RESUMEN
Uridine diphosphate (UDP)-glycosyltransferases (UGTs), which are major phase II detoxification enzymes, have been implicated in the glycosylation of lipophilic endobiotics and xenobiotics and thus potentially lead to the evolution of insecticide resistance. In this study, we identified and cloned two putative UGT genes from transcriptome data which are named UGT352A4 and UGT352A5. As demonstrated by qRT-PCR, two UGT genes were over-expressed in the thiamethoxam-resistant (THQR) strain relative to the susceptible (THQS) strain. Moreover, the induction experiment revealed that the expression of the UGT352A5 gene was significantly increased following exposure to thiamethoxam in the THQR strain. Furthermore, the expression of both UGT352A4 and UGT352A5 was downregulated after RNA interference, whereas only the silencing of UGT352A5 resulted in a noticeable increase in the mortality of THQR adults. Our results represent the first line of evidence showing that UGT352A5 might be responsible for conferring thiamethoxam resistance in B. tabaci. The results will be shed new insights for obtaining a better understanding of the role of UGTs in the evolution of insecticide resistance and developing new insect resistance management tactics within the sustainable integrated pest management framework.
Asunto(s)
Glucuronosiltransferasa/genética , Hemípteros/efectos de los fármacos , Proteínas de Insectos/genética , Resistencia a los Insecticidas/genética , Insecticidas/farmacología , Tiametoxam/farmacología , Animales , Técnicas de Silenciamiento del Gen , Glucuronosiltransferasa/deficiencia , Hemípteros/enzimología , Hemípteros/genética , Proteínas de Insectos/deficiencia , Filogenia , Interferencia de ARNRESUMEN
UGT1A1 is the only enzyme that can metabolize bilirubin, and its encoding gene is frequently mutated. UGT1A1*6 (G71R) is a common mutant in Asia which leads to the decrease of UGT1A1 activity and abnormal bilirubin metabolism. However, it is not clear whether low UGT1A1 activity-induced bilirubin metabolism disorder increases hepatocyte fragility. ugt1a+/- mice were used to simulate the UGT1A1*6 (G71R) population. Under the same CCl4 induction condition, ugt1a+/- mice showed severer liver damage and fibrosis, indicating that ugt1a1 dysfunction increased liver burden and aggravated hepatocyte damage. In the animal experiment with a continuous intraperitoneal injection of bilirubin, the ugt1a+/- mice livers had more serious unconjugated bilirubin accumulation. The accumulated bilirubin leads to hyperphosphorylation of IκB-α, Ikk-ß, and p65 and a significant increase of inflammatory factor. The α-SMA and Collagen I proteins markedly up-regulated in the ugt1a+/- mice livers. Immunofluorescence and confocal microscopy showed that hepatic stellate cells and Kupffer cells were activated in ugt1a+/- mice. Comprehensive results show that there was a crosstalk relationship between low UGT1A1 activity-bilirubin-liver damage. Furthermore, cell experiments confirmed that unconjugated bilirubin activated the NF-κB pathway and induced DNA damage in hepatocytes, leading to the significant increase of inflammatory factors. UGT1A1 knockdown in hepatocytes aggravated the toxicity of unconjugated bilirubin. Conversely, overexpression of UGT1A1 had a protective effect on hepatocytes. Finally, Schisandrin B, an active ingredient with hepatoprotective effects, extracted from a traditional Chinese medicinal herb, which could protect the liver from bilirubin metabolism disorders caused by ugt1a1 deficiency by downregulating p65 phosphorylation, inhibiting Kupffer cells, reducing inflammation levels. Our data clarified the mechanism of liver vulnerability caused by cross-talk between low UGT1A1 activity bilirubin, and provided a reference for individualized prevention of liver fragility in Gilbert's syndrome.
Asunto(s)
Bilirrubina/metabolismo , Glucuronosiltransferasa/deficiencia , Hepatocitos/metabolismo , Animales , Bilirrubina/genética , Línea Celular , Enfermedad de Gilbert/genética , Enfermedad de Gilbert/metabolismo , Enfermedad de Gilbert/patología , Glucuronosiltransferasa/química , Glucuronosiltransferasa/genética , Hepatocitos/patología , Hígado , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Estructura Secundaria de Proteína , Factores de TiempoRESUMEN
In contrast to AAV, Simian Virus 40 (rSV40) not inducing neutralizing antibodies (NAbs) allowing re-treatment seems a promising vector for neonatal treatment of inherited liver disorders. Several studies have reported efficacy of rSV40 in animal models for inherited liver diseases. In all studies the ubiquitous endogenous early promoter controlled transgene expression establishing expression in all transduced tissues. Restricting this expression to the target tissues reduces the risk of immune response to the therapeutic gene. In this study a liver specific rSV40 vector was generated by inserting a hepatocyte specific promoter. This increased the specificity of the expression of hUGT1A1 in vitro. However, in vivo the efficacy of rSV40 appeared too low to demonstrate tissue specificity while increasing the vector dose was not possible because of toxicity. In contrast to earlier studies, neutralizing antibodies were induced. Overall, the lack of a platform to produce high titered and pure rSV40 particles and the induction of NAbs, renders it a poor candidate for in vivo gene therapy.
