取代酚类物质的雌激素效应研究及相关构效分析
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摘要
随着工业化的飞速发展，越来越多的人工合成化学品进入到人类生活的环境中，这类物质中一些具有环境雌激素效应的物质由于对人类和动物的生存、繁衍存在潜在的毁灭性，引起了人们的极大关注。并且随着更多新化学品的开发，具有环境雌激素效应的物质也将随之增多，因此，科学界普遍认为研究一种切实可行的筛选雌激素效应的方法是当务之急。鱼类卵黄蛋白原(VTG)法作为当前较为流行的筛选、评价物质雌激素效应的方法具有较高的灵敏度和可行性，本文采用鲤鱼VTG的多克隆抗体作为金鱼幼鱼VTG的第一抗体，检测金鱼幼鱼的VTG；将传统的检测血清中VTG的方法改进为检测鱼体肝脏中的VTG，以便能够对小型鱼类或体型较小的鱼类就行快速取材和检测，并用此方法对多种取代酚类物质进行雌激素效应的评价，最后使用定量活性相关法多具有雌激素效应的取代酚类物质进行雌激素效应的构效分析，以寻找影响物质雌激素效应的分子参数因素，为对化学品进行简便快速筛选寻找理论依据。通过大量实验得出如下结论：
(1) 鲤鱼VTG的多克隆抗体能够识别金鱼幼鱼的VTG，采用检测肝脏中VTG的方法具有较好的灵敏度，在对体型较小的鱼类进行VTG检测时可以替代检测血清中VTG的方法。暴露药物的浓度和时间对VTG的生成积累均有一定影响。
(2) 被检测的一系列取代酚类物质，已烯雌酚、已烷雌酚、对壬基酚、对辛基酚、对戊基酚、对庚氧基酚、对叔丁基邻苯二酚、2—甲基—4—氯酚均具有较为明显的雌激素效应，而几种被检测的氯代酚类(五氯酚、2，3，5，6-四氯酚、2，3，4-三氯酚)未见有明显的雌激素效应。
(3) 对具有明显雌激素效应的9种酚类物质的雌激素效应进行构效分析，对分子特征参数与Logl／EC_(50)建立模型，结果显示分子最高占有轨道能(E_(HOMO))和二阶分子连接性指数(~(Ⅱ)X)是影响酚类物质的雌激素效应最重要的因素。R~2=0.963，表明该模型能够对酚类物质的雌激素效应做出一定的反映和预测，这为优化化学物质雌激素效应的筛选和评价方法提供了一种新的思路。
With the rapid development of industrialization, more and more man-made chemicals appeared in environment where man live, of which a kind of chemical with estrogenity attracted more attention because of their latent destroying to the living and reproduction of man and other animals. And with more new chemicals developed, more estrogenic chemicals will be detected. So it is urgent to find out a practical method to determine estrogenicity of chemicals. Method pertinent to Vitellogenin(VTG) of fishes is prevalent to determine estrogenicity of chemicals because of its feasibility and sensitivity. Polyclonal antibody raised against carp VTG was used as the first antibody in this paper to measure juvenile goldfish VTG The traditional method to measure VTG in serum was changed into detect VTG in liver, so that rapid sampling and measuring can be made to the small fish or juvenile fish, and then this method was used to evaluate the estrogenicity of many substituted phenols. QSAR Analysis was made to estimate the factors of molecule characteristic parameters affecting estrogenicity of chemicals so that some theories of estrogenic mechanism can be found. Some conclusions were achieved through lots of experiments:
1. Polyclonal antibody raised against carp VTG can identify goldfish VTG, and there exists good sensitivity to measure VTG in liver. This method is a good surrogate for measuring VTG in serum for small fish. Chemical's concentration and exposure time had impaction on the accumulation of VTG.
2. A serials of substituted phenols were tested, of which some are estrogenic such as Diethylstilbestrol(DES),Hexestrol(HES),4-nonylphenol(NP),4-tert-octylphenol(O P),4-tert-pentylphenol(PP),4-tert-butylphenol(BP),4-heptyloxyphenol(HP),4-tert-b utylcateehol(BOP),4-chloro-3-methylphenol(BClP), but others are non-estrogenic such as Pentachlorophenol(PCP) 2,3,5,6-tetrachlorophenol(TeCP) 2,3,4-trichlorophenol(TCP).
