RESUMEN
Mucopolysaccharidosis II (MPS II) is a rare lysosomal storage disease characterized by deficient activity of iduronate-2-sulfatase (I2S), leading to pathological accumulation of glycosaminoglycans (GAGs) in tissues. We used iduronate-2-sulfatase knockout (Ids KO) mice to investigate if liver-directed recombinant adeno-associated virus vectors (rAAV8-LSP-hIDSco) encoding human I2S (hI2S) could cross-correct I2S deficiency in Ids KO mouse tissues, and we then assessed the translation of mouse data to non-human primates (NHPs). Treated mice showed sustained hepatic hI2S production, accompanied by normalized GAG levels in somatic tissues (including critical tissues such as heart and lung), indicating systemic cross-correction from liver-secreted hI2S. Brain GAG levels in Ids KO mice were lowered but not normalized; higher doses were required to see improvements in brain histology and neurobehavioral testing. rAAV8-LSP-hIDSco administration in NHPs resulted in sustained hepatic hI2S production and therapeutic hI2S levels in cross-corrected somatic tissues but no hI2S exposure in the central nervous system, perhaps owing to lower levels of liver transduction in NHPs than in mice. Overall, we demonstrate the ability of rAAV8-LSP-hIDSco to cross-correct I2S deficiency in mouse somatic tissues and highlight the importance of showing translatability of gene therapy data from rodents to NHPs, which is critical for supporting translation to clinical development.
RESUMEN
During its clinical development fialuridine caused liver toxicity and the death of five patients. This case remains relevant due to the continued development of mechanistically-related compounds against a back-drop of simple in vitro models which remain limited for the preclinical detection of such delayed toxicity. Here, proteomic investigation of a differentiated, HepaRG, and proliferating, HepG2 cell model was utilised to confirm the presence of the hENT1 transporter, thymidine kinase-1 and -2 (TK1, TK2) and thymidylate kinase, all essential in order to reproduce the cellular activation and disposition of fialuridine in the clinic. Acute metabolic modification assays could only identify mitochondrial toxicity in HepaRG cells following extended dosing, 2 weeks. Toxic effects were observed around 10 µM, which is within a range of 10-15 X approximate Cmax. HepaRG cell death was accompanied by a significant decrease in mitochondrial DNA content, indicative of inhibition of mitochondrial replication, and a subsequent reduction in mitochondrial respiration and the activity of mitochondrial respiratory complexes, not replicated in HepG2 cells. The structural epimer of fialuridine, included as a pharmacological negative control, was shown to have no cytotoxic effects in HepaRG cells up to 4 weeks. Overall, these comparative studies demonstrate the HepaRG model has translational relevance for fialuridine toxicity and therefore may have potential in investigating the inhibition of mitochondrial replication over prolonged exposure for other toxicants.
Asunto(s)
Antivirales/farmacología , Arabinofuranosil Uracilo/análogos & derivados , Hepatocitos/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Arabinofuranosil Uracilo/farmacología , Línea Celular Tumoral , Replicación del ADN/efectos de los fármacos , ADN Mitocondrial/fisiología , Relación Dosis-Respuesta a Droga , Humanos , Mitocondrias/fisiologíaRESUMEN
Nonclinical rodent and nonrodent toxicity models used to support clinical trials of candidate drugs may produce discordant results or fail to predict complications in humans, contributing to drug failures in the clinic. Here, we applied microengineered Organs-on-Chips technology to design a rat, dog, and human Liver-Chip containing species-specific primary hepatocytes interfaced with liver sinusoidal endothelial cells, with or without Kupffer cells and hepatic stellate cells, cultured under physiological fluid flow. The Liver-Chip detected diverse phenotypes of liver toxicity, including hepatocellular injury, steatosis, cholestasis, and fibrosis, and species-specific toxicities when treated with tool compounds. A multispecies Liver-Chip may provide a useful platform for prediction of liver toxicity and inform human relevance of liver toxicities detected in animal studies to better determine safety and human risk.
