本文選題：己唑醇 + 氟環(huán)唑　； 參考：《中國農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：研究手性三唑類殺菌劑的生物富集行為和毒性效應(yīng)是全面評價手性三唑類殺菌劑在水生生物中的環(huán)境歸趨和毒性效應(yīng)不可或缺的部分,然而,目前對這一部分的研究十分有限。在本論文中,研究了己唑醇、氟環(huán)唑和烯唑醇在斑馬魚體內(nèi)的生物富集行為和毒性效應(yīng)。此外,利用代謝組學(xué)的研究策略和組織病理學(xué)檢查的表觀評價方法研究了己唑醇對映體和氟環(huán)唑?qū)τ丑w暴露對斑馬魚的毒性效應(yīng)。將成年斑馬魚暴露于100μgL/1和200 μg L/1己唑醇溶液中21天,研究了己唑醇在斑馬魚體內(nèi)的生物富集過程。利用LC-MS/MS建立了己唑醇對映體含量的儀器分析方法。結(jié)果得到,己唑醇在斑馬魚體內(nèi)的生物富集過程是對映體選擇性的,(-)-己唑醇要比(+)-己唑醇更容易在斑馬魚體內(nèi)發(fā)生富集。將成年斑馬魚暴露于50 μgL-1和200 μgL-1己唑醇溶液中21天,研究了己唑醇暴露處理對于斑馬魚的毒性效應(yīng)。利用抗氧化酶活性評價方法,考察了己唑醇暴露處理對斑馬魚肝臟氧化應(yīng)激發(fā)生的影響。結(jié)果表明,己唑醇溶暴露處理引起斑馬魚肝臟中SOD、CAT和GPx活性和GSH含量的顯著性下調(diào),說明己唑醇的暴露處理促使在斑馬魚肝臟中發(fā)生了氧化應(yīng)激。通過對細胞凋亡路徑上一系列相關(guān)基因表達情況進行分析,考察了己唑醇暴露處理對于斑馬魚肝臟細胞凋亡過程發(fā)生的影響。結(jié)果表明,己唑醇暴露處理引起了促細胞凋亡過程的基因p53、Puma、Apaf-1、caspase-3和caspase-9的表達顯著性上調(diào),Bcl-2/Bax的比值顯著性下調(diào),說明己唑醇暴露處理誘發(fā)了斑馬魚肝臟細胞凋亡過程的發(fā)生,且該過程的發(fā)生依賴于caspase的參與。將成年雄性斑馬魚暴露于100 μg/1和1000 μL-1的氟環(huán)唑溶液中21天,研究了氟環(huán)唑在斑馬魚體內(nèi)的生物富集過程和氟環(huán)唑暴露處理對斑馬魚的毒性效應(yīng)。利用LC-MS/MS建立了氟環(huán)唑?qū)τ丑w含量的儀器分析方法。結(jié)果得到,氟環(huán)唑在斑馬魚體內(nèi)的生物富集過程是對映體選擇性的,(+)-氟環(huán)唑要比(-)-氟環(huán)唑更容易在斑馬魚體內(nèi)發(fā)生富集。利用基于1H核磁的代謝組學(xué)分析方法,考察了氟環(huán)唑暴露處理對斑馬魚代謝輪廓的影響。結(jié)果表明,100 μg L-1和1000 μgL/1的氟環(huán)唑暴露處理引起了斑馬魚兩種相似的但不完全相同的內(nèi)源性代謝物代謝輪廓的改變,這些改變都是有關(guān)于能量代謝、脂質(zhì)代謝和氨基酸代謝的。通過對斑馬魚肝臟的線粒體呼吸鏈、ATP合成和脂肪酸β-氧化相關(guān)基因的表達情況進行分析,研究了氟環(huán)唑暴露處理擾亂斑馬魚能量代謝的機理。結(jié)果表明,氟環(huán)唑的暴露處理對于斑馬魚線肝臟的粒體呼吸鏈、ATP合成和脂肪酸的β-氧化相關(guān)基因的表達都有抑制作用。將成年雄性斑馬魚暴露于70 μg L/1和300 μg L/1的烯唑醇溶液中21天,研究了烯唑醇在斑馬魚體內(nèi)的生物富集過程和烯唑醇的暴露處理對斑馬魚的毒性效應(yīng)。利用LC-MS/MS建立了烯唑醇對映體含量的儀器分析方法。結(jié)果得到,烯唑醇在斑馬魚體內(nèi)的生物富集過程是不具有對映體選擇性的。利用基于1H核磁的代謝組學(xué)分析方法,考察了烯唑醇暴露處理對于斑馬魚代謝輪廓的影響。結(jié)果得到,相比于空白對照組,70 μgL-1的烯唑醇暴露處理引起了纈氨酸、亮氨酸、異亮氨酸、丙氨酸、乳酸和膽堿含量的顯著性上調(diào),葡萄糖、肌酸和牛磺酸含量的顯著性下調(diào);300 μg L-1的烯唑醇暴露處理引起了葡萄糖和肌酸含量的顯著性下調(diào)。以上結(jié)果說明,烯唑醇的暴露處理擾動了斑馬魚的能量代謝、氨基酸代謝和脂質(zhì)代謝。利用蘇木精-伊紅染色方法對斑馬魚肝臟、睪丸和腦進行了病理學(xué)分析,考察了烯唑醇暴露處理對斑馬魚的組織損傷情況。結(jié)果表明,在70 μg L-1和300 μg L-1的烯唑醇暴露處理組中,在肝切片中觀察到了肝細胞腫脹和空泡的形成,在睪丸切片中觀察到了精子數(shù)目的減少,在腦切片中未見病理學(xué)損傷。將斑馬魚分別暴露于100 μgL-1和1000 μgL-1的己唑醇對映體和氟環(huán)唑?qū)τ丑w溶液中21天,研究其對斑馬魚代謝輪廓和組織病理學(xué)的影響。利用基于1H核磁的代謝組學(xué)分析方法,考察了己唑醇對映體和氟環(huán)唑?qū)τ丑w分別暴露處理對于斑馬魚代謝輪廓的影響。結(jié)果得到,己唑醇對映體的暴露處理擾動了斑馬魚的能量代謝、氨基酸代謝和脂質(zhì)代謝,但(+)-己唑醇和(-)-己唑醇暴露處理引起斑馬魚代謝輪廓改變的機制存在差異;氟環(huán)唑?qū)τ丑w的暴露處理擾動了斑馬魚的能量代謝、氨基酸代謝和脂質(zhì)代謝,但(+)-氟環(huán)唑和(-)-氟環(huán)唑暴露處理引起斑馬魚代謝輪廓改變的機制存在差異。利用蘇木精-伊紅染色方法對斑馬魚腦、肝臟、睪丸和卵巢進行了病理學(xué)分析,考察了己唑醇對映體和氟環(huán)唑?qū)τ丑w分別暴露處理對于斑馬魚的組織損傷情況。結(jié)果表明,在高低兩個暴露劑量下,己唑醇對映體暴露處理都引起了雌性斑馬魚和雄性斑馬魚肝細胞的腫脹并伴隨有肝臟空泡的出現(xiàn)。并且,己唑醇對映體高低劑量的暴露處理在雄性斑馬魚中引起的肝損傷要比在雌性斑馬魚中引起的肝損傷更為嚴(yán)重。在高低兩個暴露劑量下,己唑醇對映體暴露處理都引起斑馬魚睪丸中非細胞區(qū)域的面積呈現(xiàn)濃度依賴性增大和雌性斑馬魚卵巢成熟卵細胞的增多。在高低兩個暴露劑量下,己唑醇對映體暴露處理組中,都未見斑馬魚腦切片出現(xiàn)病理學(xué)損傷。在高低兩個暴露劑量下,氟環(huán)唑?qū)τ丑w暴露處理都引起了雌性斑馬魚和雄性斑馬魚肝細胞的腫脹并都伴隨有肝臟空泡的出現(xiàn)。在高低兩個暴露劑量下,氟環(huán)唑?qū)τ丑w暴露處理都引起了斑馬魚睪丸中非細胞區(qū)域的面積增大,雌性斑馬魚卵巢的卵細胞未見有明顯改變。在高低兩個暴露劑量下,氟環(huán)唑?qū)τ丑w暴露處理組中,都未見斑馬魚腦切片出現(xiàn)病理學(xué)損傷。己唑醇對映體和氟環(huán)唑?qū)τ丑w暴露處理都擾動了斑馬魚的能量代謝、氨基酸代謝和脂質(zhì)代謝,同時都造成了肝臟和性腺的病理學(xué)損傷,說明己唑醇對映體和氟環(huán)唑?qū)τ丑w暴露處理對斑馬魚代謝輪廓和組織病理學(xué)的影響具有相似性。
