The NAD⁺ precursor nicotinic acid improves genomic integrity in human peripheral blood mononuclear cells after X-irradiation

The NAD ⁺ precursor nicotinic acid improves genomic integrity in human peripheral blood mononuclear cells after X-irradiation

Kathrin Weidele

, Sascha Beneke

, Alexander Bürkle

MolecularToxicologyGroup,DepartmentofBiology,UniversityofKonstanz,Universitaetsstr.10,78457Konstanz,Germany

Keywords:

Humanperipheralbloodmononuclearcells (PBMC)

NAD⁺ DNArepair Genomicstability Micronucleusformation Nicotinicacid

a b s t r a c t

NAD⁺isanessentialcofactorforenzymescatalyzingredox-reactionsaswellasanelectroncarrierin energymetabolism.Asidefromthis,NAD⁺consumingenzymeslikepoly(ADP-ribose)polymerasesand sirtuinsareimportantregulatorsinvolvedinchromatin-restructuringprocessesduringrepairandepige- netics/transcriptionaladaption.InordertoreplenishcellularNAD⁺levelsaftercleavage,synthesisstarts fromprecursorssuchasnicotinamide,nicotinamideribosideornicotinicacidtomatchtheneedforthis essentialmolecule.Inthepresentstudy,weinvestigatedtheimpactofsupplementationwithnicotinic acidonrestingandproliferatinghumanmononuclearbloodcellswithafocusonDNAdamageandrepair processes.

WeobservedthatnicotinicacidsupplementationincreasedNAD⁺levelsaswellasDNArepairefficiency andenhancedgenomicstabilityevaluatedbymicronucleustestafterx-raytreatment.Interestingly,rest- ingcellsdisplayedlowerbasallevelsofDNAbreakscomparedtoproliferatingcells,butbreak-induction rateswereidentical.Despitesimilarlevelsofp53proteinupregulationafterirradiation,higherNAD⁺ concentrationsledtoreducedacetylationofthisprotein,suggestingenhancedSIRT1activity.Ourdata revealthateveninnormalprimaryhumancellscellularNAD⁺levelsmaybelimitingunderconditionsof genotoxicstressandthatboostingtheNAD⁺systemwithnicotinicacidcanimprovegenomicstability.

1. Introduction

1.1. NAD⁺supplementation

Several studies have reported that administration of NAD⁺ precursors such as nicotinamide,nicotinic acid (niacin, NA) or nicotinamideribosidecanincreasetheintracellularNAD⁺concen- trationin various tissuesor cellularcompartmentsinvitro and invivo[1–3].Inthepast,nicotinicacidhasbeeninclinicaluse, atmillimolarconcentrationsinthetissue,asacholesterollower-

Abbreviations:IR,X-rayirradiation;MN,micronuclei;NA,nicotinicacid;PARP, poly(ADP-ribose)polymerase;PAR,poly(ADP-ribose);PBMC,humanperipheral bloodmononuclearcells;PHA-L,leucoagglutinin.

∗Correspondingauthorat:Dept.ofBiology,HumanandEnvironmentalToxicol- ogy,UniversityofKonstanzUniversitätsstr.10,D-78457Konstanz,Germany.

E-mailaddresses:

kathrin.weidele@item.fraunhofer.de(K.Weidele),sascha.beneke@uni-konstanz.de (S.Beneke),alexander.buerkle@uni-konstanz.de(A.Bürkle).

1 Present address: Fraunhofer Institute for Toxicology and Experimental Medicine,ProjectgroupRegensburg,Germany.

2 Presentaddress:HumanandEnvironmentalToxicology,DepartmentofBiology, UniversityofKonstanz,Germany.

3 Sharedseniorauthorship.

ingdrugdisplayingatheroprotectiveeffects [4,5].However,this treatmentwasaccompaniedbyunwantedsideeffects(reviewed in[5]),mostprominentlyprostaglandin-dependentvasodilationin theskin(flushing)[6]andhepatotoxicity[7](forreview,see[8]), withthelatterbecomingnegligiblebyapplyingniacininadiffer- entformulation[9](reviewedin[10]).Nevertheless,highniacin dosingcanresultindiscomfortingflushes[11],butniacinisstillin use[12,13]andalsomarketedasafoodsupplement.Severalstud- iesshowedprotectiveeffectsofhighNAD⁺levelsinmodelsystems aswellasinhumansundercertainpathologicalconditionsorin thecontextoftheagingprocess,possiblyduetopreservingenergy production in mitochondria [14–20]. Apart fromthe important functionofintracellularNAD⁺inmitochondrialrespirationoras coenzymeinavastrangeofredox-reactions,NAD⁺alsoparticipates inDNArepairandmaintenanceofgenomicstability.J.B.Kirkland’s groupdemonstrated the relevanceof niacin status ongenomic integrity,DNArepairandprotectionfromcarcinogenesisusingani- malmodelsfocusingonniacindeficiency[21–23].Theimportance ofNAD⁺inthiscontextwashighlightedinreportsshowingthat NA deficiencyresultsin increasedchromosomal instability[24]

andhighercancerincidence[25].Likewise,highconcentrationsof nicotinamideorNAdelayedcarcinogenesis[26],improvedrepair capacityafter␥-irradiationabove40Gyinmousemelanomacells

Konstanzer Online-Publikations-System (KOPS)

URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-2-1kukaqkhj06r89 Erschienen in: DNA Repair ; 52 (2017). - S. 12-23

https://dx.doi.org/10.1016/j.dnarep.2017.02.001

[27]andfacilitatedrecoveryofneuronalfunctionsafterhypoxia [28].Thereisalsoincreasingevidencethatmicronutrientshavean importantimpactonthemaintenanceofgrossgenomicstability.

InastudybyFenechetal.itwasshownthatlowintakeofnicotinic acidwasassociatedwithanincreasedmicronucleusfrequency[29], awidelyacceptedmeasureofgenomicstability[30].