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/patología , Dispositivos Laboratorio en un Chip , Hígado/patología , Animales , Biomarcadores/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Perros , Humanos , Macrófagos del Hígado/metabolismo , Hígado/lesiones , Hepatopatías/patología , Fenotipo , Ratas , Reproducibilidad de los Resultados , Factores de Riesgo , Especificidad de la EspecieRESUMEN
Using galactose instead of glucose in the culture medium of hepatoma cell lines, such as HepG2 cells, has been utilized for a decade to unmask the mitochondrial liability of chemical compounds. A modified glucose-galactose assay on HepG2 cells, reducing the experimental period for screening of mitochondrial toxicity to 2 to 4 hr, has been previously reported. HepaRG cells are one of the few cell lines that retain some of the important characteristics of human hepatocytes, offering advantages of working with a cell line, therefore, are considered an alternative for HepG2 cells in drug toxicity screening. A method is described here using HepaRG cells in an acute metabolic switch assay utilizing specific glucose/galactose media, a combined ATP-protein-LDH assay measuring three endpoints from one 96-well plate, and a criteria to label a compound as a mitochondrial toxin. © 2019 by John Wiley & Sons, Inc.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Medios de Cultivo/química , Galactosa/metabolismo , Glucosa/metabolismo , Hepatocitos/efectos de los fármacos , Mitocondrias Hepáticas , Bioensayo , Línea Celular , Hepatocitos/metabolismo , Humanos , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismoRESUMEN
Histone deacetylase inhibitors (HDACIs) have demonstrated effectiveness against lymphomas and myelomas in clinical practice. However, common to all currently approved broad-acting HDACIs (panHDACIs) is dose-limiting thrombocytopenia, which has prevented wider use in cancer therapy. Using CD34+ hematopoietic stem cells (HSCs), we show that megakaryocyte (MK) cell maturation and differentiation are impaired by panHDACIs, correlating to clinical thrombocytopenia. Importantly, we demonstrate that inhibitors of class II histone deacetylases (HDACs), including LMK235 and tubacin at clinically relevant concentrations, do not affect MK maturation. Furthermore, we show that HDACI-induced impairment of MK differentiation is associated with reduction of protein levels of the transcription factor GATA-1, but not tubulin hyperacetylation. Finally, we report that panHDACIs trigger a rapid loss of GATA-1 protein via a proteasome-dependent pathway. Our data support the notion that specifically targeting class II HDACs in cancer treatment is a potential strategy that would offer a safer alternative than current panHDACIs.
Asunto(s)
Anilidas/farmacología , Diferenciación Celular/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Megacariocitos/metabolismo , Anilidas/efectos adversos , Antígenos CD34/metabolismo , Línea Celular Tumoral , Factor de Transcripción GATA1/metabolismo , Células Madre Hematopoyéticas/patología , Inhibidores de Histona Desacetilasas/efectos adversos , Humanos , Ácidos Hidroxámicos/efectos adversos , Megacariocitos/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis/efectos de los fármacos , Trombocitopenia/inducido químicamente , Trombocitopenia/metabolismo , Trombocitopenia/patologíaRESUMEN
The importance of mitochondrial toxicity in drug-induced liver injury is well established. The bioenergetic phenotype of the HepaRG cell line was defined in order to assess their suitability as a model of mitochondrial hepatotoxicity. Bioenergetic phenotyping categorised the HepaRG cells as less metabolically active when measured beside the more energetic HepG2 cells. However, inhibition of mitochondrial ATP synthase induced an increase in glycolytic activity of both HepaRG and HepG2 cells suggesting an active Crabtree Effect in both cell lines. The suitability of HepaRG cells for the acute metabolic modification assay as a screen for mitotoxicity was confirmed using a panel of compounds, including both positive and negative mitotoxic compounds. Seahorse respirometry studies demonstrated that a statistically significant decrease in spare respiratory capacity is the first indication of mitochondrial dysfunction. Furthermore, based upon comparing changes in respiratory parameters to those of the positive controls, rotenone and carbonyl cyanide m-chlorophenyl hydrazone, compounds were categorised into two mechanistic groups; inhibitors or uncouplers of the electron transport chain. Overall, the findings from this study have demonstrated that HepaRG cells, despite having different resting bioenergetic phenotype to HepG2 cells are a suitable model to detect drug-induced mitochondrial toxicity with similar detection rates to HepG2 cells.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas , Metabolismo Energético , Mitocondrias/metabolismo , Adenosina Trifosfato/metabolismo , Línea Celular Tumoral , Humanos , L-Lactato Deshidrogenasa/metabolismoRESUMEN
Clinical development of Hu5c8, a monoclonal antibody against CD40L intended for treatment of autoimmune disorders, was terminated due to unexpected thrombotic complications. These life-threatening side effects were not discovered during preclinical testing due to the lack of predictive models. In the present study, we describe the development of a microengineered system lined by human endothelium perfused with human whole blood, a "Vessel-Chip." The Vessel-Chip allowed us to evaluate key parameters in thrombosis, such as endothelial activation, platelet adhesion, platelet aggregation, fibrin clot formation, and thrombin anti-thrombin complexes in the Chip-effluent in response to Hu5c8 in the presence of soluble CD40L. Importantly, the observed prothrombotic effects were not observed with Hu5c8-IgG2σ designed with an Fc domain that does not bind the FcγRIIa receptor, suggesting that this approach may have a low potential risk for thrombosis. Our results demonstrate the translational potential of Organs-on-Chips, as advanced microengineered systems to better predict human response.
