| Widespread applications of pesticides play important roles in increasing agriculture production as well as have great threats to nontarget organisms including human beings and animals. Amongst the frequently used pesticides, about 30% are chiral. It is well known that enantiomers of chiral pesticide usually show different in biological processes such as absorption, distribution, degradation and toxicity in organisms. The contents of this thesis mainly include:(1) investigating the enantioselective degradation of benalaxyl, metalaxyl and hexaconazole in rat and rabbit hepatic microsomes in vitro; (2) studying the enantioselective absorption, distribution, metabolism, excretion as well as metabolite analysis of metalaxyl in mice; (3) evaluating metabolic perturbation and toxic effect assessment of rac-metalxyl, R-metalaxyl and endosulfan using NMR and LC-MS/MS based metabolomics approaches. Such results will make important contributions in chiral pesticides risk assessments and improving pesticide safety to humans and animals.In this study, sample preparation methods including extraction, clean-up, separation and detection of benalaxyl, metalaxyl and hexaconazole enantiomers were developed based HPLC with chiral stationary phases (CSP). Such methods were successfully adopted to investigate the enantioselective degradation of these three chiral pesticides in rat and rabbit hepatic microsomes. The results showed that the degradations of benalaxyl enantiomers in rat and rabbit hepatic microsomes were enantioselective and the trend of enantioselectivity in these two species was opposite. In rabbit microsomes, R-benalaxyl degraded faster than S-benalaxyl, while S-benalaxyl degraded faster than R-benalaxyl in rat hepatic microsomes after incubating rac-benalaxyl and benalaxyl enantiomers separately. Enzyme kinetic analysis results were in accordance with the degradation consequences. The maximum degradation rate (Vmax) and degradation clearance (CLint) of R-benalaxyl was larger than S-benalaxyl in rabbit hepatic microsomes. However the maximum degradation rate (Vmax) and degradation clearance (CLint) of S-benalaxyl was larger than R-benalaxyl in rat hepatic microsomes. As for metalaxyl, the degradations of metalaxyl enantiomers in enantiomers in rat and rabbit hepatic microsomes were enantioselective and the trend of enantioselectivity in these two species was the same. In rat and rabbit microsomes, S-metalaxyl degraded faster than R-metalaxyl after incubating rac-metalaxyl and metalaxyl enantiomers separately. Enzyme kinetic analysis results were in accordance with the degradation consequences. The maximum degradation rate (Vmax) and degradation clearance (CLint) of S-metalaxyl was larger than R-metalaxyl in rat and rabbit hepatic microsomes. For hexaconazole, the degradation of hexaconazole enantiomers in rat hepatic microsomes was enantioselective and competitive inhibition between (+)-hexaconazole and (-)-hexaconazole was observed in the study. In rat microsomes, (+)-hexaconazole degraded faster than (-)-hexaconazole after incubating rac-hexaconazole and hexaconazole enantiomers individually. Enzyme kinetic analysis results were in accordance with the degradation consequences. The maximum degradation rate (Vmax) and degradation clearance (CLint) of (+)-hexaconazole was larger than (-)-hexaconazole in rat hepatic microsomes. Interaction study revealed that there was competitive inhibition between (+)-hexaconazole and (-)-hexaconazole. The IC50(+)/(-) was 34.20 μmol/L, while IC50(-)/(+) was 64.16 μmol/L, which implied the inhibition ability of (+)-hexaconazole was larger than (-)-hexaconazole. In addition, no chiral inversions were observed between benalaxyl, metalaxyl and hexaconazole enantiomers in rat and rabbit hepatic microsomes degradation.In the current contribution, the enantioselective absorption, distribution, degradation, excretion as well as metabolites analysis of metalaxyl were investigated in mice in vivo. The results indicated that the absorption, distribution, degradation and excretion processes of metalaxyl enantiomers in mice were enantioselective, implying R-metalaxyl> S-metalaxyl. The degradation rates in tissues were lung>heart>liver and the residual concentrations in urine and feces were much higher than that in tissues. Main metabolites were also determined and biotransformation reactions were hydroxylation, demethylation and didemethylation.The enantioselective health effects of rac-metalaxyl and R-metalaxyl on mice were identified using integrative NMR and LC-MS/MS based metabolomics approaches. Metalaxyl induced liver inflammation and necrosis in mice. Furthermore, R-metalaxyl caused heavier liver damage than rac-metalaxyl at the same dose. Urine 1H-NMR based metabolomics revealed that rac-metalaxyl treated groups,R-metalaxyl treated groups and control were clearly separated by partitial least-squares discriminant analysis (PLS-DA), suggesting the metabolic profiles of mice were obviously altered by rac-metalaxyl and R-metalaxyl.Rac-metalaxyl induced a total 10 metabolites and R-metalaxyl caused a total of 19 metabolites significantly changed in mice urine, which means metabolites species and quantities fluctuation induced by rac-metalaxyl and R-metalaxyl were enantioselective. Biological pathways indicated six metabolic pathways were affected, including glycine, serine and threonine metabolism; TCA cycle; pyruvate metabolism; glycolysis or gluconeogenesis; glycerophospholipid metabolism; glyoxylate and dicarboxylate metabolism. Those pathways were highly related to lipid metablolism, amino acids metabolism, energy metabolism and gut microbiota metabolism. Serum amino acids and tryptophan metabolites were accurate quantitated using stable isotope-labeled internal standards by LC-MS/MS. As a sequence,8 amino acids were significantly changed after metalaxyl exposure, which clearly indicated metalaxyl perturbed serum amino acids metabolism. In addition, metalaxyl induced the increased levels of tryptophan, serotonin, indole-3-propionic acid and decreased levels of kynurenine, indole-3-lactic acid. In addition, serum amino acids and tryptophan metabolites changes induced by rac-metalaxyl and R-metalaxyl were also enantioselective.The metabolic profiling perturbation and multitoxic effects of endosulfan on mice were evaluated using integrative H-NMR and LC-MS/MS based metabolomics approaches. Endosulfan induced weight loss, clinical biochemistry parameters changes, liver inflammation and necrosis in mice. Urine 1H-NMR based metabolomics revealed that endosulfan-treated groups and control were clearly separated by partitial least-squares discriminant analysis (PLS-DA), suggesting the metabolic profiles of mice were obviously altered by endosulfan. A total 18 metabolites were significantly changed in urine and biological pathways indicated seven metabolic pathways were affected, including glycine, serine and threonine metabolism; TCA cycle; pyruvate metabolism; glycolysis or gluconeogenesis; glycerophospholipid metabolism; glyoxylate and dicarboxylate metabolism; nicotinate and nicotinamide metabolism. Those pathways were highly related to amino acids metabolism, lipid metablolism, energy metabolism and gut microbiota metabolism. Serum amino acids and tryptophan metabolites were accurate quantitated using stable isotope-labeled internal standards by LC-MS/MS. As a sequence,10 amino acids were significantly changed after endosulfan exposure, which clearly indicated endosulfan perturbed serum amino acids metabolism. In addition, endosulfan induced the decreased levels of tryptophan, indole-3-acetic acid and increased levels of kynurenine, indole-3-propionic acid and indole-3-lactic acid. With increasing dose of endosulfan, the KYN/Trp ratio increased, which means the activity of indoleamine 2,3-dioxygenase (IDO) enzyme enhanced by endosulfan resulting in tryptophan metabolism increased through kynurenine pathway. |
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