1.2. ConsumersofNAD⁺:poly(ADP-ribose)polymerasesand sirtuins

Immediately after a genotoxic insult cells activate various responsesthatcontributetocellsurvivalordeath.Oneofthefirst reactionsispoly(ADP-ribosyl)ation(PARylation)ofproteinsmedi- atedbynuclearpoly(ADP-ribose)polymerases(PARPs)[31,32].The mostactiveenzymeinthisregardisPARP1(andtoalesserextent PARP2[33]), whichcovalentlyattachesunitsofADP-ribose ina stepwisefashiontotargetproteinsincludingitself,synthesizinga negativelychargedpolymerbyusingNAD⁺assubstrate.Depend- ing onthelevel ofDNA damage and intracellularNAD⁺ status, PARP1anditsproductpoly(ADP-ribose)(PAR)mediatetherecruit- mentofDNArepairfactorstositesoflesions,facilitateDNArepair andhelpmaintaingenomicintegrityunderconditionsofmoder- atestress[34,35].Inthisscenarioatolerableproportionoftotal cellularNAD⁺ isusedfor polymersynthesis.In contrast,drastic andirreversibleNAD⁺ depletion[36]asa resultofhyperactiva- tionofPARP1underseveregenotoxicstressconditionscanlead tocelldeath[37,38].Thisparadigmisalsoapparentininflamma- torydiseasesandneurodegenerativedisorders[39,40](reviewed in[41]).Oneparameterdeterminingthecellularresponsetostress isthelevelofavailableNAD⁺,whichiscrucialnotonlyforPAR synthesis[42],butalsofortheenzymaticactionofsirtuins(SIRTs) [43,44].Wecouldshowthatsupplementationofhumanperiph- eralbloodmononuclearcells(PBMC)withNAnotonlyraisesNAD⁺ levels,butenhancesPARformationaftergenotoxicstressandpro- tectsfromdamaged-inducednecroticcelldeath[45].Conversely, NAdeficiencyresultsinimpairedPARP1functionsinrats[46,47].

SirtuinsarehomologuesoftheyeastenzymeSir2[43,48]and haveimportantfunctionsinregulatingcellularresponsestopartic- ulartypesofsignalsandstressors[49,50].TheyuseNAD⁺inorder tode-acetylateproteins,forming2O-acetyl-ADP-riboseasprod- uct[51].BothPARPsandSIRTshavebeenimplicatedingenome stabilization[49,52–55] and crosstalkbetweenmembers ofthe twoenzymefamilieshasbeenpublished[56–58],aswasproposed alreadytenyearsago[55].Forexample,abrogationofcellularPAR formationbyParp1genedeletion,silencingorPARPinhibitorsis knowntosensitizecellstomanygenotoxicagentsandtoincrease genomicinstability[34].Incontrast,PARP1overexpressionleadsto thesuppressionofDNAdamage-inducedgenomicinstability[59].

ItwasdemonstratedthatSIRT1andPARP1sharesomeimportant tasks,i.e.bothareregulatingchromatinstructure[52,60–63]and repair[64,65],andbothdampentheactivityofthetranscription factorandstressresponseproteinp53,SIRT1byde-acetylation[66]

andPARP1bycovalentandnon-covalentmodificationbyPARyla- tion[67–69],althoughtheimpactofthismodificationislesswell understood.Interestingly,ithasbeenreportedthatalsoSIRT6and PARP1cooperateinDNArepair[70].

1.3. Mononuclearbloodcellsasamodelsystem

Human PBMC are primary cells proficient in DNA damage response cascades and repair pathways and therefore an ideal model for DNA repair studiesand most relevantto theunder- standing of biochemical and molecular mechanisms in human physiology[71–73].Furthermore,theyarewidelyusedinepidemi- ologicalstudiestoinvestigatethecorrelationofvariousparameters includingDNArepaircapacityandcancerrisk[74].AsDNArepair

mightvarythroughoutthecellcycle,analyzingrestingandpro- liferatingPBMCmightyielddifferentresults.ButquiescentPBMC in G₀ phase canbe easilystimulatedfor proliferationby treat- mentwithphytohemagglutinin(PHA-L)[75].Inthisway,cellsfrom thesamedonorcan bemonitoredfor theirresponse todiverse treatmentsintherestingandtheproliferatingstate,thusexclud- inginter-individualvariations.PBMChavebeenreportedtoutilize supplemented NAD⁺ precursors [76,77] and express nicotinate phosphoribosyltransferaseaswellasnicotinamidephosphoribo- syltransferase, making this cell type a suitable model for our approach.

1.4. Aim

Inthepresent work,we wantedtostudywhetherincreased cellular levels of NAD⁺ can influence cellular responses after genotoxictreatmentinPBMCfromhealthy,non-niacin-deficient subjects,asthereisa lackofhumandataaddressingthisissue.

WefocusedonthebiologicalconsequencesofelevatedNAD⁺lev- elsinhumanPBMCregardingPARP1/SIRTactivitiesordownstream effectsrelatedwithgenomicintegrity.Toassesshowmodulated NAD⁺levelsmaycontributetophysiologicalorpathophysiological outcomeswesetouttoinvestigatevariousendpointsincluding(i) DNAdamage,(ii)DNArepair,(iii)theinfluenceongenomicstability and(iv)sirtuin-1(SIRT1)activityaftertreatmentwithionizingradi- ation(IR)usingvaryingdoses.Itwasofparticularinteresttofind outwhethersupplementationofnicotinicacidatpharmacologi- callyrelevantconcentrationsisabletoimprovethecellularstatus inthecontextofDNAdamageinnormalhumancellsfromhealthy donors.

2. Materialsandmethods 2.1. Chemicalsandreagents

Biocoll separating solution and fetal calf serum (FCS) were purchased from Biochrom (Berlin, Germany). Antibiotics and RPMI 1640 culture medium were from Invitrogen (Darmstadt, Germany), standardchemicalsfrom Roth(Karlsruhe,Germany), Sigma-Aldrich(Munich,Germany)orMerck(Darmstadt,Germany) ifnotstatedotherwise.

2.2. Isolationofperipheralbloodmononuclearcells[45]

BloodsamplingwascarriedoutinaccordancewiththeDecla- rationofHelsinkiandwithapprovaloftheUniversityofKonstanz EthicsCommittee,fromhealthydonorsaged24–45yearsgiving informedconsent.VenousbloodwasobtainedusingtheS-Citrate- Monovette blood collection system from Sarstedt (Nümbrecht, Germany).CellswereseparatedviaBiocollgradientcentrifugation.

