Whole-genome Microarray Analysis Of Cellular Response To Benzo(a) Pyrene Diol Epoxide In Normal Human Epithelial Cells

(±)-anti-benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) is one of the metabolicproducts of benzo(a)pyrene (BaP),which belongs to the polycyclic aromatichydrocarbons (PAHs) environmental chemical pollutants.BPDE is considered as theultimate carcinogen of BaP.BPDE causing mutagenesis and carcinogenesis is not apassively accepted process by the cell.BPDE induces bulky-adduct DNA damage,which would activate intracellular nucleotide excision repair (NER) and cell cyclearrest,etc.protective mechanisms,also trigger the low-fidelity translesion replicationmechanism that leads to DNA mutations,and stimulate some tumor-promotive stresssignaling pathways,etc.Hence,a comprehensive understand of the cellular responseto BPDE,i.e.,the interaction between genes and toxicants,is essential for uncoveringthe mechanisms underlying BPDE-induced mutagenesis and carcinogenesis.We utilized Affymetrix HG-U133 Set (～33000 genes) and HG-U133 Plus 2.0(～39000 genes) whole-genome microarrays,respectively,to obtain responsive genestriggered by various doses (0.005,0.05 and 0.5μM) of BPDE at a same time point (4h) after treatment and by a single dose (0.05μM) of this carcinogen at various timepoints (1,10 and 22 h) after treatment in FL human amnion epithelial cells.Thereby,to rigorously investigate the cellular response to BPDE in normal human cells.Amedium-throughput quantitative RT-PCR validation of the microarray results based on TaqMan Low Density Array has revealed a flock of confident responsive genes.The dose-dependent effect study revealed that gene expression (H1FO,A URKA,CCNB1,CENPA,CDC20,KIF14,KIF2C,PKMYT1 and EGFR,IGF1R,PRKCA,ITPR1,ATF3,HEXIM1,MYC,SAT1,GDF15,PEA15,etc.) and the cell cycle andcytotoxictiy phenotypes were tightly linked.Transcriptional regulatory analysiscombined with experimental validation observed that a part of gene expressionchanges was due to the activation of some stress response-related transcription factors(AP-1,ATF3,NF-κB,p53,Elk-1,CREB and ATF6 etc.) by BPDE treatment.Thushelps to disclose the whole process of cellular response to BPDE from stress signalingpathways,related transcription factors to target genes.The change of a few genes,e.g.,the up-regulation of CCNE1 and CCNE2 might be associated with the mechanism ofBPDE-induced carcinogenesis.Through analysis of the cell cycle,cell growth and apoptosis-related genes (H1FO,A URKA,CCNB1,CENPA,KIF14,NEDD9,SGOL2,RCC1 and DUSP1,EIF5A,BIRC4,CTGF,ATF1,JUN,PTEN,CYR61,TOB1,MUC1,FOS,MIRN21,etc.),the time-courseeffect study revealed a model of celluar response to low-dose BPDE,i.e.,from start ofresponse,to integration and effect-generation,then to recovery.The alteration of somegenes,e.g.,up-regulation of CYR61,MUC1,FOS and MIRN21,etc.,might be relatedwith the mechanism of BPDE-induced carcinogenesis.High throughput analysis of the microarray results showed that the responsivegenes to BPDE were involved in multiple functions including cell cyle regulation,transcription regulation,RNA splicing,protein metabolism,ubiqutin cycle,lipidmetabolism,cytoskeleton,intracellular transport,cell growth,apoptosis,signaltransduction,DNA repair and DNA damage response etc.;and extensive signalingpathways including MAPK,focal adhesion,cell cycle,Wnt signaling pathway andTGFbeta.pathway etc.These observations contribute to a prospective view of thecellular response to BPDE.The relationships between some important responsive genes and pathways and the mechanism of BPDE-induced mutagenesis and carcinogenesisawait concrete experimental investigation in future.