An automated Fpg-based FADU method for the detection of oxidative DNA lesions and screening of antioxidants

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An automated Fpg-based FADU method for the detection of oxidative DNA lesions and screening of antioxidants

Nathalie Müller

^a,1

, Maria Moreno-Villanueva

^a,1

, Arthur Fischbach

^a

, Joachim Kienhöfer

^a

, Rita Martello

^a

, Peter C. Dedon

^b,c

, Volker Ullrich

^a

, Alexander Bürkle

^a,∗,2

, Aswin Mangerich

^a,b,∗∗

aUniversityofKonstanz,MolecularToxicologyGroup,DepartmentofBiology,D-78457Konstanz,Germany

bDepartmentofBiologicalEngineering,MassachusettsInstituteofTechnology,77MassachusettsAvenue,Cambridge,MA02193,UnitedStates

cCenterforEnvironmentalHealthScience,MassachusettsInstituteofTechnology,77MassachusettsAvenue,Cambridge,MA02193,UnitedStates

Keywords:

DNAdamage FADU

Genotoxicitytesting Antioxidants Highthroughput Massspectrometry

a b s t r a c t

Theoxidationofguanineto8-oxo-2-deoxyguanosine(8-oxo-dG)isoneofthemostabundantandbest studiedoxidativeDNAlesionsandiscommonlyusedasabiomarkerforoxidativestress.Overthelast decades,variousmethodsforthedetectionofDNAoxidationproductshavebeenestablishedandopti- mized.However,someofthemlacksensitivityorarepronetoartifactformation,whileothersare time-consuming,whichhamperstheirapplicationinscreeningapproaches.Inthisstudy,wepresenta formamidopyrimidineglycosylase(Fpg)-basedmethodtodetectoxidativelesionsinisolatedDNAusing amodiﬁedprotocoloftheautomatedversionoftheﬂuorimetricdetectionofalkalineDNAunwinding (FADU)method,initiallydevelopedforthemeasurementofDNAstrandbreaks(Moreno-Villanuevaetal., 2009.BMCBiotechnol.9,39).TheFADU-FpgmethodwasvalidatedusingaplasmidDNAmodel,mimick- ingmitochondrialDNA,andtheresultswerecorrelatedto8-oxo-dGlevelsasmeasuredbyLC–MS/MS.

TheFADU-FpgmethodcanbeappliedtoanalyzethepotentialofcompoundstoinduceDNAstrandbreaks andoxidativelesions,asexempliﬁedherebytreatingplasmidDNAwiththeperoxynitrite-generating moleculeSin-1.Moreover,thismethodcanbeusedtoscreenDNA-protectiveeffectsofantioxidantsub- stances,asexempliﬁedhereforasmall-molecule,i.e.,uricacid,andaprotein,i.e.,manganesesuperoxide dismutase,bothofwhichdisplayedadose-dependentprotectionagainstthegenerationofoxidativeDNA lesions.Inconclusion,theautomatedFADU-Fpgmethodoffersarapidandreliablemeasurementforthe detectionofperoxynitrite-mediatedDNAdamageinacell-freesystem,renderingitanidealmethodfor screeningtheDNA-protectiveeffectsofantioxidantcompounds.

1. Introduction

Avarietyofreactiveoxygenandnitrogenspecies(ROSandRNS) aregeneratedinbiologicalsystems,eitherendogenouslyfromcel- lularmetabolismand inﬂammatoryreactionsorexogenouslyby avarietyofchemicalsorionizingradiation(Delaneyetal.,2012;

Kasaietal.,1986).ROSandRNScanreactwithDNAmolecules, resultinginawidespectrumofdamageproducts(Delaneyetal., 2012;Tretyakovaetal.,2012).Duetothelowoxidation poten- tialofguanine,deoxyguanosine(dG)oxidationproducts,suchas

∗Correspondingauthorat:MolecularToxicologyGroup,DepartmentofBiology, UniversityofKonstanz,D-78457Konstanz,Germany.

∗∗Correspondingauthorat:MolecularToxicologyGroup,DepartmentofBiology, UniversityofKonstanz,D-78457Konstanz,Germany.Tel.:+4907531884067.

E-mailaddresses:alexander.buerkle@uni-konstanz.de(A.Bürkle), aswin.mangerich@uni-konstanz.de,amang@mit.edu(A.Mangerich).

