Sulfur and nitrogen mustards induce characteristic poly(ADP-ribosyl)ation responses in HaCaT keratinocytes with distinctive cellular consequences

Sulfur and nitrogen mustard s induce characteristic

poly(ADP-ribosyl )ation response s in HaCaT keratinocytes with distinctive cellular consequences

Aswin Mangericha ·

¹•

Malgorzata De biaka,l.3 . Matth ias Birteia ·t, Vivia ne Po nath a.

Fran k Balszuweitb, Kirsten L exa.4 . Rita Martelloa.s, Waltraud Bu rckhardt- B oera.

R o m ano Strobeltb, Markus Siegertb, Horst Th ierm annb, Dirk Ste inritzb·c . An nette Schmidtb·

²•

Alexande r Bi.irklea ·

²

·*

a University of Konstanz, Molecular Toxirology Group, Deparonent of Biology, 78457 Konstanz. Gennany

b Bundeswehr Institute of Phannacology and Toxicology. 80937 Munidt, Gennany

c Walther-Straub-lnstitute of Phannacology and Toxicology. 80336 Munich. Gennany

HIGHLIGHTS

• Detailed analysis of sulfur and nitro gen mustard induced PARylation in HaCaTcells

• PARylation participates in mustard induced stress response in a sub stance specific manner

• PARP inhibitors potentiate CEES in duced genome instability

• With limitations, CEES and HN2 can be used as surrogates to study SM related toxicity

• PARP inhibitors should be considered witb caution as SM antidotes, due to potential long term effects

Keywords:

Poly(ADP-ribose) PARP inhibitors

• Corresponding author.

GRAPHICAL ABSTRACT

ABSTRACT

Mustard agents are potent DNA alkylating agents with mutagenic, cytotoxic and vesicant properties. They include bi functional agents, such as sulfur mustard (SM) or nitrogen mustard (mustine, HN2), as well as mono functional agents, such as "half mustard" (CEES). Whereas SM has been used as a chemical warfare agent, several nitrogen mustard derivatives, such as chlorambucil and cyclophosphamide, are being used as established chemotherapeutics. Upon induction of specific forms of genotoxic stimuli, several poly (ADP ribose) polymerases (PARPs) synthesize the nucleic acid like biopolymer poly(ADP ribose) (PAR) by using NAD• as a substrate. Previously, it was shown that SM triggers cellular poly(ADP ribosyl) ation (PARylation), but so far tbis phenomenon is poorly characterized. In view of the protective effects ofPARP

E-mail address: alexander.buerkle@uni-konstanz.de {A BOrkle).

1 Shared first authorship.

2 Shared senior authorship.

3 Current address: Umweltbundesamt, 06844 Dessau-Rosslau, Germany.

4 Current address: lnstituto Gulbenkian de Ciencia, Oeiras, Portugal.

5 Current address: The Novo Nordisk Foundation Center for Protein Research, Proteomics, 2200 Copenhagen, Denmark.

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-300760

Erschienen in: Toxicology Letters ; 244 (2016). - S. 56-71 https://dx.doi.org/10.1016/j.toxlet.2015.09.010

Sulfurmustard Nitrogenmustard Genotoxicity HaCaT

Massspectrometry Micronucleus

inhibitors, the latter have been proposed as a treatment option of SM exposed victims. In an accompanyingarticle(Debiaketal.,2015),wehaveprovidedanoptimizedprotocolfortheanalysisofthe CEES inducedPARylationresponseinHaCaTkeratinocytes,whichformsanexperimentalbasistofurther analyzemustard inducedPARylationanditsfunctionalconsequences,ingeneral.Thus,inthepresent study,weperformedacomprehensivecharacterizationofthePARylationresponseinHaCaTcellsafter treatmentwithfourdifferentmustardagents,i.e.,SM,CEES,HN2,andchlorambucil,onaqualitative, quantitativeandfunctionallevel.Inparticular,werecordedsubstance specificaswellasdose andtime dependent PARylation responses using independent bioanalytical methods based on single cell immunofluorescencemicroscopyandquantitativeisotopedilutionmassspectrometry.Furthermore, weanalyzedifandhowPARylationcontributestomustard inducedtoxicitybytreatingHaCaTcellswith CEES,SM,andHN2incombinationwiththeclinicallyrelevantPARPinhibitorABT888.Asevaluatedbya novel immunofluorescence based protocol for the detectionof N7 ETE guanine DNA adducts, the excisionrateofCEES inducedDNAadductswasnotaffectedbyPARPinhibition.Furthermore,whileCEES inducedmoderatechangesincellularNAD⁺levels,annexinV/PIflowcytometryanalysisrevealedthat thesechangesdidnotaffectCEES inducedshort termcytotoxicity24haftertreatment.Incontrast,PARP inhibitionimpairedcellproliferationandclonogenicsurvival,andpotentiatedmicronucleiformationof HaCaTcellsuponCEEStreatment.Similarly,PARPinhibitionaffectedclonogenicsurvivalofcellstreated withbi functionalmustardssuchasSMandHN2.Inconclusion,wedemonstratethatPARylationplaysa functionalroleinmustard inducedcellularstressresponsewithsubstance specificdifferences.Since PARPinhibitorsexhibittherapeuticpotentialtotreatSM relatedpathologiesandtosensitizecancercells formustard basedchemotherapy,potentiallong termeffectsofPARPinhibitionongenomicstabilityand carcinogenesisshouldbecarefullyconsideredwhenpursuingsuchastrategy.

1.Introduction

Sulfurmustard(SM),bis(

b

chloroethyl)sulfidealsoknownas mustardgas,wasfirstusedinchemicalwarfareduringWWIand later on in several other military conflicts and still remains a significantthreatthroughitspotentialuseinterroristattacksor exposureofpersonnelinvolvedinthedisposalofoldSMdepots (Debiak et al., 2009; Mangerich and Esser, 2014). SM is a bi functionalalkylatingagentthatreactswithmanycellulartargets, includinglipids,proteinsandnucleicacids,formingbothintra and inter molecular cross links. It has strong vesicant and blister formingpropertiesaffectingprimarilytheskin,theeyes,andthe respiratorytract.Inparticular,basalkeratinocytesaresensitiveto SM inducedcytotoxicitycausingdetachmentofthesecellsfrom thebasementmembraneanddetachmentoftheepidermisfrom the dermis. Chronic toxicity comprises multiple skin and respiratorypathologiesandanincreasedcancerrisk(Keheetal., 2009). Several clinical approaches have been tested for the developmentof antidotes to treat SM exposed people, suchas antioxidants, scavengers,anti inflammatorydrugs and protease inhibitors,butthusfar,noneofthemyieldedasatisfyingtherapy (Debiaketal.,2009;Keheetal.,2009).Basedonitscommercial availabilityand access,theSM mono chloro derivative 2 chlor oethylethylsulfide(CEES)iswidelyusedinlaboratorysettingsasa SM substitute in experimental research. This compound also displays vesicant properties, but at 100 fold higher concen trations(Wangetal.,2012).Ironically,intheaftermathofWWIand WWII,itwasrecognizedthatnitrogenmustardgasexertsstrong cytostatic effects on thehematopoietic system. This ledtothe dawnofmoderncancerchemotherapythroughthesynthesisof several nitrogen mustards, yielding the first chemotherapeutic drugmustine(HN2,bis[2 chloroethyl]methylamine)(Gilmanand Philips,1946). When applied toskin, nitrogen mustards cause similartoxiceffectsandpathologiesascomparedtoSMtreatment (Tewari Singhetal.,2013,2014b).Severalnitrogen mustard based cytostatic compounds were developed, including chlorambucil, melphalan,andcyclophosphamide,allofwhicharestillinclinical usetodaytotreatvariousformsofleukemiaandothermalignan cies(NeidleandThurston,2005).

