nnk是煙草中特有的一種物質(zhì)

1、nnk是煙草中特有的一種物質(zhì)，可以誘導(dǎo)肺癌的發(fā)生。卷煙燃燒可產(chǎn)生4000多種化學(xué)物質(zhì)，其中40余種有明確的誘變/致癌性。主要的致癌物有煙草特有亞硝胺(TSNA)、苯并(a)芘、多環(huán)芳烴(PAH),芳香胺、苯、二嗯英、兒茶酚及致癌的醌、肼類等。TSNA是尼古丁被亞硝化的產(chǎn)物，4-(甲基亞硝氨基)-3-吡啶-1-丁酮(NNK)是已知7種TSNA中最強(qiáng)的致癌源。NNK在主流、側(cè)流煙氣及不燃燒的煙草中均大量存在。NNK是卷煙致癌的主要標(biāo)志物，尼古丁是吸煙成癮的主要原因。EfficientBioelectronicActuationoftheNaturalCatalyticPathwayofHumanM

2、etabolicCytochromeP450sSadagopanKrishnan,fDhanukaWasalathanthri,fLinlinZhao,fJohnB.Schenkman,$andJamesF.Rusling*,f,tHerein,wedescribefabricationofLbLfilmsmadebycombiningpurecytP450swithCPRmicrosomesonelectrodestoachievealargeratioofcytP450toCPR(Figure1),asinthehumanliver.1,2,40Electronsareinjectedin

3、tothefilmfromtheelectrodetoaccuratelymimicthenaturalcytP450catalyticcycleathighcatalyticturnover.WeprovideunambiguousevidenceforelectrontransferfromelectrodetoCPRtocytP450frommeasuredredoxpotentials,electrontransferrates,enzymeturnoverrates,andcarbonmonoxide(CO)binding.Resultssuggestdynamicparticipa

4、tionofaCPR-cytP450complexinakeyequilibriumredoxprocessfacilitatingefficientcatalyticturnoveroftheexcesscytP450s.Inaddition,theelectrode-driventurnoverrateforamodeloxidationreactionwasasgoodasorbetterthanwhenNADPHwasutilized.這里我們主要敘述了層層膜結(jié)構(gòu)通過將純的P450酶和P450還原酶連接到電極上來實(shí)現(xiàn)像人體中從P450酶到P450還原酶一個較大差異，電子從電極中被送到膜

5、中，準(zhǔn)確模擬再高催化轉(zhuǎn)化情況下的p450的催化過程。我們從對測量的氧化還原電勢，電子轉(zhuǎn)移速率，酶轉(zhuǎn)化速率和CO鍵合中為電子從電極傳遞到到CRP再到P450還原酶提供了有力的證據(jù)。結(jié)果顯示，在一個關(guān)鍵的氧化還原過程中CPR-cytP450的動態(tài)參與大大的促進(jìn)了大量P450酶的有效催化轉(zhuǎn)化。除此之外，電極驅(qū)動模型氧化反應(yīng)的轉(zhuǎn)化速率和使用NADPH差不多甚至更好！有資料表明，吸煙者每天吸入NNK的量約為28nmol,40年內(nèi)吸入NNK的量約為85mg(1.1mg/kg)煙草特有亞硝胺NNK與肺癌的關(guān)系3.NNK誘導(dǎo)的肺癌基因突變：在NNK誘導(dǎo)的動物肺癌中均發(fā)現(xiàn)了基因的突變，在NNK誘導(dǎo)的小鼠肺癌中發(fā)

6、現(xiàn)有Kras基因的12位密碼子GGT-GAT的轉(zhuǎn)變4。在人的肺腺癌中Kras基因12位密碼子的突變約24%50%,而在其他類型的肺癌中這種突變很少見12,13。這種突變在吸煙者和被動吸煙者比在不吸煙者中更常見。最常見的突變?yōu)?GGT-TGT(60%),其次為GGT-GAT(20%)和GGTGTT(15%)PEITC在F2344鼠和APJ鼠內(nèi)能抑制NNK誘導(dǎo)肺腫瘤,而異硫氰酸苯甲酯(BITC)在APJ鼠能抑制苯并芘(BaP)誘導(dǎo)的肺腫瘤，非毒性劑量的PEITC和BITC能有效抑制NNK和苯并芘(BaP)在鼠體內(nèi)的代謝活化和其致癌性。如果聯(lián)合PEITC和BITC，能在人體內(nèi)抑制NNK和B(a)P誘

