本文關(guān)鍵詞：基于代謝組學(xué)技術(shù)的4-壬基酚暴露生物標(biāo)志物研究　出處：《哈爾濱工業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 4-壬基酚 暴露 代謝組學(xué) 高效液相色譜-飛行時(shí)間質(zhì)譜 生物標(biāo)志物

【摘要】：4-壬基酚作為被廣泛使用的具有類雌激素活性的持久性環(huán)境污染物,其暴露引發(fā)的毒性作用已成為研究熱點(diǎn),準(zhǔn)確的暴露危害評(píng)價(jià)是對(duì)其開展風(fēng)險(xiǎn)評(píng)估的前提。本研究基于高效液相色譜-飛行時(shí)間質(zhì)譜(HPLC-QTOF-MS)技術(shù),在正負(fù)離子模式下,以4-壬基酚暴露大鼠為動(dòng)物模型,選取尿液和血漿兩種生物樣本,開展了靶向與非靶向結(jié)合的代謝組學(xué)研究。以兩種代謝組學(xué)方法的優(yōu)勢(shì)互補(bǔ)和兩種生物樣本的信息互補(bǔ)保證了所篩選的生物標(biāo)志物的可靠性。本研究采用代謝組學(xué)方法將量化的外源性刺激與內(nèi)源性代謝物的變化結(jié)合起來,獲得早期、整體和動(dòng)態(tài)的代謝物信息,找到了與4-壬基酚暴露相關(guān)的10個(gè)小分子內(nèi)源性生物標(biāo)志物:5-羥色胺、色氨酸、甘氨酸、犬尿氨酸、L-酪氨酸、甘油磷酸膽堿、丙二醛和花生四烯酸。并對(duì)以上10個(gè)代謝物所在的氧化應(yīng)激和色氨酸代謝通路進(jìn)行生物學(xué)驗(yàn)證,既證明了所篩選標(biāo)志物的準(zhǔn)確性,又對(duì)4-壬基酚的毒性機(jī)制進(jìn)行了探索。首先,建立適用于尿液和血漿樣本的非靶向代謝組學(xué)HPLC-QTOF-MS分析方法。在尿液非靶向代謝組學(xué)建立過程中,對(duì)尿液樣本進(jìn)行了四倍體積稀釋法、肌酐值校準(zhǔn)尿液體積法和肌酐值校準(zhǔn)峰面積法3種前處理方法比較研究,最后篩選了效果最好的肌酐值校準(zhǔn)峰面積法;確定了正離子模式下添加10m M甲酸、負(fù)離子模式下添加10m M乙酸銨可以大大提高代謝物檢測(cè)的覆蓋率、靈敏度及分辨率;利用尿液中常見的15種代謝物進(jìn)行方法學(xué)研究,表明方法滿足非靶向代謝組學(xué)分析的要求。在血漿非靶向代謝組學(xué)建立過程中,對(duì)血漿樣本進(jìn)行了有機(jī)溶劑除蛋白法和固相萃取法兩種前處理方法的考察,最終選定了有機(jī)溶劑除蛋白法;利用血漿中常見的13種代謝物進(jìn)行方法學(xué)研究,表明方法滿足非靶向代謝組學(xué)分析的要求。將4-壬基酚分別以0、50、250 mg/kg/d的劑量連續(xù)4天暴露于SD雄性大鼠,利用高效液相色譜串聯(lián)質(zhì)譜(HPLC-MS/MS)技術(shù)對(duì)暴露后不同時(shí)間點(diǎn)的尿液和血漿中4-壬基酚進(jìn)行定量生物監(jiān)測(cè),獲得4-壬基酚在體內(nèi)的實(shí)時(shí)暴露水平,同時(shí)證明暴露模型成功建立。利用建立的基于HPLC-QTOF-MS技術(shù)的尿液和血漿非靶向代謝組學(xué)分析方法,對(duì)對(duì)照組大鼠和高低劑量組大鼠的尿液和血漿分別進(jìn)行整體代謝輪廓分析。為了從海量的組學(xué)數(shù)據(jù)中獲得有效的生物標(biāo)志物信息,采用了主成分分析(PCA)、正交偏最小二乘判別分析(OPLS-DA)、變化倍數(shù)(Fold Change)等變量分析方法,對(duì)尿液中篩選出的36個(gè)組間差異離子和血漿中篩選出的29個(gè)差異離子進(jìn)行結(jié)構(gòu)鑒定。將HPLC-QTOF-MS獲得的一級(jí)質(zhì)譜和二級(jí)質(zhì)譜的精確質(zhì)量數(shù)與Peak View、Massbank和METLIN數(shù)據(jù)庫(kù)比對(duì),并結(jié)合離子裂解規(guī)律和標(biāo)準(zhǔn)品驗(yàn)證等方法,分別鑒定了尿液中的12個(gè)和血漿中的14個(gè)潛在生物標(biāo)志物。為了驗(yàn)證非靶向代謝組學(xué)篩選的潛在生物標(biāo)志物的有效性,本研究建立了針對(duì)尿液和血漿樣本的LC-MS/MS靶向代謝組學(xué)分析方法。對(duì)篩選的12個(gè)尿液標(biāo)志物和14個(gè)血漿標(biāo)志物進(jìn)行相對(duì)定量分析,進(jìn)一步篩選出在對(duì)照組和4-壬基酚暴露組生物樣本中具有顯著差異(獨(dú)立樣本t test檢驗(yàn),p0.05)的10個(gè)代謝物作為最終的生物標(biāo)志物,其中尿液中的5個(gè)標(biāo)志物為5-羥色胺、色氨酸、甘氨酸、甘油磷酸膽堿和丙二醛;血漿中的5個(gè)標(biāo)志物為5-羥色胺、色氨酸、犬尿氨酸、L-酪氨酸和花生四烯酸;5-羥色胺和色氨酸在兩種基質(zhì)中均存在。10個(gè)生物標(biāo)志物的生物學(xué)意義分析表明:丙二醛、甘油磷酸膽堿和花生四烯酸是4-壬基酚暴露引起機(jī)體氧化應(yīng)激反應(yīng)的結(jié)果;5-羥色胺、色氨酸、甘氨酸和犬尿氨酸作為神經(jīng)遞質(zhì)類物質(zhì)處于色氨酸代謝途徑中。對(duì)尿液中8-羥基脫氧鳥苷(8-OHd G)和丙二醛(MDA)水平測(cè)定結(jié)果表明:大鼠體內(nèi)發(fā)生了氧化應(yīng)激,且體內(nèi)4-壬基酚暴露水平與8-OHd G和MDA含量呈正相關(guān)(相關(guān)系數(shù)r2分別為0.701、0.793);對(duì)色氨酸代謝途徑中的7個(gè)活性物質(zhì)進(jìn)行定量分析,結(jié)果表明:大鼠體內(nèi)色氨酸代謝紊亂,其中5-羥基色氨酸和5-羥色胺與體內(nèi)4-壬基酚暴露水平呈正相關(guān)(相關(guān)系數(shù)r2分別為0.603、0.551)。以上結(jié)果表明:本研究篩選的10個(gè)生物標(biāo)志物具有有效性,且4-壬基酚暴露會(huì)導(dǎo)致機(jī)體的氧化應(yīng)激損傷和引起神經(jīng)遞質(zhì)損傷。綜上所述,本研究利用靶向和非靶向相結(jié)合的代謝組學(xué)方法,篩選出了大鼠尿液和血漿中的10個(gè)4-壬基酚暴露生物標(biāo)志物,可以用于4-壬基酚暴露評(píng)估,且證明4-壬基酚暴露可引起機(jī)體氧化應(yīng)激損傷,還可引起色氨酸代謝紊亂,進(jìn)而引起神經(jīng)損傷。研究結(jié)果為更準(zhǔn)確地開展4-壬基酚暴露風(fēng)險(xiǎn)評(píng)估和毒性機(jī)制研究提供了參考。逡逡