Asunto(s)
Glucuronosiltransferasa/genética , Hiperbilirrubinemia Hereditaria/patología , Virus 40 de los Simios/genética , Animales , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/metabolismo , Línea Celular Tumoral , Terapia Genética/métodos , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Glucuronosiltransferasa/deficiencia , Glucuronosiltransferasa/metabolismo , Humanos , Hiperbilirrubinemia Hereditaria/genética , Hígado/metabolismo , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , Distribución Tisular , Activación TranscripcionalAsunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Deficiencia de Dihidropirimidina Deshidrogenasa/genética , Glucuronosiltransferasa/deficiencia , Glucuronosiltransferasa/genética , Antimetabolitos Antineoplásicos/administración & dosificación , Antineoplásicos Fitogénicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Camptotecina/administración & dosificación , Camptotecina/análogos & derivados , Camptotecina/uso terapéutico , Neoplasias del Colon/diagnóstico por imagen , Femenino , Fluorouracilo/administración & dosificación , Fluorouracilo/uso terapéutico , Humanos , Irinotecán/administración & dosificación , Leucovorina/administración & dosificación , Leucovorina/uso terapéutico , Persona de Mediana Edad , Mutación/genética , Tomografía Computarizada por Rayos XRESUMEN
Mutations in human metabolic genes can lead to rare diseases known as inborn errors of human metabolism. For instance, patients with loss-of-function mutations in either subunit of propionyl-CoA carboxylase suffer from propionic acidemia because they cannot catabolize propionate, leading to its harmful accumulation. Both the penetrance and expressivity of metabolic disorders can be modulated by genetic background. However, modifiers of these diseases are difficult to identify because of the lack of statistical power for rare diseases in human genetics. Here, we use a model of propionic acidemia in the nematode Caenorhabditis elegans to identify genetic modifiers of propionate sensitivity. Using genome-wide association (GWA) mapping across wild strains, we identify several genomic regions correlated with reduced propionate sensitivity. We find that natural variation in the putative glucuronosyltransferase GLCT-3, a homolog of human B3GAT, partly explains differences in propionate sensitivity in one of these genomic intervals. We demonstrate that loss-of-function alleles in glct-3 render the animals less sensitive to propionate. Additionally, we find that C. elegans has an expansion of the glct gene family, suggesting that the number of members of this family could influence sensitivity to excess propionate. Our findings demonstrate that natural variation in genes that are not directly associated with propionate breakdown can modulate propionate sensitivity. Our study provides a framework for using C. elegans to characterize the contributions of genetic background in models of human inborn errors in metabolism.
Asunto(s)
Predisposición Genética a la Enfermedad , Glucuronosiltransferasa/genética , Propionatos/farmacología , Acidemia Propiónica/genética , Alelos , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Modelos Animales de Enfermedad , Estudio de Asociación del Genoma Completo , Glucuronosiltransferasa/deficiencia , Humanos , Mutación con Pérdida de Función/genética , Metabolismo/genética , Propionatos/metabolismoRESUMEN
Adeno-associated virus (AAV) vector-mediated gene therapy is currently evaluated as a potential treatment for Crigler-Najjar syndrome (CN) (NCT03466463). Pre-existing immunity to AAV is known to hinder gene transfer efficacy, restricting enrollment of seropositive subjects in ongoing clinical trials. We assessed the prevalence of anti-AAV serotype 8 (AAV8) neutralizing antibodies (NAbs) in subjects affected by CN and investigated the impact of low NAb titers (<1:5) on liver gene transfer efficacy in an in vivo passive immunization model. A total of 49 subjects with a confirmed molecular diagnosis of CN were included in an international multicenter study (NCT02302690). Pre-existing NAbs against AAV8 were detected in 30.6% (15/49) of screened patients and, in the majority of positive cases, cross-reactivity to AAV2 and AAV5 was detected. To investigate the impact of low NAbs on AAV vector-mediated liver transduction efficiency, adult wild-type C57BL/6 mice were passively immunized with pooled human donor-derived immunoglobulins to achieve titers of up to 1:3.16. After immunization, animals were injected with different AAV8 vector preparations. Hepatic vector gene copy number was unaffected by low anti-AAV8 NAb titers when column-purified AAV vector batches containing both full and empty capsids were used. In summary, although pre-existing anti-AAV8 immunity can be found in about a third of subjects affected by CN, low anti-AAV8 NAb titers are less likely to affect liver transduction efficiency when using AAV vector preparations manufactured to contain both full and empty capsids. These findings have implications for the design of liver gene transfer clinical trials and for the definition of inclusion criteria related to seropositivity of potential participants.
