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手性三唑类杀菌剂在 丽斑麻蜥和大鼠体内的分布、代谢和毒性研究
Alternative TitleEnantioselective tissue distribution, metabolism and toxicity of chiral triazole fungicides in Eremias argus and rats
郝伟玉
Subtype博士
Thesis Advisor李建中
2019-06
Degree Grantor中国科学院生态环境研究中心
Place of Conferral北京
Degree Name理学博士
Degree Discipline环境科学
Keyword丽斑麻蜥 ,腈菌唑,腈苯唑,降解代谢,对映选择性 Lizard, Myclobutanil, Fenbuconazole, Metabolism, EnantioselectivityLizard, Myclobutanil, Fenbuconazole, Metabolism, Enantioselectivity
Abstract

       爬行动物起源于石炭纪末期,是脊椎动物的重要组成部分,在生态系统中占据重要的地位。尽管爬行动物记录的种类在逐渐增加,但是在我国很多地区爬行动物的数量正在减少,尤其是环境污染严重的区域。因此越来越多的科学家开始关注爬行动物保护和毒理学研究,评估污染物对爬行动物的生态风险。
      三唑类杀菌剂是第二个里程碑式杀菌剂,通过抑制原菌脱甲基甾醇的合成 发挥作用。据统计, 75% 以上的三唑类农药为手性农药。尽管手性农药不同对映单体具有相似的理化性质,已有研究发现三唑类农药外消旋体和 对映单体在生物体内的行为可能存在差异 不同对映 单体在 动物 体内的毒性存在差异。如果以 单一化合物进行 手性农药的评价不仅不准确,还可能造成不可估量的损失。因此探讨手性农药的对映体差异性在农药毒理学中意义重大。
      本文主要选择结构相似的两种手性三唑类杀菌剂腈菌唑和腈苯唑,用高效液相色谱 手性 OD 3R 色谱柱制备了其对映单体,研究了外消旋体和纯对映单体在丽斑麻蜥体内的对映选择性分布和降解。单次暴露后,腈菌唑在体内的降解半衰期在 0.133 14.828 h 之间,在蜥蜴脂肪、脑 、肝脏 和肾脏中, EF 值小于 0.5腈菌 唑更容易累积。这一现象在多次暴露后更加明显, 28 d 后蜥蜴各个组织和 血液 中均可检测到 腈菌唑残留,而 腈菌唑仅在皮肤中有残留;腈苯唑在蜥蜴体内的 消除 速度明显低于腈菌唑, 脂肪可以富集腈苯唑,但是不存在对映选择性,在血液和肝脏中,(+)-腈苯唑更容易降解。另外,腈菌唑和腈苯唑在蜥蜴体内降解过程中均发现有少量的手性转化现象。
      通过定性和半定量检测腈菌唑和腈苯唑在蜥蜴体内代谢产物发现,肝脏是两种杀菌剂主要的代谢器官,腈菌唑主要代谢产物是 RH 9090 腈苯唑主要代谢产物是RH 9129&RH 9130 。 对映体的代谢产物与 对映体的代谢产物手性构型完全不同,说明腈菌唑和腈苯唑代谢产物的生成过程中也存在对映选择性。另外,外消旋体组代谢产物对映体丰度和纯对映体组不同,尤其是腈苯唑 外消旋体
组, 对映体的代谢产物 RH 6467 的最高浓度大于 对映体、产物RH 9129&RH 9130 的最高浓度小于 对映体 ,但是对映单体组的结果正好相反。这一结果说明腈菌唑和腈苯唑代谢过程中两个对映单体之间有一定的交互作用。
      进一步,以腈菌唑为例,研究暴露后蜥蜴肝脏中代谢酶相关基因的mRNA相对表达量变 化,发现 腈菌唑使细胞色素 P450 1a1 ( cyp1a1 、 cyp2d3 、 cyp2d6 、cyp3a4 和 cyp3a7 基因表达量显著上调,而 腈菌唑使 cyp1a1 、 cyp2d3 、 cyp2d6 、cyp2c8 和 cyp3a4 基因表达量显著上调,腈菌唑两个对映单体的诱导的代谢通路不同 。 抗氧化酶基因 mRNA 表达水平的研究结果表明, 腈菌唑可能会诱导更加明显的氧化应激 这一结果仍需要我们 进一步 验证。 长期暴露后,腈菌唑两个对映单体均可对蜥蜴肝脏造成明显的组织损伤。
      手性三唑类化合物外消旋体犹如一个复杂系统,在蜥蜴体内的降解代谢过程不是两个对映单体降解代谢的加和,而是存在联合作用。 为了分析这种对映选择性在爬行动物和其他物种之间的异同,分析了腈菌唑在大鼠体内的对映选择性分布和代谢。与蜥蜴类似,外消旋体和纯对映单体在体内的分布和代谢有明显区别,肝脏是腈菌唑代谢的主要场所, RH 9090 是主要的代谢产物。 在大鼠肝脏、肾脏、心脏、肺脏和睾丸中, 腈菌唑容易累积。但是,腈菌唑在大鼠体内的代谢速率明显快于蜥蜴,这主要是因为大鼠体内代谢酶基因表达量上调量明显高于蜥蜴,另外大鼠体内仅发现 腈菌唑转化为 腈菌唑。
      综上,对映单体之间的 交互作用,不仅存在于蜥蜴体内,在大鼠体内 也存在,并且手性农药的对映选择性在丽斑麻蜥和大鼠体内有明显差异 。因此,在评价手性农药对生物的毒性作用时, 不仅 应该考虑不同对映单体对生物的影响, 还应该考虑不同物种的差异。 手性农药在爬行动物 体内的对映选择性代谢行为具有其独特性 爬行动物是生态系统不可或缺的部分,因此 构建爬行动物毒理学研究体系,是势在必行的。

