Craniofacial Abnormalities In Zebrafish Induced By Clomiphene Sodium Citrate

A birth defect is defined as a major deviation from normal morphology or function that is congenital in origin. It is estimated that 3% of all newborns in the United States have a major structural malformation detectable at birth.Today, physicians are using a wider variety of drugs including aromatase-inhibitors, metformin, gonadotropin releasing hormone agonist or antagonist, estrogens, and progesterone. As much as the desire to assist infertile couple to conceive is sincere, there is a concern that these treatments may be teratogenic to the newborn.The oldest compound for the treatment of ovulatory disorder has been Clomiphene Sodium Citrate (CC) . It is a racemic mixture of two stereoisomers. There is a always a debate on Clomiphene Sodium Citrate induced neural tube defect (NTD) in the offspring. The results have been mixed, some studies do show that Clomiphene Sodium Citrate is responsible for newborns'abnormal development while others found no clear clues.Using zebrafish as a animal model to investigate drug developmental toxicity provides a new way for assessing the developmental toxicity of exposure to toxicants during early-life stages. Zebrafish is considered as an ideal model for many incomparable advantages. First of all, fish embryonic development has been shown to be sensitive to environmental stress. Also, 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. The most important thing is zebrafish genome has the close homology with the human genome as well as 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. Finally, zebrafish studies are costless, time saving with guaranteed large sample size.Application of zebrafish model in studying drug toxicity and initial screen is more and more popular. In this study, zebrafish was used as animal model to study the mechanism of Clomiphene Sodium Citrate induced abnormal craniofacial development. Also, we expect to establish a simple method for drug developmental toxicity assessment using zebrafish model.PartⅠUsing zebrafish as animal model to predict the toxicity of Clomiphene Sodium Citrate to mammalsObjective: To study LC50 value of Clomiphene Sodium Citrate on zebrafish embryo, and to evaluate the potential of 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 Clomiphene Sodium Citrate on zebrafish was 364.18 mg/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 analyze the toxicity of compounds, which calls for little amount compounds, could be highly predictive of toxicity on mammals. Objective: Study the development toxicity of Clomiphene Sodium Citrate by taking advantage of morphological observation convenience of zebrafish model. We aim to get the direct evidence for abnormal development. Method: zebrafish embryos were divided into four groups: control group and three experimental groups with Clomiphene Sodium Citrate concentration of 50, 100 and 200 mg/L separately. Drugs were exposed until 72 hpf. 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: For The hatching rate, survival rate and the malformation rate of experimental groups have changed compared with control group. Among those changes, malformation rate was significantly increased. Malformation rate in 200 mg/L group was 46±5.4%, compared with 4.7±1.8% in the control. In AO stain experiment, there is no significant phenomenon of cell apoptosis in experimental groups with lower drug concentration. Alcian-Blue stain showed clear abnormal craniofacial development. Conclusion: Clomiphene Sodium Citrate could induce cell apoptosis in experiment group with high drug concentration. Also, it's very clear that abnormal craniofacial development was induced by Clomiphene Sodium Citrate treatment. The data we got were highly consistent with those got from other animal models. Zebrafish can be an alterative animal model for rapid, directive and predictive drug toxicity assessment. Objective: To study the effects of Clomiphene Sodium Citrate on expression of abnormal craniofacial development related genes in zebrafish. Zebrafish was used as model in this study. We expect to establish a simple method for gene expression analysis on chemicals induced developmental toxicity. Method: zebrafish embryos were divided into three groups: control group and two experimental groups with Clomiphene Sodium Citrate concentration of 50 and 100mg/L separately. Chemicals were exposed until 48 hpf. Real-time PCR was used to detect the expression of craniofacial development related genes fox1, mmp13, bmp2, msx1 and p53 mRNA. Results: In Clomiphene Sodium Citrate 50 and 100mg/L groups, the mRNA levels of fox1 decreased compared with those of the control at both 24 hpf and 48 hpf time points; mmp13, bmp2 and msx1 mRNA were highly expressed in Clomiphene Sodium Citrate 50 and 100 mg/L groups; among 24 hpf and 48 hpf time points, the tendency of expression change remains the same; expression of p53 mRNA decreased at the 24 hpf time point in the drug treatment group; p53 mRNA expression slightly increased at 48hpf time point, but the change is not significant. Conclusion: Clomiphene Sodium Citrate changed the normal expression of fox1, mmp13, bmp2, msx1 and p53 and abnormal craniofacial development may be associated with changed gene expression. Because of the similiarity of protein structure and function between zebrafish and mammals, so data we got from zebrafish could provide a reliable way for assessment of drug development toxicity and safety screen.