Asunto(s)
Anticuerpos Monoclonales Humanizados/toxicidad , Enfermedades Autoinmunes/tratamiento farmacológico , Coagulación Sanguínea/efectos de los fármacos , Ligando de CD40/antagonistas & inhibidores , Diseño de Fármacos , Desarrollo de Medicamentos/instrumentación , Factores Inmunológicos/toxicidad , Dispositivos Laboratorio en un Chip , Procedimientos Analíticos en Microchip , Trombosis/inducido químicamente , Anticuerpos Monoclonales Humanizados/metabolismo , Enfermedades Autoinmunes/inmunología , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Ligando de CD40/inmunología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Factores Inmunológicos/metabolismo , Estudios Prospectivos , Receptores de IgG/metabolismo , Estudios Retrospectivos , Medición de Riesgo , Trombosis/sangreRESUMEN
TAK-875, a GPR40 agonist, was withdrawn from Phase III clinical trials due to drug-induced liver injury (DILI). Mechanistic studies were conducted to identify potential DILI hazards (covalent binding burden (CVB), hepatic transporter inhibition, mitochondrial toxicity, and liver toxicity in rats) associated with TAK-875. Treatment of hepatocytes with radiolabeled TAK-875 resulted in a CVB of 2.0 mg/day, which is above the threshold of 1 mg/day considered to be a risk for DILI. Covalent binding to hepatocytes was due to formation of a reactive acyl glucuronide (AG) and, possibly, an acyl-CoA thioester intermediate. Formation of TAK-875AG in hepatocytes and/or in vivo was in the order of non-rodents > human (in vitro only) > rat. These data suggest that non-rodents, and presumably humans, form TAK-875AG more efficiently than rats, and that AG-mediated toxicities in rats may only occur at high doses. TAK-875 (1000 mg/kg/day) formed significant amounts of AG metabolite (≤32.7 µM) in rat liver that was associated with increases in ALT (×4), bilirubin (×9), and bile acids (×3.4), and microscopic findings of hepatocellular hypertrophy and single cell necrosis. TAK-875 and TAK-875AG had similar potencies (within 3-fold) for human multi-drug resistant associated protein 2/4 (MRP2/4) and bile salt export pump, but TAK-875AG was exceptionally potent against MRP3 (0.21 µM). Inhibition of MRPs may contribute to liver accumulation of TAK-875AG. TAK-875 also inhibited mitochondrial respiration in HepG2 cells, and mitochondrial Complex 1 and 2 activities in isolated rat mitochondria. In summary, formation of TAK-875AG, and possibly TAK-875CoA in hepatocytes, coupled with inhibition of hepatic transporters and mitochondrial respiration may be key contributors to TAK-875-mediated DILI.