Other Abstract

       Reptiles originate in the late Carboniferous period. They are an important part of vertebrates, occupying an important position in the ecosystem. Although the spec ies of reptile records is increasing, the quantity of reptiles is decreasing in China,especially in areas with serious environmental pollution. Therefore, more and more scientists focus on reptile protection tox icology and ecological risk a ssessment 
      Triazole fungicide s are the sec ond milestone fungicide s and act by inhibiting the synthesis of the demethyl sterols. It has been reported that more than 75% of t hem are chiral pesticides. Although different enantiomers of chiral pesticides have similar phy sicochemical properties, the toxicity of different enantiomers o f triazole pesticides in animals is of great difference . Most current studies reported the toxicity of chiral pesticides based on the form of racemate, wheras the behavior of racemates and ena ntiomers in organisms may vary. The evaluation of chiral pesticides in the form of racemates is not only inaccurate, but may also cause immeasurable losses. Therefore, it is of great significance to explore the enantiomeric differences of chiral pesticides .

      Two kinds of chiral triazole fungicides, myclobutanil and fenbuconazole, were selected in this study. The enantiopure enantiomers were prepared by high performance liquid chromatography (HPLC) with chiral OD 3R column. The enantioselective distribution a nd degradation of racemate and enantio pure enantiomers in lizard were measured. After a single exposure, the elimination half lives of myclobutanil were between 0.133 and 14.828 h . T he enantiomer fraction (EF) value s were less than 0.5 i n lizard fat, brain and kidney , suggesting that myclobutanil was more likely to accumulate in these tissues . This phenomenon was pronounced after multiple exposures. After 28 days, ( myclobutanil were detected in all lizard tissues and blood , while myclobutanil was only detected in lizard skin. The elimination rate of fenbuconazole in lizards was significantly lower than that of myclobutanil. Fats couldould enrich fenbuconazoleenrich fenbuconazole withoutwithout enantioselectivity. enantioselectivity. In In lizard blood lizard blood and liver, (+)and liver, (+)--fenbucfenbuconazole onazole waswas enantioselectively degraded. In enantioselectively degraded. In addition, a small amount of chiral conversion was observed in the addition, a small amount of chiral conversion was observed in the eliminationelimination process process of myclobutanil and fenbuconazole in lizards.of myclobutanil and fenbuconazole in lizards.
      The results of qualitative and semi--quantitative detection of the metabolites ofquantitative detection of the metabolites of myclobutanil andmyclobutanil and fenbuconazole in lizards showfenbuconazole in lizards showeded that the liver that the liver waswas the main the main metabolic metabolic placeplace for bothfor both two fungicides. The main metabolite of myclobutanil two fungicides. The main metabolite of myclobutanil waswas RHRH--9090, and the main metabolite of fenbuconazole 9090, and the main metabolite of fenbuconazole waswas RHRH--9129&RH9129&RH--9130. The 9130. The chiral configuration ochiral configuration of the metabolites generated from (+)f the metabolites generated from (+)--enantiomer enantiomer werewere completely different from its antipode, indicating that enantioselectivitycompletely different from its antipode, indicating that enantioselectivity existexisteded during the metabolites formation. In addition, the enantiomeric enrichment of the during the metabolites formation. In addition, the enantiomeric enrichment of the metabolites in racemate group metabolites in racemate group werewere difdifferent from that of the enantiomers group, ferent from that of the enantiomers group, especially for fenbuconazole. After exposure to especially for fenbuconazole. After exposure to racrac--fenbuconazole, the highest fenbuconazole, the highest concentration of the metabolite RHconcentration of the metabolite RH--6467 produced by (6467 produced by (--))--enantiomer enantiomer waswas greater than greater than that produced by (+)that produced by (+)--enantiomer, enantiomer, whilewhile the highestthe highest concentration of the metabolite concentration of the metabolite RHRH--9129 & RH9129 & RH--9130 produced by (9130 produced by (--))--enantiomer was less than that produced by enantiomer was less than that produced by (+)(+)--enantiomer. However, the results in the lizard liver and enantiomer. However, the results in the lizard liver and bloodblood were revewere reversed. This rsed. This result indicatedresult indicated a certain interaction between the two enanta certain interaction between the two enantiomers in the metabolism iomers in the metabolism of myclobutanil and fenbuconazole in lizards.of myclobutanil and fenbuconazole in lizards.