1 Theseauthorscontributedequally.

2 SharedSeniorAuthorship.

8-oxo-2-deoxyguanosine(8-oxo-dG)and theformamidopyrimi- dineFapy-dG,areamongthemostprominentDNAlesions(Delaney etal.,2012;NeeleyandEssigmann,2006).Althoughefﬁcientrepair mechanisms for oxidative DNA lesions exist, i.e., baseexcision repair(BER),theirformationcanaffecttheefﬁciencyoftranscrip- tional processes (Khobta et al., 2010; Kitsera et al., 2011) and suchlesionscan beboth cytotoxic and mutagenic(Christmann etal.,2003;Delaneyetal.,2012;Shibutanietal.,1991).Inaddi- tiontothenucleargenome,mitochondrialDNA(mtDNA)canbe damaged by ROSand RNS generated duringmitochondrial res- piration (Kazak et al., 2012). Thus, superoxide radicals (O₂^−•) lackingfromtheelectrontransportchaincanreactwithdiffusible nitricoxide(NO)producedbynitricoxidesynthasestogenerate ONOO⁻ and severalhighly reactivemetabolites.In this respect, itwasshownthatmutationsinmtDNAplayaroleindegenera- tivediseasesofthecentralnervousandendocrinesystems,heart, kidneyandskeletalmuscle(Wallaceetal.,1998,1999).Ingen- eral,theassessmentof oxidativeDNA damagehasbeenwidely usedasa biomarkerof oxidativestressinexperimentalaswell

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-250512

https://dx.doi.org/10.1016/j.tox.2013.05.006

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16

asin epidemiological studies (Dizdaroglu, 2012; Ravanatet al., 2012).

SeveralmethodsforthedetectionofoxidativeDNAlesionshave beendeveloped,butresultsvaryconsiderablydependingonthe differentanalyticalsystemsand thesourceofDNAused(Anson etal.,2000;Ravanat,2012;RossnerandSram,2012).

High-performanceliquid chromatographycoupledwithelec- trochemicaldetection(HPLC/ECD)hasbeenoneoftheﬁrstmethods usedfor8-oxo-dGmeasurement(Floydetal.,1986;Ravanat,2012).

Themethodisbasedonthefactthatthelowoxidationpotential of8-oxo-dGallows speciﬁcand sensitivedetection in theone- electron oxidation mode. However, its disadvantages include a risk for the inductionof highlevels of artifactuallesion and a lackofinternalcalibration(Ravanat,2012).Methodsbasedongas chromatographycoupledtomass spectrometry(GC/MS)havea tendencytooverestimate 8-oxo-dGlevelsby10- to 50-fold,in partduetotheacidicconditionsoftheDNAhydrolysisaswellas hightemperatureduringthederivatizationstep(Ravanat,2012).

Pre-puriﬁcationofsamplesbyHPLC,orloweringofthederivatiza- tiontemperatureoradditionofantioxidantshavebeensuggested toavoidthistypeofartifact(HalliwellandDizdaroglu,1992).At present,oneof themostspeciﬁcand sensitivemethods forthe detectionofawide spectrumofDNAoxidation productsrepre- sentsreversephaseHPLCcoupledESItandemmassspectrometry (LC–MS/MS)whichdoesnotrequirederivatizationpriortomass spectrometricanalysis(Cadetetal.,2002;Mangerichetal.,2012;

Ravanat,2012;Taghizadehetal.,2008;Tretyakovaetal.,2012).

Incontrasttochromatographicmethods,immunologicalmeth- odsdemandlittlesamplepreparation andallowtheanalysisof apanelofsamplessimultaneously(RossnerandSram,2012).For example,ELISAtestarewidelyusedasafastandcheapmethodfor detecting8-oxo-dGinhumanﬂuids(Cookeetal.,2006).However, theusageofsuchimmuno-chemicaltechniquesremainslimited, becauseofweakantigenicityandchemicalselectivityoftheanti- bodiesused, assigniﬁcantcross-reactivitywiththeundamaged nucleobaseshasbeendescribed(Mitchelletal.,2002;Serranoetal., 1996).

