【作者】 齐素贞；

【导师】 王成菊；

【作者基本信息】 中国农业大学 ， 农药学， 2014， 博士

【摘要】 农药是农业中重要的生产物资,是保证作物产量和质量的重要手段,明确不同结构和类型农药的作用机理和作用特性,可以为开发新型高效安全农药提供支持和引导。结合当前农药领域的研究热点,本论文分别研究了手性三唑类杀菌剂戊唑醇(rac-teb)对大型溞的毒性作用差异和两种双酰胺类杀虫剂在鱼尼丁受体(RyR)上结合位点的选择特性,具体研究成果如下：1.三唑类杀菌剂戊唑醇及其对映异构体对大型溞的毒性差异研究以纤维素-三(3,5-二甲基苯基氨基甲酸酯)(CDMPC)为固定相手性拆分制得R-和S-teb,并分别测定了其对大型溞的48h急性运动抑制毒性、21d繁殖毒性和子代(F1代)14d恢复毒性差异。Rac-,R-和S-teb对大型溞的48h ECso(95%置信限)分别为3.5(3.3～3.8) mg/L,3.3(3.0～3.8)mg/L和2.7(2.3～3.1)mg/L,三者对大型溞的急性运动抑制毒性为中等且彼此之间毒性作用差异不显著。21d大型溞繁殖毒性试验中,以48h ECso值的1/10为上限,rac-, R-和5-teb的处理浓度均设定为0.01,0.05,0.10,0.20,0.30和0.40mg/Lo结果发现rac-, R-和S-teb均可显著地减少每只大型溞的总产溞数量[最低可观测效应浓度(LOEC, mg/L)分别为0.05,0.05,0.10),总繁殖胎数(LOEC分别为0.30,0.05,0.10),第一胎和第三胎的产幼溞数(LOEC分别为0.05,0.20,0.20；0.05,0.20,0.10),显著性地减少每只大型溞的蜕皮次数(LOEC分别为0.20,0.30,0.10),延长大型溞的性成熟(LOEC分别为>0.40,0.20,>0.40)和第一、第三胎产溞所需时间(LOEC分别为0.20,0.20,>0.40；0.10,0.10,0.10),使得大型溞的生长受到抑制,体长显著减少(LOEC分别为0.05,0.05,>0.40)。相同浓度处理下,rac-, R-和S-teb之间的毒性作用差异显著,强弱顺序为S->rac-≥R-teb,表明rac-, R-和5-teb之间存在显著的对映体选择毒性。14d F1代大型溞毒性恢复研究发现,当rac-teb处理F0代母溞的浓度不超过0.20mg/L时,F1代子溞的繁殖和生长发育基本可恢复,与对照组中大型溞的各项指标值差异不显著；当浓度高于0.20mg/L时,子溞的繁殖和生长会受到显著影响且不能恢复。S-teb暴露处理F0代母溞后对F1代子溞产生的影响>rac->R-teb,最小LOEC值仅为O.Olmg/L且在0.30或0.40mg/L下完全没有F2代子溞的产生。目前我国还没有水中戊唑醇残留量限值的规定,已报道的rac-teb在巴西地表水中的浓度为0.2-4.16μg/L,美国EPA报告地表水中戊唑醇最大浓度一般不超过13μg/L。本研究中最低处理浓度为O.Olmg/L时,rac-,R-和S-teb对F,代大型溞便可产生显著的不可恢复的影响,据此推测在长期暴露情况下,即使浓度低于0.01mg/L,环境中累积的rac-, R-和S-teb依然很有可能会对大型溞的繁殖和生长产生不可逆的影响,进而破坏水生生物种群稳态。根据本论文的研究发现,建议戊唑醇常规使用以外消旋体为佳,但应加强对其对映异构体在环境中的含量监测,以期建立可靠的戊唑醇及其对映异构体的环境控制标准,降低对非靶标水生生物产生的不良影响。2.双酰胺类杀虫剂结合位点的选择性研究本部分研究与加州大学-伯克利分校环境化学与毒理学实验室合作完成。自主合成新型放射性配基[3H]Chlo(氯虫苯甲酰胺),通过直接比较[3H]Ry(鱼尼丁),[3H]Chlo (氯虫苯甲酰胺)和[3H]Flu (氟虫双酰胺)与不同生物品系肌肉鱼尼丁受体(RyR)蛋白的特异性结合,推测两种双酰胺类杀虫剂在RyR上是否具体相同的作用位点,进而解释两种双酰胺类杀虫剂的物种选择特性。本论文选择龙虾(Lobster),兔子(Rabbit),四种昆虫品系包括家蝇(Musca domestica).蜜蜂(Apis mellifera),鳞翅目昆虫烟芽夜蛾(Heliothis virescens)和小地老虎(Agrotis ipsilon)制备RyR蛋白,测定ATP, Ca2+,非标记的Chlo, Cyan(氰虫苯甲酰胺),Flu, Ry对六种RyR蛋白与[3H]Ry,[3H]Chlo和[3H]Flu特异性结合的影响,结果发现[3H]Ry在六种生物品系RyR蛋白上都有结合位点,[3H]Chlo只与四种昆虫RyR有结合而[3H]Flu只与烟蚜夜蛾和小地老虎的RyR蛋白有结合。[3H]Ry与四种昆虫RyR蛋白的结合可以被Chlo, Cyan和Flu(家蝇除外)显著促进,二者龙虾与兔子体系中三者则作用不显著。在家蝇与蜜蜂体系内,Ry和Flu可促进[3H]Chlo的结合,在鳞翅目昆虫体系内,Chlo, Cyan和Flu则抑制[3H]Chlo的结合。[3H]Flu与烟蚜夜蛾和小地老虎RyR的结合可显著被Ry, Chlo, Cyan和Flu抑制。[3H]Chlo与[3H]Flu的结合对单一ATP或Ca2+的依赖度较弱,而ATP和Ca2+共同存在则可显著促进[3H]Chlo的结合反应。这说明ATP和Ca2+调控机制可能与Flu结合位点关系较弱,而与Ry和Chlo结合位点关系较强。通过烟芽夜蛾成虫RyR蛋白与[3H]Chlo和[3H]Flu的特异性结合反应判断两种双酰胺杀虫剂的结构-活性关系。结果发现在烟蚜夜蛾体系内,邻甲酰胺基苯甲酰胺类似物和邻苯二甲酰胺类似物均促进[3H]Ry的结合而抑制[3H]Chlo与[3H]Flu的结合,且两作用效果间相关性良好。在家蝇体系内,两种双酰胺类似物的作用效果不完全相同,邻甲酰胺基苯甲酰胺类似物可以促进[3H]Ry的结合而抑制[3H]Chlo的结合；邻苯二甲酰胺类似物可促进[3H]Chlo的结合而对[3H]Ry的结合影响不显著。这些发现说明两种双酰胺类杀虫剂具有明确的物种选择特性和结合位点差异性,Chlo和Flu在家蝇和蜜蜂RyR蛋白上具有相互偶联的两种不同结合位点,而在鳞翅目害虫RyR蛋白上具有相同的结合位点。本研究直接有力地证明双酰胺类杀虫剂的结合位点具有物种差异性,这对未来双酰胺类杀虫剂的结构优化和抗性机制评估具有重要的意义。更多还原