Briefly,thefreshlydrawnbloodwasmixedwithanequalvolume ofPBS(137mMsodiumchloride;10mMdisodiumhydrogenphos- phate;3mMpotassiumdihydrogenphosphate;pH7.4)andlayered on15mlofBiocollseparatingsolution,followedbycentrifugation at800×gfor15minatroomtemperature.ThePBMClayerwascol- lectedandwashedtwicewithPBS.Isolatedcellswereincubatedin standardculturemedium(RPMI1640mediumsupplementedwith 10%FCS,100U/mlpenicillinand100␮g/mlstreptomycin)at37^◦C with5%CO₂ in ahumidifiedatmosphere.NA wasaddedtothe culturemediumatafinalconcentrationof15␮Mandcellswere incubated5hbeforeDNAdamageinduction.

2.3. Mitogenstimulationofcells

ToinvestigateNAD⁺levelsandDNAstrandbreakrepairatdiffer- entcellcyclestageswechallengedcellswithX-irradiation,either

inG₀phaseor44haftermitogenicstimulationwithleucoagglu- tinin(PHA-L;Roche,Mannheim,Germany)[78].5␮g/mlPHA-Lwas addedtocellculturesinallassaysasrecommendedbytheman- ufacturer,exceptfor micronucleusinductiontests(20␮g/ml)to ensuremaximumnumberofbinucleatedcellsaftercytochalasin blockaccordingtoawell-establishedprotocolbyFenechetal.[79].

2.4. Flowcytometricanalysisofcellcycle

Methanolfixedcells(10⁶/ml)werewashedinPBSandincubated with100␮g/mlRNaseA for1hatroomtemperature.10␮g/ml Propidiumiodidewasadded.SampleswereanalyzedwithLSRII (BecktonDickinson,Heidelberg,Germany) and cellcycle distri- butionwasquantifiedusingFlowJoSoftware(TreeStar,Ashland, USA).

2.5. DetectionofintracellularNAD⁺levels

CellularNAD⁺ concentrationof proliferatingcells wasdeter- minedbyanenzymaticcyclingassayadaptedfromJacobsonetal.

[80].5×10⁵–1×10⁶cellspersamplewereirradiatedwithdoses asindicated(2.5/5/10/25Gy)in500␮lPBS,incubatedfor10min at37^◦CtoallowforPARP1activity,andprecipitatedwithice-cold perchloricacid(0.5M).Aftercentrifugationat1500×gfor10min, theNAD⁺containingsupernatantwasprocessedforcyclingassay.

Briefly,supernatantwasmixedwith350␮lof0.33MK₂HP0₄(pH 7.5),centrifuged,andfrozenat−20^◦C.Afterthawingandcentrifu- gation,40␮lofeach samplewastransferredtoa96 wellplate, mixedwith160␮lofbufferA(0.25MH₃P0₄,0.5MNaOH) and 100␮lof buffer B(0.34Mbicine-NaOH pH8.0, 2.9mg/ml BSA, 14.3mMEDTA,1.4mMMTT,1.7MEtOH,5.7mMphenazineetho- sulfate,0.14mg/mIalcoholdehydrogenase)andincubatedat30^◦C for30min.Absorptionwasmeasuredat550nm(with690nmas areferencewavelength)ina96-well-plateELlSAreader.Intracel- lularNAD⁺concentrationwascalculatedbasedondefinedNAD⁺ standardsanalyzedinparallel.

2.6. AutomatedfluorimetricalkalineDNAunwinding(FADU) assay

ForassessmentofDNAstrandbreakageandrepair,cellnum- berwasadjustedto4×10⁶cells/mlsuspendedinculturemedium.

Aliquots of 100␮l were irradiated onice (dose rate 8Gy/min;

energy70keV;1.25mmaluminumfilter) usinganX-raysource (RT100;Müller,Hamburg,Germany).Subsequently,sampleswere incubatedforDNArepairat37^◦Cforthetimeperiodsindicatedand thenkeptoniceuntilanalysisbyFADU.Amodifiedandautomated versionoftheFADUmethodwasusedfortheassessmentofinduced DNAdamageandrepairaspreviouslydescribed[81].Briefly,900␮l ofsuspensionbuffer(0.25Mmeso-inositol;10mMsodiumphos- phate,pH7.4;1mMmagnesiumchloride)wasmixedwitheach cellsampleand70␮lperwellwastransferredintriplicatesontoa 96-wellplate.Thesubsequentautomatedstepsincludedcelllysis, alkalineDNA unwindingand neutralizationofthesamples,fol- lowed byadditionof fluorescent dyeSybrGreen(Invitrogen)to monitortheamountof DNA remainingdouble-stranded. Tval- uesrepresentSybrGreenfluorescenceinsampleswithoutalkaline unwinding(totalamountofdoublestrandedDNA).P₀valuesreflect thebasallevelofbreaksingenomicDNAafteralkalineunwinding inuntreatedcontrols.PxvaluesrepresentDNAstrandbreaksafter irradiationofcellsandindicatedtimeofrepair,expressedas%of P₀.Dataareplottedonalogarithmicscale.

2.7. Westernblotanalysisofp53andac-p53status

CellsweresupplementedwithNAfor 5handwith1␮Mtri- chostatinA(EnzoLifeScience,Lörrach,Germany) for1hbefore irradiation.TrichostatinA inhibitsclass I/II histonedeacetyases (HDACs),butnotsirtuins(classIIIHDACs),thusensuringtheexclu- sivedetectionofSIRTrelateddeacylaseactivityonp53.Atdistinct time pointsafter DNAdamage, cells werecollected, washed in PBS and cellnumber wasadjusted to5×10⁵ cells per sample.