Finally,thecometassay(orsinglecellelectrophoresisassay), alkalineunwindingandalkalineelutiontechniquescanbeused todetectawidespectrumDNAlesions(GedikandCollins,2005;

Ravanat,2012; Wood et al., 2010). These methods requireuse ofglycosylases, suchasformamidopyrimidine glycosylase(Fpg) or endonuclease III (EndoIII) that specifically convert the oxi- dizedandmodifiedpyrimidineandpurinebasesintoDNAstrand breaks,whichin turncanbedetectedina very sensitiveman- ner(Pflaumet al.,1997; Ravanat,2012).Differentvariations of thesemethodshavebeendeveloped,includingcomet-basedassays thatenablehigh-throughputassessmentofcellularDNAdamage (Wood etal.,2010)and methodsthatcombinethecometassay andinsitufluorescenthybridization(FISH)todetectoxidizedbases atthesequencelevel(Shaposhnikovetal.,2011).Alimitationof glycosylase-basedmethodsisthattheyrelyonthecompleteenzy- maticprocessingofalldamagedbasespresentandthesubstrate specificityoftheglycosylase(Ravanat,2012).Forexample,Fpgis notentirelyspecificfor8-oxo-dGonly,asitwasreportedtorec- ognizealsootheroxidationproductssuchasFapy-dGandFapy-dA sites(Pougetetal.,2000)aswellasalkylationdamage(Speitetal., 2004).

Similartothecometassay,theautomatedFADUassayallows thequantiﬁcationofDNAstrandbreaksinwholecellsinahigh- throughputmanner(Brunneretal.,2012;Debiaketal.,2011;Garm etal.,2013;Kappesetal.,2008;Mangerichetal.,2010;Moreno- Villanuevaetal.,2009,2011).Thismethodisbasedonapartial denaturation/unwindingofdoublestrandedDNAundertime-and temperature-controlledalkalineconditionsina96-well-platefor- matusing an automated liquid handling device. Subsequently,

addition of the ﬂuorescent probe SYBR Green is employed to quantifythestateofDNAunwinding,i.e.,ﬂuorescenceintensities inverselycorrelatedwiththenumberofDNAstrandbreaks.The objectiveofthecurrentstudywastodevelopanFpg-basedFADU methodtodetectoxidativeDNAdamage ina highthrough-put mannerbyadaptingoftheautomatedFADUassayandtovalidate thismethodbycomparingitwithresultsfromLC–MS/MSanalysis.

2. Materialsandmethods

AllchemicalswereofanalyticalgradeandobtainedfromSigma–Aldrich,Fluka orMerck.

2.1. FADU-Fpgmethod 2.1.1. PlasmidDNAtreatment

AsaDNAmodelsystem,a14-kbpplasmid(pAcHLT-A-His6)wasampliﬁed inEscherichiacoliDH5cellsandextractedusingaDNApuriﬁcationGigaPrepKit (Qiagen).EachDNAsamplewaspreparedintriplicatescontaining104␮gofDNA.

SamplesweresupplementedwithuricacidorMnSOD(AbFrontier)andtreatedwith 100–400␮MfreshlypreparedSin-1(Calbiochem)for40minat30^◦C.Sampleswere thendividedintoaliquotsof4␮gand100␮gofDNAforFADUandLC–MSanalysis, respectively.

2.1.2. Fpgtreatment

Eachsamplewasincubatedwith8UFpgandNEB1Buffer(NewEnglandBiolabs) for30minat30^◦C.Samplesweredilutedin280␮lFADUsuspensionbuffer(250mM meso-inositol,10mMsodiumphosphate,1mMMgCl,pH7.4)andquadruplicates weretransferredintoa96-wellplatewhichwaspositionedintotheFADUpipetting robot.

2.1.3. AutomatedstepsoftheFpg-FADUassay

Theliquidhandlingdeviceaswellasitspositioninghavebeendescribedpre- viously(Moreno-Villanuevaetal.,2009).Theoriginalprotocolwasadaptedto optimizethemethodforthedetectionofFpg-generatedstrandbreaksinacell-free system.Thetemperatureofthecoolingdevicewasmaintainedat2^◦Cthroughout theentireexperiment.First,70␮lofFADUureabuffer(9Murea;10mMNaOH;

2.5mMcyclohexyl-diamine-tetraacetate;0.1%sodiumdodecylsulfate)weredis- pensedintoeachwell.Immediatelythereafter,70␮lofalkalinebuffer(0.425parts FADUureabufferin0.2MNaOH)wasadded.Thesubsequentincubationstepof theoriginalprotocolwasomittedtoavoidtotalunwindingoftheplasmid.Avol- umeof140␮lofneutralizationbuffer(14mM␤-mercaptoethanol;1Mglucose)was addedatarateof200␮l/s.Finally,156␮lofSybrGreen(MoBiTec)diluted1:8.33in waterwasaddedandsamplesweremixedbypipettingavolumeof400␮lupand downatarateof100␮l/s.Fluorescenceintensitywasmeasuredina96-well-plate ﬂuorescencereaderat492nmexcitation/520nmemission.

