, and investigate the inhibitory potential of OTS167 on UGTs. Formation of a single OTS167-glucuronide (OTS167-G) was observed in pooled human liver (HLM) (Km = 3.4 ± 0.2 µM), intestinal microsomes (HIM) (Km = 1.7 ± 0.1 µM), and UGTs. UGT1A1 (64 µl/min/mg) and UGT1A8 (72 µl/min/mg) exhibited the highest intrinsic clearances (CLint) for OTS167, followed by UGT1A3 (51 µl/min/mg) and UGT1A10 (47 µl/min/mg); UGT1A9 was a minor contributor. OTS167 glucuronidation in HLM was highly correlated with thyroxine glucuronidation (r = 0.91, P < 0.0001), SN-38 glucuronidation (r = 0.79, P < 0.0001), and UGT1A1 mRNA (r = 0.72, P < 0.0001). Nilotinib (UGT1A1 inhibitor) and emodin (UGT1A8 and UGT1A10 inhibitor) exhibited the highest inhibitory effects on OTS167-G formation in HLM (68%) and HIM (47%). We hypothesize that OTS167-G is an N-glucuronide according to mass spectrometry. A significant association was found between rs6706232 and reduced OTS167-G formation (P = 0.03). No or weak UGT inhibition (range: 0-21%) was observed using clinically relevant OTS167 concentrations (0.4-2 µM). We conclude that UGT1A1 and UGT1A3 are the main UGTs responsible for hepatic formation of OTS167-G. Intestinal UGT1A1, UGT1A8, and UGT1A10 may contribute to first-pass OTS167 metabolism after oral administration.
Asunto(s)
Antineoplásicos/metabolismo , Glucuronosiltransferasa/metabolismo , Naftiridinas/metabolismo , Inhibidores Enzimáticos/farmacología , Genotipo , Glucurónidos/metabolismo , Glucuronosiltransferasa/antagonistas & inhibidores , Glucuronosiltransferasa/genética , Humanos , Técnicas In Vitro , Mucosa Intestinal/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Hígado/enzimología , Microsomas/enzimología , Microsomas Hepáticos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Tiroxina/metabolismoRESUMEN
The aim of this study was to discover cis- and trans-acting factors significantly affecting mRNA expression and catalytic activity of human hepatic UDP-glucuronosyltransferases (UGTs). Transcription levels of five major hepatic UGT1A (UGT1A1, UGT1A3, UGT1A4, UGT1A6 and UGT1A9) and five UGT2B (UGT2B4, UGT2B7, UGT2B10, UGT2B15 and UGT2B17) genes were quantified in human liver tissue samples (n = 125) using real-time PCR. Glucuronidation activities of 14 substrates were measured in 47 livers. We genotyped 167 tagSNPs (single-nucleotide polymorphisms) in UGT1A (n = 43) and UGT2B (n = 124), as well as the known functional UGT1A1*28 and UGT2B17 CNV (copy number variation) polymorphisms. Transcription levels of 15 transcription factors (TFs) known to regulate these UGTs were quantified. We found that UGT expression and activity were highly variable among the livers (median and range of coefficient of variations: 135%, 74-217% and 52%, 39-105%, respectively). CAR, PXR and ESR1 were found to be the most important trans-regulators of UGT transcription (median and range of correlation coefficients: 46%, 6-58%; 47%, 9-58%; and 52%, 24-75%, respectively). Hepatic UGT activities were mainly determined by UGT gene transcription levels. Twenty-one polymorphisms were significantly (FDR-adjusted P < 0.05) associated with mRNA expression and/or activities of UGT1A1, UGT1A3 and UGT2B17. We found novel SNPs in the UGT2B17 CNV region accounting for variability in UGT2B17 gene transcription and testosterone glucuronidation rate, in addition to that attributable to the UGT2B17 CNV. Our study discovered novel pharmacogenetic markers and provided detailed insight into the genetic network regulating hepatic UGTs.
