【作者】 黄新；

【导师】 刘璟；

【作者基本信息】 浙江大学 ， 环境科学， 2020， 硕士

【摘要】 双酚A（Bisphenol A,BPA）是世界上产量最高的化学原料之一,全球年产量大约超过360万吨。BPA主要用于聚碳酸酯塑料和环氧树脂的制造,由于这两类塑料在工业生产和日常生活中的广泛应用,BPA在环境中几乎无处不在,在地表水、土壤、空气、食物、饮用水、机体中均有检出。BPA是典型的环境内分泌干扰物（endocrine disrupting chemicals,EDCs）,既有类雌激素活性,还能产生抗雄激素效应,能影响雄性机体的生长发育和生殖等生理功能。已有研究证明BPA是通过雄激素受体（androgen receptor,AR）介导的途径干扰AR发挥正常功能来产生抗雄激素效应,但是其分子机制尚不明确。因此我们开展BPA干扰AR活性的机制研究,为BPA的毒性机理研究和健康风险评估提供科学依据,并深入探讨典型EDCs对不同甾体激素受体产生选择性干扰作用的分子机制。本研究以BPA为研究对象,采用中国仓鼠卵巢癌细胞系CHO-K1细胞为研究模型,利用双萤光素酶报告基因实验、Western Blotting、免疫共沉淀、细胞免疫荧光等实验技术,检测BPA对AR转录活性、AR降解和稳定性、AR与伴侣蛋白（90-k Da heat shock protein,Hsp90）解离以及AR转核等功能的影响;并结合分子对接及分子动力学模拟等计算方法模拟分析BPA与AR的结合模式,来探究BPA干扰AR活性的分子机制。在本研究中,细胞活性实验结果表明,BPA在10^-10-10^-4 M浓度范围内对CHO-K1细胞无毒性。双萤光素酶报告基因实验结果表明,BPA在10^-9-10^-5 M浓度范围内对二氢睾酮（5α-dihydrotestosterone,DHT）诱导的AR转录活性具有显著的拮抗作用。分子对接及分子动力学模拟结果表明,BPA能与AR的配体结合区域（ligand binding domain,LBD）稳定结合,并形成良好的氢键作用和疏水作用,且BPA的结合可使LBD的结构趋于稳定。BPA在浓度为10^-7-10^-5 M范围时能够显著抑制由DHT引起的AR稳定化作用,且BPA在浓度为10^-5 M时抑制作用最为明显,使AR蛋白的表达量下降了48%。免疫共沉淀实验结果表明10^-7-10^-5 M浓度范围内的BPA能显著抑制由DHT诱导的AR与其伴侣蛋白Hsp90的解离。细胞免疫荧光实验结果显示了BPA能够抑制AR在DHT诱导下的转核过程。综上所述,本研究发现BPA作为一种典型EDCs,影响AR作用机制的多个环节,如配体-受体结合、AR蛋白降解与稳定化、AR与Hsp90解离、AR转核过程等,从而产生抗雄激素效应。这项研究为进一步了解BPA对AR拮抗作用的精确机制提供了新的数据。更多还原

【Abstract】 Bisphenol A（BPA）is one of the highest production volume chemicals,with an annual global production of more than 3.6 million tons.BPA is widely used in manufacturing polycarbonate plastics and epoxy resins due to remarkable properties of BPA-based products such as durable and tough.Due to its widespread use in industry and daily life,BPA can be released from products and migrate in environment.It is almost ubiquitous in the environment and has been detected in water,soil,air,food,and human tissues.BPA is a typical endocrine-disrupting chemicals（EDCs）.It has anti-androgenic effects and affects the growth and reproduction of male.The previous studies have shown that BPA interfered with the normal function of AR by the AR-mediated pathway,but molecular mechanism of its action is still unclear.In this study,we attempted to explore the molecular mechanism of BPA on AR antagonism.This study will provide scientific basis for the toxicological mechanism of BPA and health risk assessment of BPA,and provide an important reference for the molecular mechanism of EDCs selective interference with different steroid hormone receptors.In this study,the in vitro assays such as reporter gene assay,western blotting,co-immunoprecipitation and immunofluorescence experiments were carried out to determine the effects of BPA on AR transcriptional activity,degradation and stabilization of AR protein,dissociation of chaperone（90-k Da heat shock protein,Hsp90）with AR,and AR nuclear translocation.The in silico study such as molecular docking and molecular dynamics simulations were conducted to simulate the binding mode of BPA with AR.The MTS assay results indicated that BPA at concentrations of 10^-10-10^-4 M had no effects on CHO-K1 cell viability.In reporter gene assay,BPA caused a significant antagonistic effect on 5α-dihydrotestosterone（DHT）-induced AR transcriptional activity at concentrations of 10^-9-10^-5 M.The results of molecular docking and molecular dynamics simulations indicated the availability of BPA binding to the ligand binding domain（LBD）of AR and the bond of BPA made structure of LBD more stable.In addition,we found that BPA at concentrations of 10^-7-10^-5 M prevented the inhibition of receptor degradation caused by DHT binding to AR,and this effect was most pronounced at a BPA concentration of 10^-5 M,reducing the AR expression to 52%of the positive control.BPA exposure also abolished DHT-dependent dissociation of AR from its co-chaperone Hsp90 and resulted in the blockage of DHT-induced AR nuclear translocation.In conclusion,this study showed that BPA as an antiandrogen affected multiple steps of AR activation and function,which provided new data for further understanding the molecular mechanism of BPA on AR antagonism.更多还原