有机磷、有机氯农药分析方法及甲状腺内分泌干扰效应的研究

发布时间：2018-03-02 22:25

本文选题：有机磷农药　切入点：有机氯农药　出处：《昆明理工大学》2016年博士论文　论文类型：学位论文

【摘要】：近年来,甲状腺疾病的发病率有逐年上升的趋势,环境内分泌干扰物对甲状腺相关疾病和人群健康的影响越来越受到广泛关注。研究表明,甲状腺干扰物(TCDs)可影响甲状腺激素水平、干扰下丘脑-垂体-甲状腺轴稳态,造成甲状腺机能减退,甚至导致甲状腺肿瘤的发生。作为已知甲状腺干扰物的典型代表,农药类环境激素对甲状腺内分泌干扰效应的流行病学调查和作用机制研究具有重要意义。本研究选取常见的几种有机磷、有机氯农药为研究对象,首先针对农药及其代谢产物残留量低、存在基质复杂多样的特点,建立灵敏、高效的分析方法,为研究其在环境及生物体内的暴露水平,进行危害评估奠定基础；采用建立的分析方法,结合流行病学调查的手段,从宏观上了解特定人群有机氯农药体内暴露水平对甲状腺激素的影响；再初步从细胞分子水平研究农药对人甲状腺细胞全基因表达谱的影响,以探讨农药对甲状腺内分泌干扰的可能机制。主要研究内容如下：(1)采用超声辅助-分散液液微萃取样品前处理技术,建立了水样中有机磷农药残留检测的气相色谱-脉冲火焰光度检测器法。对影响萃取效率的参数包括萃取剂和分散剂种类、体积、超声时间、pH等进行了优化。在最佳条件下,7种有机磷农药(二嗪农,乙拌磷,毒死蜱,甲基对硫磷,倍硫磷,对硫磷,喹硫磷)的检测线性范围为0.1-80 ng/mL,相关系数r在0.9945～0.9995之间,方法检出限为0.01-0.04 ng/mL,定量限0.05-0.1 ng/mL,回收率为72.1%-110.0%,相对标准偏差9.5%,富集倍数为143-374。该方法具有萃取时间短,检出限低,所需溶剂少,富集倍数高等优点,适用于环境水样中痕量成分的富集和分析。(2)采用超声辅助-分散固相萃取-分散液液微萃取样品前处理技术,建立了土壤中有机磷农药残留检测的气相色谱-脉冲火焰光度检测器法。对分散固相萃取过程的条件以及分散液液微萃取的参数如萃取剂种类和体积、水相体积、超声时间等进行了优化。在最佳条件下,6种有机磷农药(二嗪农,乙拌磷,毒死蜱,倍硫磷,对硫磷,喹硫磷)在浓度为5～150 ng／g的范围内线性关系良好,相关系数r在0.9910～0.9967之间,检出限范围在0.2～0.5 ng／g,定量限0.5～1.2 ng／g。富集倍数为22-35倍,回收率在79.6%-106.8%之间,相对标准偏差范围是5.3%-7.8%。该方法将分散固相萃取与分散液液微萃取技术联用,结合两者净化和富集的优势处理复杂土壤基质；并采用轻质、低毒、对环境友好的溶剂异辛醇作为萃取剂,代替传统卤代烃类溶剂,拓展了萃取剂种类。(3)采用甲酸沉淀蛋白,C18小柱提取、 Florisil小柱净化分析物,建立固相萃取-气相色谱-电子捕获检测器法检测血清中有机氯农药残留。对色谱条件、固相萃取柱类型、洗脱溶剂种类和体积进行优化。在最佳条件下,正己烷+丙酮(9+1)作为提取和净化的洗脱剂,8种有机氯农药(α-BHC.p-BHC、γ-BHC.δ-BHC. p,p'-DDE.p,p'-DDD.α,p'-DDT.p,p'-DDT)在浓度2～200 ng/mL范围内线性关系良好,相关系数r在0.9964～0.9990,检出限为0.1～0.9 ng/mL,定量限在0.4～3.0 ng/mL,加标回收率在80.5%～112.7%之间,相对标准偏差2.1%-7.9%。该方法简单、高效,符合农残柃测要求,可用于[缸清中有机氯农药残留分析。(4)以昆明市呈贡区50～70岁农村男性居民为研究对象,共选取134例样本,采用流行病学横断面研究的方法,问卷调查与实验室分析手段相结合,应用多重线性回归、spearman相关等统计方法对数据进行处理,研究血清有机氯暴露水平与甲状腺激素水平相关性。结果表明：该人群血清有机氯农药残留以p,p'-DDE为主,中位数达357.79 ng/g lw,说明该地区有机氯来源主要是历史残留；体质指数BMI和年龄是影响血清有机氯含量的主要因素,并且呈正相关趋势；相关性分析显示,p,p'-DDE与促甲状腺激素TSH呈正相关,提示p,p'-DDE可能通过影响TSH的水平,进而影响下丘脑-垂体-甲状腺轴的稳态而干扰甲状腺功能。(5)研究p,p'-DDE和马拉硫磷对人甲状腺细胞系Nthy-ori-3-1细胞全基因表达谱的影响。MTT实验确定了不产生细胞毒性的农药剂量为1μg、mL,在该剂量下,马拉硫磷组基因芯片筛选出差异表达基因为73个,其中31个基因表达上调,42个基因表达下调,差异基因影响的通路共为35个；p,p'-DDE组筛选出差异表达基因33个,其中20个基因表达上调,13个基因表达下调,差异基因影响的通路共为14个。这些差异基因主要涉及信号转导、细胞生长和免疫调节以及细胞骨架构建、DNA合成和转录、信号转导等功能。影响细胞增殖、分化、凋亡的通路占绝大部分。与甲状腺激素分泌合成直接相关的基因并未被发现。以ACTB为内参基因,实时定量PCR随机验证马拉硫磷组11个、p,p'-DDE组4个差异基因表达,最终结果均与基因芯片结果一致。
[Abstract]:In recent years, the incidence of thyroid disease has increased year after year, environmental endocrine disruptors on thyroid related diseases and human health effect has attracted more and more attention. The research results show that thyroid disruptors (TCDs) can affect the level of thyroid hormone, disturbance of hypothalamic pituitary thyroid axis homeostasis caused by hypothyroidism, even lead to thyroid the occurrence of tumor. As a typical representative of known thyroid disruptors, has important significance in epidemiological investigation and study the mechanism of pesticide environmental hormone effects on thyroid endocrine disturbance. This study selected several common organophosphorus, organochlorine pesticides as the research object, first for the pesticide and its metabolites residues are low, characteristics of complex matrix the establishment of sensitive and efficient analysis, to investigate the environment and