Atpresenttheexactcellularmechanismsofmustard induced toxicityremainelusive,butitisthoughtthatmustardselicittheir toxiceffectsbyinductionofvariousformsofcelldeathandastrong

inflammatory response, primarily caused by unrepaired DNA damage(Keheetal.,2009;Shakarjianetal.,2010).WhenSMreacts withDNAthismainlyleadstothealkylationattheN7positionof guanine and at the N3 position of adenine resulting in the formation of N7 hydroxyethylthioethyl guanine (N7 HETE Gua) and N3 hydroxyethylthioethyl adenine (N3 HETE Ade), respec tively. In addition,bi functional mustards,suchas SM,HN2,or chlorambucilformbiadductsbetweentwoguanineN7positionsto yield bis(N7guanine ethyl) sulfide (N7 Gua ETE N7 Gua) DNA crosslinks.Quantitativeanalysisestimatedtherelativedistribution oftheseadductstobe60 88%dGmonoadducts,10 42%dG crosslinks, and 1 10% dA monoadducts depending on the methodsused,thetargetmaterial/tissueexposed, andthedose applied(Bataletal.,2013;Fidder etal.,1994;Yue etal.,2014).

Crosslinks canbedetected asearly as30minafterexposurein human lymphocytes (Debiak et al., 2011) and persist in SM exposedmousetissueforuptoseveralweeks(Bataletal.,2013).

Interestingly,aftercutaneousexposure,SM inducedDNAadducts couldalsobedetectedsystemicallyinmanyinternalorgans,such asbrain,lung,kidney,andspleen(Bataletal.,2014).Incontrastto bi functionalagents, mono functionalagents, suchas CEES,can only form monoadducts, with N7 ethylthioethyl guanine (N7 ETE Gua) beingthe mostabundantbasedamage, howeverstill sufficienttoinducegenomicinstabilityinhumancells(Bataletal., 2014;Bennettetal.,2014).Inaddition,ithasbeenreportedthat several hoursafter treatment, mustard agents induceoxidative stress via mitochondrial dysfunction, induction of ROS/RNS generating genes, and GSH depletionon a cellular level or via therecruitmentofROS/RNSgeneratingimmunecellsonatissue level,whichalsoleadstothegenerationofoxidativeDNAdamage (Inturietal.,2011;Jainetal.,2011;Steinritzetal.,2014).While mustardsarenotthoughttoproduceDNAstrandbreaksbydirect reactionwithDNA,bothDNAsingleaswellasdoublestrandbreaks are introduced during subsequent DNA repair processes and potentiallybysecondaryROSformation(Debiaketal.,2011;Inturi etal.,2011;Jainetal.,2011).Ifpersistent,mustard inducedDNA damagecancausegeneticmutationsduringcelldivisionorinduce celldeathviaapoptosisandnecrosis(Boulwareetal.,2012;Kehe etal.,2008;MatijasevicandVolkert,2007;Powelletal.,2010).

Poly(ADP ribosyl) ation (PARylation) is a post translational modificationthatoccursunderphysiologicalandpathophysiologi calconditionsandisinvolvedinthegenotoxicstressresponse.Poly

(ADP ribose)polymerases(PARPs,alsonamedARTDs)canbindto DNAsingleordoublestrandbreaksandsomeothernon canonical DNAstructures,which triggerstheirenzymatic activation(Hot tiger,2015).ActivatedPARPsuseNAD⁺asasubstratetosynthesize the nucleic acid like biopolymer poly(ADP ribose) (PAR) with chainlengths ofupto200ADP riboseunits(Gibsonand Kraus, 2012; Martello et al., 2013). PARylation refers to the covalent modification of proteinswithPAR by modifyingspecific amino acids.Alternatively,anumberofproteinscanalsononcovalently interactwithPARviadistinctPARbindingmotifs.Bythismeans, PAR modulates physico chemical properties of target proteins including PARPs themselves and several factors involved in chromatin remodeling and DNA damage response (Krietsch et al., 2013). Importantly, DNA damage induced PARylation is transientandhighlydynamic,sincePARisrapidlyhydrolyzedby poly(ADP ribose)glycohydrolase(PARG)andotherPAR degrading enzymes(Barkauskaiteetal.,2013;Hottiger,2015;Martelloetal., 2013).PARylationexertspleiotropicfunctionsingenomemainte nance,butalsoinahostofothercellfunctionssuchaschromatin remodeling, transcription, intra cellular signaling, cell cycle control, epigeneticsandregulationof celldeath(Beneke, 2012;

KrausandHottiger,2013;Robertetal.,2013).Onanorganismic level, thesefunctionslinkPARylation tomechanismsof inflam mationandmetabolismaswellastumorsuppression,forwhich PARPinhibitorsarecurrentlybeingtestedintumortherapy(Bai andVirag,2012b;CurtinandSzabo,2013;MangerichandBürkle, 2011,2012).

Papirmeister et al. (1985) were the first to propose a biochemical hypothesis on the mechanism of SM induced cytotoxicitybysuggestingthatDNAstrandbreaksthatareformed duringcellularDNArepairactivatePARPstoproducePARbythe consumption of cellular NAD⁺, thereby driving the cell into necrosis. Indeed several subsequent studies demonstrated a declineinintracellularNAD⁺dependentontimeanddoseofSM exposure. This effect could at least partially be reversed by pharmacologicalPARPinhibitorssuggestingaroleforPARylation inSM inducedNAD⁺decreases(Hinshawetal.,1999;Meieretal., 1987;Moletal.,1989;Smithetal.,1990).However,ifandhowa PARP dependentdeclineinintracellularNAD⁺levelscontributesto SM inducedcelldeathisasyetunclear:Severalstudiesrevealeda time dependent protective effect of PARP inhibitors on SM induced cytotoxicity (Meier, 1996; Meier and Johnson, 1992;

Meieretal.,2000).Ithastobenotedthat cytotoxicityinthese studieswasmeasuredbydyeexclusionandbythismethodonly necroticcellsarescoredasdeadcells.Othersdidnotobservesuch aneffect(Keheetal.,2008;Linetal.,1994;Rosenthaletal.,2001;

Smithetal.,1990),orevenshowedasensitizationofCEES induced cell deathin thepresence of PARPinhibitor treatment (Jowsey et al., 2009). Furthermore, it has been suggested that PARP inhibitioninducesaswitchinthemodeofcelldeathbyinhibiting necrosisbutdrivingcellsintoapoptosis(Keheetal.,2008;Meier andMillard,1998;Rosenthaletal.,2001).Rosenthaletal.reported thatsuchaneffectmayevenbecelltypespecific,asitwasonly observedinskinfibroblasts,butnotinkeratinocytes(Rosenthal etal.,2001).BasedontherationalethatPARPinhibitionpreserves NAD⁺levelsandcounteractsinflammation,PARPinhibitorswere testedaspotentialtherapeuticagentsforSMexposures.Interest ingly,inseveralanimalmodelsPARPinhibitionand/orboostingof NAD⁺ synthesis led to reduced pathological signs upon SM exposure (Cowan et al., 2003; Gross et al., 1985; Mol et al., 1991;Youricketal.,1991,1993;Zhangetal.,1995).