Forcelllysisanappropriatevolumeofpreheated5×SDSsample buffer(100mMTris-HClpH8.0,25%2-mercaptoethanol,5%glyc- erol,12.5%SDS,0.01%bromophenolblue)wasaddedtothecell suspensionandincubatedfor5minat95^◦C.Extractsweresoni- catedwith3–4burstsof15seach.Proteinswereseparatedona 10%SDS-PAGEgel,transferredtoanitrocellulosemembraneand probedwiththeanti-p53antibody(Calbiochem,Merck,Darmstadt, Germany)oranti-acp53(Epitomics,Biomol,Hamburg,Germany).

AnImageQuantLAS4000systemwasusedforquantitativeimag- ingofblotsbyAmershamECLchemiluminescence(bothfromGE Healthcare,Munich,Germany).

2.8. Invitrocytokinesis-blockedmicronucleustest

The inductionof micronuclei was performedaccording to a protocolfromFenech withslightmodifications[79].Aliquotsof 7.5×10⁵cellsin750␮lwereirradiatedinG₀phaseandthenstim- ulatedwith20␮g/mlPHA-L.X-irradiationofproliferatingcellswas 44hafterPHA-Lstimulation.CytochalasinBwasaddedatafinal concentrationof6␮g/mlat44hpost-irradiation(Fig.1A)or48h (1B).72hlater,cellsweretransferredtoglassslidesbycytospin at500×gfor3min.Aftermethanolfixation(10min)cellswere stainedwithGiemsasolution(Merck)dilutedinSörensenbuffer (pH6.8) (3:1)for5min.Afterdrying, thecellswereembedded withMowiol.Micronucleiwerescoredin500–1000binucleated cells(BN)persample.Micronucleusfrequencieswerecalculated bydividingthetotalnumberofMNbythetotalnumberofBNcells scored.

2.9. Datapresentationandstatisticalanalysis

Datarepresentthemean±SEMofthenumberofdonorsindi- cated,witheachdonorcontributingonedatapointpercondition.

Everysuchdatapointisthemeanofseveralreplicatesfor each donorwithinoneexperiment. Forstatisticalanalysis,datawere comparedbytwo-wayanalysisofvariances(ANOVA)andBonfer- ronipost-testusingGraphPadPrism7(LaJolla,USA),ifnotstated otherwise.Whereapplicable,sampleswereanalyzedbypairedtest tonormalizeforinter-individualvariations.P-values<0.05were consideredsignificant(*),otherP-valueswerelabeledaccordingly

**P<0.01,***P<0.001.

3. Resultsanddiscussion

WehavepreviouslyshownthatNAsupplementationreduces celldeath24hafter genotoxicstress infreshly isolated human PBMCandalsocausesashiftfromnecrosistolessharmfulapopto- sis,whichwassimilartoPARP1inhibitortreatmentandcorrelated withthelevelofNAD⁺ preservation[45].Inordertogetdeeper insight into the protective effect of NA supplementation, we determineditsimpactonDNArepair andgenomicstability, i.e.

micronucleusformation,aftertreatmentwithionizingradiation, andweanalyzedtheimpactofproliferationvs.quiescenceonthe respectiveparameters.Forclarity,thegeneraltreatmentschedule includingthetimepointsofNAsupplementation,PHA-Lstimula- tiontoinduceproliferationandirradiationissummarizedinFig.1.

Fig.1. Experimentaltreatmentschedule.

HumanPBMCwereeitherexposedtoionizingradiationasunstimulatedcells(A)or(B)44hafterPHA-Ladditionandanalyzedorculturedfortheassaysindicated.Cells weresupplemented5hbeforeirradiationwith15␮Mnicotinicacid(NA)asindicated.ForfurtherdetailsseeMaterialsandMethods.

3.1. CellcycledistributionandNAD⁺contentofunstimulatedand PHA-l-stimulatedPBMC

Weusedflowcytometrytomonitorcellcycledistributionof PBMC.AsdepictedinFig.2,freshlyisolatedPBMCdisplayadis- tinctpeakrepresentingtheDNAcontentofcellsinG₀/G₁phase,as expected.After44hofstimulationwithPHA-L,cellsarefoundin allcellcyclephases,with65–75%inG₀/G₁and20–30%inS/G₂/M, independentofsupplementation.Thus,NAdidnotinfluencethe cellcycleprofileofPBMCduringthetimeperiodofexamination.

We tested PHA-l-stimulated samples for their physiological NAD⁺ concentration(Table1).ThemeanNAD⁺ levelinprolifer- atingcellswasapproximately280␮M,whichcouldberaisedby 15␮MNAsupplementationtoupto390␮M,i.e.anearly1.4-fold increasewithin5hincubation(p=2.1×10⁻⁵).Thebasalcellular NAD⁺statusinunstimulatedPBMCwasincreased2-foldonaver- age,aswehavepreviouslyreported[45],reflectingahighergain thaninPHA-stimulatedcells.Thiscouldbeduetotheprolonged timeofmaintainingthePBMCsinstandardculturemedium,which containsitselftheNAD⁺precursorniacin,andmayalsoberesponsi- bleforthegenerallyhigherNAD⁺concentrationinstimulatedPBMC comparedtounstimulatedPBMC[45].

3.2. BaselineDNAdamageinunstimulatedversus

PHA-l-stimulatedPBMCandtheeffectofNAsupplementation ToestimatewhetherstimulationwithPHA-LorNAsupplemen- tationperseinfluencesbaselinedamageofthegenomeweanalyzed ourexperimentalFADUdatabycomparingtheP₀values(double- strandedDNAwithphysiologicalbreaks)fromPBMCof6different donors(Fig.3).

In unstimulatedcells (G₀ phase) theP₀ levels corresponded toapproximately85%oftotaldouble-strandedDNA.Proliferating cellsexhibitedhigheramountsofendogenousstrandbreaks,with themeanP₀valuesreachingonly67%oftherespectiveTvalues.

Thisisinlinewithotherstudiesshowingthatthebasallevelsof strandbreaksvaryatdifferentstagesofthecellcycle,withhighest levelsobservedduringSphase[82]duetoreplication,whichwas alsoreportedbyMayerandcolleaguesusingthecometassay[83].

OurdatarevealthatNAsupplementationchangestherespective baselineP₀valuesneitherinunstimulatednorinstimulatedPBMC.