[Abstract]:The study of the bioaccumulation and toxicity of chiral three azole fungicides is an integral part of the comprehensive evaluation of the environmental and toxic effects of chiral three azole fungicides in aquatic organisms. However, the current research on this part is very limited. In this paper, the study of hexazolyl alcohol, flurocyclozole and alkyl alcohol in zebrafish was studied in this paper. In addition, the toxic effects of ezolisol enantiomers and flurocyclic enantiomers on zebrafish were studied by metabonomics and histopathological examination, and adult zebrafish was exposed to 100 gL/1 and 200 g L/1 pyrazolol solution in 21 days. The biological enrichment process in zebrafish. The instrumental analysis of the enantiomer content of hexazolyl alcohol was established by LC-MS/MS. The results showed that the bioconcentration process of hexazolyl alcohol in zebrafish was enantiomeric, and (-) - hexazolyl alcohol was more likely to be enriched in zebra fish than (+) - hexazolyl alcohol. Adult zebrafish was exposed to 50 mu gL- The toxic effects of pyrazolol exposure on zebrafish were studied for 21 days in 1 and 200 gL-1 pyrazolol solutions. The effects of pyrazolol exposure on the oxidative stress of zebrafish liver were investigated by the methods of antioxidant enzyme activity evaluation. The results showed that the activity of SOD, CAT and GPx in zebrafish liver and GSH were caused by the treatment of pyrazolol exposure. The significant reduction in content indicated that the exposure treatment of pyrazolol induced oxidative stress in the liver of zebrafish. By analyzing the expression of a series of related genes on the apoptosis pathway, the effects of pyrazolol exposure on the apoptosis of liver cells in zebrafish were investigated. The results showed that the exposure treatment of zzzolfish was shown. The expression of p53, Puma, Apaf-1, caspase-3 and caspase-9 was significantly up-regulated, and the ratio of Bcl-2/Bax was significantly down. The apoptosis process of zebrafish liver cells was induced by pyrazolol exposure, and the occurrence of this process was dependent on the participation of caspase. The adult male zebrafish was exposed to 100. The biological enrichment process of fluosazolazole in zebrafish and the toxic effect of fluosazolazole on zebrafish were studied in 21 days in g/1 and 1000 L-1 flurozole solution. The instrumental analysis of the enantiomer content of fluosazolazole was established by using LC-MS/MS. The results showed that the bioconcentration process of fluosazolazole in the fish was enantiomers