Asunto(s)
Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Síndrome de Crigler-Najjar/terapia , Dependovirus/genética , Terapia Genética/métodos , Glucuronosiltransferasa/genética , Adolescente , Adulto , Animales , Bilirrubina/inmunología , Bilirrubina/metabolismo , Cápside/inmunología , Cápside/metabolismo , Niño , Preescolar , Síndrome de Crigler-Najjar/genética , Síndrome de Crigler-Najjar/inmunología , Síndrome de Crigler-Najjar/patología , Dependovirus/inmunología , Antagonistas de Aminoácidos Excitadores/uso terapéutico , Femenino , Expresión Génica , Glucuronosiltransferasa/deficiencia , Glucuronosiltransferasa/inmunología , Células HEK293 , Humanos , Inmunidad Innata , Inmunización Pasiva , Hígado/inmunología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Fenobarbital/uso terapéutico , Fototerapia/métodos , Plásmidos/química , Plásmidos/metabolismo , TransfecciónRESUMEN
This article studies the genetic influence of polymorphism of the UGT2B17 gen on the urinary steroid profile and its implications for the anti-doping field. The study presents the results of a triple-blind randomized placebo-controlled crossover trial with healthy athletes submitted to a single dose of 250â¯mg of testosterone cypionate. Forty urine samples were collected from each participant. Mass spectrometry-based techniques commonly used in Anti-Doping laboratories, were employed to measure the urinary concentration and the Δδ13C values of a selection of target compounds for testosterone (T) administration together with LH. Twelve volunteers were included in the study; the polymorphism was evenly distributed among them. After T administration, the most meaningful change affected the Testosterone/Epitestosterone ratio (T/E) and the urinary concentration of LH. In relation with T/E, the wild type homozygous (ins/ins) group there was a mean relative increase of 30 (CI 95%: 25.2 to 36.7); in the heterozygous mutant (del/ins) group it was 19.8 (CI 95%:15.9 to 24.7); and in the homozygous mutant (del/del) group it was 19.7 (CI 95% 14.9 to 26.2). In the case of LH, itÌs observed how LH values decrease significantly after the administration of Testex homogeneously among the three groups. The main outcome was related to the (del/del) group (homozygous mutant), where due to the depressed basal level of the steroid profile, if the longitudinal steroid profile of the athlete was not available, the analysis by GC/MS would not produce an "atypical" result according to the WADA TD2016EAAS despite the T administration. However, the genotyping of the UGT2B17 polymorphism, the follow up of LH and the use of GC-C-IRMS makes it possible to identify most of these samples as Adverse.
Asunto(s)
Glucuronosiltransferasa/genética , Antígenos de Histocompatibilidad Menor/genética , Polimorfismo Genético/genética , Testosterona/análogos & derivados , Adulto , Atletas , Estudios Cruzados , Cromatografía de Gases y Espectrometría de Masas , Glucuronosiltransferasa/sangre , Glucuronosiltransferasa/deficiencia , Humanos , Inyecciones Intramusculares , Masculino , Antígenos de Histocompatibilidad Menor/sangre , Mutación , Testosterona/administración & dosificación , Testosterona/orinaRESUMEN
Patients with combined hereditary spherocytosis (HS) and uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) deficiency have been reported sporadically. A discrepancy between the level of elevated serum bilirubin concentration and the degree of anemia may suggest the possibility of a coexistence of these conditions. In the present case report, a 20yearold female presented with congenital jaundice and anemia, but did not present with the discrepancy between hyperbilirubinemia and anemia in the patient's childhood, and was not previously diagnosed with either HS or UGT1A1 deficiency. During a followup of >10 years, the patient's hyperbilirubinemia accumulated progressively, whereas the patient's anemia became relatively mild. Upon further genetic analysis, it was determined that the patient had HS combined with UGT1A1 partial deficiency. Next generation sequencing combined with direct sequencing was used to identify a novel heterozygous mutation (c.G828T; p.Y276X) in the spectrin ß gene, which is causative for HS. Sequence analysis of the patients' UGT1A1 gene revealed a compound heterozygote with c.G211A (p.G71R) and T3279G mutations, which reduced UGT1A1 activity to 3060% of the normal level. Genetic analysis was crucial for determining the diagnosis and pathogenesis of this unusual case.