Therefore,whereasproliferationinducedhigherlevelsofphysio- logicalDNA-strandbreaks,NAsupplementationhadnoeffect.

3.3. EffectofNAsupplementationandproliferationonDNA damageinductionandrepair

TotestifelevatedNAD⁺levelscancontributetoalteredDNA repairinhumanPBMCofhealthysubjectswedamagedcellswith differentdosesofIRafter5hofNApre-incubationandmonitored DNAdamageandDNArepaircharacteristics.

3.3.1. DNAdamageinductionisnotaffectedbycellcycle

Asexpected,IRcausedanidenticaldose-dependentinduction ofDNAstrandbreakageinbothstimulatedandunstimulatedcells (Fig.4),despitethefactthatproliferatingcellshadahigherbaseline level ofstrandbreaks(Fig.3).Thus, proliferationdidnot influ- encetheabilityofX-raystoinflictDNAbreaks,leadingtothesame damageratio.

3.3.2. NiacinsupplementationmaintainsNAD⁺concentrationat highlevelsafterIR

Ofnote,NAD⁺levelsarestronglyaffectedbymassiveDNAdam- ageand subsequent PARP1 hyperactivation,as we have shown infreshlyisolatedPBMC[45],butNAD⁺levelsandPARP1activ- itybothvaryduringthecellcyclewithNAD⁺levelsbeinglowest during S/G2 phase, possibly due to PARP1 activated by natu- rallyoccurringstrandbreaksduringreplication[84].Totestthe effectsofIRonNAD⁺levelsinproliferatingPBMC,weanalyzedthe intracellularNAD⁺ concentrationinPHA-Lstimulatedcellsafter

Fig.2. CellcycledistributionofPBMC.

Flowcytometricanalysisofcellcycledistributionwithout(A/C)andafterPHA-L(B/D)stimulation.UnstimulatedPBMCrestedinG1/G0andPHA-LtreatedPBMCgrewasyn- chronously44hafterstimulation.NAsupplementationhasnoinfluenceoncellcycledistribution.DNAcontentisplottedagainstthecellcount;histogramsarerepresentative ofasingledonor.Insertedpercentagesindicatemeanvaluesfromthreedonorsinindependentexperiments.

Table1

IntracellularNAD⁺levelsin[␮M]ofPBMC44hafterPHA-Lstimulation.

Donor# NAD⁺concentration(withoutNA) NAD⁺concentration+15␮MNA Foldincrease

1 241.2 342.7 1.42

2 275.8 410.7 1.49

3 189.0 299.0 1.58

4 162.2 211.6 1.30

5 274.4 356.4 1.30

6 329.0 473.3 1.44

7 378.5 496.6 1.31

8 404.13 512.5 1.29

Mean±SEM 281.8±30.2 387.8±37.1^a 1.39±0.039

aP=2.1×10⁵bypairedT-test.

increasingdosesofX-rays.SimilartoourresultsinrestingPBMC, theimmediateandrapidcellularconsumptionofNAD⁺wasdose- dependentandmostpronouncedafter25Gyirradiation(Fig.5), withlossof53%oftotalNAD⁺innon-supplementedand40%inNA-

supplementedcellswithinthefirst10minafterdamageinduction [45]. Independent of the irradiation dose applied, the supple- mented cells always displayedsignificantly higher NAD⁺ levels comparedtonon-supplementedPBMC.Thus,NA-supplementation

Fig.3. BaselinelevelsofDNAdamageinunstimulatedandproliferatingPBMC Endogenouslevelsofstrandbreaksinundamagedcellsareexpressedaspercentage offluorescentsignalincontrolcells(P0)inrelationtothetotalamountofDNA(T).

EachbarrepresentssixindependentexperimentsusingPBMCexvivosupplemented withnicotinicacid(+NA)ornot(-NA).Statisticalanalysisbytwo-wayANOVAwith SidaksMultipleComparisontest.

Fig.4.Dose-dependentDNAdamageinductionbyIRisindependentofstimulation.

DNAstrandbreakinduction(%ofP0)ofunstimulatedandPHA-LstimulatedPBMC asafunctionofradiationdose.ThedatacorrespondtothoseshowninFigs.7and8 andrepresenttheDNAstrandbreakageinducedbyionizingradiationasanalyzed bytheautomatedFADUassay.

Fig.5. IntracellularNAD⁺levelsafterIRwithandwithoutNAsupplementation.

CellssupplementedwithNA(+NA)ornot(-NA)wereirradiatedasindicatedand incubatedat37^◦Cfor10mintoallowforpoly(ADP-ribosyl)ationbeforeNAD⁺con- centrationanalysis.Datarepresentindependentexperimentsusingfourdifferent donors.NumbersindicatetheremainingNAD⁺aspercentagecomparedtonon- irradiatedcontrols.Asterisksindicatesignificantdifferencestocontrols.

wasabletopreserveintracellularNAD⁺athighconcentrationsalso in proliferatingcells,withabsolutenumbersat 25Gymatching thoseofnon-supplementedPBMCat5Gy.Supplementationwith NAD⁺precursorsrescueddecreasingNAD⁺levelsalsoinotherset- tingslikeenhancedbasicDNAdamageinmicewithmutatedCSB

(CockayneSyndromegroupB)protein[85]andhumancellswith mutatedXPA(Xerodermapigmentosumcomplementationgroup A)protein[86],andinhumanmyocytesfrompatientswithheart failure[87].Likewise,inhibitionofPARP1duringDNAdamagein restinghumanPBMC[45]orfibroblasts[88]alsopreservedNAD⁺ levels.WeconcludethatintracellularNAD⁺concentrationcanbe maintainedathigherlevelsbylow-dosesupplementationofNA evenaftermassiveDNAdamagecomparedtonon-supplemented PBMC.

