Effects Of Exposure Of Polychlorinated Biphenyls On The Development Of Zebrafish Embryonic Heart

Congenital heart disease seriously endangers children’s lives and health. As a multifactorial genetic disease, its etiology and pathogenesis is not yet fully elucidated. That is mainly due to genetic and environmental factors of the embryonic period, which is leading to cardiovascular abnormalities. The hazards of environmental chemical pollutants on congenital heart disease get more and more attention. With the aggravation of environmental chemical pollution, the incidences of congenital heart disease are also being increased year by year. Polychlorinated biphenyls (PCBs) are global environmental pollutants with high toxicity, semi-volatile, long-term residue and bioaccumulation, which attracts more attention on the congenital diseases development of the children especially on CHD.PCBs and TCDD belong to dioxins toxins, which two have similar toxicological phenotype. Extensive experimental studies have shown that TCDD work by the AHR signaling pathway, so we hypothesized that PCBs also effect by AHR signaling pathway. The experiments are ready to prove that PCBst has a direct toxic effects on the heart developing system, especially, through AHR signaling pathway.In recent years, the zebrafish is becoming the best vertebrate model for developmental biology studies. the genetic developmental mechanisms of zabrafish is similar with mammals which is contribute to the developmental mechanisms understanding of vertebrate. We chose zebrafish as the model animal eventually. The first part is to study the effects of embryonic exposure to polychlorinated biphenyls on the development of zebrafish embryo especially on heart. The second part is to investigate the PCBs affect the regulation of heart development through the AHR, WNT and RA signaling pathway. Part I:Effects of embryonic exposure to polychlorinated biphenyls on heart morphology in zebrafishObjective:To study the effects of embryonic exposure to polychlorinated biphenyls on the development of zebrafish embryo especially on heart.Methods:Eggs of zebrafish after4hours post fertilization were exposed to five concentrations of PCBs (125μg/L、250μg/L、500μg/L、1000μg/L) dissolved with0.01%methanol into rearing solution respectively, and were reared in60mg/L sodium chloride solution as the blank control group. We study the influences of different concentrations of polychlorinated biphenyls on morphological development, mortality rate, malformation rate and percentage of hatching on different developmental stages (24hpf,48hpf,72hpf,96hpf,120hpf). Embryos were assessed at96hpf for changes in heart morphology by light and electron microscope. We describe from a histological level of PCBs on embryonic development of cardiac morphology by the whole embryo in situ hybridization with cardiac-specific markers, amhc, vmhc and cmlc2were used as cardiac-specific markers to observe cardiac morphology at24hpf,48hpf respectively. At the same time, we use Real-time PCR to support the results of situ hybridization.Results:1) Polychlorinated biphenyls (0.125mg/L) did not cause obvious changes of mortality rate at the early development, the rest concentrations all caused significant changes of morphological development, mortality rate, teratogenic rate and percentage of hatching on different developmental stages. With the concentration increasement, the hatching rate of zebrafish egg decreases while the mortality rate and the malformation rate increases obviously, that indicates an obvious dose-depending and dose-depending effect. Various concentrations of PCBs cause the performance of embryonic or larvae malformation, developmental delay, pericardial edema, spinal curvature, the linear heart tube phenotype.2) In situ hybridization revealed that PCBs have significant toxic effects on cardiac development, such as the cardiac loop incomplete, ventricular atrial developmental abnormal position, the expansion of the cardiac chambers, down-regulated expression of cardiac-specific probe, which is supported by Real-time PCR. In an conclusion, PCBs could hamper the development of zebraifsh embryo, and lead to the death and malformation of zebrafish embryo and larvae.Conclusion:Embryonic exposure to PCB induced developmental deficits in zebrafish heart morphology which suggests the value of monitoring organic contaminant.Part II Effects of embryonic exposure to polychlorinated biphenyls on the expression of photoreceptor cell-specific genesObjective:To investigate the PCBs affect the regulation of heart development through the AHR, WNT and RA signaling pathway.Methods:Eggs of zebrafish after4hours post fertilization were exposed1mg/L PCBs dissolved with0.01%methanol, and were reared in60mg/L sodium chloride solution as the blank control group. Embryos were assessed at24hpf,48hpf,72hpf and96hpf for changes in the expression of Ahr, Arnt, Cyplal, Wntl, Wnt5, and Wntll, by Real-time PCR, and in the expression of Cyplal by immunofluorescence. RA signaling pathway plays an important regulatory role in early development stage, therefore we chosen14hpf and24hpf two time points to detect changes about the expression of Raldh2and Cyp26al in Real-time PCR. At the same time, we use results of situ hybridization to support it. Results:1) AHR signaling pathway conclude three important genes Ahr2, Arnt, Cypla, the expression of which were caused significant changes in Real-time PCR. Ahr2and Arnt were up-regulated at24hpf,48hpf,72hpf and96hpf. Cypla was were up-regulated at72hpf and96hpf. We checked the expression of Cypla with immunofluorescence at24hpf,48hpf,72hpf, compared with the control group, the expression level of PCBs-treated group were up-regulated at three times.2) In WNT signaling pathway, Wntl did not have significant changes at four observation time, on the other side, Wnt5and Wntll were down-regulated at all four observation time point which were statistically significant.3) Cyp26al was down-regulated at two observation time which were statistically significant with Real-time PCR and in situ hybridization technique. Raldh2were up-regulated statistically significant.Conclusion:PCBs produce toxic effects through the AHR signaling pathway and impact WNT and RA pathway which regulate heart development.