The Inhibition And Mechanism Of Bromophenols On UGTs

Bromophenols（BPs）is a halogenated organic pollutant（HOC）,which is the raw material and intermediate of chemical materials and flame retardants.BPs,as a pollutant discharged by human and a Marine metabolite,exists widely in various environmental media.Human being have been frequently exposed to bromo-phenolic pollutant in the environment.BPs are endocrine disruptors which disrupt the body’s metabolic function.Therefore,it is necessary to study and evaluate the toxicity and relevant mechanism of bromophenol.UDP-glucuronosyltransferases（UGTs）are the most important enzymes in the phaseⅡmetabolic enzymes.UGTs can metabolize a variety of endogenous and exogenous compounds,ensuring the metabolism and development for human being,which maintain the activities of life.Purpose:To investigate the inhibition of BPs towards the metabolic activity of various isoforms of UGTs in the environment,this study investigated the toxicity and toxicological mechanism of BPs from the perspective of metabolic toxicology.Methods:1.Construct an in vitro incubation system.Eight BPs inhibitors and 11 human recombinant UGTs were selected as experimental subjects.Using 4-methylumbelliferone（4-MU）as the substrate probe,ultra performance liquid chromatography（UPLC）was used to detect the 4glucuronide（4-MUG）produced by UGTs-catalyzed reaction under the inhibition of BPs.2.The inhibition of BPs on the activity of UGTs isoform was tested using a preliminary screening.Half inhibition concentration(IC₅₀)and inhibition kinetics parameters（inhibition kinetics,inhibition parameters（K_i））were determined by.In vitro-in vivo extrapolation（IVIVE）was used to extrapolate in vivo inhibition magnitude of UGTs,and the threshold concentrations of BPs for in vivo inhibition of UGTs were calculated.3.Modeling the bromophenol molecule and the stereo-structure of UGTs protein,and using molecular docking method to simulate the optimal spatial structure of bromophenol and UGTs protein configurations.The free binding energy was calculated by using Lackmar genetic algorithm（LGA）,and the hydrogen bonds formation and hydrophobic interactions were investigated to elucidate the interaction mechanism between BPs and UGTs.Results:1.Among UGTs isoforms,UGT1A1,UGT1A3 and UGT1A7 were significantly inhibited by BPs,and 80%inhibition rate was used as the primary screening threshold for the inhibition of BPs on UGTs.Five BPs（2-BP,2,4-DBP,2,5-DBP,3,5-DBP,2,4,6-TBP）with the most significant inhibitory effect and seven specific UGTs isforms（UGT1A1,UGT1A3,UGT1A6,UGT1A7,UGT1A8,UGT1A9,UGT2B7）were selected.2.The results of enzyme inhibition kinetics were as follows:The inhibition of UGT2B7 by 2,4,6-TBP was non-competitive,and the inhibition of UGTs by bromophenol was competitive.Ki value of 2-BP on UGT1A6 was 1.74μM;K_ivalues of 2,4-DBP on UGT1A7and UGT1A9 were 1.46μM and 3.75μM,respectively.K_i value of 2,5-DBP towards UGT1A1,UGT1A7,UGT1A9 and UGT2B7 were 0.94μM,1.56μM,0.95μM and0.02μM,respectively.K_ivalues of 3,5-DBP on UGT1A6,UGT1A8 and UGT2B7 were2.46μM,2.26μM and 0.48μM.K_ivalues of 2,4,6-TBP on UGT1A3,UGT1A7 and UGT2B7 were 2.85μM,3.99μM and 31.00μM,respectively3.IVIVE indicated that moderate inhibition magnitude of 2,4,6-TBP towards UGT1A3 and UGT1A7 was found in vivo.According to evaluation standards using[I]/K_iratio（[I]/K_i>0.1）,highest concentration of 2,4,6-TBP in vivo was 0.612 m M in serum.4.Molecular docking experiments showed that 5-bromine substituents had a stronger inhibitory effect on UGT1A3 through strong hydrophobic contact,resulting in2,5-DBP and 3,5-DBP.After introducing 6-bromine substituents into UGT1A1,the binding free energy was reduced,resulting in a stronger inhibitory effect of 2,6-DBP and 2,4,6-TBP on UGT1A1.Conclusion:BPs have a strong inhibition on the metabolic activity of UGTs.Exposure to 2,4,6-TBP inhibits the metabolism of endogenous substances catalyzed by UGT1A3 and UGT1A7.The concentration of 2,4,6-TBP exposure threshold in human blood is 0.612μM,which has an adverse effect on the metabolism of endogenous substances in human body.The inhibition of 5-bromo-substituent and 6-bromo-substituent in bromophenol molecules increased the inhibition capability towards UGT1A1 and UGT1A3.