Asunto(s)
Glucuronosiltransferasa/genética , Esferocitosis Hereditaria/diagnóstico , Esferocitosis Hereditaria/genética , Biomarcadores , Análisis Mutacional de ADN , Femenino , Estudios de Asociación Genética , Pruebas Genéticas , Genotipo , Glucuronosiltransferasa/deficiencia , Humanos , Mutación , Fenotipo , Espectrina/genética , Esferocitos/patología , Esferocitosis Hereditaria/sangre , Adulto JovenRESUMEN
BACKGROUND: The human natural killer-1 (HNK-1) carbohydrate, a unique trisaccharide possessing sulfated glucuronic acid in a non-reducing terminus (HSO3-3GlcAß1-3Galß1-4GlcNAc-), is highly expressed in the nervous system and its spatiotemporal expression is strictly regulated. Mice deficient in the gene encoding a key enzyme, GlcAT-P, of the HNK-1 biosynthetic pathway exhibit almost complete disappearance of the HNK-1 epitope in the brain, significant reduction of long-term potentiation, and aberration of spatial learning and memory formation. In addition to its physiological roles in higher brain function, the HNK-1 carbohydrate has attracted considerable attention as an autoantigen associated with peripheral demyelinative neuropathy, which relates to IgM paraproteinemia, because of high immunogenicity. It has been suggested, however, that serum autoantibodies in IgM anti-myelin-associated glycoprotein (MAG) antibody-associated neuropathy patients show heterogeneous reactivity to the HNK-1 epitope. SCOPE OF REVIEW: We have found that structurally distinct HNK-1 epitopes are expressed in specific proteins in the nervous system. Here, we overview the current knowledge of the involvement of these HNK-1 epitopes in the regulation of neural plasticity and discuss the impact of different HNK-1 antigens of anti-MAG neuropathy patients. MAJOR CONCLUSIONS: We identified the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor subunit GluA2 and aggrecan as HNK-1 carrier proteins. The HNK-1 epitope on GluA2 and aggrecan regulates neural plasticity in different ways. Furthermore, we found the clinical relationship between reactivity of autoantibodies to the different HNK-1 epitopes and progression of anti-MAG neuropathy. GENERAL SIGNIFICANCE: The HNK-1 epitope is indispensable for the acquisition of normal neuronal function and can be a good target for the establishment of diagnostic criteria for anti-MAG neuropathy.
Asunto(s)
Antígenos CD57/química , Epítopos/química , Glicoproteína Asociada a Mielina/inmunología , Plasticidad Neuronal , Paraproteinemias/inmunología , Enfermedades del Sistema Nervioso Periférico/inmunología , Agrecanos/metabolismo , Animales , Autoanticuerpos/biosíntesis , Antígenos CD57/genética , Antígenos CD57/inmunología , Epítopos/genética , Epítopos/inmunología , Glucuronosiltransferasa/deficiencia , Glucuronosiltransferasa/genética , Humanos , Inmunoglobulina M/biosíntesis , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Ratones , Ratones Noqueados , Glicoproteína Asociada a Mielina/genética , Paraproteinemias/genética , Paraproteinemias/patología , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades del Sistema Nervioso Periférico/patología , Receptores AMPA/genética , Receptores AMPA/inmunologíaRESUMEN
Hyperbilirubinemia, caused by the accumulation of unconjugated bilirubin, is one of the most common clinical diagnoses in both premature and term newborns. Owing to the fact that bilirubin is metabolized solely through glucuronidation by UDP-glucuronosyltransferase (UGT) 1A1, it is now known that immaturity of UGT1A1, in combination with the overproduction of bilirubin during the developmental stage, acts as a bottleneck to bilirubin elimination and predisposes the infant to high total serum bilirubin levels. Although neonatal jaundice is mostly benign, excessively high levels of serum bilirubin in a small percentage of newborns can cause bilirubin-induced neurologic dysfunction, potentially leading to permanent brain damage, a condition known as kernicterus Although a large portion of hyperbilirubinemia cases in newborns are associated with hemolytic diseases, we emphasize here the impaired ability of UGT1A1 to eliminate bilirubin that contributes to hyperbilirubinemia-induced neurotoxicity in the developmental stage. As a series of hereditary UGT1A1 mutations have been identified that are associated with UGT1A1 deficiency, new evidence has verified that delayed expression of UGT1A1 during the early stages of neonatal development is a tightly controlled event involving coordinated intrahepatic and extrahepatic regulation. This review recapitulates the progress that has been made in recent years in understanding the causes and physiopathology of severe hyperbilirubinemia, investigating molecular mechanisms underlying bilirubin-induced encephalopathy, and searching for potential therapies for treating pathologic hyperbilirubinemia. Several animal models have been developed to make it possible to examine bilirubin-induced neurotoxicity from multiple directions. Moreover, environmental factors that may alleviate or worsen the condition of hyperbilirubinemia are discussed.