      Furthermore, the relative expression of metabolic enzyme--related genes in lizard related genes in lizard liver after exposure to myclobutanil enantiomers was studied. It was found that liver after exposure to myclobutanil enantiomers was studied. It was found that (+)(+)--myclobutanil significantly upmyclobutanil significantly up--regulated the regulated the mRNA mRNA expression of cytochrome expression of cytochrome P450 1a1 (P450 1a1 (cyp1a1cyp1a1)), , cyp2d3cyp2d3, , cyp2d6cyp2d6, , cyp3a4cyp3a4 and and cyp3a7cyp3a7 genes, while genes, while ((--))--myclobutanil significantly upmyclobutanil significantly up--regulated the expression levels of regulated the expression levels of cyp1a1cyp1a1, , cyp2d3cyp2d3, , cyp2d6cyp2d6, , cyp2c8cyp2c8 and and cyp3a4cyp3a4 genes. The result indicated that the two enantiomers of genes. The result indicated that the two enantiomers of myclobutanil induced different metabolicmyclobutanil induced different metabolic pathways. The results on the mRNA pathways. The results on the mRNA expression of antioxidant enzyme gene indicateexpression of antioxidant enzyme gene indicatedd that (+)that (+)--myclobutanil mmyclobutanil mightight induce induce more obvious oxidative stress.more obvious oxidative stress. The liver damage was obvious after 28 d exposure to The liver damage was obvious after 28 d exposure to myclobutanil enantiomers.
    The racemate of a chiral triahe racemate of a chiral triazole zole fungicide fungicide is like a complex system. The is like a complex system. The eliminationelimination in lizards is not the sum of the in lizards is not the sum of the eliminaitoneliminaiton ofof two enantiomers, but a two enantiomers, but a combined effect. combined effect. The enantioselective distribution and metabolism of myclobutanil in The enantioselective distribution and metabolism of myclobutanil in rats were studied in order to compare the srats were studied in order to compare the similarities and differences of this imilarities and differences of this enantioselectivity between reptiles and other species. Similar to lizards, the enantioselectivity between reptiles and other species. Similar to lizards, the distribution and metabolism of racemate and enantiopure enantiomers were obviously distribution and metabolism of racemate and enantiopure enantiomers were obviously different. The liver was the main metabolism site of myclodifferent. The liver was the main metabolism site of myclobutanil and RHbutanil and RH--9090 was 9090 was the main metabolite. In rat liver, kidney, heart, lunt and testis, (the main metabolite. In rat liver, kidney, heart, lunt and testis, (--))--myclobutanil myclobutanil preferentially enriched. However, myclobutanil eliminated more quickly in rats than preferentially enriched. However, myclobutanil eliminated more quickly in rats than in lizards. This may be explained by the fact that the mRNA exprin lizards. This may be explained by the fact that the mRNA expression of metabolism ession of metabolism enzymes enzymes was higher in rats. In addition, the conversion from (+)was higher in rats. In addition, the conversion from (+)--myclobutanil to myclobutanil to ((--))--myclobutanil was found in rats.myclobutanil was found in rats.
    In summary, the interaction interaction betweenbetween enantiomers enantiomers existed not onlyexisted not only in lizards, but in lizards, but also in rats. also in rats. Moreover, the enantioseMoreover, the enantioselectivity of myclobutanil was different between lectivity of myclobutanil was different between lizards and rats. lizards and rats. Therefore, the effects of different enantiomers on organisms Therefore, the effects of different enantiomers on organisms and and different species different species should be considered in assessing the toxic effects of chiral should be considered in assessing the toxic effects of chiral pesticides. pesticides. Reptiles are an indispensable Reptiles are an indispensable part of the ecosystem.part of the ecosystem. The enantioselective The enantioselective metabolic behavior of chiral pesticides in reptiles is unique. Thmetabolic behavior of chiral pesticides in reptiles is unique. Thusus it is imperative to it is imperative to constructconstruct reptile toxicology research systemreptile toxicology research systemss..
 

Pages136
Language中文
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/42205
Collection中国科学院环境生物技术重点实验室
Recommended Citation
GB/T 7714
郝伟玉. 手性三唑类杀菌剂在 丽斑麻蜥和大鼠体内的分布、代谢和毒性研究[D]. 北京. 中国科学院生态环境研究中心,2019.
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