Asunto(s)
Benzofuranos/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Hepatocitos/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Transportadores de Anión Orgánico/antagonistas & inhibidores , Sulfonas/toxicidad , Animales , Benzofuranos/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Perros , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Células Hep G2 , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Macaca fascicularis , Mitocondrias Hepáticas/fisiología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Transportadores de Anión Orgánico/genética , Consumo de Oxígeno/efectos de los fármacos , Unión Proteica , Ratas , Especificidad de la Especie , Sulfonas/metabolismoRESUMEN
PURPOSE: Pulmonary arterial hypertension (PAH) results from occlusion or vasoconstriction of pulmonary vessels, leading to progressive right ventricular failure. Dasatinib, a BCR-ABL1 tyrosine kinase inhibitor (TKI) approved for the treatment of chronic myelogenous leukemia, has been associated with PAH. In contrast, the BCR-ABL1 TKI imatinib has demonstrated anti-vasoproliferative properties and has been investigated as a potential treatment for PAH. Here we describe studies evaluating the effects of dasatinib and imatinib on cardiovascular and pulmonary functions to understand the reported differential consequences of the two TKIs in a clinical setting. METHODS: The direct effects of dasatinib and imatinib were explored in vivo to investigate possible mechanisms of dasatinib-induced PAH. In addition, effects of dasatinib and imatinib on PAH-related mediators were evaluated in vitro. RESULTS: In rats, both TKIs increased plasma nitric oxide (NO), did not induce PAH-related structural or molecular changes in PA or lungs, and did not alter hemodynamic lung function compared with positive controls. Similarly, in the pulmonary artery endothelial cells and smooth muscle cells co-culture model, imatinib and dasatinib increased NO and decreased endothelin-1 protein and mRNA. CONCLUSIONS: The results of these studies indicated that dasatinib did not induce physiological changes or molecular signatures consistent with PAH when compared to positive controls. Instead, dasatinib induced changes consistent with imatinib. Both dasatinib and imatinib induced biochemical and structural changes consistent with a protective effect for PAH. These data suggest that other factors of unclear etiology contributed to the development of PAH in patients treated with dasatinib.
Asunto(s)
Antineoplásicos/toxicidad , Dasatinib/toxicidad , Hipertensión Pulmonar/inducido químicamente , Mesilato de Imatinib/toxicidad , Inhibidores de Proteínas Quinasas/toxicidad , Animales , Antineoplásicos/farmacocinética , Dasatinib/farmacocinética , Endotelina-1/sangre , Expresión Génica/efectos de los fármacos , Hemodinámica/efectos de los fármacos , Hipertensión Pulmonar/fisiopatología , Mesilato de Imatinib/farmacocinética , Pulmón/patología , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Óxido Nítrico/sangre , Inhibidores de Proteínas Quinasas/farmacocinética , Arteria Pulmonar/efectos de los fármacos , Circulación Pulmonar/efectos de los fármacos , ARN Mensajero/sangre , Ratas , Ratas Sprague-DawleyRESUMEN
The soluble form of CD40L (sCD40L) is a platelet-derived mediator that links inflammation, hemostasis, and vascular dysfunction. Indeed, blockade of CD40L by neutralizing antibodies or genetic disruption in mice prevents atherosclerosis and atherothrombosis. Until recently, it was believed that CD40 and αIIbß3 were the only receptors on platelets responsible for binding sCD40L, leading to platelet activation and initiation of thrombotic events. Recent findings showed α5ß1 integrin as a novel platelet sCD40L receptor, with an unknown function. For the first time, using anti-α5ß1 blocking antibodies, we show that sCD40L/α5ß1 interaction leads to platelet activation as evaluated in the human whole blood. Establishing α5ß1 integrin's role in platelet activation, and therefore thrombosis will help further shed light on the etiology of thrombotic disease.