2.2. 8-oxo-dGdetectionbyLC–MS/MS

2.2.1. DNAhydrolysisanddephosphorylationofnucleotides

DNAwasdesiccatedundervacuumandhydrolyzedtonucleosidesbyacombi- nationofnucleaseP1,DNaseI,phosphodiesteraseI(USB-Affymetrix,SantaClara, CA),andalkalinephophataseinthepresenceofdeaminaseinhibitorsandantioxi- dantsasdescribedpreviously(Taghizadehetal.,2008).Enzymeswereremovedby centrifugationat16,400×gfor20minthrougha10,000MWcut-offspinﬁlter(Pall).

2.2.2. ReversephaseHPLCpre-puriﬁcation

ALC-10ATHPLCsystemfromShimadzuwasusedfor8-oxo-dGpre-puriﬁcation equippedwithaPhenomenexSynergi4-␮mHydro-RPC1880A(250mm×4.6mm) column.Asolventgradientofacetonitrilein8mMammoniumacetatewassetata ﬂowrate0.7ml/min(forgradientcompositionseeSuppl.Table1).Thedetectionwas performedusingUV–visspectroscopyat260nm.The8-oxo-dGcontainingfractions werecollectedataretentiontimeofapproximately37–42min.

2.2.3. LC–MS/MSanalysis

8-oxo-dGquantiﬁcationwasperformedwithanHPLC(Waters2695Separa- tionsModule)coupledtoatriplequadrupolemassspectrometer(WatersMicromass QuattroMicro).AreversedphaseHypersil-GoldcolumnC18(ThermoScientiﬁc;

150mm×2.1mm;3␮mparticlesize)wasusedandelutedisocraticallywith98.5%

ofH2Osupplementedwith0.1%aceticacidand1.5%ofacetonitrilesupplemented with0.1%aceticacidataﬂowrateof0.3ml/min.Detailedchromatographicsett- ingsarelistedinSuppl.Table2.22.5␮lofeachsamplewasanalyzedinasingle LC/MS–MSrun.ThemassspectrometerwasusedintheESI⁺MS/MSmodewith settingsindicatedinSuppl.Table3.Fordetection,themultiplereactionmonitoring (MRM)modewasused,andtransitionofm/z284.0→168.0for8-oxo-dGwasmoni- tored.Allmeasurementswereperformedintechnicalduplicates.Theareafromone samplewasconvertedintoamountinfmolusingexternalcalibrationcurves.The averagewascalculatedfromthetechnicalduplicatesinfmol.

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Fig.1. WorkﬂowoftheFADU-Fpgmethod.

3. Results

3.1. AnalysisofSin-1-inducedDNAdamageinaplasmidDNA model

HerewepresentanovelFpg-basedmethodtodetectoxidative DNAdamageinacell-freesystem(Fig.1),usingamodiﬁedprotocol oftheFADUassay(Debiaketal.,2011;Moreno-Villanuevaetal., 2009,2011).

A14-kbpplasmid,mimickingthemtDNAmolecule,wastreated withtheONOO⁻generator3-morpholinosydnonimine(Sin-1).Sin- 1 is thought tomimic thephysiological situationin cells as it decomposestoproduceequimolaramountsofNO^•andO₂^−•and thestable end-productSin-1C(Fig. 2).Thisresultsin a contin- ualfluxofONOO⁻ overextended periodsresultinginoxidative andnitrosativeDNAdamage(Fig.2)(Cuietal.,2013;Dedonand Tannenbaum,2004;Feelischetal.,1989;Hoggetal.,1992).Itwas shownpreviouslythattheDNAdamageprofilegeneratedbySin- 1mainlycomprisesFpg-sensitivelesionsandtoa lesserextend alsoDNAsinglestrandbreaks(Epeetal.,1996).ToconvertSin- 1-inducedoxidativeDNAlesionsintosingle-strandbreakthatcan bedetectedbytheFADUmethod,theoriginalFADUprotocolwas adaptedbyincubatingSin-1-treatedDNAwithFpg(Fig.1).There- after,samplesweredilutedinasuspensionbuffer,distributedin a96-wellplateandsubjectedtothesuccessiveautomatedaddi- tionofsolutionsbytheliquidhandlingdevice(Fig.1).ThenSYBR Greenfluorescencewasdeterminedusinga96-well-platefluores- cencereader.TheoriginalFADUprotocolwasfurthermodifiedto adaptthemethodfortheapplicationtodetectFpg-sensitiveDNA lesions.Thedilutionofthealkalinebuffer,thesuppressionofthe alkalineunwindingstepandthemaintenanceofaconstanttem- peratureat2^◦Cwerenecessarytopreventcompleteunwinding oftheDNAandtoallowasensitivedetectionoftheFpg-induced strandbreaks.Thetotalhands-ontimerequiredforthesestepsis lessthananhourandthenumberofsamplesanalyzedconcurrently isabout20inquadruplicates.Throughputcanbefurtherenhanced

