CYP Enzyme-activated Genotoxicity Of EHDPP (An Organic Phosphorus Flame Retardant) And The Influence Of Bisphenol Compounds

BackgroundAs restrictions on the use of halogen flame retardants(FRs)in many countries,organophosphorus flame retardants(PFRs)have become widely used as a new generation of flame retardants.As a substitute material for FRs,PFRs are ubiquitously present in various environmental media and human samples,which may have various toxic effects in human and other organisms,including endocrine disruption,neurological defects,and dysfunction of immune system.However,whether PFRs is genotoxic and the involvement of metabolic activation to genotoxicity remains unclear.2-Ethylhexyl diphenyl phosphate(EHDPP)as a representative PFRs is most commonly present in various environmental media and strongly toxic.At present,the genotoxicity of EHDPP and the specific human or mouse CYP enzymes responsible for its potential metabolic activation are unknown.Besides,bisphenol compounds(BPs),industrially applied as a plasticizing agent,may expose humans together with EHDPP,however,the potential combined effects have not been clarified.Previously we have reported that BPs at nanomolar concentrations drastically increased the mRNA and protein expression of various CYP enzymes,through modulating the expression of nuclear receptors.Therefore,the influence of BPs on the genotoxicity of EHDPP and its metabolic activation deserves further investigation.ObjectiveThis study was aimed at identifying the genotoxicity of EHDPP in mammalian cells,the CYP enzymes required in its metabolic activation,the difference of responsiveness in mouse versus human species,and the influence of BP compounds.Moreover,based on the screening of molecular targets of EHDPP using transcriptomics,this study was performed to reveal the influence of EHDPP on the intracellular levels of reactive oxygen species(ROS)and antioxidant substances,and its effects on cell cycle and apoptosis.Methods(1)Computer-simulated molecular docking of EHDPP to the active site of each human CYP enzyme(i.e.,CYP1A1,1A2,1B1,2B6,2E1 and 3A4,the most commonly isoforms involved in metabolic activation)was performed to analyze the affinity and substrate potential of EHDPP to each CYP.Subsequently,using cell models of Chinese hamster(V79)-derived cell lines recombinantly expressing individual human CYP enzymes(V79-Mz being used as the control cell line which had no activities of CYPs)and the human hepatoma(HepG2)cell line,micronuclei formation,identification of the presence/absence of centromere in formed micronuclei,and DNA damage(indicated by the increase of y-H2AX as analyzed by Western blotting and comet assay),to understand the mechanisms in which EHDPP is activated by specific CYP enzymes to exert genotoxicity and the form of chromosome damage.(2)To explore the responsiveness of mouse versus human species to the effect of EHDPP and the influence of BPs on nuclear receptor/CYP expression,the threedimensional structure of mouse CYP1A1、CYP1A2、CYP2B10、CYP2E1、CYP3A11 proteins were constructed using Swiss-model tool,then molecular docking of EHDPP to the active site of each CYP was performed to predict its affinity and substrate potential for each CYP enzyme,with the result of each docking and micronuclei formation assay being compared with that with its human homologous CYP.The influence of BPs on the transcriptional levels of the nuclear receptors[i.e.,aryl hydrocarbon receptor(AhR),pregnane X receptor(PXR)and constitutive androstane receptor(CAR)]and CYP enzymes in a mouse hepatoma(Hepal-6)cell line in comparison with that in HepG2 cells were determined using RT-qPCR.Then,the effects of BPA,BPF,BPS and BPAF on the micronuclei formation by EHDPP in Hepa1-6 and HepG2 cells were determined.The representability of the mouse versus human model for the study of the metabolic activated genotoxicity of EHDPP,according to the observation of the above indexes.