[Abstract]:4- nonylphenol as the estrogenic activity of persistent pollutants is widely used, its exposure to toxic effects caused has become a hot research topic, accurate exposure risk assessment is a prerequisite for the development of risk assessment. The research is based on high performance liquid chromatography time of flight mass spectrometry (HPLC-QTOF-MS) technology, in positive and negative ion mode. With 4- nonylphenol exposure rats as animal model, selection of plasma and urine of two kinds of biological samples, to carry out targeted and non targeted metabolomics studies. Combined with two kinds of metabonomics of complementary advantages and two complementary information and samples to ensure the reliability of the biomarker screening material. Using metabonomics will change exogenous stimuli and endogenous metabolites quantified together early in this study, the overall metabolic information and dynamic, found and 4- nonylphenol exposed way The 10 small molecular biomarkers: endogenous 5- serotonin, tryptophan, kynurenine, glycine, L-, tyrosine, glycerophosphocholine, malondialdehyde and four arachidonic acid. And the biological test on oxidative stress and tryptophan metabolism pathway above 10 metabolites of both, to prove the accuracy of the screening markers of 4-, and the toxicity of nonylphenol mechanism were studied. Firstly, the non target established in urine and plasma samples to metabolomics analysis method of HPLC-QTOF-MS. Establish science to metabolism in the urine of non target group, the urine samples were four times of volume dilution method, creatinine 3 pretreatment the comparative research method of calibrating the urine volume and creatinine corrected peak area method, the final screening of the best effect of creatinine corrected peak area was determined with 10m method; M acid positive ion mode, negative ion mode to add 10m M acetic acid Ammonium can greatly improve the metabolite detection coverage, sensitivity and resolution; methodological research on 15 kinds of common metabolites in urine, show that the method can meet the analysis of non target to learn metabolic requirements. Learn to establish metabolic groups in non target plasma, plasma samples of the organic solvent protein removal method and solid phase the extraction method of two pretreatment methods of investigation, and ultimately selected organic solvent except protein; were studied using 13 kinds of metabolites in plasma, shows that the method can meet the analysis of non target to learn metabolic requirements. 4- nonyl phenol respectively with 0,50250 dose of mg/kg/d for 4 consecutive days of exposure to male SD the rat, using high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) technology of plasma and urine after exposure to different time points in 4- monitoring quantitative biological nonylphenol, nonylphenol 4- in real time in the dew of violence Flat, and that the exposure model was successfully established. Using the HPLC-QTOF-MS technology based on plasma and urine analysis method to study the metabolism of non target group, the rats in control group and high dose group rat urine and plasma were analyzed the overall metabolic profile. In