本文選題：內(nèi)分泌干擾物 + 雙酚A　； 參考：《華中科技大學(xué)》2015年博士論文

【摘要】：隨著人們對(duì)環(huán)境內(nèi)分泌干擾物(Endocrine disrupting chemicals, EDCs)造成健康損害的認(rèn)識(shí)日益深入,相關(guān)國(guó)際組織適時(shí)、有針對(duì)性地制訂了部分EDCs暴露的單一限量標(biāo)準(zhǔn),如歐盟確定雙酚A及炔雌醇的每日可耐受攝入量均為50μg/kg.d、丹麥推薦壬基酚每日可耐受量為5μg/kg.d,但仍有越來越多的人們對(duì)日常生活中長(zhǎng)期、低劑量(共)暴露EDCs的潛在健康風(fēng)險(xiǎn)表示擔(dān)憂。為探討人們生活中低劑量、長(zhǎng)期(共)暴露EDCs是否可導(dǎo)致健康風(fēng)險(xiǎn),我們開展了相關(guān)研究： 第一部分市售食品中EDCs的含量與分布 目的：建立一套高效率的可用于食物中多種EDCs檢測(cè)的樣品處理、目標(biāo)物提取、凈化及儀器檢測(cè)方法體系,監(jiān)測(cè)我國(guó)多個(gè)地區(qū)常見食品中主要EDCs的含量和分布,為食品中EDCs污染來源的追溯及控制提供參考,為我國(guó)居民膳食暴露EDCs的風(fēng)險(xiǎn)評(píng)估提供基礎(chǔ)數(shù)據(jù)。 方法：按照我國(guó)河流流域的地理特點(diǎn),采集珠江、長(zhǎng)江及黃河中下游重要城市(廣州、武漢和濟(jì)南)市售食品樣品450件,樣品經(jīng)適當(dāng)前處理去除樣品中的基質(zhì),富集、濃縮目標(biāo)化合物后,用LC-MS/MS方法同步檢測(cè)樣品中壬基酚(NP),雙酚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)計(jì)算食物中總雌激素當(dāng)量(∑EEQ)并于E2檢出水平比較,衡量雌激素“共暴露”現(xiàn)象對(duì)潛在風(fēng)險(xiǎn)的影響。 結(jié)果：(1)按照優(yōu)化的前處理及LC-MS/MS檢測(cè)條件、參數(shù),可有效地實(shí)現(xiàn)前述8種EDCs的液相分離和質(zhì)譜檢測(cè),EDCs各組分標(biāo)準(zhǔn)系列均呈現(xiàn)較好的線性關(guān)系。(2)除E1外,檢測(cè)范圍內(nèi)的其它EDCs成分,在不同城市的各類食品中均有不同程度檢出,檢出率在不同食品類別中存在差別。在廣州、武漢及濟(jì)南三個(gè)城市中,EDCs檢出率的高低順位均表現(xiàn)出NPBPAEE2的現(xiàn)象。(3) Spearman相關(guān)性分析結(jié)果顯示,在眾多EDCs組分檢出水平中,NP與OP、EE2與OP、BPA與OP、EE2與NE、BPA與NP、BPA與EE2、E2與E3等均表現(xiàn)為低度正相關(guān)關(guān)系(P0.05)。(4)Wilcoxon法比較食物總雌激素當(dāng)量(∑EQ)與E2檢出水平的差別,“共暴露”時(shí)可放大接觸的雌激素劑量達(dá)到E2水平的4倍(P0.05)。(5)對(duì)應(yīng)分析(Correspondence analysis，，CA)顯示,不同類別的食物中傾向性污染的EDCs種類存在差異。 結(jié)論：本研究系統(tǒng)建立了食物中EDCs多組分同步檢測(cè)的LC-MS/MS方法,并運(yùn)用建立的方法對(duì)中國(guó)居民市售食品中EDCs的含量進(jìn)行監(jiān)測(cè)。結(jié)果顯示,市售食品中EDCs單組分的含量水平均較低,且不同地區(qū)具有相似的含量特征但不同食物種類中污染的EDCs組分存在差異性和傾向性,EDCs單組分間的正相關(guān)關(guān)系提示我們居民經(jīng)食物攝入EDCs往往是處于一種“共暴露”的狀態(tài),且“共暴露”可能放大其健康效應(yīng)。 第二部分檢出EDCs的膳食暴露概率評(píng)估 目的：基于前述監(jiān)測(cè)獲得的食品中EDCs含量數(shù)據(jù),結(jié)合2002年中國(guó)居民膳食調(diào)查報(bào)告中的食物消費(fèi)量數(shù)據(jù),運(yùn)用蒙特卡洛計(jì)算方法對(duì)中國(guó)居民經(jīng)食物暴露EDCs的水平進(jìn)行概率模擬,為風(fēng)險(xiǎn)分析及風(fēng)險(xiǎn)管理提供決策依據(jù)。 