| Paraquat?N,N?-dimethyl-4,4?-bipyridinium dichloride?is known as a widely used herbicide,but also known as the most dangerous pesticide,and many countries have banned this pesticide,but are widely used because of its obvious herbicidal effect.Paraquat can lead to multiple organ failure,especially pulmonary fibrosis,no cure for clinical treatment,with a high mortality rate.In this study,rat plasma paraquat concentration was detected by an ultra-performance liquid chromatography-tandem mass spectrometry?UPLC-MS/MS?method,and the toxicokinetics of paraquat intervention with curcumin and pirfenidone were studied.The method of UPLC-MS/MS was developed for CYP450 probe drugs,and effect of paraquat on CYP450 activity of rats was studied.The method of gas chromatography-mass spectrometry?GC-MS?was established used for separation and analysis of endogenous metabolites in rats,and serum and urine metabolomics were studied after acute paraquat poisoning.Combined with pathology and biochemical indicators,curcumin and pirfenidone intervention in paraquat poisoning were evaluate d by metabolomics.These results are of great value in analyzing the mechanism of paraquat metabolic pathway and exploring the detoxification pathways after paraquat poisoning.Based on GC-MS metabolomics could be used as an analytical tool for the diagnosis and prognosis of pesticide toxicology.The main findings are as follows:1.Toxicokinetic study of paraquatThe rats were randomly divided into paraquat group,curcumin and pirfenidone treatment group.The concentration of paraquat in rat plasma was determinated by an UPLC-MS/MS method over the range of 10-2000 ng/mL.Chromatographic separation was achieved on a BEH HILIC?2.1 mm×100 mm,1.7?m?column.The mobile phase was consisted of 10mM ammonium formate acetonitrile and ammonium formate water?0.1%formic acid?with gradient elution pumped at a flow rate of 0.4 mL/min.Protein precipitation with acetonitrile was used as sample preparation.Compared with the paraquat group,there is statistical toxicokinetics difference for curcumin treatment group and pirfenidone treatment group,AUC?0-t?decreased?p<0.05?,CL increased?p<0.05?for curcumin or pirfenidone treatment group,Cmax decreased?p<0.05?for curcumin treatment group.The results shown that treatment by curcumin and pirfenidone could relieve acute paraquat poisoning in rats.2.Effects of paraquat on enzyme activity of CYP450 in ratsA specific UPLC-MS/MS method has been described for the simultaneous determination of bupropion,metroprolol,midazolam,phenacetin,omeprazole and tolbutamide in rat plasma with diazepam as internal standard,which are the six probe drugs of the six cytochrome.The method was successfully applied to pharmacokinetic studies of the six probe drugs of the six CYP450 isoforms and used to evaluate the activities of CYP2B6,CYP2D6,CYP3A4,CYP1A2,CYP2C19 and CYP2C9 in rats.The precisions were less than 13%,and the accuracy ranged from 93.3%to 110.4%.The extraction efficiency was better than 90.5%,the matrix effects ranged from 84.3%to114.2%.The rats were randomly divided into acute paraquat poisoning group and control group.The paraquat group rats were given 36 mg/kg paraquat by intragastric administration.In the results of paraquat group compared to control group,there was statistical pharmacokinetic difference for bupropion,tolbutamide,metoprolol,midazolam and omeprazole.Acute paraquat poisoning may induce the activities of CYP2C19,and inhibit of CYP2B6,CYP2C9,CYP2D6 and CYP3A4 in rats.This may give advising for reasonable drug use after acute paraquat poisoning.The rats were randomly divided into paraquat group?Low,High?and control group.The paraquat group rats were given 9,18 mg/kg?Low,High?paraquat by intraperitoneal administration.Statistical pharmacokinetics difference for metroprolol,testosterone and tolbutamide in rats were observed by comparing paraquat group with control group.Combined with PCR results,intraperitoneal administration of paraquat induces the activities of CYP2C11 of rats.3.Metabolomics and SVM analysis of paraquat poisoningIn order to analyze the data for the serum metabolites levels of poisoning rats,and to study systematically the metabolic pathway changes induced by paraquat in male rats.Based on GC-MS metabolomics,analyzed the changes of serum metabolite levels of poisoning rats and the control group.Pattern recognition analysis,including both principal component analysis?PCA?and partial least squares-discriminate analysis?PLS-DA?revealed that acute paraquat poisoning induced metabolic perturbations.Compared with the control group,the serum level of octadecanoic acid,L-serine,L-threonine,L-valine,and glycerol in the acute paraquat poisoning group?36 mg/kg?increased,while the levels of hexadecanoic acid,D-galactose,and decanoic acid decreased.Compared to the control group,the urine level of benzeneacetic acid and hexadecanoic acid of the acute paraquat poisoning group?intragastric administration 36mg/kg?increased,while the level of butanedioic