order to learn from the mass group obtained biomarker information effective data in the principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), (Fold Change) and other changes of multiple variable analysis method, identify the structure of the ion 29 differentially screened 36 were screened out in urine between ion and plasma. The accurate mass number and Peak View HPLC-QTOF-MS received the MS and two MS, Massbank and METLIN database, and combining the ion fragmentation pattern and standard verification methods were respectively identified 14 12 in urine and in plasma Potential biomarkers. In order to verify the non targeted metabolomics screening of potential biomarkers of validity, this study established targeting LC-MS/MS urine and plasma samples to metabolomics analysis method. Screening of 12 urinary biomarkers and 14 plasma markers were further screened relative quantitative analysis. In the control group and 4- group have significant exposure to nonylphenol in biological samples (independent samples t test test, P0.05) of the 10 metabolites as the final biomarkers, including 5 markers in urine for 5- serotonin, tryptophan, glycine, choline phosphate and glycerol; 5 markers in the plasma of 5- serotonin, tryptophan, kynurenine, L- tyrosine and four arachidonic acid; 5- serotonin and tryptophan are.10 biomarker compounds analysis showed that the biological significance of the two substrates: MDA, glycerol phosphate bile Four arachidonic acid alkali and 4- nonylphenol exposure of oxidative stress response results; 5- serotonin, tryptophan, kynurenine and glycine as a neurotransmitter substance in the tryptophan pathway. The urine 8- hydroxydeoxyguanosine (8-OHd G) and malondialdehyde (MDA) levels showed that the determination results the occurrence of oxidative stress in rats in vivo, and in vivo exposure to nonylphenol 4- level and 8-OHd G and MDA were positively correlated (correlation coefficient R2 = 0.701,0.793); quantitative analysis of 7 active substances tryptophan metabolism pathway results show that the acid metabolic disorder in rats of tryptophan, the primary hydroxyl 5- ammonia acid and 5- HT and 4- in vivo exposure to nonylphenol levels were positively correlated (correlation coefficient R2 = 0.603,0.551). The results show that: the 10 biomarker screening is effective, and 4- nonylphenol exposed body will lead to oxygen Stress damage and damage caused by neurotransmitters. In conclusion, this study use the method to target and non target metabolic group to the combination of the selected 10 4- of nonylphenol in the urine and plasma of rats exposed biomarkers can be used for 4- nonylphenol exposure assessment, and show that 4- exposure can induce nonyl phenol oxidative stress, may also cause the disorder of tryptophan metabolism, thereby causing nerve damage. The results are more accurate to carry out 4- nonylphenol exposure risk assessment and toxicity mechanism. The research provides a reference from the village

【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R114

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本文編號(hào)：1359703