byincreasingthenumberof96-wellplatesprocessedatthesame timebythepipettingrobot.

TodetermineoptimalworkingconcentrationofFpg,samples wereincubatedwithincreasingconcentrationsofFpgafterDNA had beentreated with400␮MSin-1 (Fig. 3A). The number of Fpg-sensitivelesionsdetectedinthepresenceofSin-1increased until a threshold wasreachedstarting at an Fpgconcentration of 0.1U/␮l. This concentration, at which all Fpg-sensitive sites were cleaved, wasused for all subsequent FADU experiments.

It isimportanttonotethatSin-1 onlyaffectedDNAunwinding in thepresenceof Fpg(Fig.3B),thus implyingthat inoursys- tem,Sin-1generatesmainlyoxidativeDNAlesionsanddoesnot directly generate DNA singlestrand breaks in large quantities.

ThismayberelatedtothefactthatONOO⁻ reactswithCO₂ to formONOOCO2−(DedonandTannenbaum,2004).WhileONOO⁻ itself,mainlycausesdeoxyriboseoxidation(i.e.,strandbreaks)and someguanineoxidation,ONOOCO₂⁻causesmainlyguanineoxi- dation(DedonandTannenbaum,2004).ItislikelythattheSin-1 concentrationsasusedinourexperimentalsettingarenotusing upthedissolvedCO₂ inthebuffer,resultinginthegenerationof ONOOCO₂⁻.Thisinformationisofhighrelevanceasotheroxidiz- ingagents,suchasH2O2,havebeenshowntodirectlygenerate DNAsinglestrandbreaksalongsidetooxidativedamage,render- ingthespeciﬁcdetectionofoxidativeDNAlesionsinthesecases particularlybiased(unpublishedobservation).

TostudytheeffectofincreasingSin-1concentrationsonthe generationofoxidativeDNAlesions,plasmidDNAwastreatedwith Sin-1concentrationsupto400␮M.Fpg-sensitiveDNAlesionssig- niﬁcantlyincreasedwithSin-1concentrationsinadose-dependent manner(Fig.4A).Itwasanessentialpartofthisworktovalidate theFADU-Fpgmethodby comparingittoanotherstate-of-the- art analytical method used for the detection of DNA oxidation products.Tothisend,8-oxo-dGlevelswereanalyzedinthesame samplesinparallel byLC–MS/MS. Asimilarapproach wasused previouslytocalibrateanFpg-basedCometassay(Pougetetal., 2000).Previousstudiesdemonstratedthat8-oxo-dGlesionsmake

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18

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<c ^{1 \}

II!

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N- N 0 N- N 0

/ \ _ / / \ _ /

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ONOOCOi

nitrosoperoxycarbonate

··!r'> ~

... A)--\

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ONOO peroxynitrite

dO

".,~~>=0 ... A)--\

8 oxo dO dR and other damage

products

.._ _ _ _ _ _ _ _ _ No·

Sin IC

Fig. 2. Sin-1-induced formation of8-ox~G in DNA.

up of 75% of the total number Sin-1-induced Fpg-sensitive DNA damage sites (Epe et aL, 1996), suggesting that 8-oxo-dG formation gives a conservative estimate for the number of Fpg-sensitive guanine oxidation events. In our study basal levels of 8-oxo-dG, as determined by LC-MS/MS, were in the range of 1-10 lesions per 10⁶bp which is consistent with published reference values (Ravanat, 2012; Taghizadeh etal., 2008). The resulting curve ofSin- 1-induced 8-oxo-dG lesions confirms the dose-dependent increase of oxidative lesions observed in the FADU-Fpg method (Fig. 48).