Asunto(s)
Hiperbilirrubinemia Neonatal/etiología , Animales , Bilirrubina/biosíntesis , Bilirrubina/sangre , Dieta , Glucuronosiltransferasa/deficiencia , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Humanos , Hiperbilirrubinemia Neonatal/sangre , Hiperbilirrubinemia Neonatal/inducido químicamente , Hiperbilirrubinemia Neonatal/genética , Recién NacidoRESUMEN
BACKGROUND: Severe hyperbilirubinemia is toxic during central nervous system development. Prolonged and uncontrolled high levels of unconjugated bilirubin lead to bilirubin-induced neurological damage and eventually death by kernicterus. Bilirubin neurotoxicity is characterized by a wide array of neurological deficits, including irreversible abnormalities in motor, sensitive and cognitive functions, due to bilirubin accumulation in the brain. Despite the abundant literature documenting the in vitro and in vivo toxic effects of bilirubin, it is unclear which molecular and cellular events actually characterize bilirubin-induced neurodegeneration in vivo. METHODS: We used a mouse model of neonatal hyperbilirubinemia to temporally and spatially define the response of the developing cerebellum to the bilirubin insult. RESULTS: We showed that the exposure of developing cerebellum to sustained bilirubin levels induces the activation of oxidative stress, ER stress and inflammatory markers at the early stages of the disease onset. In particular, we identified TNFα and NFKß as key mediators of bilirubin-induced inflammatory response. Moreover, we reported that M1 type microglia is increasingly activated during disease progression. Failure to counteract this overwhelming stress condition resulted in the induction of the apoptotic pathway and the generation of the glial scar. Finally, bilirubin induced the autophagy pathway in the stages preceding death of the animals. CONCLUSIONS: This study demonstrates that inflammation is a key contributor to bilirubin damage that cooperates with ER stress in the onset of neurotoxicity. Pharmacological modulation of the inflammatory pathway may be a potential intervention target to ameliorate neonatal lethality in Ugt1 -/- mice.
Asunto(s)
Cerebelo/patología , Hiperbilirrubinemia Neonatal/complicaciones , Inflamación/patología , Degeneración Nerviosa/patología , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Glucuronosiltransferasa/deficiencia , Hiperbilirrubinemia Neonatal/patología , Inflamación/etiología , Inflamación/metabolismo , Ratones , Ratones Noqueados , Degeneración Nerviosa/etiología , Degeneración Nerviosa/metabolismoRESUMEN
OBJECTIVE: Hyaluronan (HA) is a polymeric glucosaminoglycan that forms a provisional extracellular matrix in diseased vessels. HA is synthesized by 3 different HA synthases (HAS1, HAS2, and HAS3). Aim of this study was to unravel the role of the HAS3 isoenzyme during experimental neointimal hyperplasia. APPROACH AND RESULTS: Neointimal hyperplasia was induced in Has3-deficient mice by ligation of the carotid artery. HA in the media of Has3-deficient mice was decreased 28 days after ligation, and neointimal hyperplasia was strongly inhibited. However, medial and luminal areas were unaffected. Cell density, proliferation, and apoptosis were not altered, suggesting a proportional decrease of both, the number of cells and extracellular matrix. In addition, endothelial function as determined by acetylcholine-induced relaxation of aortic rings, immunoblotting of endothelial nitric oxide synthase, and arterial blood pressure were not affected. Furthermore, the oxidative stress response was not affected as determined in total protein extracts from aortae. Transcriptome analysis comparing control versus ligated carotid arteries hinted toward a mitigated differential regulation of various signaling pathways in Has3-deficient mice in response to ligation that were related to vascular smooth muscle cell (VSMC) migration, including focal adhesions, integrins, mitogen-activated protein kinase, and phosphatidylinositol signaling system. Lentiviral overexpression of HAS3 in VSMC supported the migratory phenotype of VSMC in response to platelet-derived growth factor BB in vitro. Accordingly, knockdown of HAS3 reduced the migratory response to platelet-derived growth factor BB and in addition decreased the expression of PDGF-B mRNA. CONCLUSIONS: HAS3-mediated HA synthesis after vessel injury supports seminal signaling pathways in activation of VSMC, increases platelet-derived growth factor BB-mediated migration, and in turn enhances neointimal hyperplasia in vivo.