Asunto(s)
Ligando de CD40/metabolismo , Integrina alfa5beta1/antagonistas & inhibidores , Activación Plaquetaria/efectos de los fármacos , Anticuerpos Neutralizantes/farmacología , Recolección de Muestras de Sangre , Voluntarios Sanos , Humanos , Integrina alfa5beta1/inmunología , Integrina alfa5beta1/metabolismo , Solubilidad , Trombosis/etiologíaRESUMEN
BMS-986094, the prodrug of a guanosine nucleotide analogue (2'-C-methylguanosine), was withdrawn from clinical trials due to serious safety issues. Nonclinical investigative studies were conducted as a follow up to evaluate the potential for BMS-986094-related mitochondrial-toxicity. In vitro, BMS-986094 was applied to human hepatoma cells (HepG2 and Huh-7) or cardiomyocytes (hiPSCM) up to 19 days to assess mitochondrial DNA content and specific gene expression. There were no mitochondrial DNA changes at concentrations ≤10 µM. Transcriptional effects, such as reductions in Huh-7 MT-ND1 and MT-ND5 mRNA content and hiPSCM MT-ND1, MT-COXII, and POLRMT protein expression levels, occurred only at cytotoxic concentrations (≥10 µM) suggesting these transcriptional effects were a consequence of the observed toxicity. Additionally, BMS-986094 has a selective weak affinity for inhibition of RNA polymerases as opposed to DNA polymerases. In vivo, BMS-986094 was given orally to cynomolgus monkeys for 3 weeks or 1 month at doses of 15 or 30 mg/kg/day. Samples of heart and kidney were collected for assessment of mitochondrial respiration, mitochondrial DNA content, and levels of high energy substrates. Although pronounced cardiac and renal toxicities were observed in some monkeys at 30 mg/kg/day treated for 3-4 weeks, there were no changes in mitochondrial DNA content or ATP/GTP levels. Collectively, these data suggest that BMS-986094 is not a direct mitochondrial toxicant.
Asunto(s)
ADN Mitocondrial/efectos de los fármacos , Guanosina Monofosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , ADN Mitocondrial/biosíntesis , ADN Mitocondrial/fisiología , Relación Dosis-Respuesta a Droga , Femenino , Guanosina Monofosfato/metabolismo , Guanosina Monofosfato/toxicidad , Guanosina Trifosfato/metabolismo , Corazón/efectos de los fármacos , Pruebas de Función Cardíaca , Humanos , Inosina Monofosfato/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Pruebas de Función Renal , Macaca fascicularis , MasculinoRESUMEN
Breast cancer resistance protein (BCRP) is expressed in various tissues, such as the gut, liver, kidney and blood brain barrier (BBB), where it mediates the unidirectional transport of substrates to the apical/luminal side of polarized cells. Thereby BCRP acts as an efflux pump, mediating the elimination or restricting the entry of endogenous compounds or xenobiotics into tissues and it plays important roles in drug disposition, efficacy and safety. Bcrp knockout mice (Bcrp(-/-)) have been used widely to study the role of this transporter in limiting intestinal absorption and brain penetration of substrate compounds. Here we describe the first generation and characterization of a mouse line humanized for BCRP (hBCRP), in which the mouse coding sequence from the start to stop codon was replaced with the corresponding human genomic region, such that the human transporter is expressed under control of the murineBcrppromoter. We demonstrate robust human and loss of mouse BCRP/Bcrp mRNA and protein expression in the hBCRP mice and the absence of major compensatory changes in the expression of other genes involved in drug metabolism and disposition. Pharmacokinetic and brain distribution studies with several BCRP probe substrates confirmed the functional activity of the human transporter in these mice. Furthermore, we provide practical examples for the use of hBCRP mice to study drug-drug interactions (DDIs). The hBCRP mouse is a promising model to study the in vivo role of human BCRP in limiting absorption and BBB penetration of substrate compounds and to investigate clinically relevant DDIs involving BCRP.