典型污染物拟抗雌激素效应分子机制及构效关系
文献类型：学位论文
| 作者 | 黎娟 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 张爱茜 |
| 关键词 | 拟/抗雌激素活性 定量结构活性相关（QSAR） 辣椒素类物质 有机 磷酸酯（OPEs） 全氟烷酸类化合物（PFAAs） estrogenic/antiestrogenic activity quantitative structure-activity relaionship (QSAR) capsaicin analogues organophosphate esters (OPEs) Perfluoroalkyl acids (PFAAs) |
| 其他题名 | Molecular mechanism and structure-activity relationships of estrogenic/anti-estrogenic activities for selected environmental pollutants |
| 学位专业 | 环境科学 |
| 中文摘要 | 雌激素信号通路是污染物内分泌干扰效应的重要途径之一，污染物雌激素信号通路复杂多样，其表现出的拟/抗效应的分子机制尚不清晰。配体和雌激素受体(ER)结合后，会引起受体构型发生变化，进而引起不同的辅因子招募模式和继发的生物学级联调控，从而表现出拟 /抗雌激素效应。本论文结合离体实验模型和理论计算，开展基于受体介导途径的污染物拟 /抗雌激素效应分子机制的结构效应关系研究，既有望揭示环境类激素污染物与通路各靶标分子发生诱导契合作用协同调节其生物功能的分子机制，同时也为污染所致雌激素相关生态毒性风险评价提供可靠的科学参考。 辣椒素是辣椒果实中的主要活性成分，具有镇痛、抗炎、抗菌等多种药理作用。早在 1995年，辣椒素及其类似物被提出可以成为双对氯苯基三氯乙烷(DDT)作为防污涂料的替代产品。然而，由于人们把关注过多放在辣椒素类物质的抗菌活性，往往将急性毒性作为其毒性检测终点而忽略了辣椒素类物质对包括海洋环境在内的自然环境造成的潜在内分泌干扰效应。本文结合理论计算方法和离体模型，运用分子对接模拟，雌激素激动/拮抗筛选预测法(AADS)，受体多态分析(RPA)和报告基因实验，探讨了 8种辣椒素类似物的 ER转录活性，并对比其在不同物种间的 ER活性差异。分子对接和 AADS结果表明葡聚糖凝胶(6-I-CPS)具有 ER激动活性，而多巴胺(NADA)，曲古柳菌素 A (TSA)，辣椒素，辣椒平和二氢辣椒素均表现出ER拮抗活性。然而，两个分子体积较大的化合物PPAHV和PDNHV不能够进入 ER的疏水空腔中。此外，MVLN实验结果表明 6-I-CPS能够诱导荧光素酶报告基因的表，表现出拟雌激素活性；而 NADA，TSA，辣椒素，辣椒平和二氢辣椒素在单独暴露及与雌二醇 (E2)共暴露条件下均能够抑制荧光素酶报告基因的表达，表现出抗雌激素活性，这一结果与上述理论计算结果一致，在一定程度上得以相互验证，均暗示芳烃基团的加卤作用及亲酯性长碳链对辣椒素类化合物的雌激素活性有重要影响。此外，MVLN实验结果表明辣椒素类似物的拟/抗雌激素活性至少有一部分是依赖于 E2信号通路得以实现。有趣的是，RPA结果显示辣椒素类物质的拟/抗雌激素活性在人类，鳕鱼和丽龟这三个物种间具有一定差异，不同物种间 ERα活性口袋周围氨基酸有所不同，具体表现为在 ER激动构型中人类 ERα中的 ILE386和 ARG352分别被鳕鱼 ERα中 VAL319和LYS285所取代；而在拮抗构型中，人类 ERα中的 GLY344被丽龟 ERα中的THR338所取代，人类 ERα中的 ARG352和 GLY344分别被鳕鱼 ERα中的 LYS285和 THR277所取代。 有机磷酸酯（OPEs）阻燃剂是运用最为广泛的有机磷阻燃剂，主要以非化学键合方式添加到材料中，在使用中容易由材料进入环境（空气，水样和生物体）而导致暴露水平的增高，是新兴环境污染物的一种。OPEs可通过影响乙酰胆碱酯酶等受体活性产生神经毒性，但是其潜在的经由各种激素受体介导的内分泌干扰效应鲜有报道。本文以人源乳腺癌细胞为离体模型，结合理论计算，通过E-screen、ER-CALUX、荧光定量 PCR检测手段和分子模拟手段，探讨了 13种OPEs的拟/抗雌激素效应及其分子机制。结果表明，13种 OPEs在单独暴露时均不能够引起人类乳腺癌细胞(MCF-7)数目的改变，而在与 E2共暴露的情况下，均能够抑制 E2所诱导的 MCF-7细胞的增殖，且这种抑制效应随着 OPEs浓度的增大而增强，具有确定的剂量效应关系，表明这 13种 OPEs可通过干扰 E2信号通路产生生物学效应。进一步的 MVLN检测中，13种 OPEs在单独暴露时均不能够引起荧光素酶活力的改变，而在与 E2共暴露的情况下均能够抑制 E2所诱导的荧光素酶活力，且具有一定剂量效应关系。荧光定量 PCR证实 13种化合物均能够抑制雌二醇所诱导的雌激素标志基因 pS2和 EGR3的表达。荧光定量 PCR、MCF-7细胞增殖和受体报告基因结果相互验证，表明这 13种 OPEs具有抗雌激素活性，且与 ER介导通路有关。同时，两种离体实验结果均提示磷酸基团被苯环取代的 OPEs（磷酸三苯酯、磷酸三甲苯酯、膦酸二苯酯和氯磷酸二苯酯）及具有三个较长烷烃侧链的 OPEs（丁基磷酸二丁酯）具有较强的抗雌激素活性，其相应 EC50均为微摩尔级别。定量构型关系(QSAR)预测模型表明，OPEs的抗雌激素活性与分子体积、相邻分子的特征值及分子亲水性相关。随后的分子对接结果表明，三个具有较大侧链取代基团的 OPEs（磷酸三苯酯、磷酸三甲苯酯和丁基磷酸二丁酯）和典型的 ER拮抗剂 ICI 180, 782和 OHT与 ER活性口袋结合模式相似，表明这三种 OPEs与经典 ER拮抗剂具有相似的拮抗机理，即通过干扰 H12在 ER中的位置，进而引起下游转录激活的改变。 全氟烷酸类化合物(PFAAs)是一类持久性有机污染物，被广泛运用于生产生活中，所涉及产品包括食品包装材料，防水涂层及不粘锅等。目前 PFAAs在全球环境广泛检出，具有潜在的内分泌干扰效应。然而， PFAAs在雌激素信号通路中表现出的拟/抗雌激素效应却说法不一。本文以人源乳腺癌细胞为离体模型，结合理论计算，通过 ER-CALUX实验、荧光定量 PCR检测手段和分子模拟手段，探讨了 10种全氟化合物的拟/抗雌激素效应及其分子机制。在基于 ER转录激活的 MVLN检测中，3种 PFAAs：全氟丁酸(PFBA)、全氟丁基磺酸钾 (PFBS)及全氟戊酸(PFPA)，单独暴露能够抑制荧光素酶报告基因的表达，表现出抗雌激素效 应。