Asunto(s)
Regulación de la Expresión Génica , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Hígado/enzimología , Transcripción Genética , Variaciones en el Número de Copia de ADN , Variación Genética , Humanos , Receptor de la Señal 1 de Direccionamiento al Peroxisoma , Polimorfismo de Nucleótido Simple , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/genéticaRESUMEN
XK469 (NSC 697887) is a selective topoisomerase II ß inhibitor eliminated mainly by aldehyde oxidase I (AOX1). We performed a candidate gene study to investigate whether AOX1 genetic variation contributes to interindividual variability in XK469 clearance. Forty-one AOX1 single nucleotide polymorphisms (SNPs) and seven liver expression quantitative trait loci were genotyped in White patients with advanced refractory solid tumors (n=59) and leukemia (n=33). We found a significant decrease in clearance (τ=-0.32, P=0.003) in solid tumor patients with rs10931910, although it failed to replicate in the leukemia cohort (τ=0.18, P=0.20). Four other AOX1 SNPs were associated with clearance (P=0.01-0.02) in only one of the two cohorts. Our study provides a starting point for future investigations on the functionality of AOX1 SNPs. However, variability in XK469 clearance cannot be attributed to polymorphisms in AOX1.
Asunto(s)
Aldehído Oxidasa/genética , Antineoplásicos/farmacocinética , Hígado/metabolismo , Neoplasias/genética , Quinoxalinas/farmacocinética , Antineoplásicos/administración & dosificación , Ensayos Clínicos Fase I como Asunto , Estudios de Cohortes , Variación Genética , Genotipo , Humanos , Neoplasias/tratamiento farmacológico , Farmacogenética , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Quinoxalinas/administración & dosificaciónRESUMEN
BACKGROUND: Recent studies have illuminated the diversity of roles for microRNAs in cellular, developmental, and pathophysiological processes. The study of microRNAs in human liver tissue promises to clarify the therapeutic and diagnostic value of this important regulatory mechanism of gene expression. RESULTS: We conducted genome-wide profiling of microRNA expression in liver and performed an integrative analysis with previously collected genotype and transcriptome data. We report here that the Very Important Pharmacogenes (VIP Genes), comprising of genes of particular relevance for pharmacogenomics, are under substantial microRNA regulatory effect in the liver. We set out to elucidate the genetic basis of microRNA expression variation in liver and mapped microRNA expression to genomic loci as microRNA expression quantitative trait loci (miR-eQTLs). We identified common variants that attain genome-wide significant association (p < 10-10) with microRNA expression. We also found that the miR-eQTLs are significantly more likely to predict mRNA levels at a range of p-value thresholds than a random set of allele frequency matched SNPs, showing the functional effect of these loci on the transcriptome. Finally, we show that a large number of miR-eQTLs overlap with SNPs reproducibly associated with complex traits from the NHGRI repository of published genome-wide association studies as well as variants from a comprehensive catalog of manually curated pharmacogenetic associations. CONCLUSION: Our study provides important insights into the genomic architecture of gene regulation in a vital human organ, with important implications for our understanding of disease pathogenesis, therapeutic outcome, and other complex human phenotypes.
Asunto(s)
Genómica , Hígado/metabolismo , MicroARNs/genética , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad/genética , Estudio de Asociación del Genoma Completo , Humanos , Farmacogenética , Sitios de Carácter Cuantitativo/genética , ARN Mensajero/genéticaRESUMEN
OBJECTIVE: ABT-751, a novel orally available antitubulin agent, is mainly eliminated as inactive glucuronide (ABT-751G) and sulfate (ABT-751S) conjugates. We performed a pharmacogenetic investigation of ABT-751 pharmacokinetics using in-vitro data to guide the selection of genes for genotyping in a phase I trial of ABT-751. METHODS: UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) enzymes were screened for ABT-751 metabolite formation in vitro. Forty-seven cancer patients treated with ABT-751 were genotyped for 21 variants in these genes. RESULTS: UGT1A1, UGT1A4, UGT1A8, UGT2B7, and SULT1A1 were found to be involved in the formation of inactive ABT-751 glucuronide (ABT-751G) and sulfate (ABT-751S). SULT1A1 copy number (>2) was associated with an average 34% increase in ABT-751 clearance (P=0.044), an 18% reduction in ABT-751 AUC (P=0.045), and a 50% increase in sulfation metabolic ratios (P=0.025). UGT1A8 rs6431558 was associated with a 28% increase in glucuronidation metabolic ratios (P=0.022), and UGT1A4*2 was associated with a 65% decrease in ABT-751 C trough (P=0.009). CONCLUSION: These results might represent the first example of a clinical pharmacokinetic effect of the SULT1A1 copy number variant on the clearance of a SULT1A1 substrate. A-priori selection of candidate genes guided by in-vitro metabolic screening enhanced our ability to identify genetic determinants of interpatient pharmacokinetic variability.
