The Inhibition And Relevant Mechanism Of Hydroxypolychlorinated Biphenyls On UGTs

Objective:Polychlorinated biphenyls?PCBs?are persistent organic pollutants?POPs?,which are widely present in the environment and human body.Studies have shown that PCBs have a variety of toxic effects on the body.Polychlorinated biphenyls?PCBs?enter the body and are rapidly metabolized into hydroxypolychlorinated biphenyls.The concentration of OH-PCBs accounts for 10-30%of the total level of human PCBs,which is often higher than that of many single PCB congeners,and OH-PCB is an important material basis for the toxicity of PCB.So it is more meaningful for us to explain the toxic mechanism of hydroxypolychlorinated biphenyls?OH-PCBs?.Uridine diphosphate glucuronosyltransferases?UGTs?is an important phase II metabolic enzyme,which can metabolize a variety of endogenous small molecules?such as estrogen,bile acid,bilirubin,etc.?and maintain the normal physiological balance of the body.The purpose of this study was to investigate the inhibitory effect of hydroxypolychlorinated biphenyls on the activity of uridine diphosphate glucuronosyltransferase subtypes and to elucidate the toxic mechanism of hydroxypolychlorinated biphenyls from the metabolic toxicological point of view.Methods:Simulating the metabolic environment of compounds in vivo,35 representative hydroxypolychlorinated biphenyls?OH-PCBs?and 9 main human UGTs?UGT1A1,1A3,1A6,1A7,1A8,1A9,1A10,2B7 and 2B17?were selected as experimental groups,and the solvent dimethyl sulfoxide?DMSO?was used as blank control.Using recombinant UGTs catalyzed 4-methylumbelliferone?4-MU?as probe reaction,we quantitative analysis of its metabolite 4-methylumbelliferone-?-D-glucuronide?4-MUG?in vitro.By comparing the inhibitory ability of OH-PCBs with different chlorine atom number and hydroxyl position on UGT subtypes,the complex structure-activity relationship between OH-PCBs and UGT subtypes was observed,and the inhibition kinetic parameter was further determined.The inhibition types and inhibition parameters Ki,were represented by 2'-OH-PCB106 and 4'-OH-PCB106,to UGT subtypes UGT1A1,-1A7 and-2B7.The inhibitory effect of hydroxypolychlorinated biphenyls?OH-PCBs?exposuring on endogenous substance metabolism in vivo was evaluated by in vitro and in vivo extrapolation.Finally,molecular docking was used to further elucidate the inhibitory difference of OH-PCBs compounds 4'-OH-PCB9,4'-OH-PCB26,4'-OH-PCB112,4'-OH-PCB165on UGT1A1 in terms of spatial structure.Including hydrogen bonds and hydrophobic interactions.Results:OH-PCBs exhibited broad inhibition towards UGT isoforms.When we used90%inhibition potential as the threshold of initial screening,there are many OH-PCBs within this inhibition range.Therefore,two representative groups of hydroxypolychlorinatedbiphenylsandthreeuridinediphosphate glucuronosyltransferase were selected for the determination of inhibition kinetics.Concentration-dependent inhibition of 2'-OH-PCB106 on the activity of UGT1A1,-1A7 and-2B7 was demonstrated,and the IC₅₀0 value was calculated to be 1.60,15.02,and 2.76?M for the inhibition of 2'-OH-PCB106 on UGT1A1,-1A7,and-2B7.the IC₅₀0 value was calculated to be 1.56,30.47 and 5.51?M for the inhibition of4'-OH-PCB106 on UGT1A1,-1A7,and-2B7,respectively.For 2'-OH-PCB106,the inhibition kinetic parameters?Ki?were calculated to be 0.4?M for UGT1A1,1.3?M for UGT1A7,and 2.7?M for UGT2B7,respectively,for 4'-OH-PCB106,the inhibition kinetic parameters?Ki?were calculated to be 0.7?M for UGT1A1,6.8?M for UGT1A7,and 4.8?M for UGT2B7,respectively.Competitive inhibition of2'-OH-PCB106 and 4'-OH-PCB106 on UGT1A1,UGT1A7,and UGT2B7 was found.IVIVE was performed using 2'-OH-PCB106 and 4'-OH-PCB106 as the representative OH-PCBs,and the threshold value of 2'-OH-PCB106 was calculated to be 0.04?M for UGT1A1,0.13?M for UGT1A7,and 0.27?M for UGT2B7,respectively,and the threshold value of 4'-OH-PCB106 was calculated to be 0.07?M,0.68?M and 0.48?M for UGT1A1,UGT1A7 and UGT2B7,respectively.According to the