DespitepreviousreportsontheeffectofPARPinhibitorsonSM inducedNAD⁺depletion,celldeathandpathology,specificreports characterizing the actual PARylation response upon mustard treatment are incompleteand inconsistent.Using an immuno chemicalapproach,Rosenthaletal.(1998)analyzedPARformation

inkeratinocytesupontreatmentwithasingledoseSM(100

m

M) from2to24h,revealingauto modifiedPARP1peaking2hafter treatment.Using ³H NAD⁺asa substrate,Hinshawet al.(1999) analyzed PAR formation in permeabilized endothelial cells and keratinocytesupontreatmentwith250and500

m

MofSMfor2,3, and5h,showinga2 4 foldincreaseinsignalintensities(Hinshaw etal.,1999).Usingfluorescencemicroscopy, Sekeretal.(2000) showed PAR formation in MCF7 cells 24h after glufosfamide treatment (50 750

m

M). Using biotinylated NAD⁺ in an ELISA approach, Kehe et al. assessed PAR formation in permeabilzed HaCaTcells upon pulse treatmentof cells for 30minwith 10 1000

m

MSMfollowedbyfurtherincubationofcellsfor45minin SM freemediumleadingtoamaximumfour foldincreaseinsignal intensities(Keheetal.,2008).Finally,alsousingbiotinylatedNAD⁺, Paramovetal.(2011a)assessedPARPactivityinHaCaTcellextracts upontreatmentofcellsfor1hwith0.5 5mMCEES,leadingtoa maximum of a 10 fold in signal intensities at 5mM CEES treatment.

Even though the above cited literature suggests a role for PARylationinmustard inducedtoxicity,sofar,asystematicand comprehensive analysis of PARylation in response to chemical mustardexposureanditsroleduringsubsequentgenotoxicstress responseisnotavailable.However,adetailedcharacterizationof suchmechanismswouldhelptoclarifyif,ontheonehand,PARP inhibitorsmaypotentiatecarcinogeniceffectsofmustardexposure when applied as anti inflammatory antidotes to SM exposed subjects;or,ontheotherhand,ifPARPinhibitorsmaybeusedas chemosensitizersinnitrogen mustard basedcancertherapy.Inan accompanyingarticleinthisspecialissue,wehaveestablishedand validateda protocolto analyzeCEES inducedPAR formation in HaCaTcellsbymeansofsinglecellimmunofluorescencemicros copy. Moreover, we provided a detailed analysis of the CEES inducedPARylationresponseinHaCaTkeratinocytes,whichforms anexperimentalbasistostudythemolecularmechanismofPARP activationaftermustard treatment(Debiaket al., 2015).In the present study, we investigated PARylation responses and func tionalconsequencesthereofinhumanHaCaTkeratinocytesafter treatmentwithapanelofmono andbi functionalmustardagents, i.e.,SM,CEES,HN2,andchlorambucil.

2.Materialsandmethods 2.1.Laboratorysafety

Mustardsarehighlytoxicandcarcinogenicsubstances.Theuse of some of them, i.e., SM is highly restricted and its use in experimental studies requires a special license. All of those substancesmustbehandledwithutmostcaution,inanappropri atelaboratory setting by experienced personnel withadequate protection.Allexperiments withSMof thepresent studywere performed at the Bundeswehr Institute of Pharmacology and Toxicology,Munich,Germany.

2.2.TreatmentofHaCaTcellswithchemicals

ThehumankeratinocytecelllineHaCaT(Boukampetal.,1988) wasculturedinDMEM(LifeTechnologies)containing10%fetalcalf serum (PAA Laboratories) and 1% penicillin streptomycin (Life Technologies)at37C,inahumidifiedincubatorwith5%CO2.The pharmacologicalcompound ABT888 (Selleckchem) was used to inhibit PARPactivity. Stock solutions ofABT888 (2.5mM) were preparedinddH2Oandstoredat 80C.Cellsweresupplemented with 1

m

M ABT888 in fresh medium 30min prior to mustard treatment(orasindicated).Premixes(100)ofSMorCEES,orHN2 solutions were prepared in EtOH (95% v/v)/HCl (0.5% v/v) as described in Debiak et al. (2015). Briefly, directly before cell

treatment,1solutions(1%v/v,finalconcentrationsasindicated) werepreparedinpre warmedPBS(37C)andimmediatelyadded to the cells. Equal distribution of mustard agents in PBS was ensuredbyrigorouslymixingthetubes.Ifnotindicatedotherwise, controlswereincubatedwith1%(v/v)EtOH/HClinPBS.

2.3.AnalysisofPARformationbyisotopedilutionmassspectrometry

Analysis of cellular PAR levels by mass spectrometry were performedasdescribedpreviously(Martelloetal.,2013).Briefly,in ordertoextractcellularPAR,treatedcellswererinsedwithPBS(pH 7.4)andharvestedbyadditionof2ml20%TCA(w/v)andtheacid insolublematerialwasdetachedfromthedishesbyscrapingwitha rubberpoliceman.Subsequently,pellets werewashedwithice cold70%ethanol,resuspendedin225

m

l0.5MKOH,50mMEDTA andincubatedat37Cfor45min.Alkalinetreatmentwasstopped byneutralization,and5pmolof¹³C,¹⁵Nlabeled PARwasaddedas aninternalstandard.DNAandRNAweredigestedbyadditionof 44mM MgCl2, 0.5mM CaCl2, (0.1mg/ml) DNAse I (Roche) and 55

m

g/ml RNAse A (Sigma Aldrich) for 3h at 37C, following digestion with 0.2mg/ml proteinase K (Roche) overnight. PAR extractionswereperformedusingtheHighpuremiRNAisolation kit(Roche)accordingtothemanufacturer’sinstructions.Inorder topreparesamplesforLC MS/MSanalysis,PARwassubjectedto digestionwithalkalinephosphatasefrombovineintestinemucosa (AP)(Sigma Aldrich)andphosphodiesteraseI(PDE)(Affymetrix) beforeLC MS/MSanalysis.Sampleswerebroughttoafinalvolume of500

m

linasolutioncontaining1mMMgAc,30mMNH4Ac,10U AP and 0.5 U PDE. Following incubation for 3h at 37C, the enzymeswereremovedusinga10kDcut offfilter(nanosep10K, Pall)bycentrifugationfor20minat14,000g.Cellextractswere vacuum driedandresuspendedin100

m

lofmobilephasepriorto LC MS/MS analysis. HPLC separation of digestionproducts was performedusingaWaters2695separationmoduleandaHypersil GoldaQ1502.1mmparticlesize3micron(ThermoScientific)at 30C.Moleculeswereseparatedisocraticallywith1%acetonitrile supplementedwith0.1%aceticacidataflowrateof0.3ml/min.

The column effluent was coupled to a Quattro Micro mass spectrometer (Waters) operating in positive ESI mode using parametersasreportedpreviously(Martelloetal.,2013).