方法：根據(jù)專業(yè)知識(shí)確定人群膳食暴露EDCs的數(shù)學(xué)計(jì)算模型后,運(yùn)用Software Crystal Ball11.0對(duì)數(shù)學(xué)模型中的變量(EDCs水平、食物消費(fèi)數(shù)據(jù)、體重等)進(jìn)行參數(shù)模擬,以模擬的參數(shù)描述變量分布特征并納入后續(xù)計(jì)算,利用計(jì)算機(jī)按參數(shù)指定的規(guī)則模擬抽樣并迭代計(jì)算,每一次迭代計(jì)算的結(jié)果則視為樣本中某一個(gè)體的暴露水平,當(dāng)?shù)螖?shù)足夠多時(shí)則個(gè)體EDCs暴露水平的頻數(shù)分布趨于穩(wěn)定,再根據(jù)個(gè)體暴露水平的頻數(shù)分布圖進(jìn)行概率分析。 結(jié)果：經(jīng)過人群EDCs暴露水平的概率模擬,三個(gè)地區(qū)居民經(jīng)膳食平均暴露及高暴露NP、BPA和EE2場(chǎng)景下的水平并無本質(zhì)差別,且均遠(yuǎn)低于推薦的容許每日攝入量水平,如95%的居民膳食暴露BPA的水平分別為0.27μg/kg.d(廣州)、0.28μg/kg.d(武漢)和0.26μg/kg.d(濟(jì)南),遠(yuǎn)低于其推薦限量標(biāo)準(zhǔn)(50μg/kg.d)。結(jié)論：在食物中現(xiàn)有EDCs含量水平及我國(guó)居民現(xiàn)有飲食習(xí)慣的前提下,單獨(dú)考慮膳食途徑、暴露單種EDCs,尚不足以達(dá)到推薦的限量標(biāo)準(zhǔn)。綜合前述“共暴露”的客觀事實(shí)及雌激素等效性的理論,我們?nèi)杂斜匾_展多途徑、多物質(zhì)同時(shí)暴露的研究。 第三部分低劑量BPA對(duì)雄性大鼠生殖毒性的影響 目的：模擬人群長(zhǎng)期、低劑量暴露雙酚A的特征,建立低劑量雙酚A的雄性大鼠染毒模型,在觀察低劑量雙酚A致模型大鼠及其子代空間記憶行為損傷的同時(shí),進(jìn)一步探討其生殖毒性。 方法：30d齡雄性Wistar大鼠30只經(jīng)適應(yīng)性喂養(yǎng)2周后,隨機(jī)等分為對(duì)照組、10μg/kg.d及50μg/kg.d BPA等3組,對(duì)照組為玉米油溶劑,實(shí)驗(yàn)動(dòng)物按常規(guī)記錄進(jìn)食量和體重。各組大鼠干預(yù)8周后,同正常90天齡、發(fā)情期雌鼠進(jìn)行交配。交配成功1周后,所有父系大鼠進(jìn)行水迷宮測(cè)試一周后處死,取血液及睪丸、附睪組織檢測(cè)。LC-MS/MS法檢測(cè)大鼠血清BPA含量以評(píng)估動(dòng)物模型,觀察睪丸、附睪臟器系數(shù)及精子畸形率同時(shí),對(duì)睪丸組織病理形態(tài)學(xué)進(jìn)行觀察,TUNEL實(shí)驗(yàn)檢測(cè)睪丸組織細(xì)胞凋亡,免疫組化技術(shù)觀察睪丸組織PCNA的表達(dá)水平及組織蛋白泛乙�；磉_(dá)水平。 結(jié)果：(1)各組大鼠進(jìn)食量及體重增長(zhǎng)差異無顯著性(P0.05),但經(jīng)BPA干預(yù)后,各組大鼠血清BPA水平差異有顯著性(P0.05)。(2)與對(duì)照組比較,50μg/kg.d BPA染毒雄性大鼠的逃避潛伏期、游泳速度、游泳距離以及目標(biāo)象限停留時(shí)間等差異均有顯著性(P0.05),而子代大鼠僅表現(xiàn)出逃避潛伏期、游泳速度和游泳距離存在差異(P0.05);10μg/kg.d BPA染毒大鼠及其子代上述指標(biāo)差異均無顯著性。(3)各組大鼠睪丸、附睪臟器系數(shù)及精子畸形率差異并無顯著性(P0.05)。(4)HE染色、TUNEL實(shí)驗(yàn)及PCNA免疫組化結(jié)果顯示,睪丸組織細(xì)胞凋亡變化無顯著性(P0.05),但50μg/kg.d BPA染毒雄性大鼠睪丸組織生精細(xì)胞層數(shù)減少、細(xì)胞間排列疏松,且組織中PCNA表達(dá)減弱(P0.05)。(5) Western blotting實(shí)驗(yàn)結(jié)果顯示50μg/kg.d劑量BPA染毒雄性大鼠可導(dǎo)致睪丸組織在25KD處乙�；鞍妆磉_(dá)水平下降(P0.05)。 結(jié)論：50μg/kg.d劑量的BPA可損害成年雄性大鼠的空間記憶能力,并且這種損害可部分傳給下一代,其作用機(jī)制可能與其導(dǎo)致染毒大鼠睪丸組織蛋白質(zhì)乙�；较陆涤嘘P(guān)。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R114

