三种三唑类手性农药在斑马鱼体内的生物富集行为和毒性效应研究

发布时间：2018-06-10 03:53

本文选题：己唑醇 + 氟环唑　；参考：《中国农业大学》2017年博士论文

【摘要】：研究手性三唑类杀菌剂的生物富集行为和毒性效应是全面评价手性三唑类杀菌剂在水生生物中的环境归趋和毒性效应不可或缺的部分,然而,目前对这一部分的研究十分有限。在本论文中,研究了己唑醇、氟环唑和烯唑醇在斑马鱼体内的生物富集行为和毒性效应。此外,利用代谢组学的研究策略和组织病理学检查的表观评价方法研究了己唑醇对映体和氟环唑对映体暴露对斑马鱼的毒性效应。将成年斑马鱼暴露于100μgL/1和200 μg L/1己唑醇溶液中21天,研究了己唑醇在斑马鱼体内的生物富集过程。利用LC-MS/MS建立了己唑醇对映体含量的仪器分析方法。结果得到,己唑醇在斑马鱼体内的生物富集过程是对映体选择性的,(-)-己唑醇要比(+)-己唑醇更容易在斑马鱼体内发生富集。将成年斑马鱼暴露于50 μgL-1和200 μgL-1己唑醇溶液中21天,研究了己唑醇暴露处理对于斑马鱼的毒性效应。利用抗氧化酶活性评价方法,考察了己唑醇暴露处理对斑马鱼肝脏氧化应激发生的影响。结果表明,己唑醇溶暴露处理引起斑马鱼肝脏中SOD、CAT和GPx活性和GSH含量的显著性下调,说明己唑醇的暴露处理促使在斑马鱼肝脏中发生了氧化应激。通过对细胞凋亡路径上一系列相关基因表达情况进行分析,考察了己唑醇暴露处理对于斑马鱼肝脏细胞凋亡过程发生的影响。结果表明,己唑醇暴露处理引起了促细胞凋亡过程的基因p53、Puma、Apaf-1、caspase-3和caspase-9的表达显著性上调,Bcl-2/Bax的比值显著性下调,说明己唑醇暴露处理诱发了斑马鱼肝脏细胞凋亡过程的发生,且该过程的发生依赖于caspase的参与。将成年雄性斑马鱼暴露于100 μg/1和1000 μL-1的氟环唑溶液中21天,研究了氟环唑在斑马鱼体内的生物富集过程和氟环唑暴露处理对斑马鱼的毒性效应。利用LC-MS/MS建立了氟环唑对映体含量的仪器分析方法。结果得到,氟环唑在斑马鱼体内的生物富集过程是对映体选择性的,(+)-氟环唑要比(-)-氟环唑更容易在斑马鱼体内发生富集。利用基于1H核磁的代谢组学分析方法,考察了氟环唑暴露处理对斑马鱼代谢轮廓的影响。结果表明,100 μg L-1和1000 μgL/1的氟环唑暴露处理引起了斑马鱼两种相似的但不完全相同的内源性代谢物代谢轮廓的改变,这些改变都是有关于能量代谢、脂质代谢和氨基酸代谢的。通过对斑马鱼肝脏的线粒体呼吸链、ATP合成和脂肪酸β-氧化相关基因的表达情况进行分析,研究了氟环唑暴露处理扰乱斑马鱼能量代谢的机理。结果表明,氟环唑的暴露处理对于斑马鱼线肝脏的粒体呼吸链、ATP合成和脂肪酸的β-氧化相关基因的表达都有抑制作用。将成年雄性斑马鱼暴露于70 μg L/1和300 μg L/1的烯唑醇溶液中21天,研究了烯唑醇在斑马鱼体内的生物富集过程和烯唑醇的暴露处理对斑马鱼的毒性效应。利用LC-MS/MS建立了烯唑醇对映体含量的仪器分析方法。结果得到,烯唑醇在斑马鱼体内的生物富集过程是不具有对映体选择性的。利用基于1H核磁的代谢组学分析方法,考察了烯唑醇暴露处理对于斑马鱼代谢轮廓的影响。结果得到,相比于空白对照组,70 μgL-1的烯唑醇暴露处理引起了缬氨酸、亮氨酸、异亮氨酸、丙氨酸、乳酸和胆碱含量的显著性上调,葡萄糖、肌酸和牛磺酸含量的显著性下调;300 μg L-1的烯唑醇暴露处理引起了葡萄糖和肌酸含量的显著性下调。以上结果说明,烯唑醇的暴露处理扰动了斑马鱼的能量代谢、氨基酸代谢和脂质代谢。利用苏木精-伊红染色方法对斑马鱼肝脏、睾丸和脑进行了病理学分析,考察了烯唑醇暴露处理对斑马鱼的组织损伤情况。结果表明,在70 μg L-1和300 μg L-1的烯唑醇暴露处理组中,在肝切片中观察到了肝细胞肿胀和空泡的形成,在睾丸切片中观察到了精子数目的减少,在脑切片中未见病理学损伤。将斑马鱼分别暴露于100 μgL-1和1000 μgL-1的己唑醇对映体和氟环唑对映体溶液中21天,研究其对斑马鱼代谢轮廓和组织病理学的影响。利用基于1H核磁的代谢组学分析方法,考察了己唑醇对映体和氟环唑对映体分别暴露处理对于斑马鱼代谢轮廓的影响。结果得到,己唑醇对映体的暴露处理扰动了斑马鱼的能量代谢、氨基酸代谢和脂质代谢,但(+)-己唑醇和(-)-己唑醇暴露处理引起斑马鱼代谢轮廓改变的机制存在差异;氟环唑对映体的暴露处理扰动了斑马鱼的能量代谢、氨基酸代谢和脂质代谢,但(+)-氟环唑和(-)-氟环唑暴露处理引起斑马鱼代谢轮廓改变的机制存在差异。利用苏木精-伊红染色方法对斑马鱼脑、肝脏、睾丸和卵巢进行了病理学分析,考察了己唑醇对映体和氟环唑对映体分别暴露处理对于斑马鱼的组织损伤情况。结果表明,在高低两个暴露剂量下,己唑醇对映体暴露处理都引起了雌性斑马鱼和雄性斑马鱼肝细胞的肿胀并伴随有肝脏空泡的出现。并且,己唑醇对映体高低剂量的暴露处理在雄性斑马鱼中引起的肝损伤要比在雌性斑马鱼中引起的肝损伤更为严重。在高低两个暴露剂量下,己唑醇对映体暴露处理都引起斑马鱼睾丸中非细胞区域的面积呈现浓度依赖性增大和雌性斑马鱼卵巢成熟卵细胞的增多。在高低两个暴露剂量下,己唑醇对映体暴露处理组中,都未见斑马鱼脑切片出现病理学损伤。在高低两个暴露剂量下,氟环唑对映体暴露处理都引起了雌性斑马鱼和雄性斑马鱼肝细胞的肿胀并都伴随有肝脏空泡的出现。在高低两个暴露剂量下,氟环唑对映体暴露处理都引起了斑马鱼睾丸中非细胞区域的面积增大,雌性斑马鱼卵巢的卵细胞未见有明显改变。在高低两个暴露剂量下,氟环唑对映体暴露处理组中,都未见斑马鱼脑切片出现病理学损伤。己唑醇对映体和氟环唑对映体暴露处理都扰动了斑马鱼的能量代谢、氨基酸代谢和脂质代谢,同时都造成了肝脏和性腺的病理学损伤,说明己唑醇对映体和氟环唑对映体暴露处理对斑马鱼代谢轮廓和组织病理学的影响具有相似性。
[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.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ450.261

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