2.4.AnalysisofPARylationbyimmunofluorescencemicroscopy

Immuno chemicalanalysisofPARformationwasperformedas describedinDebiaketal.(2015)withsomemodifications.Cover slipswereplacedinto12 wellplatesand3.010⁵cells/wellwere seededand incubatedfor 24h priortreatment.Cells werepre incubatedwithABT888for30min(orasindicated)andtreated withdosesofSM/CEES/HN2/chlorambucilfortimesasindicatedat 37C.Controlsweretreatedwith0.1mMH2O2for5min.Negative controls were incubated with EtOH/HCl in PBS (EtOH/solvent control).CellswerewashedinicecoldPBSandfixedinicecold 100%methanol for7min.Then, cover slipswerewashed three timeswithPBSfor5min,blockedinTBScontaining5%(w/v)skim milkpowder and 0.1% (w/v) Tween20for 30minat 37C, and sampleswereincubated withanti PARprimary antibody(10H) over night at 4C in a humidified chamber. Cover slips were washed three times for 5min in PBS and incubated with an Alexa488 labeledsecondaryantibodyfor1hat37Cinthedarkin ahumidifiedchamber.Coverslipswerewashedthreetimesfor 5mininPBSandincubatedwithHoechst33342(0.2

m

g/mlinPBS) for5minatRT,washedagainthreetimesfor1mininPBS,and mounted onglass slides using Aqua PolyMount (Polysciences).

ImageswereacquiredusingaZeissAxiovert200Mepifluorescence microscopeequippedwithanAxioCam MRmcamera,and were analyzedwiththeAxiovisionorImageJsoftware.Imagingdata

were also analysed by TissueFAXS Software (3.5.5120.128) and TissueQuestSoftware(4.0.1.0127)(TissueGnostics).Laserscanning microscopypictureswereacquiredwithaZeissLSM710micro scope.

2.5.AnalysisofN7 (H)ETE guanineadductsbyimmunofluorescence microscopy

Forimmunofluorescencestainingformustard inducedguanine adducts,cellsoncoverslipswerewashedtwicewithPBSandwere fixedwithmethanol containing3% aceticacidat 20C. Then, slides were washed with PBS four times for 5min, cells were permeabilized in 0.1% sodium citrateand 0.3% TritonX100 for 10minat4C,cultureswerewashedagainwithPBSfourtimes,and were incubated in 80% formamide in PBS for 30min at 75C.

Subsequently, plateswereincubated for5minat4C andthen withtheN7 (H)ETE guaninespecificprimarymousemonoclonal antibody2F8(TNO,TheNetherlands)(Kehe etal.,2013)diluted 1:5000inDakoAntibodyDiluentat4Covernight.Then,samples werewashedfourtimesfor5minandincubatedwithanAlexa488 coupledsecondaryantibodydilutedinDakoAntibodyDiluentfor 90minat37C.Finally,sampleswerewashedfourtimes,DNAwas stainedwithHoechst33,342(0.2

m

g/mlinPBS)for5minatRT,and coverslipsweremountedonglassslidesusingAquaPolyMount (Polysciences).ImageswereacquiredusingaZeissAxiovert200M epifluorescence microscope equipped with an AxioCam MRm camera,andwereanalyzedwithAxiovisionorImageJsoftware.

2.6.Analysis

g

H2A.Xformationbyimmunofluorescencemicroscopy

For detection of

g

H2A.X an immunocytochemistry staining wasperformed.Cellswerefixedwith4%PFAatRTfor30min.For permeabilization,cells wereincubatedinPBSwith0.1%sodium citrate and 0,3% Triton X100 for 10min at 4C. After several washing steps, unspecific binding sites were saturated with blocking solution (DakoCytomation) for 30min at RT. Then sampleswereincubated withprimaryantibodysolution(Milli pore,1:600) overnightat 4C. ThesecondaryantibodyDy549 anti mouse(Vector,1:800)wasincubatedfor1.5hatRT.Allsteps wereperformed in an automatedmanner byusing a pipetting robotwithtemperaturecontrol(Tecanfreedomevo).Nucleiwere stainedwithDAPIbyincubatingsamplesfor5minwith300nM DAPI (BioLegend). Afterseveral washing steps, cells werethen embedded withProLongAntifade (Invitrogen). The fluorescence intensityin allnuclei was analyzedbya microscope systemof TissueGnosticsusingTissueQuestsoftware.

2.7.DeterminationofLC50valuesofalkylatingcompounds

HaCaTcellswereseededin96 wellplatesatadensityof25,000 cellsperwellandgrowninDMEM,supplementedwith10%FCSfor 24h. DMEM was removedandcells wereexposed toa specific agent,eitherSM,HN 2,chlorambucilorCEESatconcentrationsof 12.4

m

M,37.0

m

M,111

m

M,333

m

M,1000

m

Mand3000

m

Mfor1h.

In case of SM, the 3000

m

M concentration was omitted and concentrationsfrom4.1

m

Mto1000

m

Mwereused.Agentsused for exposure were dissolved in Minimum Essential Medium (MEM).ControlwellsreceivedpureMEM,whereas100%cytotox icity was induced by0.1% Triton X 100, dissolved in PBS. Each exposuregroupconsistedof12wells, i.e.arowonthe96 well plate.After1h,poisonedandcontrolmediumwasremovedand replacedbyfreshDMEM,supplementedwith5%FCS.Cellswere incubatedfor48h.Cellvitalitywasthendetermined,usingtheXTT assay (Roche, Switzerland). Non linear regression of dose responsecurvesanddeterminationofLC50valueswasconducted, usingGraphPadPRISMsoftware.

2.8. Annexin Vjpropidium iodide staining

To analyze the mode of cell death after CEES treatment, HaCaT cells were seeded at 6.0 x 10

⁵

cellsfwell

in 12

well

plates

and

incubated for 24 h. Prior treatment of cells with CEES for 1 h, cells

were

pre incubated

with ABT888 for 30 min. Then, cells were washed with PBS, supplemented with medium and incubated for another 23 h.

Thereafter,

medium containing cell debris and detached cells were collected in pre chilled 15

ml tubes. Cells were

trypsinized, added to the tubes as well, all cells were pelleted at 1000

rpm

for 5 min at 4 °(, washed with 5 ml cold PBS, and counted with a

CASY

cell counter (Roche). Then, cells were centrifuged at 1000

rpm for 5 min at 4

o c and r esuspended in annexin

V

binding buffer at a final titer of 2.0 x 10

⁶

cells/mJ.

In

the dark, 2.5 f.Ll of annexin

V

FITC solution (Enzo Ufesciences) was mixed with 50 f.Ll cell suspension, samples were

incubated for

15 min on ice, and 245 f.Ll annexin binding buffer

(10 mM HEPES/NaOH,

pH 7.4, 140

mM

NaCI, 2.5

mM

CaCh) and 1 f.Ll propidium iodide ( 1.0

mg/

mJ

)

were added. Subsequently, samples were subjected to flow cytometric analysis (BD

l.SR ^H).

2.9. NAD• cycling assay

NAo• levels were determined as described previously using an enzymatic cycling assay modified

from (Jacobson and jacobson, 1976). Briefly,

after

treatment,

cells were harvested and 1 x

10⁶

cells were resuspended in 1 ml PBS and immediately placed on ice.

Subsequently, 48 f.Ll of

perchloric

acid

{11.63 M)

was added,

reaction

mixtures were

incubated

for 15 min on ice, and centrifuged

for

10 min at 1500 x

g.

