中国居民EDCs膳食暴露概率评估及低剂量BPA致雄性大鼠生殖毒性的研究

发布时间：2018-06-22 02:59

本文选题：内分泌干扰物 + 双酚A　；参考：《华中科技大学》2015年博士论文

【摘要】：随着人们对环境内分泌干扰物(Endocrine disrupting chemicals, EDCs)造成健康损害的认识日益深入,相关国际组织适时、有针对性地制订了部分EDCs暴露的单一限量标准,如欧盟确定双酚A及炔雌醇的每日可耐受摄入量均为50μg/kg.d、丹麦推荐壬基酚每日可耐受量为5μg/kg.d,但仍有越来越多的人们对日常生活中长期、低剂量(共)暴露EDCs的潜在健康风险表示担忧。为探讨人们生活中低剂量、长期(共)暴露EDCs是否可导致健康风险,我们开展了相关研究：第一部分市售食品中EDCs的含量与分布目的：建立一套高效率的可用于食物中多种EDCs检测的样品处理、目标物提取、净化及仪器检测方法体系,监测我国多个地区常见食品中主要EDCs的含量和分布,为食品中EDCs污染来源的追溯及控制提供参考,为我国居民膳食暴露EDCs的风险评估提供基础数据。方法：按照我国河流流域的地理特点,采集珠江、长江及黄河中下游重要城市(广州、武汉和济南)市售食品样品450件,样品经适当前处理去除样品中的基质,富集、浓缩目标化合物后,用LC-MS/MS方法同步检测样品中壬基酚(NP),双酚A(BPA),辛基酚(OP),17-p雌二醇(E2),炔雌醇(EE2),雌酮(E1),雌三醇(E3)和己烯雌酚(DES)的含量。以雌激素等效因子(EEF)计算食物中总雌激素当量(∑EEQ)并于E2检出水平比较,衡量雌激素“共暴露”现象对潜在风险的影响。结果：(1)按照优化的前处理及LC-MS/MS检测条件、参数,可有效地实现前述8种EDCs的液相分离和质谱检测,EDCs各组分标准系列均呈现较好的线性关系。(2)除E1外,检测范围内的其它EDCs成分,在不同城市的各类食品中均有不同程度检出,检出率在不同食品类别中存在差别。在广州、武汉及济南三个城市中,EDCs检出率的高低顺位均表现出NPBPAEE2的现象。(3) Spearman相关性分析结果显示,在众多EDCs组分检出水平中,NP与OP、EE2与OP、BPA与OP、EE2与NE、BPA与NP、BPA与EE2、E2与E3等均表现为低度正相关关系(P0.05)。(4)Wilcoxon法比较食物总雌激素当量(∑EQ)与E2检出水平的差别,“共暴露”时可放大接触的雌激素剂量达到E2水平的4倍(P0.05)。(5)对应分析(Correspondence analysis，，CA)显示,不同类别的食物中倾向性污染的EDCs种类存在差异。结论：本研究系统建立了食物中EDCs多组分同步检测的LC-MS/MS方法,并运用建立的方法对中国居民市售食品中EDCs的含量进行监测。结果显示,市售食品中EDCs单组分的含量水平均较低,且不同地区具有相似的含量特征但不同食物种类中污染的EDCs组分存在差异性和倾向性,EDCs单组分间的正相关关系提示我们居民经食物摄入EDCs往往是处于一种“共暴露”的状态,且“共暴露”可能放大其健康效应。第二部分检出EDCs的膳食暴露概率评估目的：基于前述监测获得的食品中EDCs含量数据,结合2002年中国居民膳食调查报告中的食物消费量数据,运用蒙特卡洛计算方法对中国居民经食物暴露EDCs的水平进行概率模拟,为风险分析及风险管理提供决策依据。方法：根据专业知识确定人群膳食暴露EDCs的数学计算模型后,运用Software Crystal Ball11.0对数学模型中的变量(EDCs水平、食物消费数据、体重等)进行参数模拟,以模拟的参数描述变量分布特征并纳入后续计算,利用计算机按参数指定的规则模拟抽样并迭代计算,每一次迭代计算的结果则视为样本中某一个体的暴露水平,当迭代次数足够多时则个体EDCs暴露水平的频数分布趋于稳定,再根据个体暴露水平的频数分布图进行概率分析。结果：经过人群EDCs暴露水平的概率模拟,三个地区居民经膳食平均暴露及高暴露NP、BPA和EE2场景下的水平并无本质差别,且均远低于推荐的容许每日摄入量水平,如95%的居民膳食暴露BPA的水平分别为0.27μg/kg.d(广州)、0.28μg/kg.d(武汉)和0.26μg/kg.d(济南),远低于其推荐限量标准(50μg/kg.d)。结论：在食物中现有EDCs含量水平及我国居民现有饮食习惯的前提下,单独考虑膳食途径、暴露单种EDCs,尚不足以达到推荐的限量标准。综合前述“共暴露”的客观事实及雌激素等效性的理论,我们仍有必要开展多途径、多物质同时暴露的研究。第三部分低剂量BPA对雄性大鼠生殖毒性的影响目的：模拟人群长期、低剂量暴露双酚A的特征,建立低剂量双酚A的雄性大鼠染毒模型,在观察低剂量双酚A致模型大鼠及其子代空间记忆行为损伤的同时,进一步探讨其生殖毒性。方法：30d龄雄性Wistar大鼠30只经适应性喂养2周后,随机等分为对照组、10μg/kg.d及50μg/kg.d BPA等3组,对照组为玉米油溶剂,实验动物按常规记录进食量和体重。各组大鼠干预8周后,同正常90天龄、发情期雌鼠进行交配。交配成功1周后,所有父系大鼠进行水迷宫测试一周后处死,取血液及睾丸、附睾组织检测。LC-MS/MS法检测大鼠血清BPA含量以评估动物模型,观察睾丸、附睾脏器系数及精子畸形率同时,对睾丸组织病理形态学进行观察,TUNEL实验检测睾丸组织细胞凋亡,免疫组化技术观察睾丸组织PCNA的表达水平及组织蛋白泛乙酰化表达水平。结果：(1)各组大鼠进食量及体重增长差异无显著性(P0.05),但经BPA干预后,各组大鼠血清BPA水平差异有显著性(P0.05)。(2)与对照组比较,50μg/kg.d BPA染毒雄性大鼠的逃避潜伏期、游泳速度、游泳距离以及目标象限停留时间等差异均有显著性(P0.05),而子代大鼠仅表现出逃避潜伏期、游泳速度和游泳距离存在差异(P0.05);10μg/kg.d BPA染毒大鼠及其子代上述指标差异均无显著性。(3)各组大鼠睾丸、附睾脏器系数及精子畸形率差异并无显著性(P0.05)。(4)HE染色、TUNEL实验及PCNA免疫组化结果显示,睾丸组织细胞凋亡变化无显著性(P0.05),但50μg/kg.d BPA染毒雄性大鼠睾丸组织生精细胞层数减少、细胞间排列疏松,且组织中PCNA表达减弱(P0.05)。(5) Western blotting实验结果显示50μg/kg.d剂量BPA染毒雄性大鼠可导致睾丸组织在25KD处乙酰化蛋白表达水平下降(P0.05)。结论：50μg/kg.d剂量的BPA可损害成年雄性大鼠的空间记忆能力,并且这种损害可部分传给下一代,其作用机制可能与其导致染毒大鼠睾丸组织蛋白质乙酰化水平下降有关。
