RESUMEN
In our previous work using paraffin-embedded sections, we determined that Xenopus larvae exposed to 200 ppb 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 5 days from shortly after fertilization to the early larval stage showed a shortening of the digestive tract and a loss of mucosal epithelium cells due to exfoliation into the gut cavity. In the current study, we ultrastructurally examined the mucosal epithelium of the gut of TCDD-exposed Xenopus larvae 12 days after fertilization. Exfoliated cell structures at the villus tip and in the lumen were equipped with a microvillus portion and occasionally had terminal web-like structures seen by ultramicroscopy. As these exfoliated structures had nuclear fragments with condensed chromatin, they were considered to be apoptotic bodies derived from the principal cells of the epithelium. In addition, many membrane-bound cell fragments-identified as apoptotic bodies derived from the principal cells based on their ultrastructural features-were observed at the basal side of the mucosal epithelium. These apoptotic bodies were phagocytized and digested chiefly by the neighboring intact principal cells. No such cells and/or cell fragments showing apoptotic features were observed in the controls. Our observations indicate that marked apoptosis occurs in the intestinal principal cells of TCDD-exposed larvae, which may result in the shortening of the gut.
Asunto(s)
Apoptosis/efectos de los fármacos , Duodeno/efectos de los fármacos , Enterocitos/efectos de los fármacos , Contaminantes Ambientales/toxicidad , Dibenzodioxinas Policloradas/toxicidad , Xenopus laevis , Animales , Duodeno/embriología , Duodeno/patología , Embrión no Mamífero/efectos de los fármacos , Enterocitos/ultraestructura , Larva/efectos de los fármacos , Larva/ultraestructura , Microscopía ElectrónicaRESUMEN
The cAMP-responsive element (CRE) binding protein/activating transcription factor (CREB/ATF) family plays a major role in the expression of skeletal-specific genes and skeletal tissue development. We analyzed the changes of the amount, degree of phosphorylation and binding activity of the CREB/ATF family in the course of development of the murine calvarial osteoblastic cell line MC3T3-E1 as an in vitro model system of bone formation. The amount of CREB in the whole-cell extract detectable by Western blot analysis was high through all stages of development and maximal in the proliferation stage. The degree of phosphorylation estimated with anti-phosphorylated CREB antibody changed greatly and reached high levels in the proliferation stage and early mineralization stage. The ratio of phosphorylated CREB to total CREB in the CREB-CRE complex was also examined by gel shift assay. Although the binding to the consensus/CRE probe reached almost equally high levels in the proliferation stage and early mineralization stage, the relative level of phosphorylated CREB in the CREB-CRE complex was different in these two stages. In the early mineralization stage, most CREB bound to consensus/CRE was phosphorylated, while both phosphorylated and unphosphorylated CREB were bound to consensus/CRE in the proliferation stage. ATF-1 was also detected as a minor component bound to the consensus/CRE probe. The alteration of the binding of CREB to consensus/CRE over the course of osteoblast development supports the hypothesis that CREB may regulate the expression of genes defining the developmental sequence of MC3T3-E1 cells.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Osteoblastos/metabolismo , Cráneo/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Unión Competitiva , Células Cultivadas , AMP Cíclico/metabolismo , Genes fos , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Endogámicos C57BL , Oligonucleótidos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Cráneo/citología , Cráneo/crecimiento & desarrollo , Factores de TiempoRESUMEN
Xenopus embryos were exposed to 200 ppb 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 5 days from the 2- to 8-cell stage of cleavage to the early larval stage. Larvae that developed generalized edema were collected at 7 days after the end of TCDD exposure for light and electron microscopic studies. Erythrocytes in the peripheral blood of the edematous larvae were examined. Between 0.3 and 33.9% of identifiable erythrocytes of exposed larvae had dilated perinuclear cisternae. Furthermore, some had extremely condensed nuclear chromatin usually coalesced against 1 pole of the nuclear membrane and overall compacted cytoplasm. The erythrocytes showing nuclear condensation were phagocytosed by macrophages. These features are typical of cells undergoing apoptosis. Anemia is 1 symptom of TCDD toxicity in various animal species, including mammals. In this study, we demonstrate that TCDD induces apoptotic cell death in circulating erythrocytes of Xenopus larvae, which may be 1 cause of anemia in this species.
Asunto(s)
Apoptosis/efectos de los fármacos , Envejecimiento Eritrocítico/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Dibenzodioxinas Policloradas/toxicidad , Animales , Eritrocitos/patología , Eritrocitos/ultraestructura , Larva/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Mitocondrias/ultraestructura , Vacuolas/efectos de los fármacos , Vacuolas/patología , Vacuolas/ultraestructura , XenopusRESUMEN
Edema formation is a consistent feature of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) poisoning in experimental animals. Xenopus embryos exposed to 200 ppb of TCDD for 5 days from shortly after fertilization to early larval stage developed edema of the head, thorax, and abdomen, including ascites, from several days after the end of exposure. There has been no reports on liver lesions in TCDD-exposed anuran larvae. To elucidate the participation of liver in the edema formation, light and electron microscopic alterations in the livers from larvae that developed edema were examined. The marked change induced by TCDD was degeneration of the hepatocytes. Three types of degenerating hepatocytes were distinguished. The first type showed morphological features characteristic of apoptosis, including peripherally aggregated nuclear chromatin and overall cytoplasmic condensation; the cytoplasmic organelles retained their integrity and cytoplasmic blebbing. A considerable number of degenerating hepatocytes undergoing apoptosis and numerous apoptotic bodies derived from hepatocytes were observed by electron microscopy. The second type could not be determined as to which type of cell death it belonged to, and the third type was necrosis. Other alterations consisted of disturbances of the usual meshwork architecture of the liver, enlargements of bile canaliculi and the space of Disse, lipid accumulation, and appearance of phagocytizing macrophages. These morphological alterations in the liver generally correlated with the severity of edema with a few exception. These findings suggest that depressed hepatic function mainly due to hepatocyte death participates in the edema formation.
