Effects Of Exposure Of Polychlorinated Biphenyls On Development Of Zebrafish Bone During Embryonic Phase

Environmental chemical pollutants are harmful to children, which gets more and more attention. With chemical contamination of the environment on the increase, the incidence of developmental disease in children is also increasing each year. Polychlorinated biphenyls (PCBs) are global environmental pollutants with high toxicity, semi-volatile, long-term residue and bioaccumulation, which attracts more attention on the effect of PCBs on skeletal development of the children.Bone is a specialized connective tissue, which forms the framework of the body. Alterations in bone composition have previously been observed in experimental animals exposed to PCBs, as well as in natural exposure scenarios in free ranging animals. But none of these experiment or investigation had discussed the toxicity of PCBs during embryonic development period. Zebrafish (Danio rerio) is a new and popular model due to its fast development, the transparency of its embryos, its ease of manipulation at the molecular level and the availability of various mutants. The simple organization of the zebrafish skeleton and a similar developmental pattern to that of mammals makes zebrafish particularly useful for studies on skeletal development and disease.The BMPs play a crucial role in the development and differentiation of several kinds of cells. BMP-2and BMP-4as two of very important members of BMPs control early development of skeleton. And the expression of VDR, PTH, TRPV6are the controller of body calcium resorption and very sensitive to Ca2+.We apply zebrafish as the model to study the effect of embryonic exposed to PCBs on the early development from the point of the spinal morphology as well as the gross calcium and the specific genes of BMP-2, BMP-4, VDR, PTH and TRVP6associated with the development and calcification of skeleton. Part I:Effects of embryonic exposure to polychlorinated biphenyls on survival and deformity rate of zebrafishObjective:To study the effects of embryonic exposure to polychlorinated biphenyls on the development of zebrafish embryo.Methods:We study the influences of different concentrations (1,0.5,0.125mg/L) of polychlorinated biphenyls on skeletal morphological development and survival rate. Embryos were assessed at120hpf for changes in larvae spinal morphology by light microscope.Results:The spinal deformity rates of the zebrafish exposed to0.125mg/L PCB1254were infrequent, while at0.5and1.0mg/L, the numbers of larvae with spinal deformities increased significantly after72hpf. Exposure to1.0mg/L PCB1254, however, increased the percentage of deformed larvae to37.8%at120hpf. Survival rates after120hpf were97.8,100,94.4,58.9and41.1%for the control,0.01%methanol,0.125,0.5, and1.0mg/L PCB1254treatments, respectively. With the concentration and time increase, the survival rate declined, while the malformation rate increased obviously, indicating a specific time-and dose-dependent effect.Conclusion:Embryonic exposure to PCB induced developmental deficits in zebrafish survival rate and spinal morphology which suggest the value of monitoring organic contaminant. Part II Effects of embryonic exposure to polychlorinated biphenyls on the gross calcium and skeleton-related genesObjective:To study the effects of embryonic exposure to polychlorinated biphenyls on the the gross calcium and skeleton-related genesMethods:Zebrafish embryos were separately exposed to1.0,0.5,0.125mg/L PCB1254,0.01%methanol (as a control for the PCBs carrier), or egg pure water. Embryos were assessed at120hpf for changes in the expression of BMP-2, BMP-4, VDR, PTH and TRVP6mRNA by Real-time PCR.Results:1. Gross calcium:120hpf, compared to contral group, a normal level of total calcium was detected in methnaol group while significantly decreased levels of total calcium were found in0.125,0.5and1.0mg/L groups.2) Real-time PCR:120hpf, VDR PTH and TRVP6mRNA expression were all affected by PCB1254. By120hpf, the mRNA expression of VDR, PTH and TRVP6from the PCB-treated larvae were all upregulated. The expression of PTH and TRVP6were positively correlated with the level of PCBs to which the embryos were exposed.Conclusion:Embryonic exposure to PCBs could lead some influence on the gross calcium and skeleton-related genes of zebrafish. It indicates that PCBs may have some adverse effects on skeletal development of children.