[Abstract]:With the growing awareness of the health damage caused by environmental endocrine disruptors (Endocrine disrupting chemicals, EDCs), relevant international organizations have timely formulated a single limit standard for partial EDCs exposure, such as the EU determined that the daily tolerable intake of bisphenol A and estriol is 50 u g/kg.d, and Denmark recommends nonylane. There are more and more people concerned about the potential health risks of long-term, low dose (CO) exposure to EDCs in daily life. In order to explore the low dose in people's life, the long-term (CO) exposure to EDCs can lead to health risks, and we have carried out a related study.
The content and distribution of EDCs in the first part of the market
Objective: to establish a set of high efficient sample processing, which can be used for various EDCs detection in food, target extraction, purification and instrument detection system, to monitor the content and distribution of main EDCs in the common food in many regions of China, provide reference for tracing and controlling the source of EDCs pollution in food, and exposing the wind of Chinese residents to the diet of EDCs. The risk assessment provides the basic data.
Methods: according to the geographical features of the river basin in China, 450 food samples were collected from the cities of the Pearl River, the Yangtze River and the middle and lower reaches of the Yellow River (Guangzhou, Wuhan and Ji'nan). After the samples were disposed to remove the matrix, enrich and concentrate the target compounds, the nonylphenol (NP), bisphenol A (BPA) and octyl in the sample were simultaneously detected by LC-MS/ MS method. The content of phenol (OP), 17-p estradiol (E2), estriol (EE2), estrone (E1), female three alcohol (E3) and diethylstilbestrol (DES). The total estrogen equivalent (sigma) in food (EEF) was calculated by estrogen equivalent factor (EEF) and the detection level of E2 was compared to measure the effect of estrogen "CO exposure" on the potential risk.
Results: (1) according to the optimized preprocessing and LC-MS/MS detection conditions and parameters, the liquid phase separation and mass spectrometric detection of the previous 8 kinds of EDCs can be effectively realized, and the standard series of EDCs components have a good linear relationship. (2) except E1, other EDCs components in the detection range are detected in different kinds of food in different cities and detected in different degrees. Among the three cities in Guangzhou, Wuhan and Ji'nan, the high and low levels of EDCs detection rates showed NPBPAEE2 phenomenon. (3) the results of Spearman correlation analysis showed that NP and OP, EE2 and OP, BPA and OP, EE2 and NE, etc. The correlation (P0.05). (4) the difference between the total estrogen equivalent (sigma EQ) and the E2 detection level was compared with the Wilcoxon method. The estrogen dose of the amplified exposure reached 4 times (P0.05) at E2 level when "CO exposure". (5) the corresponding analysis (Correspondence analysis, CA) showed that there were different kinds of EDCs species in different types of food.
