基于代谢组学技术的4-壬基酚暴露生物标志物研究

发布时间：2017-12-31 12:10

本文关键词：基于代谢组学技术的4-壬基酚暴露生物标志物研究　出处：《哈尔滨工业大学》2017年博士论文　论文类型：学位论文

【摘要】：4-壬基酚作为被广泛使用的具有类雌激素活性的持久性环境污染物,其暴露引发的毒性作用已成为研究热点,准确的暴露危害评价是对其开展风险评估的前提。本研究基于高效液相色谱-飞行时间质谱(HPLC-QTOF-MS)技术,在正负离子模式下,以4-壬基酚暴露大鼠为动物模型,选取尿液和血浆两种生物样本,开展了靶向与非靶向结合的代谢组学研究。以两种代谢组学方法的优势互补和两种生物样本的信息互补保证了所筛选的生物标志物的可靠性。本研究采用代谢组学方法将量化的外源性刺激与内源性代谢物的变化结合起来,获得早期、整体和动态的代谢物信息,找到了与4-壬基酚暴露相关的10个小分子内源性生物标志物:5-羟色胺、色氨酸、甘氨酸、犬尿氨酸、L-酪氨酸、甘油磷酸胆碱、丙二醛和花生四烯酸。并对以上10个代谢物所在的氧化应激和色氨酸代谢通路进行生物学验证,既证明了所筛选标志物的准确性,又对4-壬基酚的毒性机制进行了探索。首先,建立适用于尿液和血浆样本的非靶向代谢组学HPLC-QTOF-MS分析方法。在尿液非靶向代谢组学建立过程中,对尿液样本进行了四倍体积稀释法、肌酐值校准尿液体积法和肌酐值校准峰面积法3种前处理方法比较研究,最后筛选了效果最好的肌酐值校准峰面积法;确定了正离子模式下添加10m M甲酸、负离子模式下添加10m M乙酸铵可以大大提高代谢物检测的覆盖率、灵敏度及分辨率;利用尿液中常见的15种代谢物进行方法学研究,表明方法满足非靶向代谢组学分析的要求。在血浆非靶向代谢组学建立过程中,对血浆样本进行了有机溶剂除蛋白法和固相萃取法两种前处理方法的考察,最终选定了有机溶剂除蛋白法;利用血浆中常见的13种代谢物进行方法学研究,表明方法满足非靶向代谢组学分析的要求。将4-壬基酚分别以0、50、250 mg/kg/d的剂量连续4天暴露于SD雄性大鼠,利用高效液相色谱串联质谱(HPLC-MS/MS)技术对暴露后不同时间点的尿液和血浆中4-壬基酚进行定量生物监测,获得4-壬基酚在体内的实时暴露水平,同时证明暴露模型成功建立。利用建立的基于HPLC-QTOF-MS技术的尿液和血浆非靶向代谢组学分析方法,对对照组大鼠和高低剂量组大鼠的尿液和血浆分别进行整体代谢轮廓分析。为了从海量的组学数据中获得有效的生物标志物信息,采用了主成分分析(PCA)、正交偏最小二乘判别分析(OPLS-DA)、变化倍数(Fold Change)等变量分析方法,对尿液中筛选出的36个组间差异离子和血浆中筛选出的29个差异离子进行结构鉴定。将HPLC-QTOF-MS获得的一级质谱和二级质谱的精确质量数与Peak View、Massbank和METLIN数据库比对,并结合离子裂解规律和标准品验证等方法,分别鉴定了尿液中的12个和血浆中的14个潜在生物标志物。为了验证非靶向代谢组学筛选的潜在生物标志物的有效性,本研究建立了针对尿液和血浆样本的LC-MS/MS靶向代谢组学分析方法。对筛选的12个尿液标志物和14个血浆标志物进行相对定量分析,进一步筛选出在对照组和4-壬基酚暴露组生物样本中具有显著差异(独立样本t test检验,p0.05)的10个代谢物作为最终的生物标志物,其中尿液中的5个标志物为5-羟色胺、色氨酸、甘氨酸、甘油磷酸胆碱和丙二醛;血浆中的5个标志物为5-羟色胺、色氨酸、犬尿氨酸、L-酪氨酸和花生四烯酸;5-羟色胺和色氨酸在两种基质中均存在。10个生物标志物的生物学意义分析表明:丙二醛、甘油磷酸胆碱和花生四烯酸是4-壬基酚暴露引起机体氧化应激反应的结果;5-羟色胺、色氨酸、甘氨酸和犬尿氨酸作为神经递质类物质处于色氨酸代谢途径中。对尿液中8-羟基脱氧鸟苷(8-OHd G)和丙二醛(MDA)水平测定结果表明:大鼠体内发生了氧化应激,且体内4-壬基酚暴露水平与8-OHd G和MDA含量呈正相关(相关系数r2分别为0.701、0.793);对色氨酸代谢途径中的7个活性物质进行定量分析,结果表明:大鼠体内色氨酸代谢紊乱,其中5-羟基色氨酸和5-羟色胺与体内4-壬基酚暴露水平呈正相关(相关系数r2分别为0.603、0.551)。以上结果表明:本研究筛选的10个生物标志物具有有效性,且4-壬基酚暴露会导致机体的氧化应激损伤和引起神经递质损伤。综上所述,本研究利用靶向和非靶向相结合的代谢组学方法,筛选出了大鼠尿液和血浆中的10个4-壬基酚暴露生物标志物,可以用于4-壬基酚暴露评估,且证明4-壬基酚暴露可引起机体氧化应激损伤,还可引起色氨酸代谢紊乱,进而引起神经损伤。研究结果为更准确地开展4-壬基酚暴露风险评估和毒性机制研究提供了参考。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

【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R114

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