本文關鍵詞： 雙酚A 氯酚 二苯甲酮-3 烷基酚 尿液 超高效液相色譜串聯(lián)質(zhì)譜 雙酚A 4-t-辛基酚 4-n-辛基酚 4-n-壬基酚 男性不育 精液參數(shù) 4-t-辛基酚 Y染色體單倍群 男性不育 易感性 代謝組學 睪丸 尿液 雙酚A 生殖 毒性　出處：《南京醫(yī)科大學》2013年博士論文　論文類型：學位論文

【摘要】：酚類物質(zhì)是一類典型的環(huán)境內(nèi)分泌干擾物,人群暴露廣泛。以往的動物和體外實驗發(fā)現(xiàn)酚類物質(zhì)暴露與男性生殖損害有關,然而這些研究缺乏人群流行病學證據(jù)支持且生殖毒性機制尚未闡明。男性不育是環(huán)境因素和基因因素共同作用的結果,故而環(huán)境-基因交互作用是目前研究化學物毒性易感性的研究熱點。另外,代謝組學作為一門新興的組學技術,在生殖毒理學研究中具有極大的潛力與價值。故而本研究關注酚類物質(zhì)暴露與男性生殖,采用環(huán)境與遺傳并進、人群加動物的整體研究策略。我們先建立了基于超高效液相色譜串聯(lián)質(zhì)譜檢測人尿液中酚類物質(zhì)的方法;并借助該方法檢測了中國普通人群尿液中酚類物質(zhì)暴露水平,探討了酚類物質(zhì)暴露與男性生殖的流行病學關聯(lián);并進一步分析了代表性酚類物質(zhì)4-t-辛基酚的生殖毒性在不同Y染色體單倍群背景下易感性的差異;最后采用代表性酚類物質(zhì)雙酚A的大鼠染毒模型,開展了多分析平臺聯(lián)合的睪丸和尿液的代謝組學研究,發(fā)現(xiàn)了雙酚A暴露的生物標志并揭示了雙酚A暴露對雄性生殖損傷的毒性機制。這些研究為酚類物質(zhì)的潛在男性生殖毒性提供了人群流行病學證據(jù)和全新的認識,為酚類物質(zhì)的男性生殖危害評價提供了基礎資料。第一部分酚類物質(zhì)暴露與男性生殖異常的流行病學研究第一節(jié)尿液中酚類物質(zhì)檢測方法的開發(fā)前言酚類物質(zhì)的暴露是否危害人類健康是重要的公共衛(wèi)生問題。然而,同時檢測多種酚類物質(zhì)的內(nèi)暴露水平的方法還很缺乏。本研究基于超高效液相色譜串聯(lián)質(zhì)譜技術首次開發(fā)了檢測人尿液中九種酚類物質(zhì)的方法,包括雙酚A,2,3,4-三氯酚、2,4,5-三氯酚、二苯甲酮-3、五氯酚、三氯生、4-t-辛基酚、4-n-辛基酚和4-n-壬基酚。方法1.采用β-葡糖苷酸-芳基硫酸酯酶酶解尿液;2.采用Envi-18固相萃取小柱提取和預濃縮目標酚類物質(zhì);3.采用超高效液相色譜串聯(lián)質(zhì)譜分離目標酚類物質(zhì)并采用多反應監(jiān)測模式進行酚類物質(zhì)定量檢測。結果1.方法的靈敏度:檢測限0.02-0.9μg/l;2.方法的穩(wěn)定性:日內(nèi)精密度2-41%,日間精密度3-38%;3.方法的回收率:78-138%;4.方法線性范圍:0.03-50μg/l;5.基于此方法檢測了325名來自中國普通人群的志愿者尿液中酚類物質(zhì)的含量,發(fā)現(xiàn)中國普通人群有較為普遍的酚類物質(zhì)暴露。結論本研究首次開發(fā)了基于超高效液相色譜串聯(lián)質(zhì)譜技術的高通量檢測尿液中酚類物質(zhì)的靈敏、準確、穩(wěn)定的方法,為酚類物質(zhì)內(nèi)暴露水平檢測和酚類物質(zhì)暴露與疾病關聯(lián)的流行病學研究提供了可行有效的檢測方法。本部分研究還首次報道了中國人群酚類物質(zhì)內(nèi)暴露水平的基線資料。第二節(jié)酚類物質(zhì)暴露與男性不育及精液參數(shù)異常的相關性研究前言酚類物質(zhì)具有內(nèi)分泌干擾效應,體外和動物實驗廣泛報道其暴露與男性生殖損傷有關,然而人群中酚類物質(zhì)暴露是否與男性不育有關依然缺乏流行病學證據(jù)。本研究采用病例對照的設計研究酚類物質(zhì)暴露與男性不育及精液參數(shù)異常的相關性。方法1.采用已開發(fā)的基于超高效液相色譜串聯(lián)質(zhì)譜技術的方法分析中國普通人群877例男性不育及713例正常生育對照尿液中酚類物質(zhì)的濃度;2.對上述人群的精液參數(shù)進行分析;3.用多元Logistic回歸和趨勢性檢驗分析酚類物質(zhì)暴露與男性不育及精液參數(shù)異常的相關性。結果1.中國普通人群的男性有較為普遍的酚類物質(zhì)的暴露,暴露水平從高到低依次是三氯生、五氯酚、二苯甲酮-3、4-t-辛基酚、4-n-壬基酚和4-n-辛基酚。2.4-t-辛基酚、4-n-辛基酚和4-n-壬基酚暴露與男性不育有關,趨勢性檢驗p值分別為0.0001,0.014和0.001。3.雙酚A暴露、4-t-辛基酚和4-n-壬基酚暴露與異常精液參數(shù)有關。4.烷基酚聯(lián)合暴露(4-t-辛基酚、4-n-辛基酚和4-n-壬基酚)與男性不育及異常精液參數(shù)有關。結論本研究首次基于大樣本人群探討了酚類物質(zhì)暴露與男性不育及精液參數(shù)異常的相關性,為酚類物質(zhì)的男性生殖毒性提供了流行病學證據(jù)。第三節(jié)代表性酚類物質(zhì)男性生殖毒性的遺傳易感性研究前言男性不育是環(huán)境因素和遺傳因素共同作用的結果。4-t-辛基酚暴露與男性不育及異常精液參數(shù)在中國普通人群具有較強的關聯(lián),故而進一步探討不同遺傳背景下的人群對4-t-辛基酚的生殖毒性的易感性差異對進一步深化對4-t-辛基酚的男性生殖毒性認識具有重要的意義。Y染色體單倍群與男性生殖密切相關,在遺傳背景研究中非常重要。本研究采用病例對照研究設計探討Y染色體單倍群對代表性酚類物質(zhì)(4-t-辛基酚)生殖毒性的易感性的影響。方法1.采用已開發(fā)的基于超高效液相色譜串聯(lián)質(zhì)譜技術的方法分析了774例男性不育及520例正常生育對照尿液中4-t-辛基酚的濃度;2.在上述人群中采用SNa Pshot方法分類了10個Y染色體單倍群;3.用似然比檢驗分析Y染色體單倍群的對4-t-辛基酚生殖毒性易感性的影響,計算假陽性率來控制多重檢驗假設帶來的假陽性錯誤率。結果1.4-t-辛基酚的暴露在本研究各Y染色體單倍群中均提高了男性不育的風險。2.Y染色體單倍群O3*人群相比非本群男性對4-t-辛基酚的潛在生殖毒性不易感(pinter=1.1e-3)。結論本研究首次分析了十種Y染色體單倍群下4-t-辛基酚生殖毒性易感性的分布情況,說明了中國男性普遍對4-t-辛基酚的生殖毒性易感,揭示了O3*單倍群一定程度上降低了4-t-辛基酚致男性不育的風險。本研究強調(diào)了以后需要更為廣泛地開展Y染色體單倍群相關的功能研究和Y染色體單倍群與化學物毒性的易感性研究。第二部分代謝組學在代表性酚類物質(zhì)致雄性生殖毒性機制研究中的應用前言流行病學研究發(fā)現(xiàn)雙酚A暴露與精子發(fā)生異常有關,而雙酚A的生殖毒性機制尚未完全闡明。代謝組學在生殖毒理學研究中具有應用潛力,尚無有關代謝組學應用于雙酚A生殖毒性研究的報道。本研究選擇雙酚A為代表性酚類物質(zhì),采用大鼠染毒雙酚A模型,分析睪丸和尿液代謝譜變化,探索雙酚A的生殖毒性機制。方法1.將18只大鼠隨機分為1個溶劑對照組和2個雙酚A染毒組(0.0005,50mg/kg/d),采用灌胃法連續(xù)染毒8周;2.