In Silico Simulation On The Metabolic Mechanisms Of Organic Flame Retardants And Polycyclic Aromatic Hydrocarbons Catalyzed By The Active Center Of P450 Enzymes

For the vast majority of xenobiotics,metabolism catalyzed by cytochrome P450 enzymes(CYPs)is an important eliminating route in biota.CYPs serve to detoxify these pollutants via increasing their molecular polarities,thus facilitating the excretion step;however,reaction intermediates or products generated therefrom may bond with biomacromolecules(e.g.proteins and nucleic acid),leading to enhanced toxic effects compared with the parent compounds.Therefore,it is of great significance to investigate the metabolic mechanisms catalyzed by CYPs for purpose of evaluating the biological fate,toxicological effects and health risk of organic pollutants.Traditional in vivo and in vitro methods towards this end are confronted with difficulties such as animal ethics,high time and labor costs,equipment-dependence and lack of chemical standards.In addition,they are incapable of qualitatively determining the products structures and probing the highly reactive intermediates,thus are hard to meet the demand of ecological risk assessment for huge and ever-increasing amounts of chemicals.For this reason,developing predictive methods for metabolic mechanisms of xenobiotic pollutants by CYPs is in urgent need.With the fast advancement of quantum chemical theories and methodologies and high performance computing capacity,in silico simulation has become an important means that can resolve the above-mentioned questions.In silico methods have demonstrated their advantages in predicting the physico-chemical properties,environmental transformation pathways,products and kinetics of organic pollutants.Previous mechanistic studies on P450 catalyzed reaction were focused on conventional chemical substrates(e.g.alkanes and benzene),endogenous hormones,drug molecules,etc.In comparison,metabolic mechanisms of numerous environmental organic pollutants with varying substituents(alkyl,aryl,halogenation)catalyzed by P450 enzymes are yet to be elucidated.By probing the metabolic transformations of selective organic contaminants(i.e.flame retardants and polycyclic aromatic hydrocarbons)catalyzed by the active species of CYPs(Compound I)with in silico simulations,this study elucidated the molecular mechanisms via which polybrominated biphenyl ethers(PBDEs)are transformed into dihydroxylated(di-HO-PBDEs)and dioxin(PBDD)products catalyzed by Compound I,predicted the metabolic pathways and products of typical organic phosphorous flame retardants(OPFRs)and investigated the metabolic profile polycyclic aromatic hydrocarbons(PAHs)and its binding with P450 enzymes.Below are the detailed research contents with corresponding conclusions:(1)By employing quantum chemical density functional theory(DFT)calculations with 2,2',4,4'-tetraBDE(BDE-47)as a model compound,the transformation mechanisms of PBDEs by Compound ? were investigated.It was found that PBDEs are first transformed by Compound ? to hydroxylated products(HO-PBDEs)wherein the ?-addition of Compound ? to the aromatic carbons is rate-limiting.The reaction proceeds preferably on non-substituted carbons,and lower-brominated PBDEs are more easily oxidized.Dihydroxylation of PBDEs starts with HO-PBDEs as precursors.The reaction proceeds via phenolic H-abstraction and hydroxyl rebound of HO-PBDEs catalyzed by Compound ?,in a manner different from that of PBDEs hydroxylation.The dihydroxylation process involves no role of epoxides intermediates,thus explaining the experimental phenomena that epoxide hydroxylase has no effects on di-HO-PBDEs formation.We also found that only heterocyclic di-HO-PBDEs with-OH linked to phenyl carbons ortho-and meta-to the ether bond can be metabolized to PBDD.Di-HO-PBDEs react with Compound ? by dual phenolic H-abstractions,and the generated diketone intermediates are then rearranged via aryl biradical coupling to HO-PBDD,the structure of which has been validated by PBDEs metabolism in rat liver microsomes.(2)We pinpointed the metabolic mechanisms and products of OPFRs by Compound ?using DFT and molecular docking calculations with triphenyl phosphate(TPHP),tris(2-butoxyethyl)phosphate(TBOEP)and tris(1,3-dichloro-2-propyl)phosphate(TDCIPP)as substrates.The results reveal that TPHP is hydroxylated on the phenyl ring,whereas TBOEP and TDCIPP are hydroxylated on the alkyl group.The metabolic reactivity of OPFRs is found to be structure-dependent,in an order of alkyl TBOEP>aryl TBOEP>chlorinated alkyl TDCIPP.The hydroxylated products of TPHP and TBOEP/TDCIPP are subjected to secondary metabolism in different manners,and the predicted products are in accordance with previous in vitro data.We found a proton-shuttle mechanism for hydroxylation of TPHP to para-OH-TPHP and a new mechanism for O-dealkylation/dearylation of OPFRs to their respective phosphodiesters(DPHP,BBOEP and BDCIPP).In addition,further metabolism of phosphodiesters is found to be affected by molecular ionic states,where ionization enhances the reactivity of DPHP and BDCIPP while decreases that of BBOEP.(3)By using DFT and molecular docking calculations,we investigated the metabolism of 16 US EPA priority PAHs by Compound ? and the binding of PAHs with human CYP 1A2.Results reveal that the ?-addition of PAHs aromatic carbons by Compound ? are site-selective.It was found that epoxides are dominant among the resulted five intermediates and products(epoxides,tetrahedral adducts,cyclohexanone,N-protonated intermediates and hydroxylated PAHs).The K-region carbons of 6 carcinogenic PAHs(chrysene,indeno[1,2,3-c,d]pyrene.benzo(g,h,i)peryene,dibenz(a,b)anthracene,benzo(a)pyrene,benzo(a)anthracene)possess lower ?-addition barriers compared with the other regions.In addition,reaction of the six carcinogenic PAHs all produce K-region epoxides,which coincides with the metabolites detected in the experiment.Molecular docking reveals that the binding affinities between PAHs and CYP 1A2 are linearly correlated with the number of rings and hydrophobicity(logKow)of PAHs.The six carcinogenic PAHs are bound with their M-region carbons oriented to the active center of CYP 1A2,thus explaining the conclusion led by the di-region theory that the M-region atoms are most easily metabolized.