Asunto(s)
Enfermedades de las Arterias Carótidas/enzimología , Glucuronosiltransferasa/deficiencia , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Neointima , Animales , Becaplermina , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/patología , Arteria Carótida Común/enzimología , Arteria Carótida Común/patología , Movimiento Celular , Proliferación Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Femenino , Eliminación de Gen , Regulación de la Expresión Génica , Genotipo , Glucuronosiltransferasa/genética , Hialuronano Sintasas , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/patología , Fenotipo , Proteínas Proto-Oncogénicas c-sis/genética , Proteínas Proto-Oncogénicas c-sis/metabolismo , Proteínas Proto-Oncogénicas c-sis/farmacología , Transducción de Señal , Transcripción Genética , TransfecciónRESUMEN
Exemestane is an aromatase inhibitor drug used for the treatment of hormone-dependent breast cancer. 17-Hydroexemestane, the major and biologically active metabolite of exemestane in humans, is eliminated via glucuronidation by the polymorphic UGT2B17 phase II drug-metabolizing enzyme. Previous microsomal studies have shown that UGT2B17 gene deletion affects the intrinsic hepatic clearances of 17-hydroexemestane in vitro. In this open-label study we set out to assess the effect of UGT2B17 gene deletion on the pharmacokinetics of 17-hydroexemestane in healthy female volunteers with and without UGT2B17. To achieve this goal, 14 healthy postmenopausal women (8 carriers of the homozygous UGT2B17 wild-type allele and 6 carriers of the homozygous UGT2B17 gene-deletion allele) were enrolled and invited to receive a single 25-mg oral dose of exemestane. Pharmacokinetics was assessed over 72 hours postdosing. Our results showed that there were statistically significant differences in plasma 17-hydroexemestane AUC0-∞ (P = .0007) and urine 17-hydroexemestane C24h (P = .001) between UGT2B17 genotype groups. Our data suggest that UGT2B17 gene deletion influences 17-hydroexemestane pharmacokinetics in humans.
Asunto(s)
Androstadienos/farmacocinética , Inhibidores de la Aromatasa/farmacocinética , Eliminación de Gen , Glucuronosiltransferasa/deficiencia , Glucuronosiltransferasa/genética , Antígenos de Histocompatibilidad Menor/genética , Androstadienos/metabolismo , Inhibidores de la Aromatasa/metabolismo , Femenino , Voluntarios Sanos , Humanos , Persona de Mediana Edad , Proyectos PilotoRESUMEN
OBJECTIVE: Articular cartilage defects commonly result from traumatic injury and predispose to degenerative joint diseases. To test the hypothesis that aberrant healing responses and chronic inflammation lead to osteoarthritis (OA), we examined spatiotemporal changes in joint tissues after cartilage injury in murine knees. Since intra-articular injection of hyaluronan (HA) can attenuate injury-induced osteoarthritis in wild-type (WT) mice, we investigated a role for HA in the response to cartilage injury in mice lacking HA synthase 1 (Has1(-/-)). DESIGN: Femoral groove cartilage of WT and Has1(-/-) mice was debrided to generate a non-bleeding wound. Macroscopic imaging, histology, and gene expression were used to evaluate naïve, sham-operated, and injured joints. RESULTS: Acute responses (1-2 weeks) in injured joints from WT mice included synovial hyperplasia with HA deposition and joint-wide increases in expression of genes associated with inflammation, fibrosis, and extracellular matrix (ECM) production. By 4 weeks, some resurfacing of damaged cartilage occurred, and early cell responses were normalized. Cartilage damage in Has1(-/-) mice also induced early responses; however, at 4 weeks, inflammation and fibrosis genes remained elevated with widespread cartilage degeneration and fibrotic scarring in the synovium and joint capsule. CONCLUSIONS: We conclude that the ineffective repair of injured cartilage in Has1(-/-) joints can be at least partly explained by the markedly enhanced expression of particular genes in pathways linked to ECM turnover, IL-17/IL-6 cytokine signaling, and apoptosis. Notably, Has1 ablation does not alter gross HA content in the ECM, suggesting that HAS1 has a unique function in the metabolism of inflammatory HA matrices.