Conclusion: This study established a LC-MS/MS method for simultaneous detection of EDCs components in food, and used the established method to monitor the content of EDCs in the food sold in China. The results show that the level of the content of the single component of EDCs in the market food is low, and there are similar content characteristics but different food types in different regions. There is a difference and tendency of the EDCs components of the pollution. The positive correlation between the single component of EDCs suggests that our residents' food intake EDCs is often in a "CO exposure" state, and "CO exposure" may amplify its health effect.
Evaluation of the probability of dietary exposure in the second part of EDCs
Objective: Based on the data of EDCs content in food obtained by the previous monitoring, combined with the food consumption data of the Chinese residents' dietary survey report in 2002, the Monte Carlo calculation method was used to simulate the level of the Chinese residents' exposure to food EDCs, so as to provide the decision basis for risk analysis and risk management.
Methods: according to the professional knowledge to determine the mathematical model of the population dietary exposure EDCs, Software Crystal Ball11.0 was used to simulate the parameters in the mathematical model (EDCs level, food consumption data, weight and so on). The parameters of the variables were described by the simulated parameters and included in the follow-up calculation, and the parameters specified by the computer were used. The results of each iteration are considered as the exposure level of a certain individual in the sample. When the number of iterations is sufficient, the frequency distribution of the individual EDCs exposure level tends to be stable, and then the probability analysis is carried out according to the frequency distribution map of the individual exposure level.
Results: through the probability simulation of population EDCs exposure, there was no essential difference between the average dietary exposure and high exposure NP in three regions, and the levels of BPA and EE2 were not essential, and were far below the recommended allowable daily intake levels, such as 0.27 mu g/kg.d (Guangzhou), 0.28 mu g/kg.d (Wuhan) and 0.. 26 mu g/kg.d (Ji'nan), far below its recommended limit standard (50 mu g/kg.d). Conclusion: under the premise of existing EDCs content level and the existing dietary habits of our residents in food, it is not enough to reach the recommended limit standard by considering the dietary route and exposing the single EDCs. In theory, it is still necessary to carry out research on multi-channel and multi material simultaneous exposure.
The third part is the effect of low dose BPA on reproductive toxicity in male rats.
Objective: to simulate the characteristics of long term and low dose exposure of bisphenol A (BPA), to establish a male rat model with low dose of bisphenol A (BPA), and to further explore its reproductive toxicity at the same time in observing the spatial memory behavior damage of rats and their offspring in the low dose of bisphenol A model.
Methods: after 2 weeks of adaptive feeding, 30 male 30d aged male Wistar rats were randomly divided into control group, 10 mu g/kg.d and 50 g/kg.d BPA, and the control group was corn oil solvent. The experimental animals were fed on regular feeding and weight. After 8 weeks of intervention, the rats were copulated with normal 90 days of age and estrus. After 1 weeks of mating success, all fathers were successfully coped. Rats were killed after a week of water maze test. The blood and testicles were taken and the epididymal tissue was detected by.LC-MS/MS to detect the BPA content of the rat serum in order to evaluate the animal model, observe the testis, epididymal organ coefficient and sperm malformation rate, observe the pathomorphology of the testis, and detect the apoptosis and immunohistochemistry of the testicular tissue by TUNEL test. The expression level of PCNA and the pan acetylation level of testicular tissue were observed.
Results: (1) there was no significant difference in the feeding and weight growth of rats in each group (P0.05), but the serum BPA level of the rats in each group was significantly different after BPA (P0.05). (2) compared with the control group, the escape latency, swimming speed, swimming distance and target quadrant residence time were significantly different from those of the control group (the control group). P0.05), while the offspring rats only showed the escape latency, the swimming speed and the swimming distance were different (P0.05); there was no significant difference between the 10 mu g/kg.d BPA rats and their offspring. (3) there was no significant difference between the testis, the epididymal organ coefficient and the sperm abnormality rate (P0.05). (4) HE staining, TUNEL test and PCNA immunization The results showed that there was no significant change in the apoptosis of the testicular tissue cells (P0.05), but the number of spermatogenic cells in the testicular tissue of 50 u g/kg.d BPA rats decreased, the intercellular arrangement was loose and the expression of PCNA decreased (P0.05). (5) the results of Western blotting experiment showed that the dose of 50 u g/kg.d in the male rats could lead to the testicular tissue in 25KD. The expression level of acetyl protein decreased (P0.05).
Conclusion: the 50 g/kg.d dose of BPA can damage the spatial memory ability of adult male rats, and this damage can be partially passed on to the next generation, and its mechanism may be related to the decrease of protein acetylation level in the testicular tissue of rats.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R114

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