5种 PFAAs：全氟己酸(PFHxA)、全氟庚酸(PFHPA)、全氟辛酸(PFOA) 、全氟任酸(PFNA)及全氟癸酸(PFDA)，单独暴露时不能够引起荧光素酶活力的改变，未表现出明显的拟/抗雌激素效应。而其中 2种 PFAAs：全氟己烷磺酸钾(PFHxS)和全氟辛基磺酸钾(PFOS)，单一暴露分别能够诱导荧光素酶报告基因的表达，具有拟雌激素效应。在复合暴露实验中，10种 PFAAs均能够减弱雌二醇所诱导的雌激素效应，具有一定抗雌激素活性，且其相应 EC50随着碳链增长而减小。基因检测结果表明，10种 PFAAs均能够抑制雌二醇所诱导的雌激素标志基因 pS2和 EGR3的表达，这一结果与 MVLN检测结果相吻合。分子对接结果表明，PFHxS和 PFOS能够与人类 ER的活性口袋相结合，与周围关键残基形成稳定氢键网络。 而其他 8种 PFAAs均不能够与 ER活性口袋稳定结合，这一结果可以进一步解释在单一暴露实验中，观察到的 PFHxS和 PFOS具有拟雌激素效应。进一步的模拟分析表明，10种 PFAAs均能够在 ER的活性口袋被 E2占据的情况下，与包括共辅因子结合位点在内的 ER表面位点相结合，这一结果解释了在共暴露实验中观察到的 10种 PFAAs的抗雌激素活性。 |
| 英文摘要 | The estrogen signaling pathway is one of the most important pathways involved in the endocrine disrupting effect induced by various pollutants. Complicated as estradiol (E2) signaling pathway is, its underlying molecular mechanism is still not very clear. Ligand-dependent mechanism plays a key role in the pathway. The interaction between the ligand and the estrogen receptor (ER) may lead to the allosteric effect of ER, and finally results in different recruitment pattern of cofactors as well as the subsequent diverse transcription regulation. In the present study, we explored the ER-mediated transcription activity of selected environmental pollutants via an integrated approach based on in silico prediction and in vitro assays, which will provide useful dada for elucidating the potential molecular mechanism of estrogenic activity of enrionmental pollutants. Capsaicin, the main active pungent ingredient in chilli peppers, has been taken into consideration as potential alternative structure template of dichlorodiphenyl trichloroethane (DDT) in antifouling paint due to its high anti-bacterial and insecticidal efficiency. However, information about the estrogenic activity of capsaicin analogues is rather limited in comparison with both DDT and its metabolites. We here explored the ER transcription activity of selected capsaicin analogues by an integrated approach based on in silico prediction and in vitro assays. Molecular simulation and the agonist/antagonist differential-docking screening identified 6-iodonordihydrocapsaicin (6-I-CPS)as potential ERα agonist, while anti-estrogenicity was expected for N-arachidonoyldopamine (NADA), capsazepine,dihydrocapsaicin, trichostatin A (TSA), and capsaicin. On the contrary, large volume chemicals such as PPAHV and PDNHV cannot fit well with ER hydrophobic cavity.The result of following MVLN assay accorded with the in silico prediction. 6-I-CPS was proved to induce luciferase gene expression in the absence of estradiol, while the other analogues of relatively small molecular volume reduced luciferase gene expression in MVLN cells both in the absence and presence of estradiol. Such suggested that the ER transcription activity of capsaicin analogues be at least partly through ERα-mediated pathway.Further toxicity study demonstrated anti-proliferation capacity of both estrogenic