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 陳同度,張昌穎;素食大鼠的貧血現(xiàn)象[J];營(yíng)養(yǎng)學(xué)報(bào);1957年04期

2 陳偉強(qiáng);趙善廣;;自制注射用大鼠固定裝置[J];上海實(shí)驗(yàn)動(dòng)物科學(xué);1992年04期

3 肖柳英,林培英,馮昭明,張丹;不同周齡的SD大鼠生理、生化及體重的正常值測(cè)定[J];中藥新藥與臨床藥理;1996年03期

4 李淑云;簡(jiǎn)易大鼠灌胃器的制作[J];錦州醫(yī)學(xué)院學(xué)報(bào);2001年04期

5 楊明智,陳積圣;一種大鼠抓取與固定的新工具介紹[J];上海實(shí)驗(yàn)動(dòng)物科學(xué);2001年03期

6 戴英,陸群;復(fù)方H_(505)對(duì)Wistar大鼠外周血的血液流變學(xué)指標(biāo)的影響[J];中國(guó)血液流變學(xué)雜志;2001年01期

7 韋應(yīng)波,孫喜慶,曹新生,姚永杰,馮岱雅,楊長(zhǎng)斌;+Gz暴露時(shí)間對(duì)大鼠記憶功能和行為的影響[J];航天醫(yī)學(xué)與醫(yī)學(xué)工程;2003年01期

8 呂學(xué)軍,郭俊生,李敏,周利梅,張永娟;暈船大鼠體內(nèi)鐵含量的變化[J];中國(guó)職業(yè)醫(yī)學(xué);2003年04期

9 湯仁仙,王迎偉,王慧,周峰;201A中藥合劑對(duì)大鼠抗腎小球基底膜腎炎病變的影響[J];徐州醫(yī)學(xué)院學(xué)報(bào);2003年06期

10 孫同柱,付小兵,翁立新,梁雪梅,陳偉;介紹一種簡(jiǎn)易的大鼠保定方法[J];上海實(shí)驗(yàn)動(dòng)物科學(xué);2004年01期

相關(guān)會(huì)議論文 前10條

1 尹音;孫振宇;胡敏;李冬霞;;持續(xù)性高正加速度對(duì)大鼠顳頜關(guān)節(jié)損傷的作用[A];第八屆全國(guó)顳下頜關(guān)節(jié)病學(xué)及（牙合）學(xué)大會(huì)論文匯編[C];2011年

2 祝~=驤;iJ梊霞;洃克琴;崔素英;文允摪;

本文編號(hào)：2051261