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High ThroughputMetabolicToxicityScreeningUsingMagneticBiocolloidReactorsandLC MS MS Anal Chem 2010 82 10172 10178 Introduction Drugs Pesticides FoodAdditives Cosmetics IndustrialChemicals EnvironmentalPollut ants humanliverandothertissues Lesstoxic excreteableforms DNA proteins andotherbiomolecules MetabolictoxicityorGenotoxicity Usingmagneticparticlescoatedwithcytosol microsome DNA lmsasbiocolloidreactorsina96 wellplateformatcoupledwithliquidchromatography massspectrometry Incorporationofbothmicrosomalandcytosolicenzymesinthe lmsprovidesabroadspectrumofmetabolicenzymesrepresentingarangeofmetabolicpathwaysforbioactivationofchemicals ReactivemetabolitesgeneratedviathisprocessaretrappedbycovalentlybindingtoDNAinthe lm TheDNAisthenhydrolyzedandnucleobaseadductsarecollectedusing ltersinthebottomforthe96 wellplateofanalysisbycapillaryliquidchromatography tandemmassspectrometry LC MS MS Themagneticparticlesfacilitatesimpleandrapidsamplepreparationandworkup Thismethodhasthepotentialforhigh throughputgenotoxicityscreening providingchemicalstructureinformationthatisComplementarytotoxicitybioassays ExperimentalSteps ProcedureforFilmPreparationThefollowingsolutionswereusedforfilmassembly salmontestesDNA 0 2mgmL 1in5mMTrisbuffer pH7 0 10mMNaCl poly diallyldimethylammoniumchloride PDDA 2mgmL 1 50mMNaCl 20mgmL 1totalproteinratlivermicrosomes in250mMsucrose andcytosol in50mMTrispH7 5 Thefollowingprotocolisdescribedintermsoftheamountofparticlesusedperreactionwell butisscalable Briefly 20 Lofmagneticparticles 20mgmL 1 wasdispersedin80 Lof5mMTrisbuffer pH7 0 5mMNaCl 100 LPDDAwasaddeddropwiselyfollowedbya20 minassemblytocoatthenegativechargedsurfacewithpositivelychargedpolyions Themagneticparticlesarepulledtowardsthemagnetandformapelletonthesideofthetube Supernatantwasdiscarded andparticleswerewashedwithTrisbuffertwicetoremovelooselyboundpolyionsanddispersedinto100 LofTrisbuffer Adropwiseadditionof40 Lmicrosomesorcytosolwasfollowed allowinga30 minassembly Thesamewashingstepswerefollowedaftereachlayer DNAfilmwascoatedastheoutmostlayerofthefilmbyadding200 Lmagneticparticledispersioninto200 LofDNAsolutionwitha20 minassembly ThemodifiedbioreactorsweredispersedinTrisbuffer pH7 0 toafinalvolumeof200 Landstoredat0 tilluse ReactionsusingmagneticPDDA DNAbiocolloidswithoutenzymeswererunsimultaneouslyinthe96 wellplateat37 forvarioustimeswithstyreneoxide SO themajormetaboliteofstyrene ProductionscansofN7 SO guanine m z272 andN3 SO adenine m z256 afterreactionusingmagneticPDDA DNAbiocolloidswithoutenzymeswith5mMstyreneoxidefor5min Fragmentationpatternsareillustratedas N7 SO guanineand N3 SO adeninesincethesearethetwomajoradductspresentingthehighestformationrates refermaintextfordetails Totesttheapplicabilityofenzymehydrolysistomagneticbiocolloidreactorsinthe96 wellplates biocolloidswithPDDA DNA lmswereenzymaticallyhydrolyzedfor17handthehydrolysatewasanalyzedbyLC MS MS demonstratethesuccessfulreleaseofthefournativeDNAnucleosidesfeaturingasignatureneutrallossofsugarmoiety 赴美生子好不容易终
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