7、導(dǎo)的肺腫瘤，將會為吸煙導(dǎo)致肺癌的預(yù)防帶來新的防治措施吲哚232甲醇(I3C)也是人類食物中的一種成分，它在一些十字花科蔬菜中以結(jié)合形式存在，它主要通過增加肝臟清除NNK的能力來阻斷NNK誘導(dǎo)的實(shí)驗(yàn)鼠的肺腫瘤，同樣能增加吸煙者肝臟代謝NNK的能力和尿排泄NNAL和NNAL2Gluc的能力。還有研究提示一些藥物如維生素C、維生素E、阿司匹林及黃綠色蔬菜水果等均能減少和預(yù)防動物肺腫瘤的發(fā)生4。維生素E是通過調(diào)節(jié)多胺的代謝，而阿司匹林是通過抑制環(huán)氧酯酶的活性來實(shí)現(xiàn)其抑制NNK的致癌性的NNK和NNAL在體內(nèi)的代謝:NNK在體內(nèi)的半衰期極短，很快代謝轉(zhuǎn)化為NNAL、NNAL2G1UC和其他產(chǎn)物。NNK在

8、動物體內(nèi)主要有三條代謝途徑:碳基還原反應(yīng)、吡啶氮氧化反應(yīng)和a2羥化反應(yīng)1.NNK和NNAL在體內(nèi)的代謝：NNK在體內(nèi)的半衰期極短，很快代謝轉(zhuǎn)化為NNAL、NNAL2Gluc和其他產(chǎn)物。NNK在動物體內(nèi)主要有三條代謝途徑:碳基還原反應(yīng)、吡啶氮氧化反應(yīng)和a2羥化反應(yīng)NNKrequiresmetabolicactivationtoexertitscarcinogeniceffects(3).MetabolicactivationofNNKoccurslargelyviacytochromeP450catalyzedhydroxylationofthecarbonatomsadjacenttothen

9、itrosomoiety(i.e.,Rhydroxylation)oNNK-N-oxideoP450-MediatedHydroxyl呂tionNNINAIL0oa-MethyleneHydroxylation嚴(yán)OHhi3c-nnohCH2OHch2oa-MethylHydroxylation；0OHOPBDNA(/-methylguanine7-metliylguanine/-methylthymineNNKrequiresmetabolicactivationtoexertoN-N-OHOHN=NO-pyridyloxobutylguanine7-pyridyloxobutylgiiani

10、ne.V*-nvnclvlAk門hiitvlunsmmINHPRitscarcinogeniceffects.MetabolicactivationofNNKoccurslargelyviacytochromeP450-catalyzedhydroxylationofthecarbonatomsadjacenttothenitrosomoiety(i.e.,a-hydroxylation).AscanbeseeninSchemel,hydroxylationofthea-methylenea-hydroxynitrosaminemethanediazohydroxidecarbonofNNKg

11、eneratesanunstablethatspontaneouslydecomposestoandOPB.Methanediazohydroxide,orthemethyldiazoniumion,reactswithDNAbasestoformmethyladductssuchasO6-mGand7-mG.Hydroxylationofthea-methylcarbonofNNKgeneratestheunstablemetabolite4,whichspontaneouslybreaksdownto5andformaldehyde(Scheme1).Thediazoniumion6for

12、medfromthispathwayisapyridyloxobutylatingagent;itreactswithDNAbasestoformpyridyloxobutyladductsorwithwatertoformHPBapreponderanceofhydroxylationandthe3HcexperimentaldatainA/Jmiceimplicatesa-methylenesubsequentformationofO6-mGascrucialfactorsforNNKtumorigenicityNNAL-N-OXIDE使用A/J老鼠收集實(shí)驗(yàn)數(shù)據(jù)很大優(yōu)勢是可以得出a-met

13、hylenehydroxylation（a-亞甲基羥基化）和隨后產(chǎn)生的O6-mG（6-甲氧基鳥嘌吟）是NNK致癌的關(guān)鍵！TheinitialP450-mediatedmetabolicactivationofNNKisclearlyanimportanteventinthegenerationofamethylatingspecies,theformationofapromutagenicbase,and,ultimately,thedevelopmentofA/Jmouselungtumors.NNK的代謝最初以P450為媒介是甲基化的重要步驟，是導(dǎo)致A/J小鼠產(chǎn)生肺癌腫瘤的基礎(chǔ)和最終原因！