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Neoplasias/metabolismo , Xenobióticos/farmacocinética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Animales , Disponibilidad Biológica , Biotransformación/efectos de los fármacos , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Interacciones Farmacológicas , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Sustitución del Gen , Humanos , Absorción Intestinal/efectos de los fármacos , Masculino , Moduladores del Transporte de Membrana/sangre , Moduladores del Transporte de Membrana/metabolismo , Moduladores del Transporte de Membrana/farmacocinética , Moduladores del Transporte de Membrana/farmacología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Distribución Tisular/efectos de los fármacos , Xenobióticos/sangre , Xenobióticos/metabolismo , Xenobióticos/farmacologíaRESUMEN
The objective of this study was to evaluate potential protective effects of vehicles containing d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), which may impact nonclinical safety assessments of oxidative processes. This was achieved by evaluating plasma, liver and adrenal gland concentrations of d-α-tocopheryl succinate (TS) and d-α-tocopherol as well as oxidative status of plasma following oral dosing of TPGS-containing vehicles, intraperitoneal (IP) dosing of TS or ex vivo treatment of blood with H2O2. Male and female rats were dosed orally with formulations containing 5% or 40% TPGS (70 or 550 mg kg(-1) day(-1) TS, respectively) for 1 week. A control group was dosed orally with polyethylene glycol-400 (PEG-400; no vitamin E) and positive control animals received a single 100 mg kg(-1) day(-1) IP injection of TS. Whole blood from untreated animals was treated ex vivo with 5 or 50 mm H(2)O(2), with or without TS (0.5, 5, 50 or 500 µm) or ascorbate (1 mm), for 1 h. Oral TPGS treatments did not affect d-α-tocopherol concentrations in plasma or adrenal glands and caused only transient increases in liver. Concentrations of TS in plasma, liver and adrenal glands were undetectable in control animals, but increased in all other groups. Oral administration of TPGS did not reduce plasma lipid peroxidation in vivo. Substantially greater TS concentrations used ex vivo (100× greater than in vivo) were also unable to reduce lipid peroxidation in H2O2 -treated whole blood. These results provide evidence that administration of oral TPGS vehicles is unlikely to impact nonclinical safety assessments of pharmaceuticals.
Asunto(s)
Portadores de Fármacos/farmacología , Estrés Oxidativo/efectos de los fármacos , Vitamina E/análogos & derivados , Glándulas Suprarrenales/química , Animales , Portadores de Fármacos/farmacocinética , Femenino , Hígado/química , Masculino , Oxidación-Reducción/efectos de los fármacos , Polietilenglicoles/farmacocinética , Polietilenglicoles/farmacología , Ratas , Ratas Sprague-Dawley , Tiobarbitúricos/farmacología , Vitamina E/sangre , Vitamina E/farmacocinética , Vitamina E/farmacología , alfa-Tocoferol/análisis , alfa-Tocoferol/sangreRESUMEN
The corticotrophin releasing factor (CRF) receptor I antagonist, BMS-764459 (evaluated as a potential treatment of affective disorders), was orally dosed to female Sprague-Dawley rats once daily for 2 weeks (vehicle control or 175mg/kg/day). To investigate the mechanism of BMS-764459-related liver weight increases, total liver RNA was isolated and evaluated for mRNA gene expression by microarray analysis (assessing the expression of approximately 24,000 genes) from snap-frozen tissue. Subsequently, mRNA and miRNA (microRNA) were also analyzed 5 years later from FFPE (Formalin Fixed Paraffin Embedded) samples via RT-PCR (about 800 miRNA evaluated). Genomic analyses showed that BMS-764459 induces AhR target genes with additional inductions of CYP2B, CYP3A, and Abcc3 consistent with the gene expression pattern of atypical CYP1A1 inducers. Analysis of miRNA expression identified a number of significantly affected miRNAs. To further evaluate their role in atypical CYP1A1 induction, an in silico evaluation of differentially expressed miRNA was performed and their putative mRNA 3'-UTR (untranslated region) binding sequences were evaluated. MiR-680 and miR-29a were identified as potential regulators and biomarkers of atypical CYP1A1 induction by regulating Abcc3, CYP3A and CYP2B as well as a number of AhR targeted genes.