formula,when the ratio of[I]/Ki was greater than 0.1 as the standard for inhibiting endogenous metabolism of hydroxypolychlorinated biphenyls?OH-PCBs?.the thresholds of 2'-OH-PCB106 for UGT1A1,UGT1A7 and UGT2B7were 0.04?M,0.13?M and 0.27?M,respectively,The thresholds of 4'-OH-PCB106for UGT1A1,UGT1A7 and UGT2B7 were 0.07?M,0.68?M and 0.48?M,respectively.When the concentration of 2'-OH-PCB106 and 4'-OH-PCB106 in vivo exceed the above thresholds,the metabolism of endogenous substances will be inhibited.According to the results of molecular docking with spatial configuration,the binding free energies of four hydroxyl polychlorinated biphenyls?4'-OH-PCB9,4'-OH-PCB26,4'-OH-PCB112 and 4'-OH-PCB165?were calculated to be-6.25kcal/mol,-7.89 kcal/mol,-7.41 kcal/mol and-7.29 kcal/mol,respectively,that is,4'-OH-PCB9<4'-OH-PCB26⁴'-OH-PCB112>4'-OH-PCB165,the results are consistent with the inhibitory ability of the primary screening test.This inhibition mechanism can be explained by hydrogen bonds and hydrophobic interactions.The results showed that a hydrogen bond?TYR-312?was formed between 4'-OH-PCB9and amino acid residues of UGT1A1,and hydrophobic connections were formed with LEU-81,GLY-314,PRO-290,PHE-80,THR-315 and SER-287.Two hydrogen bonds?ASN-259?were formed between 4'-OH-PCB26 and the amino acid residues of UGT1A1,which were associated with LEU-256,TRP-329,GLN-332 and PRO-331.Two hydrogen bonds?GLN-332 and LEU-330?were formed between 4'-OH-PCB112and amino acid residues of UGT1A1,and hydrophobic connections were formed with LEU-16,SER-284,ARG-311,TRP-329 and SER-13.There was no hydrogen bond between the amino acid residues of 4'-OH-PCB165 and UGT1A1,and hydrophobic connection is formed with PHE-79 and HIS-72.Conclusions:1.Hydroxypolychlorinated biphenyls?OH-PCBs?,as environmental persistent organic pollutants,have a strong and extensive inhibitory effect on phase II metabolic enzyme uridine diphosphate glucuronosyltransferases superfamily?UGTs?.This process is closely related to the properties of hydroxypolychlorinated biphenyls?the number of chlorine atoms and the position of hydroxyl groups?.Of the 35hydroxypolychlorinated biphenyls screened in this experiment,when the number of chlorine atoms is 3,4,5 and the hydroxyl group is located in the para-position,the inhibition ability is the strongest,and then we further elucidated the strong inhibitory ability of some representative hydroxypolychlorinated biphenyls?2'-OH-PCB106 and4'-OH-PCB106?on UGTs by means of half inhibitory concentration IC₅₀0 and inhibition kinetic parameter Ki,The smaller the IC₅₀,the stronger the inhibition.2.When the concentration of hydroxypolychlorinated biphenyls exceeds a certain threshold in vivo,it will inhibit the metabolism of endogenous substances.Researchers usually use the ratio of[I]/Ki greater than 0.1 as the standard for the inhibition of endogenous substance metabolism by hydroxypolychlorinated biphenyls?OH-PCBs?.For example,the thresholds of 2'-OH-PCB106 for UGT1A1,UGT1A7and UGT2B7 were 0.04?M,0.13?M and 0.27?M,respectively.The thresholds of4'-OH-PCB106 for UGT1A1,UGT1A7 and UGT2B7 were 0.07?M,0.68?M and0.48?M,respectively.Therefore,we should prevent the concentration in vivo from reaching this threshold and fundamentally avoid the inhibition of endogenous substance metabolism.3.OH-PCBs have a complex structure-activity relationship with UGTs.In this experiment,with the help of molecular docking software,the internal mechanism of the inhibitory effect of OH-PCBs on UGTs was verified from the spatial configuration,which may be caused by hydrogen bond and hydrophobic interaction.The results were consistent with the primary screening.The lower the binding free energy value,the more hydrogen bond,the stronger the hydrophobic interaction,the stronger the inhibition ability of OH-PCBs.4'-OH-PCB26 and 4'-OH-PCB112 had two hydrogen bonds,while 4'-OH-PCB9 and 4'-OH-PCB165 have only one hydrogen bond.The result of this experiment was as follows:4'-OH-PCB9<4'-OH-PCB26⁴'-OH-PCB112>4'-OH-PCB165.