14、在!KTR&T人類原發(fā)性肺腺癌中存在,0U基因密碼子F!的突變，但其他類型肺腫瘤中幾乎未發(fā)現(xiàn)過=F,!K,!&?。這些突變在吸煙者和被動吸煙者中比在不吸煙者中更常見，表明這些突變可能是由煙草煙氣中的某種成分引起在!KTR&T人類原發(fā)性肺腺癌中存在v,0U基因密碼子F!的突變，但其他類型肺腫瘤中幾乎未發(fā)現(xiàn)過=卩,!K,!&?。這些突變在吸煙者和被動吸煙者中比在不吸煙者中更常見，表明這些突變可能是由煙草煙氣中的某種成分引起的=!#?。最常觀察到的突變是CCV!VCV,非常典型，占密碼子F!突變的#T，其次是CCV!CV（!T）和CCV!CVV（F&T）。C!V突變的普遍性導(dǎo)致這樣一種推測，這些突變

15、是起因于苯并芘，后者可通過二醇氧化物代謝活化途徑誘發(fā)這些突變=!#?。然而，C!V突變也可由;；vOE（;實(shí)施B甲基羥基化后與醋酸形成的酯）誘發(fā)研究表明，異硫氰酸鹽抑制SK&U酶是其抑制;誘發(fā)小鼠肺腫瘤的主要機(jī)制=!$G%F?。這導(dǎo)致O#B7C的形成和腫瘤被抑制。當(dāng)被加至小鼠肺微粒中孵化時，S（WVX通過競爭和非競爭機(jī)制抑制；氧化InorderforNNKtoexertitscarcinogenicity,itmustbemetabolicallyactivated.ThemetabolicactivationofNNKinvolvesa-hydroxylationofthemethylorm

16、ethylenecarbon,leadingtotheformationofelectrophiles,whichcanpyridyloxobutylateandmethylateDNA,respectivelyNNK的代謝活化被認(rèn)為是誘發(fā)癌變的首要條件22DNAadductformationfromtobacco-specificN-nitrosaminesStephenS.Hecht)UnilersityofMinnesotaCancerCenterBox806,Mayo,420DelawareStreetSEMinneapolis,MN55455,USAThispaperwilldesc

17、ribeDNAadductformationfromtobacco-specificnitrosamines.AlmostallstudiestodatehavebeencarriedoutwithNNK,NNAL,andNNN.Althoughsomeoftheadductsarehighlyspecificandcanbederivedonlyfromtobacco-specificnitrosamines,othershaveavarietyofsources.Inthispaper,metabolicactivationandDNAadductformationbyNNK,NNAL,a

18、ndNNNwillbesummarized.AnextensivereviewoftheliteratureonthistopichasrecentlybeencompletedFig.3.LntemiediatesandproductsformeduponsolvolysisofetoxTiiethylmtrosamuioJ-1-(3-pyndyl)-1-butanone(NNK-oAimilafila，cnPb曲apecursorto曲論丫電珂幽囲ijDiagohydroxide(CNPB).WlienY=OH,8ringopensto5.3yieldsdiazoniumion4which

19、hasthreefates:reactionwithnucleophilesY:.producing5,formationofthecyclicoxoniumion6,orlossofN2andH+yieldingthea,卩-unsaturatedketone7.Thelattertworeactwithnucleophilestoformproducts8and9.ExtensivestudiesconclusivelydemonstratethatthemajorDNAadducts.formedbythispathwayinvitroandinvivo,accountingforatl

20、east50%oftheDNAbind-ing,releasestheketoalcoholHPBFig.2.uponacidorneutralthermal,butnotbasehydrolysis.Thisadducts.isproducedviaintermediates3,4and/or6butnotbyHPBitself.TheHPBreleasingadducts.havedifferingstabilitiesinDNA,beingreleasedinatriphasicmannerNNK羰基還原反應(yīng)的主要催化劑不是細(xì)胞色素P450，而是11B-羥基類固醇脫氫酶，它是一種微粒體酶

