本文選題：甲霜靈　切入點(diǎn)：苯霜靈　出處：《中國農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：本論文利用代謝組學(xué)的研究策略,確證了酰胺類手性農(nóng)藥甲霜靈在大鼠肝微粒體及苯霜靈在小鼠肝微粒體中的代謝物,進(jìn)而在對映體水平上研究了這兩種農(nóng)藥在大鼠和小鼠體內(nèi)及體外模型中的選擇性代謝行為,并從多個(gè)層面評價(jià)了其毒性效應(yīng)。利用高分辨質(zhì)譜和穩(wěn)定同位素標(biāo)記的方法對甲霜靈在大鼠肝微粒體中的代謝產(chǎn)物進(jìn)行鑒定,結(jié)果確證了3種代謝物。利用體外代謝的方法制備了13C標(biāo)記的甲霜靈代謝物標(biāo)樣。對選擇性代謝的研究結(jié)果表明,在肝微粒體中,S-體優(yōu)先降解,代謝物的生成也具有明顯的選擇性；在肝細(xì)胞中,R-甲霜靈的消除速度快于S-體,代謝物生成的選擇性一致,都是S-體的生成量多于R-體。對肝細(xì)胞的毒性測定結(jié)果顯示,毒性大小依次為rac-甲霜靈S-甲霜靈R-甲霜靈,且S-甲霜靈造成的氧化損傷效應(yīng)也強(qiáng)于R-體。利用基于LC-MS的非靶向代謝組學(xué)及靶向代謝組學(xué)相結(jié)合的策略,對苯霜靈在小鼠肝微粒體中的代謝產(chǎn)物進(jìn)行了鑒定,結(jié)果確證了7個(gè)代謝物,其中有2個(gè)是首次報(bào)道的。利用體外代謝的方法制備得到了苯霜靈代謝物標(biāo)樣。對選擇性代謝的研究結(jié)果表明,在肝微粒體中,R-苯霜靈優(yōu)先降解,大部分代謝物的生成也具有一定的選擇性；在肝細(xì)胞中,S-苯霜靈的消除快于R-體,代謝物生成的選擇性不都一致。對肝細(xì)胞的氧化損傷測定結(jié)果表明,苯霜靈及兩對映體均引起了肝細(xì)胞SOD和CAT酶活性的下調(diào),但兩對映體之間差異不顯著。采用灌胃的給藥方式,研究了苯霜靈在小鼠體內(nèi)的立體選擇性代謝及排泄。結(jié)果顯示,rac-苯霜靈給藥后,在血漿、心臟、肝臟、脾臟、肺臟、腎臟、肌肉、腦組織中的降解都有一定的選擇性,都是R-苯霜靈優(yōu)先降解；在糞便和尿液中也是S-苯霜靈的量多于R-體,說明排泄過程也具有選擇性;兩對映體都是在糞便和尿液中的殘留量遠(yuǎn)遠(yuǎn)大于血漿和組織,說明苯霜靈易于排泄。R-和S-苯霜靈分別灌胃給藥后,在糞便中檢測到的代謝物種類和數(shù)量都最多,且代謝物的生成也具有一定的選擇性；在血漿中檢測到了兩對映體相互轉(zhuǎn)化的現(xiàn)象,且R-體向S-體轉(zhuǎn)化的趨勢強(qiáng)于S-體向R-體的轉(zhuǎn)化。通過30天連續(xù)灌胃的方式評價(jià)了苯霜靈對小鼠的亞慢性毒性效應(yīng)。表觀層面上,組織病理切片結(jié)果顯示苯霜靈沒有造成肝臟和腎臟的明顯損傷。從酶的層面對氧化損傷的評價(jià)結(jié)果顯示,苯霜靈引起了小鼠腎臟和肝臟中MDA含量的顯著上調(diào)以及CAT活性一定程度的上調(diào)。代謝物層面上,基于1HNMR的非靶向代謝組學(xué)研究結(jié)果表明,苯霜靈主要影響了小鼠能量代謝、脂質(zhì)代謝、維他命B代謝、尿素循環(huán)及氨基酸代謝的穩(wěn)態(tài)；基于LC-MS的靶向代謝組學(xué)研究結(jié)果表明,苯霜靈引起了血漿中天冬酰胺的顯著上調(diào)以及組氨酸、賴氨酸和天冬氨酸的顯著下調(diào)。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：TQ450.2

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