RESUMEN
Although "genomically" humanized animals are invaluable tools for generating human disease models as well as for biomedical research, their development has been mainly restricted to mice via established transgenic-based and embryonic stem cell-based technologies. Since rats are widely used for studying human disease and for drug efficacy and toxicity testing, humanized rat models would be preferred over mice for several applications. However, the development of sophisticated humanized rat models has been hampered by the difficulty of complex genetic manipulations in rats. Additionally, several genes and gene clusters, which are megabase range in size, were difficult to introduce into rats with conventional technologies. As a proof of concept, we herein report the generation of genomically humanized rats expressing key human drug-metabolizing enzymes in the absence of their orthologous rat counterparts via the combination of chromosome transfer using mouse artificial chromosome (MAC) and genome editing technologies. About 1.5 Mb and 700 kb of the entire UDP glucuronosyltransferase family 2 and cytochrome P450 family 3 subfamily A genomic regions, respectively, were successfully introduced via the MACs into rats. The transchromosomic rats were combined with rats carrying deletions of the endogenous orthologous genes, achieved by genome editing. In the "transchromosomic humanized" rat strains, the gene expression, pharmacokinetics, and metabolism observed in humans were well reproduced. Thus, the combination of chromosome transfer and genome editing technologies can be used to generate fully humanized rats for improved prediction of the pharmacokinetics and drug-drug interactions in humans, and for basic research, drug discovery, and development.
Asunto(s)
Citocromo P-450 CYP3A/genética , Edición Génica , Glucuronosiltransferasa/genética , Inactivación Metabólica/genética , Animales , Técnicas de Transferencia de Gen , Genoma , Humanos , Tasa de Depuración Metabólica/genética , Ratones , Ratones Transgénicos , RatasRESUMEN
Drug-induced liver injury (DILI) is one of the most common adverse drug reactions. DILI is often accompanied by skin reactions, including rash and pruritus. However, it is still unknown whether DILI-associated genes such as S100 calcium-binding protein A and interleukin (IL)-1ß are involved in drug-induced skin toxicity. In the present study, most of the tested hepatotoxic drugs such as pioglitazone and diclofenac induced DILI-associated genes in human and mouse keratinocytes. Keratinocytes of mice at higher risk for DILI exhibited an increased IL-1ß basal expression. They also showed a higher inducibility of IL-1ß when treated by pioglitazone. Mice at higher risk for DILI showed even higher sums of DILI-associated gene basal expression levels and induction rates in keratinocytes. Our data suggest that DILI-associated genes might be involved in the onset and progression of drug-induced skin toxicity. Furthermore, we might be able to identify individuals at higher risk of developing DILI less invasively by examining gene expression patterns in keratinocytes. Copyright © 2017 John Wiley & Sons, Ltd.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/etiología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/genética , Sustancias Peligrosas/toxicidad , Queratinocitos/efectos de los fármacos , Preparaciones Farmacéuticas , Adulto , Anciano , Animales , Proteínas de Unión al Calcio , Femenino , Humanos , Interleucina-1beta , Hígado/fisiopatología , Masculino , Ratones , Persona de Mediana EdadRESUMEN
Anticonvulsants can increase the risk of developing neurotoxicity in infants; however, the underlying mechanism has not been elucidated to date. Thyroxine [3,5,3',5'-l-tetraiodothyronine (T4)] plays crucial roles in the development of the central nervous system. In this study, we hypothesized that induction of UDP-glucuronosyltransferase 1A1 (UGT1A1)-an enzyme involved in the metabolism of T4-by anticonvulsants would reduce serum T4 levels and cause neurodevelopmental toxicity. Exposure of mice to phenytoin during both the prenatal and postnatal periods significantly induced UGT1A1 and decreased serum T4 levels on postnatal day 14. In the phenytoin-treated mice, the mRNA levels of synaptophysin and synapsin I in the hippocampus were lower than those in the control mice. The thickness of the external granule cell layer was greater in phenytoin-treated mice, indicating that induction of UGT1A1 during the perinatal period caused neurodevelopmental disorders. Exposure to phenytoin during only the postnatal period also caused these neurodevelopmental disorders. A T4 replacement attenuated the increase in thickness of the external granule cell layer, indicating that the reduced T4 was specifically associated with the phenytoin-induced neurodevelopmental disorder. In addition, these neurodevelopmental disorders were also found in the carbamazepine- and pregnenolone-16-α-carbonitrile-treated mice. Our study is the first to indicate that UGT1A1 can control neurodevelopment by regulating serum T4 levels.