Results obtained from the FADU-Fpg and the LC-MS/MS analy- sis showed similar sensitivity and a highly significant positive correlation (r2 of 0.998) (Fig. 4C) indicating that the FADU-Fpg method indeed detects oxidative DNA lesions in a sensitive and dose dependent manner.

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3.2. Scavenging ofSin-1 oxidation in a plasmid DNA model

The automated FADU-Fpg method requires little sample prepa- ration and offers a rapid and reliable measurement of oxidative lesions, thus making it a suitable method for screening the antioxidant properties of enzymes and small molecules.

To study the antioxidant properties of MnSOD, which catalyze the dismutation of superoxide into oxygen and hydrogenperoxide, plasmid DNA was supplemented with increasing concentrations of MnSOD (0.94-75 ng/~tl) prior to Sin-1 incubation. Both FADU-Fpg and LC-MS/MS measurements highlight the ability of MnSOD to scavenge ONoo- and concur in demonstrating a dose-dependent protection of the DNA against Sin-1-induced oxidation by MnSOD (Fig. SA and B). Again, a highly positive correlation between results

B 5'

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Fig. 3. (A) Fpg treatment leads to dose-dependent strand break formation in plasmid DNA treated with Sin-!. Plasmid DNA was treated with or without 400 ~M Sin-1 and then incubated with increasing concentrations of Fpg ( 4-800 mU/~1). (B) Samples treated without or with 400 ~M Sin-1 (without Fpg incubation). Experiments were performed in quadruplicates and data are expressed as means± SEM.

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FADU

0 100 200 300 400 0

10 20 30 40 50

*

Conc. Sin-1 [μM]

Decreaseinfluorescence[%]

LC-MS/MS

0 100 200 300 400 0

5 10 15

20 *

Conc. Sin-1 [μM]

Foldinductionof8-oxo-dG comparedtocontrol

Correlation FADU vs LC-MS/MS: Sin-1

0 2 4 6 8 10 12 14 16 0

10 20 30 40

50 r²=0.998

Fold induction of 8-oxo-dG compared to control

A B C

Fig.4.Sin-1inducesFpg-sensitiveDNAlesionsand8-oxo-dGinplasmidDNAinadose-dependentmanner.PlasmidDNAwastreatedwithincreaseinconcentrationsofSin-1 (50–400␮M).DNAdamagewasanalyzed(A)byFADU-Fpgand(B)byLC–MS/MS(8-oxo-dG).(C)Alinearregressionanalysiswasperformed.Samplesofeachexperiment weredistributedintoaliquotsforFADU-FpgandLC–MS/MSmeasurements,respectively.Datarepresentmeans±SEMof3independentexperiments.FADUexperimentswere performedintechnicalquadruplicates,LC–MS/MSmeasurementsintechnicalduplicates.r²representsthelinearregressioncoefﬁcient.Statisticalanalysiswasperformed byone-wayANOVAtestfollowedbyBonferroni’sposttest,*P<0.05.

frombothmethodswasobserved,withr²=0.89(Fig.5C),however,theFADU-Fpgmethodshowedhigherreproducibilitythan LC–MS/MSmeasurements.

Uricacidisproducedfromxanthinebyxanthineoxidase.Itis knowntobeapowerfulantioxidantthatisabundantlypresentin humanblood(Amesetal.,1981).Afterapre-incubationofplasmid DNAwithuricacidinconcentrationsupto100␮M,sampleswere treatedwith100␮MSin-1.BothFADU-FpgandLC–MS/MSmea- surementsrevealedadose-dependentdecreaseinSin-1-induced oxidativeDNAlesionsstartingatauricacidconcentrationof25␮M (Fig.6AandB).CompleteprotectionofDNArequireduricacidcon- centrationsabove50␮M.Valuesmeasuredbybothmethodsagain correlatedwellwithanr²=0.96(Fig.6C).