3.3.3. NAsupplementationdiminishesacetylationofinducedp53 afterIR

AsNAD⁺ servesassubstrateforbothPARPs andsirtuins,we analyzeddeacetylationactivityofSIRT1onitsclassicaltargetp53 [66].UponcellularstressorssuchasDNAdamage,p53is stabi- lizedandactsastranscriptionalregulator[89].Deacetylationof p53bySIRT1reducesitsactivityandrenderscellslesssusceptible toapoptoticstimuli[90].AsdisplayedinFig.6,NAsupplementation ofPBMCdidnotinfluencep53accumulationafterlow-doseirra- diation,butreduceditsacetylation.Asimilarfindingonp53has beenreportedforAROS,astimulatorofSIRT1activity[91].Thus maintainedNAD⁺levelsinoursupplementationexperimentsalso inducedhigherSIRT1activity,atleastasdeducedfromreducedlev- elsofacetylatedp53,aclassicaltargetofSIRT1.Thisobservationhas alsobeenreportedforapplicationofPARP1inhibitors,whichabol- ishtheconsumptionofNAD⁺byPARP1.Thus,maintainedNAD⁺ levelsinoursupplementationexperimentsprobablyalsoinduce higherSIRT1activity,afactthathasbeenreportedforapplication ofPARP1inhibitors,whichabolishtheconsumption ofNAD⁺by PARP1[55,58].

AlthoughtotalcellularlevelofNAD⁺isstillabout50%afterhigh- doseirradiation(Fig.5),whichcorrelatestoabout140␮MNAD⁺in non-supplementedcells(Table1),concentrationsinspecificcellu- larcompartmentscanbelowerthanthat.Whereasmitochondria haveahighNAD⁺content(about400␮M)([92]),whichcansum upto70%oftotal NAD⁺ [93],cytosolicaswellasnuclearNAD⁺ concentrationsreachonly100–150␮M.Thelattertwocompart- mentsdisplayidenticalNAD⁺levelsasthereisfreeexchangeof thismoleculeacrossthenuclearpore.Incontrast,mitochondrial NAD⁺seemstobecompletelyseparatedfromtheotherpools,at leastinhumans[94].Thereportedk_M valueofhumanPARP1is about30–60␮M[95], whereas thek_M of SIRT1 is 150–200␮M (reviewedin[96]),whichcorrespondstoNAD⁺concentrationsjust atlimitingconditionsforSIRT1activityeveninnucleiofundis- turbedcells.IncaseofevenratherlowDNAdamage,PARP1may readilydepletethelocallyavailablenuclearpoolsofNAD⁺,prevent- ingSIRT1fromfulfillingitsfunctionastranscriptionalregulator and chromatin organizer, and mayalso decreaseSIRT1 expres- sionby a sofarunknownmechanism [85].In linewiththis, it hasbeenreportedthatcellschallengedbyDNAdamagingagents [88],underpathophysiologicalconditions[85–87]orduringaging [97]displayhighPARlevels,alowerNAD⁺contentanddecreased SIRT1activity.Importantly,thiscanberescuedbyPARP1inhibi- tionorreplenishingthecellularNAD⁺ poolbysupplementation withNAD⁺precursors.Ofnote,acetylationofp53inresponseto PARP1inhibitionorNAD⁺supplementationisreducedcompared tosituationswithlowNAD⁺content[87,88],suggestingthatpre- servingsufficientlyhighNAD⁺concentrationisaprerequisitefor SIRT1-mediatedde-acetylationofp53.OurresultsinhumanPBMC areinsupportofthesedata.

3.3.4. NAsupplementationenhancesDNArepairefficiency independentofproliferation

HavingshownthatNAsupplementationpreservesoverallNAD⁺ levelsandenhancesSIRT1activityafterirradiation,wesetoutto monitortheimpactofNAonDNArepairafterexposureofPBMC

Fig.6. Totalp53accumulationandK382-acetylationinresponsetoIR.

PBMCwerepre-treated−/+NA,TSAandirradiatedwith10Gy.Levelsofp53andac-p53wereanalyzedinwholecelllysatesviaimmunoblotanalysisatindicatedtime pointsafterirradiation.Arepresentativeblotisshownin(A)andquantitativeanalysisofbandintensitiesfornormalizedacetylatedp53levelswereshownfor6independent experimentsin(B).Asignificantdecreaseofp53-acetylationlevelisdetectableat3hinNA-supplementedcellscomparedtocontrol-irradiatedcells.

toincreasingdosesofX-ray.Asexpected,in unstimulatedcells 2.5GyinducedmoderatedamagetoDNA(Fig.7A),whereasmassive strandbreakinductionwasachievedwithhigh-doseirradiation (25Gy;Fig.7D).Within40minrepairtime,rejoiningofDNAbreaks wasevident from thepartial recovery of fluorescent signals in PBMCfromalldonorstested.Strandbreakformationinsupple- mentedPBMCwasverysimilarcomparedtonon-supplemented cells(Fig.7A-D,comparevaluesat0min),butinterestingly,repair ofstrandbreakswasslightlymoreefficientinsupplementedcells comparedtocellswithoutadditionalNAtreatmentinadose-and time-dependentmanneralreadyafterapplicationof2.5Gy.

InordertostudytheroleofcellcycleprogressioninDNAdam- ageformationandrepair,westimulatedcellsfor44hwithPHA-L andinducedDNAdamagebyIRintheseproliferatingPBMC(Fig.8), identicaltotheexperimentalsetupinFig.7.Thedataobtainedfrom PHA-stimulatedculturesdisplayedaverysimilarpictureasquies- centPBMC.Again,NAsupplementationslightly,butsignificantly improvedDNArepairincorrelationtotheapplieddoseandtimeof repair,althoughthedifferencewaslesspronouncedcomparedto restingcells,i.e.reachingsignificanceat5Gyorhigher.

Insummary, usingtheautomatedFADUassay[81] wewere ableto showthat PHA-stimulationinducedproliferation ledto higher basal levels of strand breaks and NA-supplementation did not impact on this. NA-supplementation increased consis- tentlytheNAD⁺concentrationincellsandmaintaineditathigh levels even at the supralethal dose of 25Gy irradiation com- paredto non-supplemented PBMC.These resultsare similarto dataobtained fromquiescentPBMC[45].Mostinterestingly,NA treatmentenhancedefficiencyofDNArepairindependentofpro- liferationstatus,butwasslightlymoreeffectiveinquiescentcells.