Asunto(s)
Antineoplásicos/farmacocinética , Sulfonamidas/farmacocinética , Moduladores de Tubulina/farmacocinética , Adulto , Anciano , Arilsulfotransferasa/genética , Femenino , Dosificación de Gen , Variación Genética , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Sulfotransferasas/genética , Sulfotransferasas/metabolismoRESUMEN
OBJECTIVE: Circulatory metabolites are important biomarkers for many diseases, especially metabolic disorders. The biological mechanism regulating circulatory levels of metabolites remains incompletely understood. Focusing on the liver as the central organ controlling metabolic homeostasis, we investigated the potential function of nine polymorphisms associated with serum metabolomic traits in a recent GWAS. MATERIALS/METHODS: The mRNA levels of the associated genes were measured by real-time PCR and correlated with genotypes in normal liver tissue (n=42). Genotype and mRNA data were also correlated with total hepatic lipid content (HLC). Our findings were also compared with the previously published gene expression quantitative traits loci (eQTL) data in the liver. RESULTS: We found that seven out of nine genes were highly expressed in hepatic tissue, while expression of four genes was significantly or marginally associated with genotypes (SPTLC3 vs rs168622, P=.002; ACADS vs rs2014355, P=.016; PLEKHH1 vs rs7156144, P=.076; ACADL vs rs2286963, P=.068). The SNP rs168622 at the SPTLC3 locus was also significantly correlated with HLC (P=.02). HLC was significantly correlated with FADS1 (r=-0.45; P=.003) and ETFDH (r=0.33; P=.037) expression. When compared with published eQTL data, SNPs in SPTLC3, ACADS, ELOVL2 and FADS1 were also in strong linkage disequilibrium (R(2)≥0.41, D'≥0.96) with eQTLs significantly affecting expression of these genes (P≤1.74×10(-5)). CONCLUSIONS: Our study suggests that genetic variants affecting serum metabolites levels may play a functional role in the liver. This may help elucidate the mechanism by which genetic variants are involved in metabolic diseases.
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Regulación de la Expresión Génica/genética , Metabolismo de los Lípidos , Hígado/metabolismo , Metabolómica , Polimorfismo de Nucleótido Simple , ARN Mensajero/genética , Transcripción Genética/genética , delta-5 Desaturasa de Ácido Graso , Humanos , Sitios de Carácter Cuantitativo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The discovery of expression quantitative trait loci ("eQTLs") can help to unravel genetic contributions to complex traits. We identified genetic determinants of human liver gene expression variation using two independent collections of primary tissue profiled with Agilent (nâ=â206) and Illumina (nâ=â60) expression arrays and Illumina SNP genotyping (550K), and we also incorporated data from a published study (nâ=â266). We found that â¼30% of SNP-expression correlations in one study failed to replicate in either of the others, even at thresholds yielding high reproducibility in simulations, and we quantified numerous factors affecting reproducibility. Our data suggest that drug exposure, clinical descriptors, and unknown factors associated with tissue ascertainment and analysis have substantial effects on gene expression and that controlling for hidden confounding variables significantly increases replication rate. Furthermore, we found that reproducible eQTL SNPs were heavily enriched near gene starts and ends, and subsequently resequenced the promoters and 3'UTRs for 14 genes and tested the identified haplotypes using luciferase assays. For three genes, significant haplotype-specific in vitro functional differences correlated directly with expression levels, suggesting that many bona fide eQTLs result from functional variants that can be mechanistically isolated in a high-throughput fashion. Finally, given our study design, we were able to discover and validate hundreds of liver eQTLs. Many of these relate directly to complex traits for which liver-specific analyses are likely to be relevant, and we identified dozens of potential connections with disease-associated loci. These included previously characterized eQTL contributors to diabetes, drug response, and lipid levels, and they suggest novel candidates such as a role for NOD2 expression in leprosy risk and C2orf43 in prostate cancer. In general, the work presented here will be valuable for future efforts to precisely identify and functionally characterize genetic contributions to a variety of complex traits.
