RESUMEN
As drug-induced seizures have severe impact on drug development, evaluating seizure induction potential of candidate drugs at the early stages of drug discovery is important. A novel assay system using zebrafish has attracted interest as a high throughput toxicological in vivo assay system, and we tried to establish an experimental method for drug-induced seizure liability on the basis of locomotor activity in zebrafish. We monitored locomotor activity at high-speed movement (> 20 mm/sec) for 60 min immediately after exposure, and assessed seizure liability potential in some drugs using locomotor activity. However this experimental procedure was not sufficient for predicting seizures because the potential of several drugs with demonstrated seizure potential in mammals was not detected. We, therefore, added other parameters for locomotor activity such as extending exposure time or conducting flashlight stimulation (10 Hz) which is a known seizure induction stimulus, and these additional parameters improved seizure potential detection in some drugs. The validation study using the improved methodology was used to assess 52 commercially available drugs, and the prediction rate was approximately 70%. The experimental protocol established in this present study is considered useful for seizure potential screening during early stages of drug discovery.
Asunto(s)
Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Actividad Motora/efectos de los fármacos , Convulsiones/inducido químicamente , Toxicología/métodos , Pez Cebra/fisiología , Animales , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Estimulación LuminosaRESUMEN
The Hand1- and Cmya1-ESTs are novel short-term tests for embryotoxic chemicals using genetically engineering mouse ES cells for luciferase reporter gene assays. These ESTs allow convenient determination of differentiation toxicity and cell viability in a short duration with high throughput 96-well microplates for prediction of embryotoxicity of chemicals. To assess the Hand1-EST technical protocol, we firstly compared reporter gene assay and cytotoxicity test data for a representative compound (hydroxyurea) from four different laboratories with tests carried out under the same experimental conditions. Extensive investigations of the Hand1- and Cmya1-ESTs were then performed to explore reproducibility by comparing a set of 6 well-known test chemicals, including hydroxyurea, across the laboratories. The results gave good correspondence in all four laboratories, indicating that transferability, intra-laboratory variability and inter-laboratory variability of the present technical protocols of the ESTs were sufficient to conduct further validation studies.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Unión al ADN/genética , Células Madre Embrionarias/citología , Laboratorios , Proteínas Nucleares/genética , Animales , Animales Modificados Genéticamente , Diferenciación Celular , Proteínas del Citoesqueleto , Determinación de Punto Final , Etiquetas de Secuencia Expresada/química , Marcadores Genéticos , Ratones , Reproducibilidad de los ResultadosRESUMEN
The embryonic stem cell test (EST) is a validated method and a useful screening tool for drug discovery. EST requires microscopic observation of beating cells to be considered cardiomyocytes as an endpoint assay. However, this procedure is time-consuming and limits the throughput performance. Instead of microscopic observation, we previously established a novel assay method based on cardiac field potential as an endpoint. However, cardiac specificity of this field potential is not yet clarified, because beating cells have not been rigorously evaluated as skeletal or cardiomyocyte. Here, we investigated the relationships between field potential, beating, and cardiac troponin T (cTnT) expression, selected as a cardiomyocyte-specific marker, and evaluated suitability of the field potential as a marker for cardiomyocyte in vehicle or 5-fluorouracil treated embryo bodies. Embryoid bodies of mouse embryonic stem cells (D3) were differentiated in a chamber with multi-electrode array for 5 days, and field potential and beating were measured at the end of differentiation. In addition, these chambers were immunohistochemically stained with anti-cTnT antibody, and the correlation between field potential, beating, and cTnT expression was examined. These results indicated the area of field potential or beating mainly coincided with that of cTnT expression. 5-fluorouracil treatment decreased not only the number of field potential detecting electrodes and beating area, but also cTnT expression, and the area of these parameters was also nearly identical. These results indicate that field potential can be used as a suitable cardiac differentiation marker, and can be a promising parameter of EST.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Determinación de Punto Final , Miocitos Cardíacos/efectos de los fármacos , Pruebas de Toxicidad/métodos , Alternativas a las Pruebas en Animales , Animales , Biomarcadores/análisis , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Electrofisiología , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Fluorouracilo/toxicidad , Inmunohistoquímica , Ratones , Microelectrodos , Microscopía Fluorescente , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Teratógenos/toxicidad , Troponina T/biosíntesisRESUMEN
The embryonic stem cell test (EST) is a validated in vitro method to assess the embryotoxic potential of compounds and is a promising tool for drug screening. EST requires microscopic observation of beating cardiomyocytes differentiated from embryonic stem cells as a toxicological endpoint. However, this process is time-consuming and lacks throughput performance. To improve the analysis, we introduced an electrophysiological method with a microelectrode array system for the evaluation of differentiated cardiomyocytes. Embryotoxic (valproic acid, verapamil, and 5-fluorouracil) and non-embryotoxic (penicillin G, d-camphor, and isoniazid) compounds were assessed with the system. Mouse embryonic stem cells were differentiated into cardiomyocytes and treated with each compound during the differentiation process. The embryotoxicity of each compound was then assessed by measuring the field potentials of differentiated cardiomyocytes using the microelectrode array system, as well as by microscopic evaluation. All the embryotoxic compounds dose-dependently inhibited the field potential formation and the myocardial beating of differentiated cells, while the non-embryotoxic compounds did not affect either endpoint. The detection capabilities of the two assay methods were similar. These results indicated that the field potential measurements can be used as an alternative endpoint of EST. Moreover, the field potential can be measured automatically, introducing a high throughput performance compared to the conventional microscopic observation. We therefore concluded that the endpoint analysis with the microelectrode array system improves the original EST and can be useful for the assessment of the embryotoxic potential of compounds.