acid,pentanedioic acid,altronic acid decreased.Based on these urinary metabolomics data,support vector machine?SVM?was applied to discriminate the metabolomic change of paraquat groups from control group which achieved 100%classification accuracy.In this study,the metabolomics of15 paraquat poisoned patients with plasma paraquat concentrations in excess of0.1?g/mL and 16 healthy volunteers were investigated.The plasma samples were evaluated through the use of GC-MS and analyzed by PLS-DA.Based on the metabolomics data,a SVM discrimination model was developed.The results showed the plasma levels of urea,glucose and L-phenylalanine decreased and cholesterol increased in paraquat poisoned patients in comparison to healthy volunteers.In conclusion,metabolic pathways including the urea cycle,and amino acid,glucose,and cholesterol metabolism were impaired after paraquat poisoning.An SVM discrimination model,based on metabolomics data,was established and may become a new powerful tool for the diagnosis of paraquat poisoning.4.Metabolic changes of acute paraquat poisoning intervention with curcuminIn this study,we developed a urine,serum,tissue?lung,liver,kidney,heart?metabolomics method by GC-MS to evaluate the effect of curcumin in rats of acute paraquat poisoning.The acute paraquat poisoning group rats were given 36 mg/kg of paraquat by intragastric administration?ig?.PLS-DA revealed that intragastric administration of curcumin induced metabolic perturbations of acute paraquat poisoning.When treatment by curcumin,compared to the acute paraquat poisoning group,there biomarkers changed in urine,serum,tissue?lung,liver,kidney,heart?of acute paraquat poisoning intervention of curcumin group.When curcumin intervened,biomarkers had changes in in urine,serum,tissue?lung,liver,kidney,heart?in curcumin intervention in acute paraquat poisoning group.The biochemical indicators after curcumin intervention,alanine aminotransferase,aspartate aminotransferase,alkaline phosphatase,creatinine have a certain reduction in curcumin intervention acute paraquat poisoning group.Paraquat-induced histological changes,intervention with curcumin,bleeding,edema,alveolar septal inflammatory cell infiltration and thickening diminished.Compared with control group,the metabolites change of urine,blood and tissue in paraquat poisoning group and curcumin intervention group increased or decreased,which indicated that paraquat poisoning or curcumin intervention had energy metabolism,fatty acid metabolism,amino acid metabolism,urea cycle disturbance.The urine,blood and tissue metabolomics results showed that the control group,the paraquat poisoning group and the curcumin intervention group could be clearly separated.Combined with the results of histological and biochemical indexes,indicating that curcumin intervened with paraquat,there is a certain treatment effect.The results indicated that metabolomic method by GC-MS may be useful to elucidate the effect of curcumin in rats of acute paraquat poisoning.5.Metabolic changes of acute paraquat poisoning intervention with pirfenidoneWe developed a urine metabolomic method by GC-MS to evaluate the effect of pirfenidone in rats of acute paraquat poisoning.The acute paraquat poisoning group rats were given 20 mg/kg of paraquat by intraperitoneal administration?ip?.PLS-DA revealed that intragastric administration of pirfenidone induced metabolic perturbations of acute paraquat poisoning.When acute paraquat poisoning group treatment by pirfenidone in the fourth day,compared to the acute paraquat poisoning group,the level ofà-DL-lyxofuranoside,arabinofuranose,octadecanoic acid,benzeneacetic acid,pentanedioic acid in urine increased in Low pirfenidone?20 mg/kg,ig?treatment group,while galactaric acid decreased;the level of glycerol,N-phenylacetyl glycine,octadecanoic acid,benzeneacetic acid in urine increased in the High pirfenidone?40mg/kg,ig?treatment group.When treatment by pirfenidone in the seventh day,compared to the acute paraquat poisoning group,the level of creatinine in urine decreased in Low pirfenidone?20 mg/kg,ig?treatment group;the level of d-ribose,creatinine,D-galactose in urine decreased in the High pirfenidone?40 mg/kg,ig?treatment group.Compared to the acute paraquat poisoning group in fourth day,the pentanedioic acid increased in both Low and High pirfenidone treatment group,while d-ribose increased in the Low pirfenidone?20 mg/kg,ig?treatment group,ethanedioic acid increased in the High pirfenidone?40 mg/kg,ig?treatment group,andà-D-glucopyranoside,glycerol decreased in the High pirfenidone?40 mg/kg,ig?treatment group.The changes in tissue metabolomics of rats treated with pirfenidone were similar to serum and urine.Histopathologic and biochemical results found that pirfenidone intervention on paraquat has a certain effect.The results showed that the effect of pirfenidone on acute paraquat poisoning could be identified by GC-MS metabolomics combined with histopathologic and biochemical results. |
PDF Full Text Request