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Genoma Humano , Hígado/metabolismo , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Regiones no Traducidas 3' , Factores de Edad , Población Negra , Mapeo Cromosómico , Clonación Molecular , Femenino , Perfilación de la Expresión Génica , Vectores Genéticos , Estudio de Asociación del Genoma Completo , Genotipo , Células Hep G2 , Hispánicos o Latinos , Humanos , Mediciones Luminiscentes , Masculino , Análisis de Componente Principal , Regiones Promotoras Genéticas , Reproducibilidad de los Resultados , Factores Sexuales , Transfección , Población BlancaRESUMEN
The degree of macrovesicular steatosis is typically evaluated in liver biopsies by visual estimation, which is subject to intraobserver and interobserver variations. Computer morphometry and biochemical measurement may provide more accurate results. Our aim was to develop a morphometry method and compare its results with visual and biochemical measurements. Twenty-six fresh frozen liver specimens were each divided into 4 aliquots. Three aliquots were processed biochemically to extract fat, and the fat content was defined as the weight percentage of fat. One aliquot was fixed in formalin, from which hematoxylin and eosin slides were made and reviewed by 3 pathologists to estimate fat content. Digital images of slides were analyzed by computer morphometry, which defined fat content as the percentage of area occupied by fat droplets. The results showed that individually, each method produced highly precise and reproducible measurements. Compared with each other, they showed very strong correlations (correlation coefficient r = 0.81-0.95). The range of fat content in all 26 specimens was 2.2% to 15% by biochemical, 0.8% to 82.5% by visual, and 0.3% to 19.6% by morphometry method. Visual estimation appeared to have a systematic bias, giving results nearly 4-fold higher than other methods. This may be because visual estimation denotes the fraction of hepatocytes containing fat droplets, instead of the true fraction of fat. Strong correlations between different methods suggest that all 3 are valid methods for measuring steatosis. Computer morphometry is easy to implement and not affected by the bias seen in visual estimation. It may serve as a potential supplemental or alternative method.
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Hígado Graso/patología , Hepatocitos/patología , Procesamiento de Imagen Asistido por Computador/métodos , Hígado/patología , Biopsia , Criopreservación , Grasas/análisis , Hígado Graso/metabolismo , Hepatocitos/química , Humanos , Modelos Lineales , Hígado/química , Reproducibilidad de los ResultadosRESUMEN
High interindividual pharmacokinetic variability was observed in phase 1 studies of vorinostat (suberoylanilide hydroxamic acid), an oral histone deacetylase inhibitor. Thus, we hypothesized that the variability can be explained by genetic variants of the uridine 5'-diphosphate-glucuronosyltransferases (UGTs) involved in vorinostat metabolism. Baculosomes expressing human UGTs and 52 human liver microsomes were screened for vorinostat glucuronidation activity to identify the potential enzymes and functional variants. UGT2B17 had the largest activity. Human liver microsomes with at least one copy of UGT2B17 showed significantly greater enzymatic activity than those with UGT2B17 null genotype (P<0.004). UGT2B17 plays an important role in vorinostat hepatic glucuronidation and the gene deletion polymorphism may influence vorinostat biotransformation and clearance. The clinical impact of this UGT2B17 genetic variant on vorinostat metabolism and drug effect is unknown.