Selective, (+) - flurozole is more likely to be enriched in zebrafish than (-) - flurozole in zebrafish. Using 1H NMR based metabonomics analysis, the effect of fluorocarbzole exposure on zebrafish metabolic profile was investigated. The results showed that 100 g L-1 and 1000 gL/1 fluorocarbzole exposure treatment caused two similar but not complete zebrafish. All the same metabolic profiles of endogenous metabolites were changed, these changes were related to energy metabolism, lipid metabolism and amino acid metabolism. The expression of mitochondrial respiratory chain, ATP synthesis and the expression of fatty acid beta oxidation related genes in zebrafish liver were analyzed, and the energy metabolism of flurocyclic exposure treatment disrupted zebrafish energy metabolism. The results showed that the exposure treatment of flurocyclozole inhibited the expression of the granular respiratory chain of the liver of zebrafish line, ATP synthesis and the expression of the beta oxidation related genes of fatty acids. The adult male zebrafish was exposed to 70 g L/1 and 300 g L/1 in the alkazolol solution for 21 days, and the bioaccumulation of allazolyl alcohol in zebrafish was studied. The toxic effects of the process and the exposure treatment of azolzol on zebrafish. The instrumental analysis of enantiomers was established by using LC-MS/MS. The results showed that the bioaccumulation process of enolisyl alcohol in zebrafish was not enantioselective. Using the 1H NMR based metabolite analysis method, the exposure of alkyl alcohol was investigated. The effect of treatment on the metabolic profile of zebrafish. Results obtained, compared with the blank control group, 70 UX gL-1 exposure treatment resulted in significant up-regulation of valine, leucine, isoleucine, alanine, lactic acid and choline content, glucose, creatine and taurine decreased significantly; the exposure treatment of allolyl alcohol by 300 mu L-1 A significant reduction in glucose and creatine content was played. The above results showed that the exposure treatment of alkyl alcohol disturbed the energy metabolism, amino acid metabolism and lipid metabolism of zebrafish. The liver, testis and brain of zebrafish were pathologically analyzed by the hematoxylin eosin staining method, and the tissue of zebrafish was examined by the exposure treatment of alkyl alcohol. The results showed that in the exposure treatment group of 70 g L-1 and 300 g L-1, the hepatocyte swelling and the formation of vacuoles were observed in the liver slices. The number of sperm was reduced in the testicular slices and no pathological damage was found in the brain slices. Zebrafish was exposed to 100 mu and 1000 mu of hexazolyl alcohol. The effects of enantiomers and fluosazole enantiomers on the metabolic profile and histopathology of zebrafish were studied for 21 