Then, the supernatant was

A

Bis(2-chlorethyl)-sulfide (SM, sulphur mustard)

CI~S~CI

4-[bis(2-chlorethyl)amino]-benzenebutanoic acid (Chlorambucil)

Cl~

B

Cl

~·

.c

!!

>

100

50 ... .

0

OH

mixed with 700 f.Ll of 0.33 M K

2

HP04

(pH 7.5 ), centrifuged, snap frozen in liquid nitrogen and stored at 80°C. After thawing and

centrifugation, 40

f.Ll

samples were mixed with 160 f.Ll of buffer A

(0.25 M H3P04, 0.5 M NaOH) and 100 f.Ll of buffer B [0.34 M bicine

NaOH (pH 8.0), 2.9mgfml BSA, 14.3 mM EDTA, 1.4mM MTT, 1.7M EtOH, 5.7 mM phenazine ethosulfate, 0.14 mgfml ADH ). After a 30 min incubation

period

at 30°C, absorption was measured at 550 nm

(with

690 nm as a reference wavelength) in a 96 well plate ELISA reader.

2.10. Proliferation assay

Cells were seeded in 12 well

plates in

triplicates and treated with ABT888 and CEES as described above. For time series experiments,

non treatment controls were included for every time

point. At the time points of

interest, cells were trypsinized and cell

numbers were determined using a

CASY

cell counter (Roche).

2.11. Cytokinesis block micronucleus assay

The assay was performed based on a protocol

published

by

(Fenech, 2007)

with some modifications. Briefly, 3

x 10⁵

cells were seeded on cover slips and incubated in medium supplemented or not with 1 f.LM ABT888, 30min prior to mustard exposure for 1 h.

Thereafter, samples were washed twice with PBS and cells were

incubated

for another 24 h

in

medium containing 6 1-Lg/ml cytochalasin B

(Sigma

Aldrich) to block cytokinesis

in the absence

or presence of ABT888. After 24 h, supernatant was removed and slides were washed twice and fixed with pre cooled methanol at 20 o c for 7 min. Subsequently, samples were washed three times

2-Chloroethyl-ethyl-sulfide (CEES, half mustard)

c1~

⁵

'-.../

Bis(2-chloroethyl)-methylamine (HN2)

CI~N~CI I

~ Sulfur mustard (LCSO: 96 IJM) oo@o CEES (LCSO: 1443 11M)

,;;;;.. HN-2 (LCSO: 88 IJM)

Chlorambucil (LCSO: 377 IJM)

10° 1 0 ¹ 10² 10³ 10⁴

Concentration [IJM)

Fig. 1. Mustard agents and their cytotoxicity profiles. A Chemical structures of sulfur and nitrogen mustards as used in this study. CEES is a mono-functional agent. whereas sulfur mustard, HN2, and chlorambucil are bi-functional agents. B. Cytotoxicity profiles and LCSO values of substances as shown in A HaCaTcells were treated with chemicals in concentrations as indicated and subjected to cytotoxicity testing 48 h post treatment. Data represent means± SO from 6-7 independent experiments.

Fig.2. Sulfurandnitrogenmustardsinducedose-dependentPARformationinHaCaTcells.A.DoseresponserelationshipofHN2treatmentandPARformationasevaluatedby immunofluorescencemicroscopy.CellsweretreatedwithHN2atconcentrationsasindicatedfor20minorwith100mMH2O2for5minaspositivecontrolandsubjectedto immunofluorescencestaining.Scalebarindicates20mm.Densitometricevaluationofmicroscopicdata;meansSDof3independentexperiments.C.Characterizationof H2O2-,CEES-andSM-inducedPARylationinHaCaTcellsusinglaserscanningmicroscopy.Cellsweretreatedwith3mMCEESand0.3mMSMfor10minorwith1mMH2O2for 5min.D.Dose-dependentinductionofPARformationaftertreatmentwithlowdosesofSM,CEES,andHN2asanalyzedbyquantitativeisotope-dilutionmassspectrometryas describedin(Martelloetal.,2013).DatarepresentmeansSDfromatleast3independentexperiments.Statisticalanalysiswasperformedusingone-wayANOVAtesting followedbyDunnett’smultiplecomparisontesting.*P<0.05,**P<0.01;***P<0.001.

for 5min,DNAwas stained withHoechst33342, and coverslips mounted on glass slides using Aqua PolyMount (Polysciences).

Counting of micronucleiwas performed bya blindedevaluator usingaZeissAxiovert200Mepifluorescencemicroscopefollowing thecriteriabyFenech(Fenech,2007).

2.12.Clonogenicsurvivalassay

The assaywas performed based ona protocol publishedby Franken and colleagues (Franken et al., 2006) with some modifications. Briefly, HaCaT cells (210⁵cells/ml) were

incubated for 10min in the presence or absence of 1

m

M ABT888beforeadditionof 10

m

lofa100CEESstockorEtOH/

HClcontrolsolutions.After30minincubation,100

m

lofthecell suspensionsweretransferredinto9.9mlofPBS,resultinginacell titerof2cells/

m

l.Cellswereseededinadequatecellnumbersin6 cmplatesandincubatedfor2weeksinthepresenceorabsenceof 1

m

MABT888.Then,mediumwasremovedandcolonieswerefixed and stained for 30min using a solution of 6% glutaraldehyde (Sigma Aldrich)mixedwith0.5%crystalviolet.Theculturedishes werewashedwithPBSandcoloniesconsistingofatleast50cells werecountedusingastereomicroscope(Leica).

Fig.3.Mustardsinduceahighlydynamicandsubstance-specificPARylationresponses.A.Time-dependentcellularPARylationinHaCaTcellsaftertreatmentwithmustards asanalyzedbyepi-immunofluorescencemicroscopy.CellsweretreatedwithSM,CEES,andHN2,fixedattimepointsasindicated,andstainedforcellularPARusingananti- PAR-specificmAB(10H).H2O2(100mM)treatmentfor5minservedasa positivecontrol.Scale barsindicate50mm.B.Signalintensitiesofindividualcellswere densitometricallyevaluated.DatarepresentmeansSD.Statisticalanalysiswasperformedusingone-wayANOVAtestingfollowedbyDunnett’smultiplecomparisontesting.

*P<0.05,**P<0.01;***P<0.001.

2.13.Statisticalanalysis

All experiments were performed in replicate numbers as indicatedanddataanalyzedwithGraphPadPrismSoftware,using statisticaltestsasindicated.

3.Results

3.1.Mustardsexhibitsubstance specificcytotoxicityprofiles

Epidermal keratinocytes are considered one of the primary targetsformustard inducedpathologies(Keheetal.,2009).Inthe present study, we used HaCaT cells as an established human keratinocytemodel(Boukampetal., 1988)tostudythePARylation dependent molecular toxicology of mustards. To define LC50values ofmustardsin thesecells andunder experimental conditions as used in this study, we performed a comparative cytotoxicitystudyonapanelofmustardagents,includingmono andbi functionalnitrogenaswellassulfurmustards(Fig.1).Asis evidentfromcytotoxicityprofiles,thebi functionalmustardsSM andHN2exhibitthelowestLC50values(100

m

M)whereasthe pharmacologicallyrelevantbi functionalnitrogenmustardchlor ambucil displayed an LC50 value in the sub millimolar range (377

m

M)andthemono functionalhalfmustardCEESatconcen trations>1mM.BasedonthefindingofsimilarcytotoxicityofSM andHN2aswellasthestructuralsimilarityofSMwithCEES,we selectedSManditsanaloguesHN2andCEESasmodelsubstances forfurtherin depthanalysis.However,oneneedstokeepinmind

that the CEES toxicity depends on specific conditions of the treatment protocol.Thestudiesof CEES induced cytotoxicityin HaCaTcellsreportedLC50valuesdifferingbyatleastoneorderof magnitudewithinarangeof0.25to2mMdependingontreatment timeandconditionsapplied(Paromovetal.,2011a,b;Tewari Singh etal.,2010,2014a).