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Glucurónidos/genética , Glucurónidos/metabolismo , Ácidos Hidroxámicos/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Genotipo , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Glicosilación/efectos de los fármacos , Humanos , Cinética , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Farmacogenética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Regresión , Especificidad por Sustrato/efectos de los fármacos , VorinostatRESUMEN
PURPOSE: Werner's syndrome (WS) is a recessive disorder of premature onset of processes associated with aging. Defective DNA repair has been reported after exposure of cells isolated from WS patients to DNA-damaging agents. The germline 4330T>C (Cys1367Arg) variant in the WS gene (WRN) has been associated with protection from age-related diseases, suggesting it has a functional role. We studied whether the 4330T>C variant confers altered drug sensitivity in vitro. METHODS: 4330T>C was genotyped in 372 human lymphoblastoid cell lines (LCLs) from unrelated healthy Caucasian individuals using a TaqMan-based method. The study was powered to detect the effect of the 4330T>C genotypes after exposure to camptothecin (based upon preliminary data). The effect of the 4330T>C variant on the cytotoxicity of etoposide, carboplatin, cisplatin and daunorubicin was also tested. WRN expression in 57 LCLs was measured by microarray. RESULTS: No significant difference between the IC50 of the cells was observed among genotypes (P = 0.46) after exposure to camptothecin. No association was also observed for etoposide, carboplatin, cisplatin, and daunorubicin (ANOVA, P > 0.05). WRN expression also did not vary across genotypes (ANOVA, P = 0.37). CONCLUSION: These results suggest that this nonsynonymous variant has relatively normal function at the cellular level.
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Linfocitos B/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Exodesoxirribonucleasas/genética , Compuestos de Platino/farmacología , Polimorfismo de Nucleótido Simple/genética , RecQ Helicasas/genética , Inhibidores de Topoisomerasa , Síndrome de Werner/genética , Antibióticos Antineoplásicos/farmacología , Antineoplásicos/farmacología , Antineoplásicos Fitogénicos/farmacología , Camptotecina/farmacología , Carboplatino/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cisplatino/farmacología , Daunorrubicina/farmacología , Etopósido/farmacología , Genotipo , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Helicasa del Síndrome de WernerRESUMEN
OBJECTIVE: UDP-glucuronosyltransferase 2B7 (UGT2B7) plays a central role in the liver-mediated biotransformation of endogenous and exogenous compounds. The genetic basis of interindividual variability in UGT2B7 function is unknown. This study aimed to discover novel gene variants of functional significance. METHODS: Caucasian human livers (n=54) were used. UGT2B7 was resequenced in 12 samples [(six highest and six lowest for the formation of morphine-3-glucuronide (M3G)]. Haplotype-tagging single nucleotide polymorphisms were genotyped in the entire sample set. Samples were phenotyped for mRNA expression. RESULTS: 10 haplotype-tagging single nucleotide polymorphisms were identified and their haplotypes were inferred. Haplotype 4 (-45597G; -6682_-6683A; 372A; IVS1+9_IVS1+10A; IVS1+829T; IVS1+985G; IVS1+999C; IVS1+1250G; 801T; IVS4+185C) (frequency of 0.12) was associated with an increase in enzyme activity and gene expression. The 1/4 and 4/6 diplotypes had higher M3G formation compared with 1/1 (P<0.05) and 2/3 (P<0.01) diplotypes. Diplotypes containing haplotype 4 resulted in a significant 45% average increase in the formation of M3G compared with diplotypes without haplotype 4 (P=0.002). There was also an association between haplotype 4 and increased mRNA expression. IVS1+985A>G, 735A>G, and 1062C>T are the putative functional variants of haplotype 4. We also identified two mRNA splicing variants (UGT2B7_v2 and UGT2B7_v3) splicing out exon 1, 4, 5, and 6 but sharing exons 2 and 3 with the involvement of additional 5' exons. UGT2B7_v2 was detected in all livers tested, but UGT2B7_v3 was present at much lower levels compared with UGT2B7_v2. The UGT2B7 reference sequence mRNA is now named UGT2B7_v1. CONCLUSION: UGT2B7 haplotype 4 is functional and its effects on the biotransformation of UGT2B7 substrates should be tested in controlled clinical trials. Biochemical studies should investigate the functional role of the newly discovered mRNA splicing variants.