days. The effects of ezolisol enantiomers and fluosazole enantiomers on the metabolic profile of zebrafish were investigated by 1H NMR based metabolomics analysis. The results were obtained, and the exposure of ezolisol enantiomers was obtained. The energy metabolism, amino acid metabolism and lipid metabolism of zebrafish were disturbed, but the mechanisms of the metabolic profile of zebrafish caused by (+) - hexazolyl alcohol and (-) - hexazolyl alcohol exposure treatment were different. The exposure treatment of fluorocarbzole enantiomers disturbed the energy metabolism of zebrafish, amino acid metabolism and lipid metabolism, but (+) - fluonazole and (-) - Fluoro ring. The mechanisms of metabolic profiles of zebrafish caused by azoles were different. The brain, liver, testis and ovaries of zebrafish were pathologically analyzed by hematoxylin eosin staining, and the tissue damage of ezoloxalin enantiomers and fluoxazole enantiomers to zebrafish was investigated. The results showed that two of them were high and low. Exposure to exposed doses of ezolisol enantiomers caused swelling of the hepatocytes of female zebrafish and male zebrafish and accompanied by the presence of liver vacuoles. And the liver damage caused by high and low doses of pyrazolol enantiomers in male zebrafish was more severe than that in female zebrafish. Under the low two exposure doses, the area of the non cell area in the testicle of zebrafish showed a concentration dependence and the increase of female zebrafish ovary mature egg cells. In the two exposure doses, there was no pathological damage in the zzzoli exposure treatment group. Under high and low two exposure doses, the enantiomer exposure treatment of flurocyclic zebrafish and male zebrafish caused swollen and hepatic vacuoles in both female zebrafish and male zebrafish. At high and low two exposure doses, the enantiomer exposure treatment of fluosacrozole caused the increase in the area of the non cell areas in the testicles of zebrafish, female zebrafish No obvious changes were found in the ovaries of the ovaries. No pathological damage was found in the brain slices of the zebra fish in the enantiomer exposure treatment group with two exposure doses, and the energy metabolism of zzanfish was disturbed by the enantiomers of pyrazolol and the enantiomers of fluocyclic zolozole, the metabolism of ammonia acid and the metabolism of lipid, and the liver caused the liver. The pathological damage of the visceral and gonadal glands showed that the effects of the enantiomers and the enantiomers of pyrazolol on the metabolic profile and histopathology of zebrafish were similar to those of the enantiomers of the enantiomers and fluocyclic enantiomers.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：TQ450.261

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