睪丸和尿液采用液相色譜串聯(lián)四級桿飛行時間質(zhì)譜進行非靶標代謝組學分析,采用精確分子量、同位素分布、數(shù)據(jù)庫比對、標準品比對等多種方法鑒定生物標志;3.根據(jù)睪丸代謝譜提示的信息,用分光光度法試劑盒測定睪丸超氧物歧化酶、谷胱甘肽過氧化物酶和過氧化氫酶活性;4.尿液采用毛細管電泳串聯(lián)飛行時間質(zhì)譜進行高通量代謝譜分析,采用標準品庫定性內(nèi)源性化學物;5.肝臟蛋氨酸腺苷轉移酶Iα(Mat1a)和蛋氨酸腺苷轉移酶IIα(Mat2a)的m RNA和蛋白水平分別采用RT-PCR和Western blot檢測;6.計算假陽性率來控制多重檢驗假設帶來的假陽性錯誤率,用KEGG數(shù)據(jù)庫對結果進行生物信息學分析。結果1.雙酚A染毒組尿液中葡萄糖醛酸苷結合型雙酚A顯著上升;2.睪丸代謝譜:采用多種方法鑒定出了雙酚A暴露大鼠睪丸的潛在生物標志-亞油酸和花生四烯酸,雙酚A暴露對大鼠睪丸不飽和脂肪酸的代謝產(chǎn)生了影響使得花生四烯酸上升,亞油酸下降,花生四烯酸/亞油酸比值上升,大鼠超氧物歧化酶活性在雙酚A染毒后發(fā)生顯著下降,谷胱甘肽過氧化物酶和過氧化氫酶活性出現(xiàn)了下降趨勢;3.尿液代謝譜:雙酚A導致甲基化氨基酸、甲基化嘌呤上升,尿素循環(huán)增強,核酸降解增強,水溶性維生素排出增多,膽堿相關代謝增強,肝臟編碼膽堿代謝通路關鍵酶的基因mat1a和mat2a的m RNA和蛋白水平均顯著上升。結論本研究發(fā)現(xiàn)了代表性酚類物質(zhì)雙酚A暴露的睪丸生物標志,雙酚A影響了睪丸的脂肪酸代謝,降低了睪丸的抗氧化能力;通過增強膽堿代謝相關關鍵代謝酶的基因表達,增強膽堿代謝引起了體內(nèi)高甲基化環(huán)境導致異常的蛋白降解和DNA損傷效應從而可能引起生殖毒性。本研究首次從代謝組學角度為雙酚A的生殖毒性提供了新的視角,并說明了代謝組學在生殖毒理學研究領域的潛在意義和應用價值。
[Abstract]:Phenol is a kind of typical environmental endocrine disruptors, population exposure widely. The previous animal and in vitro experiments found that the damage of phenolics exposure and male reproductive related, however these studies lack of epidemiological evidence supports and reproductive toxicity mechanism has not been elucidated. Male infertility is the result of interactions between environmental factors and genetic factors, and environment gene interaction is a hotspot of the research on susceptibility to toxicity of chemicals. In addition, metabolomics is an emerging technology, has great potential and value in the study of reproductive toxicology. Therefore this research focuses on the phenolics exposure and male reproductive, with genetic and environmental research, the overall strategy and the crowd animal. We first established a method of ultra performance liquid chromatography tandem mass spectrometry in human urine based on phenolic compounds; and using this method to detect Chinese general population of phenolic substances in the urine level of exposure and discusses the phenol exposure epidemiological association with male reproduction; and further analyzes the differences in the reproductive toxicity of representative phenols 4-t- octylphenol in different Y chromosome haplogroup background susceptibility; the poisoning rats model representative of phenolic compounds of bisphenol A the group carried out metabolic analysis platform with testicular and urine studies, found biomarkers of exposure to bisphenol A and bisphenol A exposure reveals the toxic mechanism of injury to male reproductive potential. These studies for male reproductive toxicity of phenolic substances to provide epidemiological evidence and new understanding, provide the basic data for male reproductive hazard evaluation of phenolic compounds. The first part phenols exposed examination phenolic compounds and epidemiological studies of male reproductive abnormalities section in urine The development of exposure measurement methods of phenolic compounds are harmful to human health is an important public health problem. However, the method of simultaneous detection of multiple exposure levels of phenolic compounds in is still lacking. This