3. QSAR analysis was made for 9 substituted phenols with obvious estrogenicity. Equations were founded between molecule parameters and log1/EC_(50), and the results showed that they were the most important factors to affect estogenicity of substituted phenols. R~2 is 0.963, which showed that the Equation has some ability to reflect and dope out the estrogenicity. This provided a new idea to filter and evaluate the estrogenicity of chemicals.
(1) 鲤鱼VTG的多克隆抗体能够识别金鱼幼鱼的VTG，采用检测肝脏中VTG的方法具有较好的灵敏度，在对体型较小的鱼类进行VTG检测时可以替代检测血清中VTG的方法。暴露药物的浓度和时间对VTG的生成积累均有一定影响。
(2) 被检测的一系列取代酚类物质，已烯雌酚、已烷雌酚、对壬基酚、对辛基酚、对戊基酚、对庚氧基酚、对叔丁基邻苯二酚、2—甲基—4—氯酚均具有较为明显的雌激素效应，而几种被检测的氯代酚类(五氯酚、2，3，5，6-四氯酚、2，3，4-三氯酚)未见有明显的雌激素效应。
(3) 对具有明显雌激素效应的9种酚类物质的雌激素效应进行构效分析，对分子特征参数与Logl／EC_(50)建立模型，结果显示分子最高占有轨道能(E_(HOMO))和二阶分子连接性指数(~(Ⅱ)X)是影响酚类物质的雌激素效应最重要的因素。R~2=0.963，表明该模型能够对酚类物质的雌激素效应做出一定的反映和预测，这为优化化学物质雌激素效应的筛选和评价方法提供了一种新的思路。
With the rapid development of industrialization, more and more man-made chemicals appeared in environment where man live, of which a kind of chemical with estrogenity attracted more attention because of their latent destroying to the living and reproduction of man and other animals. And with more new chemicals developed, more estrogenic chemicals will be detected. So it is urgent to find out a practical method to determine estrogenicity of chemicals. Method pertinent to Vitellogenin(VTG) of fishes is prevalent to determine estrogenicity of chemicals because of its feasibility and sensitivity. Polyclonal antibody raised against carp VTG was used as the first antibody in this paper to measure juvenile goldfish VTG The traditional method to measure VTG in serum was changed into detect VTG in liver, so that rapid sampling and measuring can be made to the small fish or juvenile fish, and then this method was used to evaluate the estrogenicity of many substituted phenols. QSAR Analysis was made to estimate the factors of molecule characteristic parameters affecting estrogenicity of chemicals so that some theories of estrogenic mechanism can be found. Some conclusions were achieved through lots of experiments:
1. Polyclonal antibody raised against carp VTG can identify goldfish VTG, and there exists good sensitivity to measure VTG in liver. This method is a good surrogate for measuring VTG in serum for small fish. Chemical's concentration and exposure time had impaction on the accumulation of VTG.
2. A serials of substituted phenols were tested, of which some are estrogenic such as Diethylstilbestrol(DES),Hexestrol(HES),4-nonylphenol(NP),4-tert-octylphenol(O P),4-tert-pentylphenol(PP),4-tert-butylphenol(BP),4-heptyloxyphenol(HP),4-tert-b utylcateehol(BOP),4-chloro-3-methylphenol(BClP), but others are non-estrogenic such as Pentachlorophenol(PCP) 2,3,5,6-tetrachlorophenol(TeCP) 2,3,4-trichlorophenol(TCP).
3. QSAR analysis was made for 9 substituted phenols with obvious estrogenicity. Equations were founded between molecule parameters and log1/EC_(50), and the results showed that they were the most important factors to affect estogenicity of substituted phenols. R~2 is 0.963, which showed that the Equation has some ability to reflect and dope out the estrogenicity. This provided a new idea to filter and evaluate the estrogenicity of chemicals.
引文
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