and anti-estrogenic compounds with a dose dependent manner, the structure-activity relationship of which can be encoded using geometrical descriptors QXXs. Moreover, receptor polymorphism analysis indicated that species difference in estrogenic activity can result from the replacement of ILE386 and ARG352 in agonist-bound hERα by VAL319 and LYS285 in codERα, GLY344 in antagonist-bound hERα by THR338 in turERα, ARG352 and GLY344 in antagonist-bound hERα by LYS285 and THR277 in codERα, respectively. Therefore,capsaicin analogues may exhibit diverse species selectivity for human beings and marine species such as Gadus morhua and Lepidochelys olivacea. There is significant increase in application of organophosphate esters (OPEs) as flame retardants due to the ban of brominated flame retardants. As a kingd of emerging environmental pollutants, OPEs can exhibit various acute toxicities. Inhibition of acetylcholinesterase (AChE) is the primary toxicological effect of OPEs. Unfortunately, the low-dose endocrine disrupting effects mediated by hormone receptors are commonly underestimated for OPEs. Recent cohort studies indicated that OPEs exposure may be associated with altered hormone level. Therefore, a structure orientated research was carried out to explore the estrogenic/antiestrogenic effect of selected OPEs and potential mechanism. Various in vitro assays were made such as E-screen assay, MVLN assay and the detection of estrogen-responsive genes in MCF-7 cells. Besides, in silico modeling was also performed to further probe the interaction between OPEs and human ERα. All tested OPEs exerted anti-estrogenic activities in both E-screen assay and MVLN assay. Specifically, OPEs with bulky substitute group such as phenyl rings (triphenyl phosphate, tritolyl phosphate, diphenylphosphoryl chloride, diphenylphosphite) or relative long chain alkyl (dibutylbutylphosphonate) exerted a comparatively strong ER antagonism potency at micromolar concentrations. The established QSAR model indicates that the anti-estrogenic activities of OPEs mainly depend on the volume, leading eigenvalue, and hydrophilicity of the molecules. Furthermore, molecular docking revealed that 3 tested OPEs with the most bulky substitute groups on the phosphate ester group (triphenyl phosphate, tritolyl phosphate and dibutyl butanephosphonate) have a similar interaction mode as the classical ER antagonists, ICI 180, 782 and OHT. The correlation between their anti-estrogenic activity and the corresponding ER binding affinity is statistically significant, strongly suggesting that they may possess the classical antagonism mechanism, interfering with positioning of H12 in ER. Clearly, the structural features of phosphate ester group are crucial for the anti-estrogenic potency of OPEs. Perfluoroalkyl acids (PFAAs) are widely used in the many products. Numerous studies have focused