行政院国科会环保署空污费科技计画成果报告

:NSC89-EPA-Z-007-004:8878912:(:)benzo[a]pyreneERKJNKp38MAPkinasesB[a]Pp38kinaseERKJNKkinaseB[a]Pp38CL3B[a]Pp38ERKp38ERK(0.1μM)4B[a]PcytochromeP450reductasec-ablc-fosbadcyclooxygenase1cyclooxygenase1B[a]P4244cytochromeP450reductasecyclooxygenase1B[a]PB[a]Psmad1Furinuracil-DNAglycosylasegadd153rad50msh2rad50msh2(30-500μM)ERKJNKp38PD98059ERKMEK1/2ERKPD98059hprtERKERKkinsaeABSTRACT(Keywords:leadbenzo[a]pyrenehumanlungcancerornormalcells)Theexpressionofgenescouldbeinducedorsuppressedtomakepropercellularresponsessuchasdetoxification,DNArepair,cellgrowtharrestandapoptosiswhencellsareexposedtotoxicants.Manyresearcheshaveindicatedthattheexpressionofgenesaremarkedlydefferencebetweencancerandnormalcells.Investigationoftheeffectsofenvironmentaltoxicantsongeneexpressionsisthusimportantforcancerdetection,preventionandtherapy.Leadandpolycyclicaromatichydrocarbons(PAHs)areubiquitousenvironmentalpollutantsthatarehighlyassociatedwithincreasedriskforlungcancers.Thisresearchprojectisdesignedtoinvestigatethealterationofgeneexpressionsinducedbyleadandbenzo[a]pyreneinhumanlungcancerornormalcellsusingacDNAmicroarrayassay.WewillfocusonexaminingtheexpressionofhundredsofgenesincludingthoseforDNArepair,metabolicactivationofPAHs,freeradicalsmetabolismandcatabolism,regulatingcellproliferation,apoptosis,transformation,andmetastasis.Wewillalsostudythesynergisticeffectsofleadandbenzo[a]pyreneongeneexpressions.Theroleofbenzo[a]pyrene-inducedp38pathway(anapoptosisrelatedsignal)ingeneexpressionwillbeinvestigatedusingcellscontainingdominantnegativeformofp38andthecDNAmicroarrayassay.Furthermore,wewilldeterminecytotoxicityandmicronucleiinducedbyleadandbenzo[a]pyrenetorevealtheirgenotoxicity.TheadoptionofthisnovelcDNAmicroarrayassaytoscreentheexpressionofmanygenesatthesametimewouldallowustorapidlyidentifymarkergenesalteredbyenvironmentalpollents,andprovidedetectionandpreventionways.TheresultsobtainedfromthisresearchshouldprovidemorebasictheoryonthegenotoxicityandlungcancerinducedbyleadandPAHs.p38p38(1)(1)(2-6)(7)(8)(proteinkinaseC)(9,10)(cysteine)(GSH)(carboxylicgroup)(asparticacid)(glutamicacid)(cytochromeP450)(11)(calmodulin)(12,13)(δ-aminolevulinicaciddehydrogenase)(superoxidedismutase)invivoinvitro(14,15)(thymidinekinase)2-3(16)(90%)(V79cells)()(hypoxanthine-guaninephosphoribosyltransferase;hprt)(17)(0-5μM)V79hprt(18,19)DNA(alkalineelution)V79DNADNA-DNADNA-(17)(nucleoidsedimentation)HeLaDNA(18)invitroDNAβRNADNA(20,21)80%15%CHO-K1()(0.5-1.5mM)50hprt()hprt8(polymerasechainreaction;PCR)hprtcDNAhprtCHO-K1hprt(basesubstitution)54%(splicemutations)18%DNA(deletions)25%GGAT30%GTA27%GCG20%(22)HFW(softagar)(anchorageindependence)(23)HFW(23)X-(reactiveoxygenspecies,ROS)(24,25)(24,25)(1O2)(OH)(O2-)(H2O2)H2OFentonreactionM(n-1)++H2O2Mn++OH-+OH(26)O2-+Mn+M(n-1)++O2Haber-WeissreactionH2O2+O2-OH-+OH+O2Haber-WeissreactionH2O2+O2-OH-+OH+1O2(27)(28-30)(31)C8-hydroxydeoxyguanosine(8-OHdG)(catalase)C8-OHdG(32,33)dibenzo[a,l]pyrene(DB[a,l]P),benzo[a]pyrene(B[a]P)(34-36)(37,38)p450(cyclooxygenaseprostaglandinHsynthase)(39-43)B[a]Pbenzo[a]pyrenediolepoxide(BPDE)guanineN2adenine(44)B[a]P(45)thymineglycol(46)B[a]P8-OHdG8-OHdG(47)B[a]Pp4501A1,p4502A2,cyclooxygenase-2,glutathioneS-transferaseP1-1,c-Ha-ras(48-51)B[a]PB[a]P(52)caspase-3c-JunNH2-terminalJNKkinases1(JNK1)(53)B[a]PBPDELyn,Syktyrosinekinases(54)BPDESP-1,E2FBPDE(55,56)BPDEp53(57,58)TPABPDEp53(59)10(60)(PCR)biotindigoxigenin(60)interferons,serum(61,62)CL3B[a]PmRNAbiotin-16-dUTPdigoxigenin-11-dUTPcDNAp38p38B[a]Pp38dominantnegativep38p38p38MAPkinasesB[a]Pp38JNK,ERK6-TGhprtB[a]PCL360(72)RPMI164010%0.22%w/v100U/ml(penicillin)100mg/ml(Streptomycin)0.03%w/v(L-glutamine)5%CO2371x106p10010%DMSO1(60)IMAGEPCRlhcI,rbcL,rcaCL3GAPDH96107(mRNA)oligo-dT1-2μgmRNA(200U,MuMLVreversetranscriptase)oligo-d(T)20degeneratepromers,0.5mMdATP/dCTP/dGTP,40μMdTTP,40μMbiotin-16-dUTPdigoxigenin-11-dUTP,10mMDTT,0.5U/mlRNasin40cDNA995.5μl,3MNaOH50mRNAcDNA5.5μl,3Macetate50μl,7.5Mammoniumacetate,20μglinearpolyacrylamide,375μlabsolutealcohol,525μl2%dextransulfate0.3%BSA,10mMTris-HCl,pH7.4,150mMNaClstreptavidin/β-galactosidaseanti-digoxigenin/alkalinephosphatase3-10x105p100B[a]P60mm200-100095%10%Giemsa(22)CL31×106p100p1001×10540μM(6-TG)p60200-1214(denovopathway)(salvagepathway)HPRT6-TGHPRT6-TG;hprtHPRT6-TG6-TGhprtMAPkinasesWCE(20mMHepes,pH7.6,75mMNaCl,2.5mMMgCl2,0.2mMEDTA,0.1%TritonX-100,0.5mMDTT,0.1mMNa3VO4,50mMNaF,0.5μg/mlleupeptin,1μg/mlaprotinin,100μg/ml4-(2-aminoethyl)benzenesulfonylfluoride)10000rpm10BSAGST-cJun(1-79)JNKkinases(74)100μg10μgGST-cJun(1-79)20μlglutathione-Sepharose4B43Hepes(20mMHepes,pH7.6,50mMNaCl,2.5mMMgCl2,0.1mMEDTA,0.05%TritoxX-100)kinases(20mMHepes,pH7.6,20mMMgCl2,2mMDTT,0.1mMNa3VO4)30μl20μMATP,3μCi[γ-32P]ATPkinases30Hepes30μl1.5XLaemlli510%SDS-polyacrylamideERKp38ERK,p38kinases10-50μg10%SDS-polyacrylamidePVDFERKp38horseradishperoxidase-conjugatedPVDFERKp38ERKp38NewEnglandBioLabsJNK,ERKp38JNK,ERKp38PharmingenSantaCruz(ICP-AES)1020.5%NP-40,0.25Msucrose,10mMTris-HCl,pH7.4,0.1mMDTT,10mMKCl1.5mMMgCl22,500rpm0.5-(Inductivelyco