Asunto(s)
Apoptosis/efectos de los fármacos , Hígado/patología , Dibenzodioxinas Policloradas/toxicidad , Xenopus laevis , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas , Edema/inducido químicamente , Edema/patología , Femenino , Larva/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/ultraestructura , Hepatopatías/patología , Masculino , Orgánulos/efectos de los fármacos , Orgánulos/patología , Orgánulos/ultraestructuraRESUMEN
To examine the developmental toxicity of caffeine, Xenopus larvae just after hatching, were continuously exposed to 100-2,000 mg/L caffeine for 48 hours. Caffeine interfered with development of Xenopus larvae at a concentration of 100 mg/L and above in a concentration-dependent manner. Characteristic external abnormalities, such as shortened body with wavy fins, were observed, the severity of which was clearly concentration dependent. These larvae were frequently accompanied by abnormal body flexure and edema in the fin. Light microscopy revealed that exposure to caffeine induced severe damage in the myotome and neural tube, and at higher concentrations, the epidermal tissue was also affected. Myoblasts showed wide intercellular spaces, and their cytoplasm lost uniform staining. Ultrastructural studies of myoblasts revealed distinct myofibril disorganization and degeneration, and mitochondrial alterations. In the neural tube, cells at the dorsal part of tube showed wide intercellular spaces and some of them were segregated to the peripheral region. Furthermore, vacuole-like structures of various sizes appeared in the white matter. The outer layer of epithelial cells in the epidermis were vacuolated and swollen. With regard to the pathogenesis of myofibril damage, caffeine appeared to cause a disturbance of intracellular calcium regulation, by releasing calcium ions from the sarcoplasmic reticulum, and the mitochondrial changes observed in myotomal cells were considered to be reflective of this increased intracellular calcium ion levels. It is speculated that caffeine interferes with cell adhesion in the myotome and neural tube by affecting calcium ion regulation.
Asunto(s)
Anomalías Inducidas por Medicamentos , Cafeína/toxicidad , Embrión no Mamífero/efectos de los fármacos , Teratógenos/toxicidad , Animales , Relación Dosis-Respuesta a Droga , Embrión no Mamífero/ultraestructura , Larva , Microscopía Electrónica de Rastreo , Músculos/efectos de los fármacos , Músculos/embriología , Músculos/ultraestructura , Notocorda/efectos de los fármacos , Notocorda/patología , Piel/efectos de los fármacos , Piel/embriología , Piel/ultraestructura , Xenopus laevisRESUMEN
Fourteen- and 15-day mouse embryos treated with triamcinolone on day 11 of gestation were examined for the presence of variant rugae. Nontreated mouse embryos served as controls. Variant rugae found were classified into five types. All five types of variations (bifurcation, division, supernumerary, shortness and cross) were observed in triamcinolone-treated embryos, and shortness was most frequently seen. Supernumerary, bifurcation and division were ranked next, following by cross. Variant, rugae, except the cross, were also observed in non-treated embryos in low frequencies, but more than one-half of them were the bifurcation of the second ruga. Divided rugae ranked next, and supernumerary and shortness were found occasionally. Except for the bifurcated and supernumerary rugae, the greater part of the variant rugae were found in the fifth and fourth ruga in the triamcinolone-treated groups and in the fifth ruga in the nontreated groups. As the incidence of variant rugae in the triamcinolone-treated embryos was significantly higher than that in the nontreated, it was regarded as one of the changes induced by the corticoid. Based on the characteristic features of the rugal region, it is speculated that the formation of variant rugae is associated with the disturbance of normal epithelial-mesenchymal interaction which may be controlled by the nerve fibers appearing at the time of rugal formation. The relationship between the increased appearance of variant rugae and the failure of palatal shelf elevation was examined, but no direct evidence was obtained.
Asunto(s)
Anomalías Inducidas por Medicamentos/embriología , Hueso Paladar/efectos de los fármacos , Triamcinolona Acetonida/toxicidad , Animales , Fisura del Paladar/inducido químicamente , Fisura del Paladar/embriología , Femenino , Edad Gestacional , Ratones , Ratones Endogámicos ICR , Microscopía Electrónica de Rastreo , Hueso Paladar/anomalías , Hueso Paladar/embriología , EmbarazoRESUMEN
Morphological studies of secondary palate formation, with special reference to the development of rugae, were carried out on Jcl:ICR mouse embryos. Three rugae were observed on the anterior part of the future oral surface of the vertically developing palatal shelves in 13-day embryos. Rugae increased in number as the development of the palatal shelves proceeded, and five to six prominent rugae were observed in 14-day embryos just prior to shelf elevation. The folding of these five to six rugae progressed in conjunction with the formation of a sharp, valley-like groove at the base of the anterior two-fifths of the vertical palatal shelves. As palatal shelves elevated, the groove disappeared gradually, and, accordingly, the folding of rugae loosened. In the groove region, the superficial epithelial cells were roundish, while the basal ones were elongated. Such characteristic features were no longer observed when the disappearance of the groove was completed. Eight rugae were observed on the future hard palate of 14-day embryos with already completed palatal fusion. An additional ruga was frequently found in 15-day embryos, and the pattern then was almost the same as that of an adult. Epithelial thickening and condensation at the rugae region, as well as mesenchymal condensation under the epithelium of the rugae, were confirmed in embryos both before and after elevation of the palatal shelves. There is a possibility that these structural characteristics observed in the epithelial and mesenchymal cells of the rugae and groove regions may be related to palatal shelf elevation.