3.2.SulfurandnitrogenmustardsinducePARylationinHaCaTcellsin adoseandtimedependentmanner

Despitetheexistenceofsporadicreportsofmustard induced cellularPARylationresponse(seeabove,Section1),thisphenom enonispoorlycharacterized.Inanaccompanyingarticle(Debiak etal.,2015),weestablishedareliableprotocolforthetreatmentof HaCaT cells with CEES and subsequent analysis of cellular PARylationresponseusingimmunofluorescencemicroscopy.Here weappliedthisprotocoltoexaminesystematicallydose andtime dependentPARylation dynamicsupon treatmentof HaCaTcells withdifferent bi functional mustardderivatives (SM,HN2,and chlorambucil). We analyzed cellular PAR formation using two completelyindependentbioanalyticalapproaches(Figs.2and3).

Ourdataobtainedwithsemi quantitativesinglecellimmunofluo rescencemicroscopyusingthePAR specificantibody10Hrevealed that both nitrogen and sulfur mustards induce cellular PAR formation in living cells in a dose dependent manner (Figs.2and3).Asexpected,mustardagentsinducedPARformation atalowerlevelthantheoxidizinggenotoxicagentandstrongPARP activator H2O2,usedas a positive control(Fig.2A C). We used

Fig.4.Dose-andtime-dependentchlorambucil-inducedPARylationinHaCaTcells.A.Doseresponserelationship.Cellsweretreatedwithchlorambucilatconcentrationsas indicatedfor90minandsubjectedtoimmunofluorescencestaining.B.Time-dependentcellularPARylationaftertreatmentwithchlorambucil(2mM)fortimesasindicated followedbyimmunofluorescencestaining.H2O2(100mM)treatmentfor5minservedasapositivecontrol.Scalebarsindicate20mm.

A

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>

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~ _c

<

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(/)

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1 .50 1 .2 5 1.0 0 0.7 5 0.5 0

• - ABT888 ] •••

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^{+ ABT888}

0.25 •

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CEES

Viability

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=' 2.0 0

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; 30

_~

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i ^J

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0.0 0.5 1.0 1.5 2.0 CEES [mM]

+

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0 1 2 3 4 5 6 7 8 9 10 H202 [mM]

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^~

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+

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Time [h]

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Cont

~ Cont + ABT888

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24 Time [h]

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~ 2S u

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+ ·

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·--··

0.0 0.5 1.0 1.5 2.0 CEES [mM)

+

-ABT888 ]-

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+ ABT888 •••

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/ ^!~£

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~~0/

0 1 2 3 4 5 6 7 8 9 10 H20z [mM]

0.1 1

CEES [mM]

Fig. s. Analysis of shon-tenn cytotoxidty, NAD• levels, and cell protiferation after combined treatment of HaCaTcells with CEES and PARP inhibitor ABT888. A and B. CEES treatment induces a moderate decrease in intracellular NAD• levels in HaCaTcells that can be rescued by PARP inhibitor treatment. Cells were pre-incubated± ABT888 (1 IJ.M) for 30 min and then treated with genotoxins as indicated. A Dose-response relationship of CEES treatment and intracellular NAD• levels. Cells were treated with CEES at concentrations as indicated for 10 min. H202 (1 mM) treatment served as a positive control. Control indicates EtOHfHCI solvent control. Data represent means± SO from 3 independent experiments. B. Time~ourse analysis of intra-cellular NAD• levels after low~ose CEES treatment (250 IJ.M) for 1 h. Data represent means±SD from

>3 independent experiments. C. CEES-induced shon-term cytotoxicity does not dependent on cellular PARylation. Cells were pre-incubated±ABT888 (11J.M) for 30 min, treated with CEES and H202 at concentrations as indicated for 1 h. and subjected to cell death analysis 23 h later using Annexin Vjpropidium iodide staining and flow cytometric analysis. Data represent means± SEM from > 3 independent experiments. Viable cells refer to annexin V /PI~ouble negative cells; apoptotic cells to annexin V- positive. PI-negative cells; and necrotic and late-apoptotic cells to annexin VfPI-double positive cells (ratios compared to total cell numbers). D and E. PARP inhibition

confocalmicroscopytocharacterizeaccuratelytheintra cellular distributionofPARin HaCaTcells uponmustardtreatment.SM, CEESandH2O2treatmentledtoexclusivelynuclearPARformation atdistinctfoci,stronglyindicatinganactiveroleofPARylationin mustard inducedgenotoxicstressresponse.(Fig.2C).Analysisof cellularPAR formationupon low dosemustard treatmentusing quantitative isotope dilution mass spectrometry as published recently (Martello et al., 2013) proved the dose dependent formation of PAR molecules upon mustard treatment with unequivocal chemical specificity (Fig. 2D). Specifically, these experiments revealed that bi functional mustards, i.e., SM and HN2,areabletoinducecellularPARylationinthelowtomedium micromolar range, whereas millimolar concentrations of the mono functionalagentCEESarenecessarytoinducePARylation significantly.This isconsistentwiththe factthat CEES induced cytotoxicityis>10 foldlowerthanthoseofSMandHN2(Fig.1).

Interestingly,PARylationdynamicsconsiderablydifferedbetween bi and mono functional mustard derivatives, indicating major differencesinthetoxicokineticand/ortoxicodynamicpropertiesof thesecompounds(Fig.3).AfterCEEStreatment, PAR formation peaked at 10min post treatment and declined close to the thresholdofsensitivityafter45min.NosecondwaveofPARylation wasobservedupto6haftertreatment(Fig. S1).However,it is important to note that even 6h after treatment, PAR signal intensitywas abovebackground, indicating sustained low level stimulationofPARPactivity(Fig.S1).Incontrast,thebi functional compounds clearly induced a biphasic PARylation response, showingamoderatefirstwaveofPARPactivationwithinthefirst 2 10min,whichdeclinedclosetothethresholdofsensitivityafter 30min,beforeinducinga secondwaveof PARylationwithpeak valuesafter60min.Ofnote, thepharmacologically relevantbi functionalcompoundchlorambucilalsoinducedPARformationin HaCaTcells withsimilardynamicsasobservedforSMandHN2 (Fig. 4), suggesting a role for mustard induced PARylation in chlorambucil relatedcancertherapy.

AsshowninDebiakatal.(2015),whencellswerepre incubated for30minwith1

m

MofthePARPinhibitorABT888,PARformation couldbestronglyinhibitedaftergenotoxictreatmentwithCEESor H₂O₂.To this end, we used this protocol toexplore functional consequencesofmustard inducedPARformationasreportedin thefollowingsections.