21、，主要作用是將活潑的11-羥基糖皮質(zhì)激素轉(zhuǎn)化為不活潑的11-羰基式吡啶-N-氧化反應(yīng)吡啶-N-氧化反應(yīng)只在體外實(shí)驗(yàn)中被發(fā)現(xiàn)，根據(jù)實(shí)驗(yàn)動物種類和組織的不同，NNK產(chǎn)生N-氧化產(chǎn)物也不同。NNK在大鼠和小鼠肺微粒體中主要代謝生成NNKN-oxide，而在未經(jīng)預(yù)處理的大鼠肝微粒體、小鼠肝微粒體和大鼠鼻粘膜微粒體中則僅為次要反應(yīng)或不能被檢測到。吡啶-N-氧化反應(yīng)依賴細(xì)胞色素P450的催化。在動物組織體外實(shí)驗(yàn)中,CYP4502B1主要表現(xiàn)出催化NNK形成NNKN-oxide的活性還有實(shí)驗(yàn)表明CYP4503A4在人肝微粒體中有催化作用POB-DNA加合物還會影響O6-烷基鳥嘌吟-DNA烷基轉(zhuǎn)

22、移酶(AGT)對DNA的修復(fù)作用。AGT是一種重要的DNA修復(fù)酶，它可通過復(fù)原DNA鏈上烷化劑導(dǎo)致的鳥嘌吟O6位烷基化而達(dá)到修復(fù)DNA的目的oA:Oa-MedGuo;B:7-MedGuo;C:O4-MedThd;D:Oe-(POB-l-yl)dGuo;E:7-(POB-l-yl)dGiio;F:N2-(POB-l-yl)dGu0；G:N2-(POB-2-yl)dGtio;H:O2-(FOB-1-yl)dCyd;I:O+-(FOB-1-yl)dThdhowever,evidencehasaccumulatedthat1,4-phenylenebis(methylene)selenocyanate

23、(p-XSC)isabletopreventtheinitiationphaseofcarcinogenesiscausedbyDMBAandNNKbyinhibitingtheformationofDNAadducts.(Effectsofdietaryl,4-phenylenebis(methylene)selenocyanateon4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone-inducedDNAadductformationinlungandliverofA/JmiceandF344rats)Cardnogenesisvol.17no.4

24、pp.749-753,19964hoursLiver-11escvaaeuvoE=L96hoursNNKFig,LEffectsofXSCandxliumseleniteonlevelsofNIVK-inducedtX-methylguanineinheliverundlungsofA/Jmice.為201510呂oNNK+5ppmSeleniteNNK+5ppmp-XSCNNK+15ppmp-XSCWhileSohnetal.havedemonstratedthatp-XSC（苯二甲硒氰）pretreatmenthasnoeffectonphaseIenzymessuchasP4501A1,

25、2B1,2E1and3A4intheliverofmaleF344ratsandoffemaleSprague-Dawleyrats,theeffectsofp-XSContheinductionofsuchenzymesinextrahepatictissues,suchasthelung,havenotbeenstudied（通過膳食補(bǔ)充）Bothdosesofp-XSCinhibitedtheformationof（AmGuaand7-mGuainmouseliver.Thehighestinhibitionof06-mGuawasobservedwithp-XSCatthe15p.p.

26、m.Sedose.InhepaticDNA,dietarysupplementationofp-XSCatthislevelresultedin69.5%inhibitionofthislesionafter4hand73.8%after96h(Figure1)SuppressiveoligodeoxynucleotidesreducelungcancersusceptibilityinmicewithsilicosisCarcinogenesisvol.00no.00p.1of6,2014doi:10.1093/carcin/bgu005AdvanceAccesspublicationJan

27、uary8,2014KineticsofO6-Pyridyloxobutyl-2-deoxyguanosineRepairbyHumanO6-alkylguanineDNAAlkyltransferaseBiochemistry2013,52,4075-4088WefoundthatHBECcellswerecapableofremovingO6-POB-dGlesions,andtherepairratesweresignificantlyreducedinthepresenceofanAGTinhibitor(O6-benzylguanine).O6-Alkylguanine-DNAalk

28、yltransferase(AGT)proteincandirectlyremovetheO6-alkylgroupfromO6-alkylguaninesinDNA,restoringnormalguanine.AGTproteinbindstotheminorgrooveofDNAviathehelix-turn-helixmotif,inducingflippingofO6-Alk-dGoutoftheDNAhelixtoentertheproteinactivesite.Cysteine-145thiolwithintheactivesiteoftheAGTproteinisdepro

29、tonatedviainteractionswithotheractivesiteresidues,andtheresultingCys-145thiolateanionundergoesnucleophilicattackatthea-carbonoftheO6-alkylgroup,leadingtoitstransferfromDNAtotheprotein.6甲氧基鳥嘌吟DNA甲基轉(zhuǎn)移酶(AGT)蛋白能夠直接將6甲氧基基團(tuán)從6甲氧基鳥嘌吟移除，使鳥嘌吟恢復(fù)正常，AGT蛋白根據(jù)DNA的雙螺旋結(jié)構(gòu)直接鍵合在小溝中，誘導(dǎo)O6-Alk-dG從DNA螺旋中翻轉(zhuǎn)出去，從而進(jìn)入蛋白活性點(diǎn)，半胱氨酸的