on whether or not PFAAs may exhibit estrogenic or antiestrogenic effect. However, there is insufficient information on both PFAAs-ER interaction and the potential biological function induced by such receptor binding. Moreover, estrogenic activities of PFAAs are in controversy, whereas, either estrogenic effects or antiestrogenic effects of PFAAs can present potential hazard to human health. Therefore, ER-mediated transcription activity of PFAAs was analyzed under both single exposure and co-exposure with E2 in human breast cancer cells. The estrogenic/antiestrogenic effects of PFAAs were investigated using the MVLN assays and the evaluation of estrogen-responsive genes in MCF-7 cells. In order to gain insight into the molecular mechanism of PFAAs induced ER regulation, in silico simulation was also performed to elucidate the interaction between PFAAs and human ERα. 3 tested PFAAs, including heptafluorobutyric acid (PFBA), potassium nonafluoro-1-butanesulfonate (PFBS) and perfluoropentanoic acid (PFPA), reduced luciferase gene expression in MVLN cells in the absence of estradiol.. On the contrary, tridecafluorohexane-1-sulfonic acid potassium salt (PFHxS) and potassium heptadecafluoro-1-octanesulfonate (PFOS) induced luciferase activity in MVLN cells under both single exposure. There was no significant response observed for other PFAAs in MVLN cells without estradiol coexposure. However, all tested PFAAs attenuate E2-stimulated luciferase activity and estrogen-responsive genes (TFF1 and EGR3) expression in MCF-7 cells. Furthermore, PFAAs incubation with E2 and ICI 180, 782 caused further downregulation of luciferase activity, and such additional reduction in transcription activity can be observed for PFAAs at nanomolar concentrations, depending on the concentration of ICI 180, 782. The in silico results shown that PFOS and PFHxS were typical ER agonist, and all PFAAs could bind with ER in its surface area for coactivator when the ligand binding domain of ER has already been occupied by E2. The developed QSAR model showed good prediction ability and mechanism interpretability. The estrogenic/antiestrogenic effects of PFAAs are dependent on the structural features of sulfonic substitution and chain length. Our results suggested that direct interact with ER ligand binding pocket is not the only pathway involved in the estrogenic/antiestrogenic activity of PFAAs. The present research also provides important evidence to illustrate the estrogenic/antiestrogenic effects of short chain PFAAs, especially for PFBS, which has been reported to be safe alternative for PFOS. |
| 公开日期 | 2015-07-07 |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/15619]  |
| 专题 | 生态环境研究中心_环境化学与生态毒理学国家重点实验室 |
| 推荐引用方式 GB/T 7714 | 黎娟. 典型污染物拟抗雌激素效应分子机制及构效关系[D]. 北京. 中国科学院研究生院. 2014. |
入库方式： OAI收割
来源：生态环境研究中心
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