research method based on ultra performance liquid chromatography tandem mass spectrometry was developed for the first time to detect the urine of nine kinds of phenolic substances. Including bisphenol A, 2,3,4- three 2,4,5- three two chlorophenols, chlorophenols, benzophenone -3, PCP, three triclosan, 4-t- 4-n- octylphenol, nonylphenol and Octylphenol 4-n-. Methods 1. the beta glucuronide - arylsulfatase enzyme urine; 2. using Envi-18 solid phase extraction column extraction and preconcentration of target phenols material; 3. using ultra performance liquid chromatography tandem mass spectrometry was used to separate target phenols and phenolic compounds detected using multiple reaction monitoring mode. The sensitivity results of 1. methods: the detection limit of 0.02-0.9 g/l; the stability of 2. methods: Date Precision in 2-41%, precision 3-38%; 3. methods of recovery rate: 78-138%; 4. linear range: 0.03-50 g/l 5.; based on this method to detect the content of phenolic compounds in 325 from the general population China volunteers in the urine, ordinary people have found China phenols more general conclusions of this exposure. The first study developed a high-throughput tandem mass spectrometry UPLC detection of phenols in urine based on sensitive, accurate, and stable method for phenols in exposure level detection and phenolics exposure and disease associated epidemics provide a feasible and effective method to detect research. This part of the study it was first reported that the baseline data on exposure levels of phenolic compounds in the Chinese population. The second section phenolics exposure and male infertility and abnormal semen parameters to study the correlation between the phenolic compounds with endocrine disturbance Effect of in vitro and animal experiments reveal the widely reported damage is related to male fertility, however phenols crowd exposure is associated with male infertility is still a lack of epidemiological evidence. This study used a case-control design study on phenolics exposure associated with male infertility and abnormal semen parameters. Methods 1. methods using ultra performance liquid chromatography chromatography tandem mass spectrometry has been developed based on the analysis of China general population of 877 cases of male infertility and 713 cases of normal birth control the concentration of phenols in urine; analysis of semen parameters of 2. of this population; 3. with multivariate Logistic regression and trend test analysis of phenolic substances exposure associated with male infertility and abnormal semen parameters results 1. of the general population. China men exposed phenols widely, the exposure level from high to low are three triclosan, PCP, two Benzophenone -3,4-t- octylphenol, nonylphenol and Octylphenol 4-n- 4-n-.2.4-t- octyl phenol, octyl phenol 4-n- and 4-n- nonylphenol exposure associated with male sterility, trend test p values were 0.0001,0.014 and 0.001.3. exposure to bisphenol A, 4-t- octylphenol and 4-n- nonylphenol exposure and abnormal semen parameters related.4. alkylphenol combined exposure (4-t- octylphenol 4-n- 4-n-, octylphenol