Craniofacial Dysplasia In Zebrafish Induced By Dexamethasone Sodium Phosphate

Glucocorticoids are a class of steroid hormones that are produced endogenously under the control of the hypothalamus-pituitary-adrenal axis. These hormones regulate cellular growth, differentiation, and apoptosis. Glucocorticoids are also one of the most commonly prescribed drugs worldwide, primarily used for their anti-inflammatory or immune-suppressive effects. These compounds are used to treat asthma, dermatitis, rheumatoid arthritis, and autoimmune diseases, as well as to prevent graft rejection. Glucocorticoids are potent teratogens, with the most common defect observed being cleft palate.Most commonly, both human diseases and possible drug candidates have been modeled in mammals because of close homology between the mammalian genomes. However, establishing mammalian models is often expensive and time consuming, and there remains a clear disconnection between in vitro and in vivo data preventing reliable predictive effects on toxicity.One of the most promising in vivo model systems for toxicity studies is Danio rerio, commonly known as zebrafish. Zebrafish is considered as an ideal model for assessing the developmental toxicity of exposure to toxicants during early-life stages. Fish embryonic development has been shown to be sensitive to environmental stress. Zebrafish embryo assays are also regarded to be pain-free in vivo tests and are gradually being accepted as a good replacement for other types of animal experiments. Utility of zebrafish for toxicity screens is largely based upon the close homology with the human genome. The genetic parallels impart physiological and anatomical similarities. When introduced to xeno-substances, zebrafish and mammals demonstrate a similar physiologic response. Furthermore, the biology and optical clarity of zebrafish embryos allows testing at all stages of embryonic development, such as organogenesis, vasculogenesis, and deleterious effects during anatomical development. Costs are significantly lowered because zebrafish are much smaller and require less expensive husbandry/housing than do mammals. Using zebrafish as animal model for studying drug toxicity and initial screen is now newly developed in China and abroad. In this study, zebrafish was used as animal model to study LC50 value, morphological changes, and changes of related genes expression. To further investigate mechanism of Dexamethasone Sodium Phosphate induced abnormal craniofacial development, we carried out this research. And we also hope to establish a simple method for drug developmental toxicity assessment using zebrafish modle.PartⅠLC50 Value of Dexamethasone Sodium Phosphate on Zebrafish EmbryoObjective: To study LC50 value of Dexamethasone Sodium Phosphate on zebrafish embryo, and to assess the potential for zebrafish model to predict chemical toxicity on mammals. Method: LC50 value was determined by using KARBER Method. Relationship between zebrafish LC50 value and LD50 values of other animal models was studied by using linear regression analysis. Results: LC50 value of Dexamethasone sodium phosphate on zebrafish was 443.86mg/L. Linear regression analysis suggests there is a good linear relationship on LC50 values between zebrafish and other animal models. Conclusion: Using zebrafish as animal model to study drug toxicity, which calls for little amount drug, could be highly predictive of toxicity on mammals.PartⅡDexamethasone Sodium Phosphate Induced Abnormal Craniofacial Development in ZebrafishObjective: To study Dexamethasone Sodium Phosphate induced abnormal craniofacial development by using developing zebrafish as animal model, aiming to establish a rapid, direct and predictive assessment system for drug-induced developmental toxicity. Method: zebrafish embryos were divided into four groups: control group and three experimental groups with Dexamethasone Sodium Phosphate concentration of 5, 10 and 50mg/L separately. Chemicals were exposed until 96hpf. Hatching rate, survival rate, malformation rate and heart rate of each group were determined; morphological changes were studied with stereomicroscope; abnormal craniofacial development and apoptosis were observed by Alcian-Blue Stain and AO stain. Results: The hatching rate, survival rate of experimental groups have no significant difference while the malformation rate and heart rate were changed compared with control group. Among those changes, malformation rate was significantly increased. Malformation rate in 50 mg/L group was 71.33±6.5%, compared with 4.67±1.8% in the control. In AO stain experiment, cell apoptosis existed in all experimental groups. Alcian-Blue stain further conformed Dexamethasone Sodium Phosphate-induced abnormal craniofacial development. Conclusion: Using zebrafish as animal model to explore Dexamethasone sodium phosphate-induced abnormal craniofacial development, the data was highly consistent with those got from other animal models. Zebrafish can be an alterative animal model for rapid, directive and predictive drug toxicity assessment. PartⅢThe Effect of Dexamethasone Sodium Phosphate to Expression of Craniofacial Development Related Genes mRNA in ZebrafishObjective: To study the effects of Dexamethasone sodium phosphate to expression of abnormal craniofacial development related genes egf,tgfβ1 and mmp-13 mRNA in zebrafish. Suppose to use zebrafish model establishing a simple method for gene expression analysis on chemicals induced developmental toxicity. Method: zebrafish embryos were divided into four groups: control group and three experimental groups with Dexamethasone Sodium Phosphate concentration of 5, 10 and 50mg/L separately. Chemicals were exposed until 96hpf. Real-time PCR was used to detect the expression of craniofacial development related genes egf, tgfβ1 and mmp-13 mRNA. Results: In Dexamethasone sodium phosphate 5, 10 and 50mg/L groups, the mRNA levels of egf, tgfβ1 and mmp-13 increased at 48, 72, 96 hour endpoint compared with those of the control. Conclusion: Dexamethasone sodium phosphate increases expression of egf, tgfβ1 and mmp-13 and abnormal craniofacial development may be associated with increased gene expression. Protein structure and function in zebrafish are highly homologous with mammals, so that can provide stable and reliable data for drug development toxicity study and safety screen.