3.3.Analysisofmustard inducedshortandlong termcytotoxicityasa functionofPARPactivity

PreviouslyitwasreportedthatSMtreatmentledtoalowering ofNAD⁺levels,aneffectthatinsomestudiescouldbeinhibitedby PARPinhibitortreatment(seeabove,Section1).WhileKeheetal.

(2008)observednochangesinoverallcytotoxicityuponSMand PARP inhibitor treatment, their results suggested that PARP inhibitioninducesaswitchinthemodeofcelldeathbyinhibition ofnecrosisbutpromotionofapoptosis.Totestifthesameholds trueuponCEEStreatmentweexposedHaCaTcellstoincreasing dosesofCEESanddeterminedintra cellularNAD⁺levelsusingan enzymaticNAD⁺cyclingassay(JacobsonandJacobson,1976).As expected,treatmentofcellswith1mMH2O2for10minledtoa massive lowering of NAD⁺ levels, an effect that was almost completely abrogated by pre incubating cells with ABT888 (Fig. 5A). In contrast, short term treatment of HaCaT cells for 10minwith CEESin concentrations up to3mM reduced NAD⁺ levels only by 10%. Despite the unexpected finding that this

reductionshowednodose dependency,reducedNAD⁺levelswere mostlikelycausedbyPARPactivation,since theeffectcouldbe completelyabrogatedbyABT888treatment.Thesefindingsarein linewithtime courseexperimentsdemonstratingthatlow dose CEEStreatment(250

m

M)leadstoasignificantdropinNAD⁺levels by40%at3 6haftertreatment,followedbypartialrecoveryat24h aftertreatment.Onceagain,applicationofABT888preventedthe CEES inducedloweringofNAD⁺levels(Fig5B).Next,totestifthese moderatedecreasesinNAD⁺levelsmayaffectcellularsurvival,we treatedHaCaTcells withincreasingconcentrationsof CEESand performed cytotoxicity analyses. As expected, annexin V in combinationwithpropidiumiodidestainingrevealedthatPARP inhibitionsignificantlyreducedapoptoticandnecroticcelldeath uponH2O2treatment,mostlikelyduetoinhibitionofPARP1and consequentlypreservationofNAD⁺levels(Fig.5C).Itisinteresting tonote that at lower concentrations of H2O2 treatment (upto 3mM)PARPinhibitionsignificantlypreventedapoptosisinduc tion,whileathigherconcentrationsofH2O2treatment(>5mM) inhibition ofnecroticcelldeathbecamemoreprevalent. Inline with therather moderate reduction of NAD⁺ levelsupon CEES treatment, but contrary to previous reports on SM induced cytotoxicity, no effect of PARP inhibition on CEES induced cell deathwasobservedatall,i.e.neitherforapoptoticnorfornecrotic celldeath(Fig.5C).Totestiflessdrasticendpointsthancelldeath wereaffectedbyPARPinhibition,wedeterminedtheproliferation capacity ofHaCaTcellsupon CEES treatmentin theabsenceor presenceofABT888.WhileABT888treatmentaloneslightly,but non significantly, affected cell proliferation, CEES treatment significantly impaired cell proliferation, as expected, at sub millimolarconcentrations(Fig.5DandE).Importantly,thiseffect waspotentiatedbyABT888.Theseresultsindicatethatmustard inducedPARylationindeedplaysafunctionalroleincellularstress response. In order to characterize this further, we examined clonogenic survival of HaCaT cells upon mustard and PARP inhibitor treatment. The clonogenic survival assay is a very sensitive long term toxicity test, which apart from cell death, takesintoaccountimpairedcelladhesionandproliferationeffects.

Fig. 6 demonstrates that the soletreatmentwith SM, CEES,or HN2 ledto dose dependent impairment of clonogenic survival withEC50valuesforSMandHN2inthelowmicromolarrange,and for CEES in the high micromolar range. Interestingly, while ABT888 treatment alone did not affect clonogenic survival (Fig. 6B), it significantly sensitized cells to mustard induced toxicityforallthreemustardstested,withthelargestsensitization observedforSMtreatment(Fig.6).

3.4.Analysisofmustard inducedgenotoxicityasafunctionofPARP activity

AkeyquestionofthisstudywastoanalyzeifPARPinhibition sensitizesHaCaTcellstomustard inducedgenotoxiceffects.Ifthis were thecase it would imply that PARP inhibitors are contra indicatedforSM exposedsubjects,duetopotentialpotentiationof SM induced carcinogenesis.To this end, we analyzedtwo end points of mustard induced genotoxicity, i.e., DNA adducts and micronucleiformationrates.

3.4.1.ExcisionofCEES inducedguanineadductsisnotaffectedby PARPinhibition

Totest ifPARylationalreadyaffects early stagesof mustard induced DNAdamageresponse, we analyzedifPARP inhibition

combinedwithCEEStreatmentimpairsproliferationofHaCaTcells.CellswerepretreatedABT888(1mM)for30minandthentreatedwithCEESfor1h.D.AfterCEES treatment(0.5mM),sampleswereincubatedfortimepointsasindicated(ABT888)andthensubjectedtocellcounting.Theinhibitoralonedidnotinfluencethecellgrowth.

DatarepresentmeansSEMfromthreereplicates.E.AfterCEEStreatmentatconcentrationsasindicated,cellswereincubatedfor72h(ABT888)andsubjectedtocell counting.DatarepresentmeansSEMfrom3independentexperiments.Statisticalevaluationwasperformedusingtwo-wayANOVAtestingfollowedbySidak'smultiple comparisontesting.*P<0.05,**P<0.01;***P<0.001.

affectstheexcisionrateofmustard inducedguaninebasedamage.

To this end, wemodifieda recently reportedimmuno slot blot protocolforthedetectionofSM andCEES inducedN7 (H)ETE guanine base damage (Kehe et al., 2013) for usage in an immunofluorescencesetting.FigS2demonstratesthatthisassay is able to detect CEES induced N7 ETE guanine adducts at a concentration of >100

m

M CEES and SM induced N7 HETE guanine adductsat a concentrationof>10

m

M.Using thesame assay, we monitoredthe excision kineticsof damaged bases in HaCaTcells after a single dosetreatment of CEES for thetime courseof2days.Interestingly,eventwodaysafterCEEStreatment, N7 ETE guanine derivedsignalswereclearlyabovebackground, indicatingthatnotallbaselesionsarerepairedduringthisrepair period (Fig. 7). No significant effect of PARP inhibition on the removalN7 ETE guanineadductswasobservedwithin48hafter mustard treatment.This isconsistentwiththenotionthat PAR formationisinducedbyDNAstrandbreaksthatoccurduringthe repair of DNA adducts. Although this hypothesis needs to be analyzedin greaterdetail,

g

H2A.Xstaining(Fig. S3)aswell as previous reports using an automated DNA alkaline unwinding assay(i.e.,FADUassay,(Debiaketal.,2011))supportthenotionthat DNAstrandbreaksareformedduringSM inducedgenotoxicstress response,whichcanleadtoPARP1bindingandactivation.

