| In recent years, the incidence of thyroid disease shows an increasing trend, Thyroid disrupting chemicals (TDCs) in environment draw a widely public attention because its inflence to thyriod disease and population health. Epidemiologic study to indicate, TDCs can effect thyroid hormone levels, influent stable of hypothalamic-pituitary-thyroid axis, and cause hypothyreosis, even thyroid tumor. As typical TCDs know, to study thyroid endocrine disrupting effects of pesticides, including epidemiologic survey and its disrupting mechanism is most important.Organophosphorus pesticides (OPPs) and Organochlorine pesticides (OCPs) were selected as objective in the research. Firstly, for residues of pesticide and its meiaboliu is very luw concentration and exist in complex matrix. The sensitive and quick analytical methods were established to detect its exposure levels and evaluate its dangerous. Next, epidemiological investigation methods and laboratory testing were combined to study the effect of OCPs on thyroid hormone levels among special population. Then, impact of pesticides on the expression profile of genes of human thyroid cells was studied to explore the possible mechanism that pesticides disturb the thyroid. The main research contents of this thesis were as follow:(1) Ultrasound-assisted dispersive liquid-liquid microextraction (UA-DLLME) technique combine with gas chromatography-flame photometric detection (GC-PFPD) was developed to analysis OPPs in water samples. Some parameters affecting the extraction efficiency, including the types and volumes of extractant and dispersant, sample pH, and ultrasonic time were systematically evaluated. Under optimum conditions, seven OPPs (diazinon, disulfoton, chlorpyrifos, parathion-methyl, fenthion, parathion, and quinalphos) showed good linearity in the range of 0.1-80 ng/mL with a correlation coefficient between 0.9945-0.9995. Enrichment factors (EF) varied from 143 to 374 fold. The limits of detection (LOD) and limit of quantification (LOQ) were 0.01-0.04 ng/mL and 0.05-0.1 ng/mL, respectively. Recovery studies were carried out at two concentration levels in real samples within the range of 72.1 ～ 110.0%, and RSD were less than 9.5%. The proposed methods have the advantages of low cost, high enrichment, easy operation and environmental-friendliness. The method is suitable for enrich and determine trace analyses in water samples.(2) A new method has been developed to determine trace levels of OPPs in soil samples by using dispersive solid-phase extraction (DSPE) combined with dispersive liquid-liquid microextraction (DLLME), followed by gas chromatography pulsed-flame photometric detection (GC-PFPD) analysis. The critical parameters affecting the extraction efficiency including the types and volumes of extractant, aqueous phase volume, sample pH, and ultrasonic time were systematically evaluated. Under optimum conditions, six OPPs (diazinon, disulfoton, chlorpyrifos, fenthion, parathion, and quinalphos) showed good linearity in the range of 5-150 ng/g with a correlation coefficient between 0.9910-0.9967. The enrichment factors (EF) varied from 22-35 fold. The limit of detection (LOD) and the limit of quantification (LOQ) were 0.2-0.5 ng/g and 0.5-1.2 ng/g, respectively. The recoveries at the two spiking levels were in the range of 79.6% ～ 106.8%, and the relative standard deviations (RSDs) were 5.3%-7.8%. The technique combining DSPE with DLLME not only pre-concentrates the analytes from soil samples, but it also reduces the matrix effects. The advantage of the procedure was the use of a less toxic, low-density solvent. The proposed method provides a sensitive, convenient, and eco-friendly process for determining OPPs in soil samples.(3) A Solid-phase extraction combined with gas chromatography pulsed-flame photometric detection (GC-ECD) analysis method was proposed to determine the OCPs in serum. The critical analytical parameters include chromatography condition, the type of extraction column, elution solvent volume and species were systematically evaluated. Under the best conditions, Formic acids were used to sediment the protein and followed Cis column extract OCPs in serum, then Florisil column to clear the extract. Acetone+hexane (9+1) as elution solvent have the good extraction efficiency. Eight OCPs(a-BHC、β-BHC、 γ-BHC、δ-BHC、 p,p’-DDE, p,p’-DDD、 o,p’-DDT、 p,p’-DDT) showed good linearity in the range of 2-200 ng/g with a correlation coefficient between 0.9944-0.9976. The limit of detection (LOD) and the limit of quantification (LOQ) were 0.1-0.9 ng/mL, and 0.4-3.0 ng/mL, respectively. The recoveries at the two spiking levels were in the range of 80.5% ～ 112.7%, and the relative standard deviations (RSD) were between 2.1% ～ 7.9%. The developed methods had the merit of simple and effective, meet the requirement of pesticide residue analysis, and can be used to analysis trace OCPs in serum.(4) 134 of 50～70 years old man live in Chenggong district, Kunming city were recruited for assessment of the association between OCPs and thyroid hormone levels in serum. In the research, questionnaire survey and analytical methods were adopted to get the data, statistic methods include multiple linear regressions and spearman correlations were used to deal with the data. The results showed that p,p’-DDE was the main exposure OCPs for the population and the median was 357.79 ng/g lw, it suggest the resource of OCPs in serum mostly come from history residues; Both the BMI and age are two main factors influent the level of OCPs in serum, and their relationship is positive correlation; Serum concentration of p,p’-DDE were associated with increased TSH, it indicated p,p’-DDE can disturb the stable of hypothalamic pituitary thyroid axis via influent the TSH level, and then finally disturb the function of thyroid.(5) Effection of malathion and p,p’-DDE on the expression profile of genes of human thyroid cells Nthy-ori-3-1 were rtudied. MTT method was applied to study the effect on cell proliferation. The results showed both pesticides have no significant change on cell proliferation at the dose of 1μg/ml. Under this dosage, Single standard Agilent expression profiling microarray screen 73 different express gene in malathions group, including 31 up-regulated genes and 42 down-regulated genes. For p,p’-DDE group,33 different express gene was found, including 20 up-regulated genes and 13 down-regulated genes. Most of the changed gene connected with cell signal transduction, cell growth and immune regulation, DNA synthesis and transcription. The pathway influent the function of cell proliferation, differentiation and apoptosis.The gene effect the thyroid hormone was not found.11 genes of malathion group and 4 genes of p,p’-DDE were seleced to verify the results by Real time quantitative PCR technique. The qPCR results were consistent with the microarray. |
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