Thismaybeduetothefactthatproliferatingcellsaremoreprone tocelldeathinductionifchallengedbyIR[82,83]independentof NAD⁺pools.Asaconsequence,severelyaffectedcellsdonotrepair thedamageanymoreanddie,whereasquiescentcellsarelesslikely toinduceapoptosisundertheseconditions.Theoverallmildstimu-

lationofrepairbyNA-supplementationcannotbesolelydependent onmaintainingNAD⁺athighlevelsdespitePARP1activity,aslow dosesthatdonothyperactivatePARP1,i.e.2.5and5Gy,displayonly amoderatereductioninNAD⁺asexpected,butrespectivesamples stillshowasignificantincreaseinefficiency.Thiscouldbedueto differentmechanisms:

ThestructureofPARsynthesizedbyPARP1canvarydependent onseverityofDNA-damage[98,99],ontherespectiveinteraction partner[69,100],andalsoonthelocalNAD⁺concentration[100].

Whereasseverityofdamage(seeFig.4)isthesame,NAD⁺levels differbetweenNA-supplementedandnon-supplementedcellsby thefactorof1.4(Table1).Thereforesubtlechangesintheresulting PAR-structure(length,branching)mayrecruitrepairfactorswitha differentefficiency,leadingtotheobserveddifferencesinrepair.

Sirtuinsregulateamultitudeofpathways,withSIRT1involved inchromatinrestructuringandregulationofrepair[49],directly bytargeting histonesand indirectlyby inactivatingotherregu- latorproteinssuch asp53or p300,theacetylasetargetingp53 [101,102].P53isthecentraltranscriptionfactorinstress-response [90,103–105].Inourexperimentalsetup,p53isinducedafterIRas expected.Highlevelsofp53notonlystopthecellcycletoorches- traterepair,butcanalsoinduceapoptosis[106,107].SIRT1reduces theacetylatedformofp53,thusdampentheadverseeffectsofhigh p53activation[87,88].

Alternatively,restructuredchromatinduetoadifferentacety- lationpatternmaymoresusceptibletorecognitionand/orrepair ofdamage.Althoughgeneralchromatinacetylationisimplicated inrepair,therearealsosomereportsaboutinvolvementofHDAC in earlystepsof DNA repair (reviewed in[108]).Whether this isrelevant forourapproach,and whetherSIRT1orothersirtu- insareinvolvedremainstobedetermined.Inaddition,chromatin isorganizedbyPARylation(reviewedin[61,62]).DNAbreakage isan immediatetriggerofPARP1 activity,whichleadstocom- plexre-structuringofthechromatinatsitesofdamage,facilitating recruitment of repair proteins and activation of signaling cas-

Fig.7. DNAdamageandrepairinunstimulatedPBMC.

CellswereirradiatedinG0.StrandbreakformationandsubsequentDNArepairwithinthefirst40minafterDNAdamagewasmeasuredusingtheautomatedFADUassay.

ValuesrepresentthemeanfluorescenceofdoublestrandedDNAinrelationtonon-irradiatedcontrols(P0)asobtainedinindependentexperimentscovering(A)n=12donors (B)n=13donors(C)n=11donors(D)n=10donors.

cades(forreview,see[109]).Afaster openingofthechromatin withquickerrecruitmentofrepairenzymesduetolocallyhigher availabilityofNAD⁺andthereforedifferentlystructuredPARmay accounttotheslightincreaseinDNAbreakresealingweobserved.

3.4. EffectofNAsupplementationongenomicstability

Studies have demonstrated a positive role of poly(ADP- ribosyl)ationinthemaintenanceofgenomicintegrity[59]anda negativeinfluencebyniacindeficiency[24].Thereforeweinves- tigated theimpactof NA supplementation ontheformation of micronuclei in the low dose range of IR (1–5Gy). The basal micronucleus frequency in undamaged cells was low, but MN frequencyclearlyincreasedinresponsetoDNAdamage(Fig.9).

Non-irradiatedcontrolcellsdisplayedMNfrequenciesof0.7%and 0.5%(Fig.9Aunstimulated/0Gy,±NA)and1.5%and1.1%(Fig.9B stimulated/0Gy, ±NA).Irradiation ofcells in G0 inducedhigher MNfrequenciescomparedtocellsdamagedduringproliferation.

NA-treatmentreduced MNfrequencyinproliferatingaswellas quiescentcellscomparedtonon-supplementedPBMC,butthiswas morepronouncedinproliferatingcells.Forexample,MNfrequency wasnotaffectedbyNAsupplementationinrestingPBMCat3Gy, butwassignificantlyreducedfrom24%to20%inPHA-Lstimulated cells.At5Gy,NAsupplementationreducedMNfrequencyforboth conditions,fromnearly70%to60%incellsinG₀,andfrom36%to 30%inproliferatingPBMC.Theseresultsareinlinewiththedata obtainedfromourDNAbreakresealingmeasurements.Asrepairis

moreefficientinsupplementedcellsMNformationisslightlybut significantlyreduced.

AlthoughtherewasnosignificantdifferenceinbasalMNforma- tionorafter1Gyirradiation,atdoses≥3GylowerMNfrequencies wereobservedincellsdamagedduringproliferationcomparedto PBMCirradiatedinG₀.Thisfindinghasbeendescribed forcells damagedduringadistinctcellcyclephase[110]orwithdosesofIR leadingtomitoticarrest[111].Bothconditionsdelaytheappear- ance micronuclei,asthesecells undergoa (temporary)division blockandarethereforenotincludedinthescoringofbinucleated cells.ThedegreeofIRinduceddivisiondelayisdependentonthe cellcyclepositionatthetimeofirradiation,withIRdamageinS/G2 phaseleadingtoastrongerdelay[112,113]andgenerallyresulting inlowerMNfrequencies,whichisalsoapparentinourstudy.This effectwasalsoreportedforDNAdamageinductionincombina- tionwithPARPinhibitors,showingthatrestingcellsweremuch lessaffectedincomparisontoproliferatingcells[114].Thelatter displayedreducedmutationratesbuthighertoxicity.