4. Discussion

The main drawback of available Fpg-based and LC–MS/MS- basedmethodsistherequirementofelaboratesamplepreparation andprocessingsteps.Wehavebeenabletoovercomethislimita- tionbyestablishinganFpg-basedmethodfordetectingoxidative DNA damagelesions inisolated DNA onthebasis of the highthroughput FADU method (Brunner et al., 2012; Debiak et al., 2011; Garmet al., 2013;Kappes et al., 2008; Mangerichet al.,

2010;Moreno-Villanuevaetal.,2009,2011).TheFADU-Fpgmethod wasvalidatedusingaplasmidDNAmodelmimickingmtDNAin combinationwiththeONOO⁻generatorSin-1(Fig.2).AnFpgpre- incubation step hasbeen added to the original FADU protocol thatallowsthedetectionofoxidativeDNAlesions,bygenerating strandbreaksthatcanbedetectedbytheFADUmethod.Wehave controlledthevalidityofthemethodbymeasuring8-oxo-dGlev- elsusingLC–MS/MSanalysiswhichallowsunambiguouschemical selectivity(Taghizadehetal.,2008).

AnotableadvantageofthemodifiedFADU-Fpgmethodover the HPLC-based methods is the quantity of DNA necessary for theanalysis.While25–50␮gisrequiredforLC–MS/MSanalysis, only 1␮gof plasmid DNA per measurement was sufficient for theFADU-Fpgmethod.Moreover,theLC–MS/MS-basedmethodis unfavorableforscreeningpurposes,becauseitisrathercostlyand time-consuming.Inparticular,samplesmustundergoanumberof criticalprocessingsteps,i.e.,digestion,dephosphorylation,filter- ing,vacuum-concentration,pre-purificationbyHPLC,andanalyte detectionbyLC–MS/MS.Also,samplesarepurifiedandanalyzed sequentiallybyHPLCandLC–MS/MS,demandingadditionaltime.

Foranumberof20samplesmeasuredinquadruplicates,atime periodofaround3daysisrequired.Incontrast,thesameamountof samplescanbemeasuredwiththeFADU-Fpgmethodinlessthan

FADU

0 5 10 15 20 25

0.1 1 10 100

0

* * *

Conc. MnSOD [ng/μl]

LC-MS/MS

1 2 3 4

0.1 1 10 100

0

Conc. MnSOD [ng/μl]

Correlation FADU vs LC-MS/MS: MnSOD

1.5 2.0 2.5 3.0 3.5 0

5 10 15 20 25 r²=0.89

A B C

Fig.5. MnSODprotectsDNAagainstSin-1-inducedDNAdamageinadosedependentmanner.PlasmidDNAwastreatedwith100␮MSin-1andsupplementedwithincreasing concentrationsofMnSOD.Oxidativedamagewasanalyzed(A)byFADU-Fpgand(B)byLC–MS/MS(8-oxo-dG).(C)Alinearregressionanalysiswasperformed.Samplesofeach experimentweredividedintoaliquotsforFADU-FpgandLC–MS/MSmeasurements.Datarepresentmeans±SEMof3independentexperiments.FADU-Fpgexperimentswere performedintechnicalquadruplicates,LC–MS/MSmeasurementsintechnicalduplicates.r²representsthelinearregressioncoefﬁcient.Statisticalanalysiswasperformed byone-wayANOVAtestfollowedbyBonferroni’sposttest,*P<0.05.

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20

FADU

0 5 10 15 20 25 30 35

1 10 100

0

****

Conc. uric acid [μM]

LC-MS/MS

1.0 1.5 2.0 2.5 3.0

1 10 100

0

* ***

Conc. uric acid [μM]

Correlation FADU vs LC-MS/MS: Uric Acid

1.0 1.5 2.0 2.5 3.0 0

10 20 30 40 r²=0.96

A B C

Fig.6. UricacidprotectsDNAagainstSin-1-inducedDNAdamageinadose-dependentmanner.PlasmidDNAwastreatedwith100␮MSin-1andsupplementedwith increasingconcentrationsofuricacid.Oxidativedamagewasanalyzed(A)byFADU-Fpgand(B)byLC–MS/MS(8-oxo-dG).(C)Alinearregressionanalysiswasperformed.

SamplesofeachexperimentweredividedintoaliquotsforFADU-FpgandLC–MS/MSmeasurements.Datarepresentmeans±SEMof3independentexperiments.FADU-Fpg experimentswereperformedintechnicalquadruplicates,LC–MS/MSmeasurementsintechnicalduplicates.r²representsthelinearregressioncoefﬁcient.Statisticalanalysis wasperformedbyone-wayANOVAtestfollowedbyBonferroni’sposttest,*P<0.05.

halfaday.ThefactthatintheFADU-Fpgmethodmanysamples canbeprocessedina96-wellplatesimultaneouslybytheauto- matedliquidhandlingdevicenotonlyincreasesthroughput,but alsoensuresequaltreatmentofsamples.