organism level of exposure and risk of And lay the foundation for the assessment; the establishment of analytical methods, combined with epidemiological investigation methods, to understand the effect of organochlorine pesticide exposure level of specific groups of thyroid hormone from the macro; and preliminary from cellular and molecular level of spectrum of the effects of pesticides on the expression of human thyroid cell gene, to explore the possible mechanism of pesticide endocrine disturbance the main contents are as follows: (1) using ultrasound assisted dispersive liquid-liquid microextraction sample pretreatment technique, was developed for the detection of the residues of organophosphorus pesticides in water gas chromatography pulsed flame detector. The method of parameters affecting the extraction efficiency including the extraction agent and dispersing agent, volume, ultrasonic time, pH were optimized. Under the optimal conditions, 7 kinds of organophosphorus pesticides (two diazinon, disulfoton, chlorpyrifos, methyl parathion, parathion, fenthion, quetiapine P) linear detection The range of 0.1-80 ng/mL, the correlation coefficient r is 0.9945 ~ 0.9995, the detection limit is 0.01-0.04 ng/mL 0.05-0.1 ng/mL, the limit of quantification, the recovery rate was 72.1%-110.0%, the relative standard deviation of 9.5% 143-374., enrichment factor is the method has short extraction time, low detection limit, required less solvent, high enrichment factor, enrichment and analysis suitable for trace elements. (2) using ultrasound assisted dispersive solid-phase extraction and micro - Extraction Pretreatment of dispersion liquid, established the detection of residues of organophosphorus pesticides in soil gas chromatography pulsed flame photometric detector. The dispersive solid phase extraction process conditions and dispersive liquid-liquid microextraction the parameters such as extraction solvent type and volume, water volume, ultrasonic time were optimized. Under the optimal conditions, 6 kinds of organophosphorus pesticides (two diazinon, disulfoton, chlorpyrifos, parathion, sulfur and phosphorus times, quintiofos) At the concentration of 5~150 ng / g range a good linear relationship, correlation coefficient r is 0.9910 ~ 0.9967, the detection limit in the range of 0.2 ~ 0.5 ng / g, the quantitative limit of 0.5 ~ 1.2 ng / g. concentration ratio of 22-35 times, the recovery rate was 79.6%-106.8%, the relative standard deviation is in the range of 5.3% -7.8%. in the method dispersive solid phase extraction and dispersive liquid-liquid microextraction coupled with both purified and enriched the advantages of dealing with complex soil matrix; and the use of lightweight, low toxic, environmentally friendly solvent of isooctyl alcohol as extractant, instead of halogenated hydrocarbon solvent, development of extractants. (3) the acid precipitated protein, C18 column extraction, analyte purification Florisil column, a solid phase extraction and gas chromatography with electron capture detector to detect serum organochlorine pesticide residues method. The chromatographic conditions, solid phase extraction column