4. Conclusion

Niacinsupplementationisgenerallythoughttobebeneficial in conditions of pathologies and aging withtheability topre- servemitochondrialenergyproduction[16,17]withsomecaveats regardingside-effectsofhigh-doseregimens.Inaddition,thereare severalreportsonpositiveeffectsongenomestabilityornegative impactofNAD⁺deficiency[26–28].Inthisstudy,weaddressedthe

Fig.8. DNAdamageandrepairinproliferatingPBMC.

CellswereirradiatedafterPHA-Lstimulation.Repairofstrandbreaksaftergenotoxictreatmentwasmeasuredwithinthefirst40minbyusingtheautomatedFADUassay.

ValuesrepresentthemeanfluorescenceofdoublestrandedDNAinrelationtonon-irradiatedcontrols(P0)asobtainedinindependentexperimentscovering(A)n=8donors (B)n=10donors(C)n=8donors(D)n=9donors.

Fig.9. GenomicstabilityinhumanPBMCexposedtoDNAdamageasassedbymicronucleiformation.

(A)Unstimulatedor(B)PHA-LstimulatedcellswerechallengedwithlowdosesofIR.Percentageofmicronucleusfrequencyinbinucleatedcellswasdeterminedfor(A)n=7 and(B)n=9donors.

questionofwhetherDNArepairisalsoaffectedbyNAsupplemen- tationinnormalhumanPBMC.Basedonanautomatedversionof theFADUtechnique[81]wemonitoredDNAstrandbreakforma- tioninPBMCafterIRandtheearlyphaseofDNArepairafterdamage todiscoverimmediateeffects.AlthoughtheFADUassaycannotdis- criminatebetweensingleanddoublestrandbreaks,theformerare knowntobeseveralfoldmorefrequentthanthelatterafterirradi-

ation[115].Ourdataindicatethatevenashorttreatmentwithlow dosesofniacin(15␮M)isabletoincreaseNAD⁺levelsinprolifer- atingPBMCsimilartorestingPBMC[45].Wecouldnotobserve anyinfluenceof NA-supplementationontherateof DNA dam- ageinflictedbyirradiation.Interestingly,repairrateswereslightly butsignificantlyenhancedifNAD⁺levelswereincreasedinrest- ingaswellas inproliferatingPBMC.Accordingly,micronucleus

formationwasmarginallyreducedafterNAsupplementation,sug- gestingaslightlybetterpreservationofoverallgenomicstability.

ThisslightdifferencecouldbebasedonthelowerNAD⁺levelsavail- ableinS-phase,whichconstrainstherepairactivityofproliferating cells.Theobservedresultsmostlikelydependontheavailabilityof NAD⁺ assubstratefortwopathways,i.e.poly(ADP-ribosyl)ation, whichisthemainconsumerofNAD⁺ aftergenotoxicstress,and sirtuin-dependentproteinde-acetylation.Theseenzymaticreac- tionsregulatetheDNArepairmachineryandchromatinstructure [63,64,85].EspeciallyPARPsandPARhaveapivotalroleinpreserv- ingthegenomicintegrityastheyregulateDNArepairprocesses andtelomerelengthandcanbedetectedatcentrosomesandcen- tromeres,thusensuringcorrectdistributionofthegeneticmaterial duringcelldivision[116].Butthereisampleevidencethatalso SIRT1isinvolvedinmaintainingchromatinintegritybyregulating differenttargets(reviewedin[117]).Inlinewiththis,knockoutof SIRT1and/orPARP1ledtochangesinchromatinstructureandalter- ationsofhistonemarks[63].Inourstudy,thenon-supplemented cellswerenotdeficientinNAD⁺,soitisunlikelythata general restructuringofchromatintookplaceinsupplementedPBMC.It canbespeculatedthatthehigheravailabilityofNAD⁺atthetimeof damagefacilitatesspecificremodelingofthechromatinatthesite ofdamageandthusleadstoenhancedrepair.Innon-supplemented PBMC,PARP1consumessubstantialamountsofNAD⁺presentin thenucleusafterbreak induction,stillworkingeffectively even atconcentrationsofNAD⁺thatdisfavorSIRT1activity[96].PARP1 activitylimitsbythisprocessthesubstrateavailabilityforSIRT1, blockingde-acetylationoftargetproteins.Inlinewiththis,wepro- videevidencethatNA-supplementationpreservesSIRT1enzymatic activityalsoinnormalhumanPBMCasindicatedbyreducedlevels ofacetylatedp53.Soitcanbespeculatedthatmaintainingactivity ofSIRT1isimprovingrepairefficiency,i.e.alsobyenhancingsur- vival[87].AsitwasshowninmicethatinactivationofPARP1leads toaccelerationofaging,shortenedlifespanandincreasedsponta- neouscarcinogenesis[118],itcanbehypothesizedthatmaintained PARformationbyNAsupplementationisapotentialstrategyto counteracttheseeffects.Notably,wemonitorednoadverseimpact ontheinvestigated parameters whenNAD⁺ levelswere modu- lated.Basedonourdata,furtherusageofNAcouldbeconceivable, as weshowed that applicationof a low concentration(15␮M) NAD⁺ precursorhasapositiveeffectongenomeintegrityinour setting.NAD⁺dependentenzymesPARPandSIRTbothinfluence genomicstabilityandmetabolism,andadequateNAD⁺availabil- ityislikelytopreservetheiractivitiesandfunctions.Thiscouldbe favorableforhumansubjectswithdecreasedPARPactivityorlow basalNAD⁺ levels,whichoccursduringtheagingprocess[119], inthepathogenesisofdiseases[120]orcancertherapy[121,122].

HighcellularNAD⁺levelsasachievedbyNAsupplementationto maintainpoly(ADP-ribosyl)ationandsirtuinactivitythushavea plethoraofpositiveeffects,includingenhancedrepairandchro- mosomalstability.

Conflictofintereststatement

Theauthorsdeclarethattherearenoconflictsofinterest.

Acknowledgements

ThisworkwassupportedbytheDeutscheForschungsgemein- schaft through the Research Training Group 1331 (fellowship awarded to K.W.). We thank Anja Göder for her assistance in determiningNAD⁺levelsandProf.ChristofR.Hauck,Universityof Konstanz,forkindlygivingusaccesstotheflowcytometer.

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