Asunto(s)
Cartílago Articular/patología , Regulación de la Expresión Génica , Glucuronosiltransferasa/deficiencia , Glucuronosiltransferasa/genética , Articulación de la Rodilla/patología , Osteoartritis de la Rodilla/enzimología , ARN/genética , Animales , Cartílago Articular/enzimología , Cartílago Articular/lesiones , Enfermedad Crónica , Modelos Animales de Enfermedad , Fibrosis/enzimología , Fibrosis/patología , Glucuronosiltransferasa/biosíntesis , Hialuronano Sintasas , Inflamación/enzimología , Inflamación/genética , Inflamación/patología , Articulación de la Rodilla/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Osteoartritis de la Rodilla/genética , Osteoartritis de la Rodilla/patología , Reacción en Cadena de la PolimerasaRESUMEN
UDP-Glucuronosyltransferases (UGTs) conjugate a glucuronyl group from glucuronic acid to a wide range of lipophilic substrates to form a hydrophilic glucuronide conjugate. The glucuronide generally has decreased bioactivity and increased water solubility to facilitate excretion. Glucuronidation represents an important detoxification pathway for both endogenous waste products and xenobiotics, including drugs and harmful industrial chemicals. Two clinically significant families of UGT enzymes are present in mammals: UGT1s and UGT2s. Although the two families are distinct in gene structure, studies using recombinant enzymes have shown considerable overlap in their ability to glucuronidate many substrates, often obscuring the relative importance of the two families in the clearance of particular substrates in vivo. To address this limitation, we have generated a mouse line, termed ΔUgt2, in which the entire Ugt2 gene family, extending over 609 kilobase pairs, is excised. This mouse line provides a means to determine the contributions of the two UGT families in vivo. We demonstrate the utility of these animals by defining for the first time the in vivo contributions of the UGT1 and UGT2 families to glucuronidation of the environmental estrogenic agent bisphenol A (BPA). The highest activity toward this chemical is reported for human and rodent UGT2 enzymes. Surprisingly, our studies using the ΔUgt2 mice demonstrate that, while both UGT1 and UGT2 isoforms can conjugate BPA, clearance is largely dependent on UGT1s.
Asunto(s)
Glucuronosiltransferasa/deficiencia , Glucuronosiltransferasa/genética , Microsomas Hepáticos/metabolismo , Xenobióticos/metabolismo , Animales , Compuestos de Bencidrilo/metabolismo , Compuestos de Bencidrilo/farmacología , Inactivación Metabólica/fisiología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Microsomas Hepáticos/efectos de los fármacos , Fenoles/metabolismo , Fenoles/farmacología , Xenobióticos/farmacologíaRESUMEN
PURPOSE: Given the toxic nature and narrow therapeutic index of traditional chemotherapeutics, better methods of dose and therapy selection are critical. Pharmacological methods, including pharmacogenomics and pharmacokinetics, offer a practical method to enrich drug exposure, reduce toxicity, and improve quality of life for patients. METHODS: PubMed and key abstracts from the American Society of Clinical Oncology (ASCO) and American Association for Cancer Research (AACR) were searched until July 2015 for clinical data relating to pharmacogenomic- and/or pharmacokinetic-guided dosing of anticancer drugs. RESULTS: Based on the results returned from a thorough search of the literature and the plausibility of utilizing pharmacogenomic and/or pharmacokinetic methods to personalize chemotherapy dosing, we identified several chemotherapeutic agents with the potential for therapy individualization. We highlight the available data, clinical validity, and utility of using pharmacogenomics to personalize therapy for tamoxifen, 5-fluorouracil, mercaptopurine, and irinotecan, in addition to using pharmacokinetics to personalize dosing for 5-fluorouracil, busulfan, methotrexate, taxanes, and topotecan. CONCLUSION: A concerted effort should be made by researchers to further elucidate the role of pharmacological methods in personalizing chemotherapy dosing to optimize the risk-benefit profile. Clinicians should be aware of the clinical validity, utility, and availability of pharmacogenomic- and pharmacokinetic-guided therapies in clinical practice, to ultimately allow optimal dosing for each and every cancer patient.
