硝酸盐对中华大蟾蜍母源性和内源性甲状腺激素干扰效应及其作用机理的研究

Concentrations of nitrate in water body are increasing dramatically around the world due to nitrogen input from agriculture and industry. Previous study showed that excessive nitrate impaired thyroid hormone acting process. Thyroid hormone is crucial to regulate amphibian embryonic development and tadpoles metamorphosis. Generally,embryonic development is regulated by maternal thyroid hormones deposited in egg yolk, while tadpole metamorphosis is regulated by endogenous thyroid hormone. However, the disrupting effects of nitrate on thyroid hormone during the process of amphibian embryonic development and tadpoles metamorphosis are still unclear. Here, Bufo gargarizans were used as test animals to examine the acting mechanisms and disrupting effects of nitrate on maternal and endogenous thyroid hormone. Firstly,morphological indicators of metamorphosis ratios,developmental stage, weight and total length of embryos and tadpoles were investigated to evaluate individual responses to nitrate stress. Secondly, thyroid activity was assessed using histomorphological features to detect nitrate’s ability to disrupt thyroid. Finally, the expression and precise localization of thyroid hormone receptor (TR)and deiodinase (D2 and D3) mRNA in target organs were detected using in situ hybridization and real time polymerase chain reaction (RT-PCR) to reveal molecular responses to nitrate in embryos and tadpoles. The study will be essential to assess ecological risks of nitrate in aquatic environments.

由于工农业的快速发展，氮排放迅速增加，从而导致水域内硝酸盐含量攀升。研究表明，硝酸盐具有甲状腺激素干扰效应。两栖动物的发育受甲状腺激素调控，来源于卵黄的母源性甲状腺激素调控胚胎发育，而内源性甲状腺激素则调节幼体发育。然而在两栖动物胚胎和幼体发育过程中硝酸盐对甲状腺激素的干扰效应鲜有研究。本课题拟从个体、组织和分子三个水平阐明硝酸盐对母源性和内源性甲状腺激素的干扰效应和作用途径。通过硝酸盐水体暴露中华大蟾蜍(Bufo gargarizans)胚胎和幼体，综合变态率，发育分期，体长和体重等形态指标反映硝酸盐胁迫下的个体水平响应；通过幼体甲状腺组织发生和超微结构反映硝酸盐胁迫下甲状腺激 素合成水平；并采用原位杂交和RT-PCR技术分析靶器官中甲状腺激素受体和脱碘酶mRNA等表达分布及表达水平，以反映硝酸盐胁迫下胚胎和幼体发育的分子响应。本课题的实施，可为水体硝酸盐污染的生态风险评估提供理论依据。

由于工农业的快速发展，氮排放迅速增加，从而导致水域内硝酸盐含量攀升。研究表明，硝酸盐具有甲状腺激素干扰效应。本研究对中华大蟾蜍（Bufo gargarizans）胚胎持续进行硝酸盐暴露，通过个体、组织和分子三个水平研究了硝酸盐对中华大蟾蜍胚胎发育和变态过程的影响，以此来阐明硝酸盐对母源性和内源性甲状腺激素的干扰效应和作用途径。结果表明，在胚胎阶段，硝酸盐暴露能够造成胚胎畸形发育，并且能够增加胚胎的体重与体长。通过对中华大蟾蜍胚胎进行转录组与qRT-PCR分析，硝酸盐暴露能够诱导氧化应激，并影响母源性甲状腺激素信号通路中相关基因的表达。同时，不同发育时期的中华大蟾蜍胚胎原位杂交结果表明，D2、D3、TRα和TRβ的表达模式与发育时期相关，没有受到硝酸盐暴露的影响。中华大蟾蜍变态发育阶段，硝酸盐暴露能够造成蝌蚪变态迟滞，体重、体长减少。同时，组织学结果表明，硝酸盐暴露能够造成蝌蚪甲状腺中滤泡上皮细胞增厚、胶质减少，肝脏中胆小管纤维化、肝细胞降解和脂滴减少。电镜观测肝脏的超微结构变化，说明肝细胞中线粒体和脂滴都受到了硝酸盐暴露的影响。对变态高峰期蝌蚪的肝脏、尾和后肢进行qRT-PCR分析，发现硝酸盐干扰了内源性甲状腺激素信号通路中相关基因的表达。此外，变态高峰期蝌蚪肝脏的转录组和qRT-PCR结果表明，硝酸盐能够对肝脏中脂代谢造成影响。本研究的结果有助于为水环境硝酸盐污染对两栖动物母源性和内源性甲状腺激素的影响和潜在机理提供一定的理论依据。