【Abstract】 Pesticides play an important role in the process of agriculture practice, and they are used to improve the production and quality of the agricultural products. Studies of the mode of action and characteristics of pesticides can provide support and guidance for developing effective and safe new agrochemicals. Consulting the current hot research issues in pesticides, enantioselective toxicity of chiral fungicide tebuconazole to non-target aquatic organism Daphnia manga and binding sites differences of two diamides on RyRs are studied in the present study and the main findings are listed below:1. Studies of toxicity differences of the tebuconazole and its enantiomers’to Daphnia magnaEnatiomers of tebuconazole, R-and5-teb were prepared using HPLC on CDMPC column and the48h acute immobilisation,21d reproduction and14d offspring recovery tests were performed to tell the toxicity differences of rac-, R-and5-teb. The48h EC50s of rac-,R-and5-teb to Daphnia were3.5(3.3-3.8) mg/L,3.3(3.0-3.8) mg/L and2.7(2.3-3.1) mg/L respectively. They are all medium toxic to Daphnia and have no significant toxicity differences. Based on the1/10of48h EC50s, neonates were exposed to0.01,0.05,0.10,0.20,0.30and0.40mg/L of rac-, R-and S-teb for21d reproduction test. During these exposures, rac-,R-and S-teb significantly reduce the total number of offspring [lowest-observed-effective-concentration (LOEC, mg/L) were0.05,0.05,0.10respectively), total number of broods (LOEC-0.30,0.05,0.10respectively), and size of the1st and3rd brood for per daphnia (LOEC-0.05,0.20,0.20and0.05,0.20,0.10respectively). Otherwise, the development of Daphnia was also impacted seriously as the molting retes (LOEC-0.20,0.30,0.10respectively) were reduced and the age at maturity (LOEC->0.40,0.20,>0.40respectively), days to the1st and3rt brood reproduction (LOEC-0.20,0.20,>0.40and0.10,0.10,0.10respectively) were all significantly delayed as well as the body legth (LOEC0.05,0.05,>0.40respectively). Under the same concentration, there are significant differences among the three tebs and the toxicity order is S>rac-b≥R-teb, which indicating the obvious stereoselective toxicity among tebs. The14d F1offspring-generation Daphnia recovery test showed that the impact of rac-teb on F1offsprings can generally recover when exposure concentrations to Fo Daphnia lower than0.20mg/L, but can’t be recoverd when concentrations higher than0.20mg/L. The toxic effects of S-tebs on F1offspring is>rac-≥R-teb. The lowest LOEC of S-teb to observed paramets is just0.01mg/L and no F2offsprings were found under0.30and0.40mg/L exposure. By now, China has no standards limiting the residue amount of teb in water but0.2-4.16μg/L of rac-teb was calculated in Brazil groundwater and as by USEPA records, teb in water generally should be lower than13μg/L. Comprehensively considering the significant unrecovery effects of rac-, R-and D-teb on the F1offspring-generation Daphnia at0.01mg/L, it is reasonable to speculate that rac-, R-and S-teb may impact the fecundity and development of daphnia in long-time exposure