Asunto(s)
Antineoplásicos/administración & dosificación , Neoplasias/tratamiento farmacológico , Medicina de Precisión , Alelos , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Biotransformación/genética , Sistema Enzimático del Citocromo P-450/deficiencia , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/fisiología , Deficiencia de Dihidropirimidina Deshidrogenasa/genética , Dihidrouracilo Deshidrogenasa (NADP)/genética , Dihidrouracilo Deshidrogenasa (NADP)/fisiología , Relación Dosis-Respuesta a Droga , Hipersensibilidad a las Drogas/genética , Femenino , Predicción , Glucuronosiltransferasa/deficiencia , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/fisiología , Humanos , Masculino , Metiltransferasas/genética , Metiltransferasas/fisiología , Errores Innatos del Metabolismo de la Purina-Pirimidina/genéticaRESUMEN
Recent advances in medical genomics open new perspectives for personalized medicine through the identification of genetic variants that influence drug response and/or the risk of side effects. Today, the clinical applications of pharmacogenetics remain scarce as a consequence of the cost and turn-around-time of genetic tests. However, a few tests are recommended, for instance before the prescription of some anti-cancer agents or the anti-retroviral agent abacavir. In the future, we will probably move either towards rapid targeted tests or towards a large screening, before any diagnosis, of all the genetic factors influencing the therapeutic response. In that case, physicians will have to consult the patient genomic data before drug prescription in order to personalize the choice of the therapeutic agent or its dosage. However, such a genomic approach brings economical and ethical questions and will require further progress in our capacity to interpret and store the personal genomic data without compromising their confidentiality.
Asunto(s)
Pruebas Genéticas/estadística & datos numéricos , Farmacogenética/métodos , Medicina de Precisión/tendencias , Biomarcadores Farmacológicos/análisis , Didesoxinucleósidos/uso terapéutico , Deficiencia de Dihidropirimidina Deshidrogenasa/diagnóstico , Deficiencia de Dihidropirimidina Deshidrogenasa/genética , Hipersensibilidad a las Drogas/diagnóstico , Hipersensibilidad a las Drogas/genética , Pruebas Genéticas/tendencias , Glucuronosiltransferasa/deficiencia , Glucuronosiltransferasa/genética , Antígenos HLA-B/genética , Humanos , Neoplasias/genética , Neoplasias/terapia , Medicina de Precisión/métodos , Errores Innatos del Metabolismo de la Purina-Pirimidina/diagnóstico , Errores Innatos del Metabolismo de la Purina-Pirimidina/genéticaRESUMEN
Crigler-Najjar syndrome presents as severe unconjugated hyperbilirubinemia and is characteristically caused by a mutation in the UGT1A1 gene, encoding the enzyme responsible for bilirubin glucuronidation. Here we present a patient with Crigler-Najjar syndrome with a completely normal UGT1A1 coding region. Instead, a homozygous 3 nucleotide insertion in the UGT1A1 promoter was identified that interrupts the HNF1α binding site. This mutation results in almost complete abolishment of UGT1A1 promoter activity and prevents the induction of UGT1A1 expression by the liver nuclear receptors CAR and PXR, explaining the lack of a phenobarbital response in this patient. Although animal studies have revealed the importance of HNF1α for normal liver function, this case provides the first clinical proof that mutations in its binding site indeed result in severe liver pathology stressing the importance of promoter sequence analysis.
Asunto(s)
Síndrome de Crigler-Najjar/genética , Síndrome de Crigler-Najjar/metabolismo , Glucuronosiltransferasa/deficiencia , Glucuronosiltransferasa/genética , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Adulto , Secuencia de Bases , Sitios de Unión/genética , Receptor de Androstano Constitutivo , Síndrome de Crigler-Najjar/clasificación , Femenino , Homocigoto , Humanos , Hígado/metabolismo , Datos de Secuencia Molecular , Mutagénesis Insercional , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares/metabolismo , Análisis de Secuencia de ADN , Transcripción Genética/efectos de los fármacosRESUMEN
Hepatocyte transplantation has the potential to cure inherited liver diseases, but its application is impeded by a scarcity of donor livers. Therefore, we explored whether transplantation of hepatocyte-like cells (iHeps) differentiated from human induced pluripotent stem cells (iPSCs) could ameliorate inherited liver diseases. iPSCs reprogrammed from human skin fibroblasts were differentiated to iHeps, which were transplanted into livers of uridinediphosphoglucuronate glucuronosyltransferase-1 (UGT1A1)-deficient Gunn rats, a model of Crigler-Najjar syndrome 1 (CN1), where elevated unconjugated bilirubin causes brain injury and death. To promote iHep proliferation, 30% of the recipient liver was X-irradiated before transplantation, and hepatocyte growth factor was expressed. After transplantation, UGT1A1+ iHep clusters constituted 2.5%-7.5% of the preconditioned liver lobe. A decline of serum bilirubin by 30%-60% and biliary excretion of bilirubin glucuronides indicated that transplanted iHeps expressed UGT1A1 activity, a postnatal function of hepatocytes. Therefore, iHeps warrant further exploration as a renewable source of hepatocytes for treating inherited liver diseases.