Despite the reported approximate speciﬁcity of Fpg toward DNAoxidationandalkylationdamage(Pougetetal.,2000;Speit etal.,2004), theside-by-sidecomparison oftheFADU-Fpg and theLC–MS/MSmethodsinourstudyresultedinhighlysigniﬁcant positivecorrelationsofFpg-sensitiveDNA lesionsand8-oxo-dG levels(Figs.4–6).ThisstronglyindicatestheabilityoftheFADU-Fpg methodtodetectoxidativeDNAdamage.

Oneimportantlimitationofourmethodisthat,sofar,itcan- notbeappliedtodetermineoxidativeDNAdamageincells,due tohighconcentrationsofureaintheFADUcelllysisbuffer,which isnecessaryforcompletecelllysis,butwhichinterfereswithFpg activity.Moreover,currentmethodsusedforextractionofgenomic andmtDNAfromcellsintroducesigniﬁcantlevelsofDNAstrand breaks,whichisincompatiblewithFADU-Fpgmeasurements.For thisreason,theFADU-Fpgmethodiscurrentlyrestrictedtothe plasmidDNAmodelasreportedinthisstudy.Furtheroptimization andadaptationsofthemethodareindicatedtoovercomethese limitations.

Ontheotherhand,theFADU-Fpgmethodinitscurrentstateis asimple,rapidandreliablehigh-throughputalternativeformea- suringrelativelevelsofoxidativeDNAdamageinplasmidDNA.A comparabletechniquethathasbeenusedformanyyearsusesiso- latedDNAfromthebacteriophagePM2incombinationwithDNA glycosylasesandelectrophoreticseparationofsupercoiled,circu- larandlinearDNA(Epeetal.,1993,1996).Thismethodallowseasy andreliabledetectionofawiderangeofDNAlesions,howeverin itspresentform,isnotavailableinanautomatedversion.Asan alternativetothismethod,theFADU-Fpgmethodisparticularly suitableforscreeningpurposes,e.g.,tostudyprotectiveeffectsof antioxidantsubstances,asourexperimentswithMnSODanduric aciddemonstrate(Figs. 5and6).Theprotective effectsofthese compoundsfollowcomplexchemicalmechanisms:Whileuricacid actsasageneralscavengerofROS(Amesetal.,1981),theprotec- tiveeffectofMnSODmayberelatedtoreduceddecompositionof Sin-1,duetoMnSOD-mediatedscavengingofsuperoxide(Feelisch etal.,1989),ordirectDNAprotectiveeffectsbybindingofMnSOD toDNA(Kienhoferetal.,2009).

Recentlythe valueof theFADU-Fpgmethod intoxicological andpharmaceuticalresearchhasbeendemonstratedin astudy

inwhichthebiochemicalmechanismsoftheneuroprotectiveand anti-inﬂammatoryeffectsofminocycline,asemisyntheticderiva- tiveoftetracycline,wereinvestigated(Schildknechtetal.,2011).In thisstudy,theFADU-Fpgmethod,whichisdescribedandvalidated hereindetail,wasusedtodeterminetheperoxynitrite-scavenging propertiesofminocycline.Theloweffectiveconcentrationof5␮M for 50% inhibition determined in that study corresponds with standardconcentrationspresentinbrainafterrepeatedoralintake, asdeterminedinclinicalstudies(Schildknechtetal.,2011).

Inconclusion,theFADU-Fpgmethodcombinesthesensitivity andreliabilityofanFpg-basedassayforthedetectionofoxidative DNAlesionswiththedecreasedoperationtimeandhighthrough- putof an automated procedure. For this reason, theFADU-Fpg methodrepresentsausefultoolforscreeningDNA-damagingand antioxidantpropertiesofsubstancesintoxicology,pharmacology, andinthefoodindustry,wherethedevelopmentofnutraceuticals iscurrentlyofhighpriority.

Conﬂictofinterest

Theauthorshavedeclarednoconﬂictofinterest.

Acknowledgments

ThisworkwassupportedbyGrantBU698/6-1fromtheDeutsche Forschungsgemeinschaft(DFG).FundingwasalsoprovidedbytheUS NationalInstitutesofHealth(GrantsES002109andCA026731).

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An automated Fpg-based FADU method for the detection of oxidative DNA lesions and screening of antioxidants