and nonylphenol) associated with male infertility and abnormal semen parameters. Conclusion this is the first population-based study based on phenol exposure associated with male infertility and abnormal semen parameters, to provide epidemiological evidence for male reproductive toxicity of phenolic compounds. The third section represents the genetic susceptibility of the male sterility phenolics in male reproductive toxicity is the interaction of environmental and genetic factors results.4-t- octylphenol exposure and male infertility and abnormal semen parameters Has a strong correlation in the general population China, therefore to further explore the different genetic background of the population differences in susceptibility to reproductive toxicity in 4-t- of octyphenol further deepening of male reproductive toxicity of 4-t- octylphenols understanding of the significance of.Y chromosome haplogroup and male reproductive is closely related to the genetic background is very important in the research. The a case-control study design to investigate the Y chromosome haplogroups of representative phenolic compounds in this study (4-t- octylphenol) affect susceptibility to reproductive toxicity. Methods 1. methods using ultra performance liquid chromatography tandem mass spectrometry has been developed based on the analysis of 774 cases of male infertility and 520 cases of normal birth control 4-t- concentration of Octyl phenol in urine 2. in the crowd; using SNa Pshot method for the classification of 10 Y chromosome haplogroup; 3. with likelihood ratio test analysis of Y chromosome haplogroup of 4-t- octyl Effect of phenol on reproductive toxicity susceptibility, calculate the false positive rate to control the false positive error rate caused by multiple hypothesis testing. The results of octylphenol exposure of 1.4-t- compared to the non potential reproductive toxicity of this group of male 4-t- octylphenol in this study are not susceptible to the Y chromosome haplogroups were increased the risk of male infertility.2.Y chromosome group O3* group (pinter=1.1e-3). Conclusion this is the first study to analyze ten kinds of distribution of Y chromosome haplogroup 4-t- octylphenols reproductive toxicity susceptibility, the China men generally on the reproductive toxicity of octylphenol 4-t- susceptible, haplogroup O3* reveals to some extent reduce the risk of male infertility caused by 4-t- octylphenol. This study highlights the need for more extensive future study on susceptibility of Y chromosome haplogroup related function research and the Y chromosome haplogroup and the toxicity of chemicals. In the second part, metabolism Study on representative phenols induced by the application of epidemiological study on Mechanism of male reproductive toxicity study found in exposure to bisphenol A is associated with abnormal sperm, and the reproductive toxicity mechanism of bisphenol A has not been fully elucidated. Metabonomics has potential application in the study of the reproductive toxicology, there is no relevant application of metabonomics to study the reproductive toxicity of bisphenol A reported. This study is representative of bisphenol A phenol, bisphenol A by rat model, analysis of the changes of testis and urine metabolic profiles, reproductive toxicity mechanism of bisphenol A. Methods 1. 