Study On Enantioselective Behavior And Hepatotoxicity Of Bromuconazole

Due to the excellent protective,curative,and eradicant power against a wild-spectrum of crop diseases,triazoles are becoming the most popular class of agricultural fungicides.It is known that most triazole fungicides are chiral and their optical isomers exhibit different bioactivity and toxicity.After entering into the environment through its application,multiple organisms may stereoselectively take up and biotransform the different enantiomers,resulting in unique fate and disposition of these stereoisomers.The contents of this thesis mainly include:(1)investigating the enantioselective degradation of paclobutrazol in rat liver microsomes and bromuconazole in rat,mouse,rabbit,dog and human liver microsomes;(2)studying the enantioselective absorption,distribution,metabolism and transcriptional responses of bromuconazole in rat;(3)evaluating metabolic perturbation,toxicity effect and mechanism of bromuconazole using metabolomics and transcriptomics approaches.Such results will make important contributions in chiral pesticides rick assessments and improving pesticide safety to humans and animals.The degradation of the enantiomers of(2RS,4RS)-and(2RS,4SR)-bromuconazole by phase I metabolism were investigated using rat,rabbit,dog,mouse and human liver microsomes.Bromuconazole isomers were quantified using HPLC-MS/MS(ESI⁺)after separation on a combination of a reversed phase and a chiral analytical column.Moreover,the absolute configuration of four enantiomers had been determined based on comparisons of the vibrational circular dichroism experimental spectra with the theoretical curve obtained by density functional theory calculations.The degradation of all four isomers followed first-order kinetics and the(2R,4S)-bromuconazole displays a significantly longer half-life during microsomal incubation than other antipodes.Moreover the metabolic rates of four isomers in human and mouse to be much slower than that in rat,rabbit and dog,indicating species-specific differences in the metabolism of bromuconazole.The V_max values for(2S,4R)-bromuconazole were obviously higher than those for its antipode in all liver microsomes studied,the V_max values for(2R,4R)-bromuconazole were significantly higher than those for(2S,4S)-bromuconazole in all liver microsomes studied except human.Enantioselective degradation of paclobutrazol was investigated in rat liver microsomes in vitro.The t_1/2 of(2R,3R)-paclobutrazol is 18.60 min,while the t_1/2 of(2S,3S)-paclobutrazol is 10.93 min.Such consequences clearly indicated that the degradation of paclobutrazol in rat liver microsomes was stereoselective and the degradation rate of(2S,3S)-paclobutrazol was much faster than(2R,3R)-paclobutrazol.In addition,significant differences between two enantiomers were also observed in enzyme kinetic parameters.The V_max of(2S,3S)-paclobutrazol was more than 2-fold of(2R,3R)-paclobutrazol and the Cl_int of(2S,3S)-paclobutrazol was higher than that of(2R,3R)-paclobutrazol after incubation in rat liver microsomes.The associated enantioselective absorption,distribution,metabolism and transcriptional responses of bromuconazole in rat were determined following a single-dose(13.8 mg·kg^-1 body weight)exposure to rac-,(2R,4R)-,(2S,4S)-,(2R,4S)-or(2S,4R)-bromuconazole.The enantiomer fractions(EFs)and diastereoisomer fractions(DFs)were deviation of 0.5(0.56)with time in the intestine,liver,lung,heart,kidney and testis,suggesting enantioselective absorption,distribution of bromuconazole in these tissues.In addition,the degradation of bromuconazole in rat tissues was consistent with first-order kinetics,and the dissipation half-life was 0.56?6.48 h.Bromuconazole has different stereoselectivity in different tissues.For(2RS,4RS)-bromuconazole,the concentration of(2R,4R)-bromuconazole was higher in intestine,lung,heart,kidney and testis,while the concentration of(2S,4S)-bromuconazole was higher in liver.For(2RS,4SR)-bromuconazole,the concentration of(2R,4S)-bromuconazole was higher in intestine,liver and testis,while the concentration of(2S,4R)-bromuconazole was higher in lung and kidney.At the same time,the concentration of(2RS,4RS)-bromuconazole was lower than(2RS,4SR)-bromuconazole.Main metabolites were also determined and biotransformation reactions were hydroxylation,dihydroxylation and hydroxyl replace bromine.Moreover the relative concentration of bromuconazole metabolites in the intestine was the highest and the types of metabolites in the lung were relatively abundant.(2S,4S)-bromuconazole has a few types of metabolites and a low relative content of metabolites,which may be related to its preferential degradation in the intestines,lungs,heart,kidneys,and testes.(2R,4S)-bromuconazole has more types of metabolites and higher relative content of metabolites,which may be related to its high content in tissues and the slowest degradation rate.The transcriptional level of target genes(cyp1a2,cyp2d4,cyp2e1,cyp3a1)showed significant up-regulation in livers of bromuconazole treatment compared with the solvent control,suggesting that all of these enzymes may contribute to the metabolism of bromuconazole.In addition,(2R,4S)-bromuconazole had a relatively low influence transcriptional level of the metabolic genes,while(2R,4R)-bromuconazole had a relatively high influence.The influence of rac-bromuconazole on cyp2e1 and cyp3a1 transcriptional level was generally lower than that of(2S,4S),(2R,4R)-bromuconazole,indicating that different enantiomers had different effects on the transcriptional level of metabolic genes in rat liver.The metabolic profiling perturbation,toxicity effect and mechanism of bromuconazole on rat were evaluated using metabolomics and transcriptomics approaches.Male adult rats were exposed to 0,13.8,32.8 and 65.6 mg/kg/d bromuconazole for 10 days by oral gavage.Then,the integrative metabolomics and transcriptomic analysis were used together to elucidate the complex pathophysiological and toxic pathways related to bromuconazole exposure.We observed that short time bromuconazole exposure not only caused liver histological damage but also change the levels of some hepatic phycological parameters including aspartate transaminase(AST),triglyceride(TG),pyruvate(Pyr)and total cholesterol(TC),indicated that BROMU caused hepatotoxicity in rats.In addition,according to transcriptomics and metabolomics analysis,a total of 58 metabolites and 259 genes changed significantly in high-dose bromuconazole treated group.Although several different pathways involved,lipid metabolism and bile acids metabolism related pathways highlighted both in metabolomics and transcriptomics analysis.More importantly,further validation proved that bromuconazole not only could interact with PPAR-?but also significantly decreased its protein and gene expression in the liver,supporting that bromuconazole decreased the TG synthesis via inhibiting PPAR-?pathway.From all of the above experiments,we made a very clear point that short time BROMU exposure could lead to hepatotoxicity at the physiological,metabolomics,and transcriptomic levels in rats.These findings would provide some important steps toward understanding the mechanism underlying bromuconazole-induced mammalian hepatic toxicity.