Effects Of Exposure To Bisphenol Substances On Neurotransmitters Metabolism Based On UHPLC-MS/MS

Part one: Establish a UHPLC-MS/MS method for neurotransmitters and their precursors and metabolitesObjectives: The main purpose was to establish a derivatized method for detecting neurotransmitters and their precursors and metabolites in the serum and brain samples based on ultra-high-performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS).Methods: 23 neurotransmitters and their precursors and metabolites were analyzed by internal standard method.Three sulfonyl chloride derivatization reagents were compared,and the p H of buffer solution,reaction temperature,reaction time and concentration of derivatization reagent were systematically optimized.The analytes in serum were extracted by methanol and the analytes in brain samples were extracted by an extracting solution(methanol: water = 3:1,v/v).After the extracting solution was evaporated,the analytes were derivatized by dansyl chloride.Then the analytes were separated by gradient elution on an Acquity UPLC BEH-C18 column and detected by mass spectrometry.Results: The limit of detections of the analytes were between 0.05-5 ng/m L,and the limit of quantifications were between 0.1-10 ng/m L;calibration curves for each analyte were in the ranges of 10-5000 ng/m L for 4-aminobutyric acid(GABA),glutamic acid(Glu),glycine(Gly),aspartic acid(Asp),tryptophan(Trp),Tyrosine(Tyr)and adenosine(Ado),1-500 ng/m L for kynurenine(KYN),3-hydroxykynurenine(3-HK),3-hydroxyanthranilic acid(3-HAA),5-hydroxyindoleacetic acid(5-HIAA),nacetyl-5-hydroxytryptamine(NAS),melatonin(MLT),L-dopa,3,4-dihydroxyphenylacetic acid(DOPAC),3-methoxytyramine(3-MT),norepinephrine(NE),3-methoxy-4-hydroxyphenylethylene glycol(MHPG),vanillylmandelic acid(WMA),dopamine(DA)and homovanillic acid,(HVA),2-500 ng/m L for 5-hydroxytryptamine(5-HT)and epinephrine(E);the linearity of the method was good and the correlation coefficient of the standard curve was greater than 0.99;the relative standard deviations of the intra-and inter-day precision of analytes in brain tissue and serum were less than 15%;the spiked recoveries in brain tissue and serum were 85.5-114.8% and 85.3-115.0% respectively.Conclusion: The established UHPLC-MS/MS detection method of 23 neurotransmitters and their precursors and metabolites exhibited high sensitivity and good stability,which can provide potent technical support for the study on neurotransmitters metabolism.Part two: Effects of BPA,BPS and BPF exposure on neurotransmitters metabolismObjectives: The main purpose was to investigate the effects of bisphenol A(BPA),bisphenol S(BPS)and bisphenol F(BPF)exposure on neurotransmitters metabolism in mice based on UHPLC-MS/MS.Methods: C57BL/6J male mice were randomly divided into four groups: control,BPA,BPS and BPF group.The mice were subcutaneously injected for 30 days at dose of 50 μg/kg/day.After the mice were sacrificed,the serum,prefrontal cortex and hippocampus were taken and the neurotransmitters and their precursors and metabolites were detected by UHPLC-MS/MS.The multivariate statistical models(principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA))and one-way variance were used to analyze the results.Results: 1.PCA and OPLS-DA models were used to analyze the overall changes of neurotransmitters metabolism.In the PCA models,the separation of exposure groups and control group was significantly greater in the serum than in the prefrontal cortex and hippocampus.In the OPLS-DA model,the separation of BPS group in the prefrontal cortex and hippocampus,and BPF group in the serum were completely separated with the control group.2.The levels of neurotransmitters and their precursors and metabolites were analyzed.In the prefrontal cortex,L-dopa was significantly increased in all the three exposure groups;DA and Asp were increased while Ado was decreased in BPS and BPF group;3-HK and Glu were increased in BPS group.In the hippocampus,5-HT was increased and Asp was decreased in BPA group;DOPAC and GABA were increased and Ado was decreased in BPS group;Trp and 3-HK were increased in BPF group.In the serum,BPA increased 5-HIAA,DOPAC,GABA and Glu and decreased KYN and 3-HAA;BPS increased 5-HIAA,Glu and VMA and decreased KYN and 5-HT;BPF increased GABA and VMA and decreased KYN,5-HT,Trp,NE and Gly.3.The biosynthesis and metabolism of neurotransmitters were analyzed.BPS and BPF decreased HVA/DOPAC,3-MT/DA and GABA/Glu radios in the prefrontal cortex;BPF increased 3-HK/KYN ratio in both the prefrontal cortex and hippocampus;in the serum,BPA,BPS and BPF increased 3-HK/KYN ratio and decreased KYN/Trp ratio,meanwhile,BPS and BPF increased 5-HIAA/5-HT and NAS/5-HT ratios.Conclusion: The neurotransmitters metabolism in the mice prefrontal cortex,hippocampus and serum was differently affected by BPA,BPS and BPF exposure,and the alterations in the serum were more pronounced than in the prefrontal cortex and hippocampus.Moreover,the effects of BPS and BPF on neurotransmitters metabolism in the prefrontal cortex,hippocampus and serum were more pronounced than BPA.