18 rats were randomly divided into 1 control groups and 2 bisphenol A exposure group (0.0005,50mg/kg/d), by gavage method after 8 weeks; 2. testis and urine series four stages of rod using time-of-flight mass spectrometry non target metabolomic analysis of liquid chromatography, the accurate molecular weight, isotope distribution, ratio of database The sign of a variety of methods, comparison and identification of biological standards; 3. according to testicular metabolism spectrum prompt information, spectrophotometric determination kit testicular superoxide dismutase, glutathione peroxidase and catalase activity; 4. urine by capillary electrophoresis for high-throughput metabolic profiling of tandem time-of-flight mass spectrometry, using the standard library qualitative endogenous chemicals; 5. liver methionine adenosyltransferase I alpha (Mat1a) and methionine adenosyltransferase II alpha (Mat2a) m RNA and protein level respectively by RT-PCR and Western blot detection; 6. calculation of false positive rate to control the false positive error rate caused by multiple hypothesis testing, bioinformatics analysis of results with the KEGG database. The glucuronide 1. bisphenol A exposure group in urine with bisphenol A increased significantly; 2. testicular metabolic spectrum: using a variety of methods to identify bisphenol A exposure rats The potential biomarkers in testis of linoleic acid and four arachidonic acid, exposure to bisphenol A on rat testis unsaturated fatty acid metabolism affected the four arachidonic acid increased, linoleic acid decreased four arachidonic acid / linoleic acid ratio increased, rat superoxide dismutase activity in bisphenol A after exposure to a significant decline, glutathione peroxidase and catalase activity decline; 3. urine metabolic profiles: bisphenol A causes methylated amino acids, methylation of purine nucleic acid increased, the urea cycle increased, enhanced degradation of water soluble vitamin excretion increased, choline metabolism enhanced liver choline metabolism pathway key enzyme encoding genes mat1a and mat2a m RNA and protein levels were significantly increased. Conclusion this study found testis biomarkers representative phenols exposure to bisphenol A, bisphenol A of fatty acid metabolism in testis, reduced testosterone Pill antioxidant capacity; by enhancing the expression of key metabolic enzymes choline metabolism genes, increased choline metabolism caused in high methylation environment lead to abnormal protein degradation and DNA damage which may cause reproductive toxicity. This is the first study from the aspect of metabonomics provides a new perspective for the reproductive toxicity of bisphenol A, and the metabonomics in reproductive toxicology research potential significance and application value.

【學位授予單位】：南京醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R114

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