三种手性农药对映体选择性种间CYP450代谢差异研究

发布时间：2018-05-17 23:44

本文选题：手性农药 + 对映体选择性作用　；参考：《浙江大学》2015年硕士论文

【摘要】：研究背景：手性农药的广泛使用,使其不可避免的进入到各种环境系统,进而可通过各种介质和途径进入生物体内,造成伤害。同一手性农药的不同对映体,由于生化特性的不同,进入环境后往往具有不同的环境行为,其对生物及人体的生理作用也具有对映体选择性差异,从而导致一系列截然不同急慢性毒性作用。但长期以来,外消旋的手性农药都被当作一种化合物来研究其环境行为和生态毒性效应。近年来有关手性农药对映体选择性环境毒性的研究已经引起了人们的热切关注。另外,由于不同物种对进入其体内的手性化合物的对映体选择性降解存在明显的种间差异,导致其在不同物种体内呈现不同的浓度和ER值,从而造成的急慢性毒性效应也会存在种间差异。当前的研究已经关注到这一现象,但没有深入探讨,关于手性农药对映体选择性种间代谢差异的原因,也未见报道。研究方法：实验选取目前世界上使用最为广泛,且能代表杀虫、除草和杀菌三种主要类型的手性农药：氟虫腈、禾草灵和甲霜灵为研究对象,以人肝癌细胞系(HepG2)、大鼠肝癌细胞系(H4IIE)、鸡肝癌细胞系(LMH)和草鱼肝脏细胞系(L8824)这几种能代表高等脊椎动物的细胞系为研究模型。分别研究这三种手性农药在上述物种细胞内代谢时主要的CYP450酶的诱导情况,看是否存在明显的种间差异。进而以甲霜灵为例,用UPLC-MS/MS测定其经过这四种细胞代谢24h后的残留及ER值的变化情况,比较其对映体选择性种间代谢差异,和上述酶活实验结果的种间差异性相互映证。并通过添加不同的CYP450的特异性抑制剂,来进一步验证上述对映体选择性种间代谢差异的产生与特定的CYP450酶被诱导之间的关系。研究结果：酶活实验中,不同的手性农药在同种物种细胞内代谢时,细胞内主要的CYP450酶的诱导情况是不一样的,且同种手性农药在不同物种细胞体内代谢时,诱导的CYP450酶的种类也存在种间差异,添加特异性的CYP450抑制剂后,这些诱导的酶活可以得到相应的抑制。分析实验中,甲霜灵经四种物种细胞代谢后,两种对映异构体的残留和ER值的情况变化存在明显的种间差异。HepG2、H4IIE和LMH细胞选择性代谢S-甲霜灵,L8824细胞选择性代谢R-甲霜灵。并且,添加上述酶活实验中被诱导的CYP450酶的特异性抑制剂后,这些差异和变化都得到了相应的抑制。研究结论：本研究表明,手性农药在不同物种细胞内代谢时存在明显的对映体选择性种间代谢差异,经过不同物种细胞代谢后,手性农药会呈现不同的ER值；并且种间代谢差异的产生与特定的CYP450酶被诱导有关,抑制相应的CYP450酶活后,这些对映体选择性种间代谢差异情况也会得到抑制。说明这些手性农药在不同物种体内可能是由不同的CYP450酶来进行对映体选择性代谢的。由于不同的物种细胞对手性农药存在对映选择性种间代谢差异,使得手性农药在不同物种体内呈现不同的浓度和ER值,进而可能对这些物种细胞造成不同的毒性作用,所以在评价手性农药的生态风险时应充分考虑对映异构体的种间代谢差异。
[Abstract]:Research background: the wide use of chiral pesticides makes it inevitable to enter various environmental systems, and can then enter organisms through various mediums and ways, causing injury. The different enantiomers of the same chiral pesticide have different environmental behavior after entering the environment because of the different biochemical characteristics. The physiological action also has the selectivity difference in enantiomers, which leads to a series of distinct acute and chronic toxicity. But for a long time, the chiral pesticides in raceme have been used as a compound to study their environmental behavior and ecotoxicity. In recent years, the study of the environmental toxicity of chiral pesticides enantiomers has been caused by people. In addition, there are significant differences in the selective degradation of enantiomers of chiral compounds entering their bodies, resulting in different concentrations and ER values in different species, and there are interspecies differences in acute and chronic toxic effects. But there is no in-depth discussion on the reasons for the differences in the interspecific metabolic difference between chiral pesticides enantiomers. Research methods: the experiment selected three main types of chiral pesticides in the world, which are the most widely used in the world, which represent insecticidal, herbicidal and germicidal types: fluonitrile, grasses and metalaxyl, and human hepatoma cells. Line (HepG2), rat hepatoma cell line (H4IIE), chicken liver cancer cell line (LMH) and grass carp liver cell line (L8824), the cell lines that can represent higher vertebrates, are the research models. The induction of the main CYP450 enzyme in the intracellular metabolism of these three chiral pesticides in the above species is examined to see whether there is an obvious interspecific difference. Taking formalin as an example, the changes in the residual and ER values of these four cells after 24h metabolism were measured by UPLC-MS/MS, and the differences in the interspecific metabolic difference between the enantiomers and the results of the above enzyme activity were compared. And the specific inhibitors of different CYP450 were added to further verify the enantiomers. The relationship between the production of selective interspecific metabolic differences and the induction of specific CYP450 enzymes. Results: in the enzyme activity experiment, the main CYP450 enzyme induction in the cell is different when different chiral pesticides are metabolized in the same species cell, and the same chiral pesticides are induced in the cells of different species in the cells of CYP450. There are also interspecies differences in the species of enzymes. After adding specific CYP450 inhibitors, these induced enzyme activities can be suppressed accordingly. In the analysis of experiments, after the metabolism of four species of metalaxyl, there are obvious interspecific differences in the changes of the residual and ER values of the enantiomers, and the selective metabolism of S- in H4IIE and LMH cells, S-. Metalaxyl, L8824 cells selectively metabolize R- metalaxyl. And these differences and changes have been suppressed after the addition of the specific inhibitors of the CYP450 enzyme induced in the above enzyme activity experiments. Conclusions: This study shows that there is an obvious enantioselective metabolism of enantiomeric pesticides in the intracellular metabolites of different species. The difference is that after the cell metabolism of different species, the chiral pesticides present different ER values; and the production of interspecific metabolic differences is related to the induction of specific CYP450 enzymes. After inhibiting the corresponding CYP450 enzyme activity, these enantioselective interspecific metabolic differences in the enantiomers will also be suppressed. Selective metabolism of enantiomers is carried out by different CYP450 enzymes. Due to the differences in the enantioselective metabolism of the chiral pesticides in different species, the chiral pesticides present different concentrations and ER values in different species, and may cause different toxic effects on these species, so the chiral agriculture is evaluated. The interspecific metabolic differences of enantiomers should be fully considered in the ecological risk of drugs.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X592

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