type, the optimal solvent type and volume of elution . under the optimum conditions, hexane and acetone (9+1) as the extraction and purification of eluent, 8 kinds of organochlorine pesticides (alpha gamma delta -BHC. -BHC.p-BHC, -BHC. P, p'-DDE.p, p'-DDD. alpha, p'-DDT.p, p'-DDT) in the concentration range of 2~200 ng/mL good linear relationship, the correlation coefficient r between 0.9964 and 0.9990. The detection limit is 0.1 ~ 0.9 ng/mL, the limit of quantitation in 0.4 ~ 3 ng/mL, the recovery rate is from 80.5% to 112.7% between the standard, the relative standard deviation of the 2.1%-7.9%. method is simple, efficient, meets the measurement requirements for pesticide residues Eurya, analysis of organochlorine pesticide residues in the bathtub. "(4) in Kunming District of Chenggong city at the age of 50~70 rural male residents as the research object, a total of 134 cases of samples, cross-sectional study using epidemiological methods, questionnaire survey and laboratory analysis methods combined, using multiple linear regression, Spearman correlation statistical methods for data processing, the study of serum organochlorine exposure dew Flat the level of thyroid hormone and correlation. The results showed that the serum organochlorine pesticide residues in P, p'-DDE, median reached 357.79 ng/g LW, to indicate the source of organic chlorine in the region is mainly historical residue; body mass index BMI and age are the main factors affecting the organic chlorine content in serum, and the tendency of positive correlation; correlation analysis showed P, p'-DDE, correlation, and promoting positive thyroid hormone TSH suggested that P, p'-DDE may influence the level of TSH, thereby affecting the hypothalamic pituitary thyroid axis and the steady state interference. The thyroid function (5) of P, p'-DDE and malathion on effects of.MTT of the experimental spectra confirmed that the pesticide dose does not produce cytotoxicity was 1 g, Nthy-ori-3-1 cell gene expression in human thyroid cell line mL in the dose, malathion group gene microarray selected153differentially expressed 73 genes, including 31 up-regulated genes and 42 A gene expression pathway, differential gene effects for a total of 35; P, p'-DDE group identified 33 differentially expressed genes, including 20 up-regulated genes and 13 downregulated genes, gene pathway effects for a total of 14. These differentially expressed genes were mainly involved in signal transduction, cell growth and immune regulation and cell skeleton construction, DNA synthesis and transcription function, signal transduction etc. affect cell proliferation, differentiation, apoptosis pathway andcontainermetaphoraccountforthemajorpartofthetotalmetaphorsinthedata. Thyroid hormone secretion and directly related to the synthesis of the gene has not been found. Using ACTB as the reference gene, real time quantitative PCR random verification malathion group 11, P group, p'-DDE gene expression in 4 a difference in the end result results with the gene chip.

【学位授予单位】：昆明理工大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R581;X592

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