两种酰胺类手性农药在动物体内及体外的代谢及毒性研究

发布时间：2018-03-19 06:16

本文选题：甲霜灵　切入点：苯霜灵　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

【摘要】：本论文利用代谢组学的研究策略,确证了酰胺类手性农药甲霜灵在大鼠肝微粒体及苯霜灵在小鼠肝微粒体中的代谢物,进而在对映体水平上研究了这两种农药在大鼠和小鼠体内及体外模型中的选择性代谢行为,并从多个层面评价了其毒性效应。利用高分辨质谱和稳定同位素标记的方法对甲霜灵在大鼠肝微粒体中的代谢产物进行鉴定,结果确证了3种代谢物。利用体外代谢的方法制备了13C标记的甲霜灵代谢物标样。对选择性代谢的研究结果表明,在肝微粒体中,S-体优先降解,代谢物的生成也具有明显的选择性；在肝细胞中,R-甲霜灵的消除速度快于S-体,代谢物生成的选择性一致,都是S-体的生成量多于R-体。对肝细胞的毒性测定结果显示,毒性大小依次为rac-甲霜灵S-甲霜灵R-甲霜灵,且S-甲霜灵造成的氧化损伤效应也强于R-体。利用基于LC-MS的非靶向代谢组学及靶向代谢组学相结合的策略,对苯霜灵在小鼠肝微粒体中的代谢产物进行了鉴定,结果确证了7个代谢物,其中有2个是首次报道的。利用体外代谢的方法制备得到了苯霜灵代谢物标样。对选择性代谢的研究结果表明,在肝微粒体中,R-苯霜灵优先降解,大部分代谢物的生成也具有一定的选择性；在肝细胞中,S-苯霜灵的消除快于R-体,代谢物生成的选择性不都一致。对肝细胞的氧化损伤测定结果表明,苯霜灵及两对映体均引起了肝细胞SOD和CAT酶活性的下调,但两对映体之间差异不显著。采用灌胃的给药方式,研究了苯霜灵在小鼠体内的立体选择性代谢及排泄。结果显示,rac-苯霜灵给药后,在血浆、心脏、肝脏、脾脏、肺脏、肾脏、肌肉、脑组织中的降解都有一定的选择性,都是R-苯霜灵优先降解；在粪便和尿液中也是S-苯霜灵的量多于R-体,说明排泄过程也具有选择性;两对映体都是在粪便和尿液中的残留量远远大于血浆和组织,说明苯霜灵易于排泄。R-和S-苯霜灵分别灌胃给药后,在粪便中检测到的代谢物种类和数量都最多,且代谢物的生成也具有一定的选择性；在血浆中检测到了两对映体相互转化的现象,且R-体向S-体转化的趋势强于S-体向R-体的转化。通过30天连续灌胃的方式评价了苯霜灵对小鼠的亚慢性毒性效应。表观层面上,组织病理切片结果显示苯霜灵没有造成肝脏和肾脏的明显损伤。从酶的层面对氧化损伤的评价结果显示,苯霜灵引起了小鼠肾脏和肝脏中MDA含量的显著上调以及CAT活性一定程度的上调。代谢物层面上,基于1HNMR的非靶向代谢组学研究结果表明,苯霜灵主要影响了小鼠能量代谢、脂质代谢、维他命B代谢、尿素循环及氨基酸代谢的稳态；基于LC-MS的靶向代谢组学研究结果表明,苯霜灵引起了血浆中天冬酰胺的显著上调以及组氨酸、赖氨酸和天冬氨酸的显著下调。
[Abstract]:In this paper, the metabolites of amides in rat liver microsomes and benzyl in mouse liver microsomes were confirmed by the strategy of metabonomics. Then the selective metabolic behaviors of the two pesticides in rats and mice in vivo and in vitro were studied at enantiomeric level. High resolution mass spectrometry and stable isotope labeling were used to identify the metabolites of Metalaxyl in rat liver microsomes. Results three metabolites were confirmed. The 13C-labeled metabolites of Metalaxyl were prepared by the method of in vitro metabolism. The results of selective metabolism showed that the S- body was preferentially degraded in liver microsomes. The formation of metabolites also showed obvious selectivity. In hepatocytes, the elimination rate of R- methylalaxyl was faster than that of S- body, and the selectivity of metabolite production was consistent, the amount of S- body production was higher than that of R- body. The toxicity test of hepatocytes showed that the toxicity of R- Metalaxyl was higher than that of S- body. The order of toxicity was rac-metalaxyl S-methylalaxyl, and the oxidative damage caused by S- methylalaxyl was stronger than that of R-body. The strategy of combination of non-target metabolomics and targeted metabolomics based on LC-MS was used. The metabolites of benazepine in mouse liver microsomes were identified and 7 metabolites were confirmed. Two of them were reported for the first time. Standard samples of benzyl metabolites were prepared by in vitro metabolism. The results of selective metabolism showed that R- benzalazine was degraded preferentially in liver microsomes. The formation of most metabolites was also selective, and the elimination of S- benzyl in hepatocytes was faster than that of R- bodies, and the selectivity of metabolites formation was not the same. The results of oxidative damage to hepatocytes showed that, The enzyme activities of SOD and CAT in hepatocytes were down-regulated by benazepine and two enantiomers, but there was no significant difference between the two enantiomers. The stereoselective metabolism and excretion of benzyl in mice were studied. The results showed that the degradation of BYL was selective in plasma, heart, liver, spleen, lung, kidney, muscle and brain. Both of them are preferentially degraded by R- benzalaxyl; in feces and urine there are also more S- benzyl than R- bodies, indicating that the excretion process is also selective; both enantiomers have far greater residues in faeces and urine than in plasma and tissue. The results showed that benzyl was easy to be excreted. R- and S- benzyl were respectively administered by stomach, the kinds and quantities of metabolites detected in feces were the most, and the formation of metabolites had certain selectivity. The transformation of two enantiomers was detected in plasma, and the tendency of transformation from R- body to S- body was stronger than that from S- to R- body. Histopathological findings showed that benzyl did not cause significant damage to the liver and kidney. The evaluation of oxidative damage at the enzyme level showed that, Bentalin induced a significant increase in the content of MDA in kidney and liver and an increase in CAT activity to a certain extent. On the metabolite level, the results of non-target metabolic studies based on 1H NMR showed that benzyl mainly affected the energy metabolism of mice. Homeostasis of lipid metabolism, vitamin B metabolism, urea cycle and amino acid metabolism; targeted metabolic studies based on LC-MS showed that benzalazine caused a significant up-regulation of asparagine and histidine in plasma. Significant down-regulation of lysine and aspartic acid.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TQ450.2

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