30、巰基在AGT蛋白的活性點(diǎn)中通過與其他殘基活性點(diǎn)相互作用去質(zhì)子化，具有親核作用的半胱氨酸巰基陰離子在攻擊6甲氧基基團(tuán)上的a碳原子是6甲氧基基團(tuán)從Jdieme2.AGTRepairofO鼻PyridyloxobutylMGAdductsDNA上轉(zhuǎn)移到蛋白質(zhì)中。Onepossiblemechanismfortheincreasedmutagenesisatthesesitesinvolvesinefficientrepairoftobaccocarcinogen-inducedDNAadductssuchasO6-Me-dGandO6-POB-dG,leadingtotheiraccumulatio

31、natmethylatedCpGsequences.DeterminationofDNAMeltingTemperatures.DNAduplexescontainingsite-specificO6-POB-dGadducts(3nmol)weredissolvedinsodiumphosphatebuffer(10mM,pH7.0)containing50mMsodiumchloride(9.7“MDNA).DNAmeltingtemperatureswereobtainedwithaVarianCary-100BioUVvisiblespectrophotometerusingatemp

32、eraturegradientbetween30and90C.Temperatureincrementsordecrementsof0.5C/minwereused.Theexperimentwasrepeated46timestodetermineDNAmeltingtemperaturesusingCaryWinUVThermalsoftwareComparisonoftheChemopreventiveEfficaciesof1,4-phenylenebis(methylene)selenocyanateandSelenium-EnrichedYeaston4-(Methylnitros

33、amino)-1-(3-pyridyl)-1-butanoneInducedLungTumorigenesisinA/JMouseArunangshuDas,DhimantDesai,BrianPittman,ShantuAmin&KaramInbiochemicalstudies,p-XSCwasshowntosignificantlyinhibitformationofO6-methylguanine(O6-MG)and7-methylguanine(7-MG)inthelungsandliversofmicetreatedwithNNK.NNKwaspurchasedfromChemsy

34、nScienceLaboratories(Lenexa,KS).BenzylmorpholineAnalogsasSelectiveInhibitorsofLungCytochromeP4502A13fortheChemopreventionofLungCancerinTobaccoUsersReceived:14January2013/Accepted:2April2013/Publishedonline:12June201Biochemicalapplicationsofultrathinfilmsofenzymes,polyionsandDNAReceived(inCambridge,U

35、K)15thJune2007,Accepted3rdAugust2007FirstpublishedasanAdvanceArticleontheweb30thAugust2007DOI:10.1039/b709121bConstructionbeginsbyadsorbinganinitiallayerofchargedpolyionfromsolutionontoanoppositelychargedsolidsurface.Looselyboundpolyionsareremovedbywashingwithwater,thenasecondlayerofpolyionsoftheopp

36、ositechargetothefirstisadsorbed.起初通過吸附通過靜電力結(jié)合與固體表面上結(jié)合的聚離子層上，用水將松散的聚離子洗掉，然后帶相反電荷的第二層聚離子再吸附第一層。Hereinwepresentashortaccountofourbiochemically-relatedapplicationsutilizingLbLfilms,firsttomakestablefilmsofenzymesandpolyionsforbiocatalysis,andsecondtomakefilmsofDNA,metabolicenzymesandpolyionsfortoxicitys

37、creeningLvovandIspecificallywishedtoapplytheLbLmethodtofacilitatedirectelectrochemicalactivationofmetabolicenzymesonsolidelectrodesAkeyistoplacetheproteininafilmoronasurfacethatprotectsitfromdenaturingandfoulingtheelectrodeAsecond,non-stereoselectivepathwayislikelytoinvolveolefinoxidationbyaperoxylr

38、adicalonanamino-acidresidueattheproteinssurface.MajoradvantagesincludegreatlyenhancedenzymestabilityandthetinyamountofenzymerequiredTheLbLfilmnanoreactoronthesensorsynthesizesreactivemetabolitesinthevicinityoflargeconcentrationsofDNAinthefilm.TherateofDNAdamagefrommetabolite-nucleobaseadductformationisameasureofrelativegenotoxicity,andcanbedetec