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Variación Genética , Glucuronosiltransferasa/genética , Polimorfismo de Nucleótido Simple/genética , Empalme Alternativo/genética , Secuencia de Bases , Epirrubicina/biosíntesis , Exones/genética , Regulación Enzimológica de la Expresión Génica , Haplotipos , Humanos , Hígado/enzimología , Datos de Secuencia Molecular , Derivados de la Morfina/metabolismo , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Regresión , Análisis de Secuencia de ADNRESUMEN
Interindividual variability in the glucuronidation of xenobiotics metabolized by UDP-glucuronosyltransferase 1A9 (UGT1A9) suggests the presence of functional UGT1A9 variants. The aim of this study was to evaluate whether the putative functionality of the UGT1A9 variants-118T(9>10) (rs3832043), I399C>T (rs2741049), -275T>A (rs6714486), and-2152C>T (rs17868320) could be confirmed in an independent study. UGT1A9 genotypes and UGT1A9 activity (i.e., flavopiridol and mycophenolic acid glucuronidation) were determined in 46 Caucasian human livers. mRNA levels were quantitated by real-time polymerase chain reaction in 35 of these livers. In addition, samples from 60 unrelated Caucasians belonging to the HapMap Project were also genotyped to confirm the allele frequencies and linkage disequilibrium (LD) pattern observed in our Caucasian livers. The allele frequencies of the-118T(9>10), I399C>T, -275T>A, and-2152C>T variants were 0.39, 0.39, 0.02, and 0.02 in the livers, respectively. The I399C>T variant was in complete LD (r(2) = 1) with-118T(9>10) (linked alleles: C and T(9), respectively). Complete LD between these two variants was also found in the HapMap samples (frequencies of-118T(9>10) and I399C>T = 0.38). I399C>T and-118T(9>10) correlated with neither UGT1A9 activities nor mRNA levels. Because of the low frequencies of the-275T>A and-2152C>T variants, an effect on phenotype could not be evaluated. Our data demonstrate that the common I399C>T and-118T(9>10) polymorphisms do not explain interindividual variation in hepatic UGT1A9 activity and mRNA expression and are in complete LD in the donor liver samples we studied.
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Regulación Enzimológica de la Expresión Génica , Glucuronosiltransferasa/genética , Hígado/enzimología , Polimorfismo de Nucleótido Simple , Línea Celular , Estudios de Cohortes , Flavonoides/metabolismo , Frecuencia de los Genes , Genes Reporteros , Genotipo , Glucurónidos/metabolismo , Glucuronosiltransferasa/metabolismo , Humanos , Desequilibrio de Ligamiento , Luciferasas de Luciérnaga , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/metabolismo , Fenotipo , Piperidinas/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/análisis , Especificidad por Sustrato , Transfección , UDP Glucuronosiltransferasa 1A9RESUMEN
The effects of green tea compounds on the metabolism of irinotecan have never been investigated. We aimed to study whether catechins [(-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC), (-)-epicatechin] affect the inactivation metabolism of irinotecan into 7-ethyl-10-[4-N-(1-piperidino)-1-amino]carbonyloxycamptothecin (NPC) (by CYP3A4) and 7-ethyl-10-hydroxycamptothecin (SN-38) into 7-ethyl-10-hydroxycamptothecin glucuronide (SN-38G) (by UGT1A1). Human liver microsomes, hepatocytes and Hep G2 cells were incubated with catechins and treated with irinotecan and/or SN-38. NPC and SN-38G formation was measured by high-performance liquid chromatography. UGT1A1 mRNA levels were measured by real-time polymerase chain reaction. In human liver microsomes, a concentration-dependent decrease in the formation of NPC and SN-38G was observed. In human hepatocytes, a significant increase in SN-38G production was observed in 33% (EGCG), 44% (ECG), and 44% (EGC) of the hepatocyte preparations. Phenobarbital increased the formation of SN-38G in 100% of the same hepatocyte preparations. In Hep G2 cells, no increase in SN-38G formation was observed. With the exception of ECG in one liver, catechins did not increase UGT1A1 mRNA levels. NPC production was also significantly increased in 40% of the hepatocyte preparations for each catechin. However, the production of 6beta-hydroxytestosterone remained unaffected in other hepatocyte preparations. At pharmacologically relevant concentrations, catechins are unlikely to inhibit the formation of irinotecan inactive metabolites when administered concomitantly. The induction effect of catechins on UGT1A1 seems to be modest and highly variable. Catechins do not induce CYP3A4 activity. The effect of acute and prolonged use of green tea on the pharmacokinetics of irinotecan in patients remains to be evaluated.