(3)Using the transcriptomic technology the molecular targets related to EHDPPinduced human hepatocyte toxicity were screened,which were further verified by RTqPCR and Western blot assay;moreover,using immunofluorescence labeling,substrate color development methods and flowcytometry,the changes in the levels of ROS,antioxidant substances,cell cycle disturbance and apoptosis induced by EHDPP were analyzed,and their relevance to the CYP enzymes were clarified.Results(1)The results of molecular docking indicated that all the studied CYP enzymes(human CYP1A2,1B1,2B6,2E1 and 3A4),expect for CYP1A1,were valid for potentially metabolizing EHDPP as a substrate;subsequent micronucleus tests with EHDPP(5～40μM)in V79-derived cell lines expressing various CYP enzymes gave rise to results seemingly in accordance to the molecular docking results,i.e.,except for V79-Mz and V79-hCYP1A1,all the other V79-derived cell lines(expressing other CYPs than 1A1)were positive toward EHDPP.Moreover,pretreatment of HepG2 cells with BPAF potentiated EHDPP-induced formation of and double-strand DNA breaks.Immunofluorescence staining of centromere protein B(CENP-B)showed that the micronuclei formed by in V79-hCYP2E1-hSULT1A1 and V79-hCYP3A4-hOR cells were predominantly centromere-free,and that in BPAF-pretreated HepG2 were unexceptionally centromere-free(similar to the effect of ethyl methanesulfonate,an known clastogen),which suggests that the effect of EHDPP on chromosomes is featured by clastogenic changes.(2)The three-dimensional structure of each mouse CYP enzyme was constructed.The results of molecular docking showed that EHDPP is potentially a substrate for all mouse CYP enzymes(i.e.,mouse CYP1A1,1A2,2B10,2E1,and 3A11),expect for CYP1B1.Furthermore,the metabolic activation of EHDPP by mouse CYP2E1 was verified by the micronuclei formation with EHDPP in V79-mCYP2E1 cells,in comparison to the negative result in V79-Mz cells.Besides,EHDPP induced the micronucleus formation in murine hepatoma Hepa1-6 cells,which was attenuated by selective inhibitors of mouse CYP1A1,CYP2E1,CYP2B10 enzymes,while no influence of specific mouse CYP1B1,CYP1A2 and CYP3A11 inhibitors.Further experiments indicated that BPs(including BPA、BPS、BPF,and BPAF)at nanomolar concentrations drastically elevated the expression of nuclear receptors(AhR and PXR)and various CYP enzymes in both Hepa1-6 and HepG2 cells;meanwhile,pretreatment of both lines of cells with each BP compound led to significantly enhanced induction of micronuclei and double-strand DNA breaks.Moreover,gastric gavage of both BPAF and PCB126(positive control)to female mice increased the hepatic expression of CYP enzymes and their total activity,and promoted the metabolizing rate of EHDPP in mouse liver microsomal metabolic system(substrate consumption).These results suggest that mouse CYP2E1 enzyme has a potential similar to its human homologous to activate EHDPP,and bisphenols may increase the expression of both mouse and human CYP enzymes,thus to enhance the genotoxicity of EHDPP in both mouse and human cells.(3)The results of transcriptome sequencing showed that EHDPP(10 μM)induced changes in the transcription of multiple genes in HepG2 cells,including genes involved in regulating gluconeogenesis,oxidative stress,and cell cycle arrest,et al.In V79hCYP3A4-hOR,V79-hCYP2E1-hSULT1A1,HepG2 and human hepatocyte(L-02)cells,EHDPP induced the elevation of intracellular ROS,with no effect in V79-Mz cells;meanwhile,in HepG2 and L-02 cells EHDPP elevated the levels of reduced glutathione(GSH),malondialdehyde(MDA),and increased the activity of superoxide desmutase(SOD),which were either blocked or attenuated by ABT coexposure.Moreover,the rate of apoptosis in HepG2 and L-02 cell line was both promoted,and the cell cycle was arrested at G1 phase;simultaneously,the protein expression of two critical molecules in the p53 signaling pathway,p53 and p21,were both increased,while that of Bcl2 was decreased.Conclusions(1)EHDPP may be metabolically activated by CYPs for inducing chromosome and DNA breaks in mammalian(including human)cells,primarily by human CYP3 A4,2E1 and 2B6.(2)BPs may activate some mouse nuclear receptors and upregulate the expression of their downstream CYP enzymes in mouse hepatocytes and mouse liver(in vivo model),thus potentiate the genotoxicity of EHDPP as mediated by metabolic activation,which are generally similar to responses in human hepatocytes.Therefore,the mouse species as a model for the study of the effects of indirect mutagens by enhancing the expression of relevant CYP enzymes may be representative of the human species,at least to some extent.(3)EHDPP may induce multiple toxic effects in human hepatocytes,including DNA damage,oxidative stress,cell cycle arrest and apoptosis,in which the metabolic activation of EHDPP by CYP enzymes plays a critical role.At the molecular level,the activation of p53 signaling pathway might be an important mechanism mediating the toxicity of EHDPP in human hepatocytes.