The Inhibition Of Chlorophenols On Human CYP3A4 And Differences Among Various Species

Objective:As serious persistent organic pollutants,exposure of chlorophenols（CPs）and their derivatives cause serious adverse effects on human health and ecological environment,such as acute toxicity,histopathological changes,mutagenicity,and cancer.Cytochromes P450（CYPs/P450）are the most important phaseⅠmetabolizing enzymes involved in the oxidation and hydroxylation of endogenous and exogenous compounds.CYP3A4 is the most important CYP subfamily in humans,being involved in the phase I metabolism of more than 50%of clinical drugs.At present,animal models have been commonly used to predict relevant studies in the human body.Therefore,it is very important to detect species differences among different animals.This study aims to determine the inhibitory potential of fourteen CPs towards the activity of human CYP3A4.In addition,in order to obtain the results of species differences,the inhibitory effects of CPs towards liver microsomes from different animal species（monkey,rat,dog,pig）were examined.And the most suitable animal models were selected for in vivo experiments to explain the clinical effects.Methods:The incubation mixture,with a total volume of 200μL,consisting of reduced nicotinamide adenine dinucleotide phosphate（NADPH）-generating system,liver microsomes,testosterone and Tris-HCl buffer.Using CYP3A4 catalyzed testosterone as probe reaction to quantitative analysis of metabolites 6β-hydroxytestosterone by ultra-performance liquid chromatography（UPLC）.First,enzyme activity determinations and metabolic kinetics tests were performed.Secondly,fourteen CPs were selected as the experimental groups,to conduct preliminary screening tests.An equal volume of ketoconazole was used as the positive control,and dimethyl sulfoxide was used as the negative control.For CPs showing more than 80%inhibition,the concentration-dependent inhibition tests and inhibition kinetics determinations were carried out.The molecular docking was used to explain the difference in the inhibition of CYP3A4 by different structures of CPs in terms of the spatial structure.In addition,CPs with an inhibition rate of more than 80%were also selected for the determinations on various animal（monkey,rat,dog,pig）liver microsomes,including inhibition activity,concentration-dependent inhibition tests and inhibition kinetics determinations.The inhibition results were compared with those of HLMs to obtain species differences.Result:Among fourteen CPs,2.3.4-TCP,3.4.5-TCP and 2.3.4.5-TECP inhibited CYP3A4 by more than 80%.Therefore,the above three CPs were selected for the next determinations.IC₅₀ values of these three CPs towards CYP3A4 were 20.5μM,8.0μM,and 6.0μM,respectively.The inhibition kinetic types of these three CPs on CYP3A4were non-competitive inhibition,and K_ivalues were 26.4μM,13.5μM,8.8μM,respectively.By testing the inhibitory effects of the above three CPs towards monkey,rat,dog,pig liver microsomes,it showed that the three CPs have strong inhibitory effects towards monkey and dog liver microsomes.Subsequently,the concentration-dependent inhibition tests and inhibition kinetics determinations were performed.IC₅₀values of these three CPs on Monkey liver microsomes（My LMs）were 37.1μM,10.4μM,and 10.5μM.2.3.4-TCP inhibited monkey CYP3A by competitive inhibition with K_ivalue of 4.9μM.3.4.5-TCP and 2.3.4.5-TECP inhibited My LMs by non-competitive inhibition with K_ivalues of 8.1μM and 28.7μM.IC₅₀ values of 2.3.4-TCP,3.4.5-TCP,and 2.3.4.5-TECP on Dog liver microsomes（DLMs）were 31.6μM,11.9μM,and 38.9μM.The inhibition kinetic types of these three CPs on DLMs were non-competitive inhibition,and K_ivalues were 13.8μM,0.6μM,6.1μM.According to the result of molecular docking between CPs and CYP3A4,the binding free energy of 2-CP,2.4-DCP,2.4.6-TCP and CYP3A4 were-5.29 kcal/mol,-5.99 kcal/mol,-5.88 kcal/mol,respectively.In the binding pocket,2-CP formed one hydrogen bond and 5 hydrophobic contacts with CYP3A4.2.4-DCP formed one hydrogen bond and 6 hydrophobic contacts with the activity cavity of CYP3A4.2.4.6-TCP formed two hydrogen bonds and 5 hydrophobic contacts with the activity cavity of CYP3A4.Conclusion:The inhibition potential of 14 CPs towards the activity of human CYP3A4 existed some structure-activity relationships.The inhibitory capability of CPs towards CYP3A4 would increase significantly when one chlorine atom was added in the fourth site.When one chlorine atom was added in sixth site,the inhibition potential towards CYP3A4 will significantly decrease.In addition,comprehensive analysis of the results of inhibition kinetic types and K_i values,the monkey was more suitable as animal model to evaluate the inhibition of 3.4.5-TCP towards CYP3A,the dog was the more suitable animal model to evaluate the inhibition of 2.3.4-TCP and 2.3.4.5-TECP towards CYP3A.