Asunto(s)
Aminopiridinas/farmacología , Citocromo P-450 CYP1A1/biosíntesis , Hígado/efectos de los fármacos , MicroARNs/metabolismo , Pirazinas/farmacología , Animales , Inducción Enzimática , Femenino , Formaldehído/farmacología , Perfilación de la Expresión Génica , Hígado/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Adhesión en Parafina/métodos , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Notch signaling pathways are involved in the regulation of cell differentiation and are highly conserved across species. Notch ligand binding leads to gamma-secretase-mediated proteolytic cleavage of the Notch receptor releasing the Notch intracellular domain, resulting in its subsequent translocation into the nucleus and gene expression regulation. To investigate the level of expression of Notch signaling pathway components in microanatomic regions following renal injury, kidneys from untreated, vehicle control, and puromycin aminonucleoside (PA, 150 mg/kg)-treated rats were evaluated. Frozen tissue sections from rats were microdissected using laser capture microdissection (LCM) to obtain glomeruli, cortical (proximal) tubules, and collecting ducts, and relative gene expression levels of Presenilin1, Notch1 and Hes1 were determined. In untreated rats, the Notch1 expression in glomeruli was higher than in the proximal tubules and similar to that in collecting ducts, whereas Presenilin1 and Hes1 expressions were highest in the collecting ducts, followed by cortical tubules and glomeruli. Following PA-induced renal injury, Hes1 gene expression increased significantly in the glomeruli and tubules compared to the collecting ducts where no injury was observed microscopically. Although these data present some evidence of change in Notch signaling related to injury, the expression of Presenilin1, Notch1, and Hes1 in the microanatomic regions of the kidney following PA treatment were not significantly different when compared to controls. These results demonstrate that there are differences in Notch-related gene expression in the different microanatomic regions of the kidneys in rats and suggest a minimal role for Notch in renal injury induced by PA. In addition, this work shows that LCM coupled with the RT-PCR can be used to determine the relative differences in target gene expression within regions of a complex organ.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/fisiología , Expresión Génica/efectos de los fármacos , Riñón/efectos de los fármacos , Puromicina/farmacología , Receptor Notch1/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Femenino , Proteínas de Homeodominio/genética , Riñón/enzimología , Riñón/metabolismo , Riñón/cirugía , Presenilina-1/genética , Ratas , Ratas Sprague-Dawley , Factor de Transcripción HES-1RESUMEN
In this in vitro model of hepatocyte multinucleation, separate cultures of rat Clone 9 cells are labeled with either red or green cell tracker dyes (Red Cell Tracker CMPTX or Vybrant CFDA SE Cell Tracer), plated together in mixed-color colonies, and treated with positive or negative control agents for 4 days. The fluorescent dyes become cell-impermeant after entering cells and are not transferred to adjacent cells in a population, but are inherited by daughter cells after fusion. The mixed-color cultures are then evaluated microscopically for multinucleation and analysis of the underlying mechanism (cell fusion/cytokinesis). Multinucleated cells containing only one dye have undergone cytokinesis failure, whereas dual-labeled multinucleated cells have resulted from fusion.
Asunto(s)
Células Clonales/citología , Citocinesis/efectos de los fármacos , Colorantes Fluorescentes , Hepatocitos/efectos de los fármacos , Células Híbridas/efectos de los fármacos , Coloración y Etiquetado/métodos , Animales , Bioensayo/métodos , Fusión Celular , Células Cultivadas , Células Clonales/efectos de los fármacos , Hepatocitos/citología , Ratas , Pruebas de Toxicidad/métodos , Xenobióticos/toxicidadRESUMEN
Actin filaments play a critical role in the normal physiology of lenticular and retinal cells in the eye. Disruption of the actin cytoskeleton has been associated with retinal pathology and lens cataract formation. Ocular toxicity is an infrequent observation in drug safety studies, yet its impact to the drug development process is significant. Recognizing compounds through screening with a potential ocular safety liability is one way to prioritize development candidates while reducing development attrition. Lens epithelial cells from human, dog, and rat origins and retinal pigmented epithelium cells from human, monkey, and rat origins were cultured and investigated with immunocytochemical techniques. Cells were treated using noncytotoxic doses of the compound, fixed, stained for actin with rhodamine phalloidin, and counterstained for nuclei with TOTO-3, followed by confocal imaging. Tamoxifen and several experimental compounds known to be in vivo lens and retinal toxicants caused a reduction in F-actin fluorescence at noncytotoxic concentrations in all cells tested as observed by confocal microscopy. Developing an assay that predicts ocular toxicity helps the development process by prioritizing compounds for further investigation. Drug-induced cytoskeletal alterations may be useful as a potential safety-screening marker of retinal and lens toxicity. The knowledge of actin molecular biology and the application of other mechanistic screens to toxicology are discussed. Reducing this work to a high-throughput platform will enable chemists to select compounds with a reduced risk of ocular toxicity.