Asunto(s)
Glucuronosiltransferasa/biosíntesis , Trastornos del Neurodesarrollo/enzimología , Animales , Animales Recién Nacidos , Encéfalo/metabolismo , Encéfalo/patología , Carbamazepina/química , Carbamazepina/farmacología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Inducción Enzimática/efectos de los fármacos , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genes del Desarrollo , Humanos , Ratones Endogámicos C57BL , Ratones Transgénicos , Leche Humana/metabolismo , Trastornos del Neurodesarrollo/sangre , Trastornos del Neurodesarrollo/genética , Fenitoína/química , Embarazo , Carbonitrilo de Pregnenolona/farmacología , Efectos Tardíos de la Exposición Prenatal/sangre , Efectos Tardíos de la Exposición Prenatal/genética , Efectos Tardíos de la Exposición Prenatal/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Prueba de Desempeño de Rotación con Aceleración Constante , Tiroxina/sangre , Tiroxina/químicaRESUMEN
Although UDP-glucuronosyltransferases (UGTs) are important phase II drug-metabolizing enzymes, they are also involved in the metabolism of endogenous compounds. Certain substrates of UGTs, such as serotonin and estradiol, play important roles in the brain. However, the expression of UGTs in the human brain has not been fully clarified. Recently, humanized UGT1 mice (hUGT1 mice) in which the original Ugt1 locus was disrupted and replaced with the human UGT1 locus have been developed. In the present study, the expression pattern of UGT1As in brains from humans and hUGT1 mice was examined. We found that UGT1A1, 1A3, 1A6, and 1A10 were expressed in human brains. The expression pattern of UGT1As in hUGT1 mouse brains was similar to that in human brains. In addition, we examined the expression of UGT1A1 and 1A6 in the cerebellum, olfactory bulbs, midbrain, hippocampus, and cerebral cortex of hUGT1 mice. UGT1A1 in all brain regions and UGT1A6 in the cerebellum and cerebral cortex of 6-month-old hUGT1 mice were expressed at a significantly higher rate than those of 2-week-old hUGT1 mice. A difference in expression levels between brain regions was also observed. Brain microsomes exhibited glucuronidation activities toward estradiol and serotonin, with mean values of 0.13 and 5.17 pmol/min/mg, respectively. In conclusion, UGT1A1 and UGT1A6 might play an important role in function regulation of endogenous compounds in a region- and age-dependent manner. Humanized UGT1 mice might be useful to study the importance of brain UGTs in vivo.
Asunto(s)
Química Encefálica/genética , Proteínas de Transporte de Monosacáridos/genética , Adulto , Envejecimiento/metabolismo , Animales , Carbamazepina/farmacología , Estradiol/metabolismo , Femenino , Glucurónidos/metabolismo , Humanos , Isoenzimas/biosíntesis , Isoenzimas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microsomas/metabolismo , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Persona de Mediana Edad , Carbonitrilo de Pregnenolona/farmacología , Serotonina/metabolismoRESUMEN
Glycyrrhetinic acid (GA) is an active metabolite of glycyrrhizin, which is a main constituent in licorice (Glycyrrhiza glabra). While GA exhibits a wide variety of pharmacological activities in the body, it is converted to a toxic metabolite GA 3-O-glucuronide by hepatic UDP-glucuronosyltransferases (UGTs). To avoid the development of the toxic metabolite-induced pseudohyperaldosteronism (pseudoaldosteronism), there is a limitation in maximum daily dosage of licorice and in combined usage of other glycyrrhizin-containing natural medicine. In this study, we investigated the inhibitory effects of various polyphenols and triterpenoids on the UGT-mediated GA 3-O-glucuronidation. In human liver microsomes, UGT-mediated GA glucuronidation was significantly inhibited by protopanaxadiol with an IC50 value of 59.2 µM. Isoliquiritigenin, rosmarinic acid, alisol B, alisol acetate, and catechin moderately inhibited the GA glucuronidation with IC50 values of 96.4 µM, 125 µM, 160 µM, 163 µM, and 164 µM. Other tested 19 polyphenols and triterpenoids, including liquiritigenin, did not inhibit UGT-mediated GA glucuronidation in human liver microsomes. Our data indicate that relatively higher dosage of licorice can be used without a risk of developing pseudohyperaldosteronism in combination of natural medicine containing protopanaxadiol such as Panax ginseng. Furthermore, supplemental protopanaxadiol and isoliquiritigenin might be useful in preventing licorice-inducing pseudoaldosteronism.