even the concentration of teb is lower than10μg/L. Thus, in general use of teb, it is not recommended to separate the enantiomers to improve the safety to non-target aquatic organisms and it is necessary to establish monitoring standards of teb in water.2Selective studies of binding sites of diamide insecticidesThis part of study was achieved by collaborations with Environmental Chemistry and Toxicology Laboratory at University of California at Berkeley. New radiolabeled anthranilic diamide insecticide [N-C3H3]chlorantraniliprole (Chlo) was synthesized at high specific activity and compared with phthalic diamide insecticide [3H]flubendiamide (Flu) and [3H]ryanodine (Ry) in radioligand binding studies with muscle membranes of different species to chosed to define species differences in the degree and mechanisms of diamide selective action at the binding sites.Lobster, rabbit and four insect species, house fly (Musca domestica), honey bee(Apis mellifera), tobacco budworm(Heliothis virescens) and black cutworm(Agrotis ipsilon) are used to prepare RyR membarnes, and the effect of ATP, Ca2+, Ry, Flu, Chlo and Cyan on three radioligands binding are calculated. Through the radioligand specific binding studies, the [3H]Ry site is observed in muscle of lobster, rabbit and four insect species whereas the [3H]Chlo site is evident in the four insects and the [3H]Flu site in only the two lepidoptera.[3H]Ry binding is significantly stimulated by Chlo, Cyan and Flu with the insects (except Flu with Musca) but not the lobster and rabbit.[3H]Chlo binding is stimulated by Ry and Flu in Musca and Apis but not in the lepidoptera, while Flu and Cyan are inhibitory.[3H]Flu binding is strongly inhibited by Chlo and Cyan in Agrotis and Heliothis.[3H]Chlo and [3H]Flu binding are not dependent on added Ca2+or ATP whereas the other radioligand-receptor combinations are enhanced by Ca2+and ATP. A possible relationship is therefore evident between the Flu site but not the Ry and Chlo sites in the Ca2+and ATP modulation mechanisms. Using Heliothis with [3H]Chlo and [3H]Flu allows binding site structure-activity correlations for anthranilic and phthalic diamides. The anthranilic and phthalic diamide analogs stimulation of [3H]Ry binding greatly parallels their inhibition of [3H]Chlo and [3H]Flu binding. In Musca adults, anthranilic diamides stimulate [3H]Ry binding and inhibit [3H]Chlo binding. Phthalic diamide stimulate [3H]Chlo binding but has no significant effect on [3H]Ry binding. These results showed that two diamides have obvious species selectivity, Chlo and Flu have two different but coupled binding sites on Musca and Apis RyR, but they have same binding site on lepidoptera insect RyR. These observations establish species differences in diamide target site specificity and are important in considering diamide structure optimization and evaluation of resistance mechanisms.更多还原