3.4.2.PARPinhibitionpotentiatesCEES inducedmicronuclei formation

TotestifPARylationaffectslaterstagesofthemustardinduced genotoxic stress response we performed the cytokinesis block micronucleus assay. Micronucleus formation is a sensitive

biomarker for major genomic rearrangements that occur in dividing cells and is commonly used in genotoxicity testing (Fenech, 2007) (Fig. 8A). While mustard or ABT888 treatment alonedidnotsignificantlyincreasemicronucleusfrequenciesin HaCaTcells,culturestreatedwithmustardsincombinationwith ABT888showedamoderateincreaseinmicronucleusfrequencies, whichreachedstatisticalsignificanceinsamplesco treatedwith CEES and ABT888 (Fig. 8Band D) Theseresults providestrong evidence for an active role of cellular PARylation in mustard inducedgenotoxicstressresponseinordertoprotectandrestore cellular function upon the genotoxic insult. In conclusion, our resultsshowthattheuseofPARPinhibitorsascountermeasuresto treat mustard induced injuries must becarefully consideredin viewofrisk benefitanalysisandmightberestrictedtotreatment ofacute inflammatoryconditions;however, they mayraise the possibilitytousePARPinhibitorsaschemosensitizingagentsfor mustard basedcancertherapy.

4.Discussion

Inanaccompanyingarticle,wehaveestablishedatreatment protocol and provided a detailed analysis of the CEES induced PARylationresponse inHaCaT(Debiak etal.2015). Thepresent studyprovidesnowasystematicandcomparativecharacterization ofthemustard inducedPARylationresponsesanditsfunctional consequenceswithrespecttomustard inducedcytotoxicityand genotoxicityinHaCaTcellsaftertreatmentwithapanelofmono andbi functional mustardagents.Ourdata revealedsubstance specific, dose and time dependent PARylation dynamics after Fig.6.PARPinhibitioncombinedwithmustardtreatmentimpairsclonogenicsurvivalinasubstancespecificmanner.Cellswerepre-incubatedABT888(1mM)for10min andthentreatedwithmustardsatconcentrationsasindicatedfor30min.Afterincubationfor2weeksABT888(1mM),samplesweresubjectedtoclonogenicsurvival analysis.A.RepresentativeexperimentshowingclonogenicsurvivalofHaCaTcellsaftersulfurmustardtreatment.B.ABT888treatmentalonedidnotsignificantlyinfluence platingefficiencyofHaCaTcells(notsignificantcomparedto100%asevaluatedbyone-samplet-test).C-E.Dose-responsecurvesforcellstreatedwithSM(B),CEES(C),and HN2(D).DatarepresentmeansSDfrom3independentexperimentsperformedintechnicaltriplicates.Statisticalevaluationwasperformedusingapairedtwo-way ANOVAtestfollowedbySidak’smultiplecomparisontesting(*P<0.05,**P<0.01;***P<0.001)orStudent’st-test(^##P<0.01).

treatmentofHaCaTcellswithfourdifferentsulfur andnitrogen mustardsusing completely independent bioanalytical methods based on immunofluorescence microscopy and quantitative isotope dilution mass spectrometry. These results demonstrate thatbi functionalagents,suchasSMandHN2induceabiphasic PARylation response with peak values after 60min, whereas mono functionalCEESinducesonlyasinglewaveofPARylation withmuchlowerpotencythatpeaksat10 15min.Furthermore, weprovideevidencethatPARylationhasfunctionalconsequences inmustard inducedcellularstressresponse,sincePARPinhibition sensitizes cells to mustard treatment in terms of clonogenic survival (SM, CEES, and HN2), cell proliferation (CEES), and micronucleus formation (CEES). These results have several important implications in terms of mechanisms of toxicity of nitrogenandsulfurmustardsaswellastowardsthepotentialof PARPinhibitorstotreatmustard inducedinjuries,ontheonehand, ortosupportmustard basedchemotherapy,ontheotherhand.

4.1.(i)CEESandHN2asSMsurrogates

SMisclassifiedasaSchedule1chemicalwarfareagentandits useisregulatedbytheChemicalWeaponsConvention.Researchon SM relatedmolecularmechanismsisofhighinterestforseveral reasons(MangerichandEsser,2014):(i)mustardexposureisstilla considerablethreat,asthis mayoccurduringthedestructionof existing SM stockpiles or SM release by terroristic attacks or uncontrolledmilitaryactions.Inordertodevelopefficientmedical countermeasures for mustard induced pathologies, a detailed understanding of the underlying toxicological mechanisms is essential.(ii)SM derivatives,i.e.,several nitrogenmustards,are being used as chemotherapeutics for the treatment of various typesofcancers.Tothisend,anunderstandingofthemolecular mechanisms of mustard compounds may help render such therapiesmoreefficientandtolerable.Forlegalreasons,research partners (e.g., universities or commercial enterprises) of the above mentionedexpertlaboratoriescannotconductexperiments withSMandthus,thereisasignificantneedtodefinethemost appropriatesurrogate substancesfor SMfor theirresearch. The mono functionalalkylatingagentCEESiswidelyusedassucha substitute,becauseitrepresentsthemono functionalanalogueto SM[Fig.1,(Inturietal.,2011;Jainetal.,2011;Jowseyetal.,2009;

Sayeretal.,2010;Wangetal.,2012)].Inaddition,thebi functional nitrogenmustardisusedasasurrogateagent,becauseittakesinto accountthebi functionalreactionmechanismofSM[Fig.1,(Jain etal.,2013;Tewari Singhetal.,2013;Wangetal.,2012)].However, whichofthetwosubstancesrepresentsthemoreappropriateSM substitute is a matterof debate. Our data suggest that HN2is actuallythepreferablesubstitutetostudySM relatedmolecular mechanisms. Like SM, it is a bifunctional agent and its simple structure withoutany voluminoussubstituents facilitates rapid reactionwithbiologicalmacromoleculesandthus,highcytotoxic ity.Incontrast,chlorambucilisstillabifunctionalagent,butalarge substituenthasbeenintroducedtolimititstoxicityandasshown inFig. 1,anapprox.4 foldconcentration(comparedtoSMorHN 2) is required to induce a similar 50% cytotoxicity. CEES is a monofunctionalalkylatingagentanditstoxicityprofileinHaCaT cellsisverydifferentfromallothercompoundstested:concen trations of up to333

m

Mweretolerated withoutany apparent effectoncellvitality,even 1000

m

Monly induceda minor,10%

decline,whereas3000

m

MCEESresultedinalmost100%celldeath under the conditions used in experiments shown in Fig.1. In summary,ourresultsshowthat withsomelimitations HN 2,a bifunctional alkylating agent with a simple structure closely resemblingSM,representsanappropriatesurrogatetostudySM relatedtoxicity.

Similarresults wereachievedin clonogenic survival(Fig.6) cytotoxicity profiles, in which SM and HN2 exhibit similar LD50 valuesin HaCaTcells,which are>10 foldlowerthanthat ofCEES.Furthermore,thePARylationresponsesinducedbySMand HN2aremuchmoresimilarthantheoneinducedbyCEES.This holdstrue,bothforquantitativeaspectsofPARformation(Fig.2D) aswellasfortime dependentPARylationdynamics(Fig.3).This may be attributed to the fact that SM and HN2 induce DNA crosslinks,whereasCEESdoesnotformthiskindofdamage.These invitroresultsareconsistentwiththetissuepathologyinducedby mustard compounds. Thus, while SM and HN2 display similar pathologicalpotenciesinmice,CEESneedstobeappliedin100 foldhigherconcentrationstoobservecomparableeffects(Tewari Singhetal.,2013;Wangetal.,2012).Furthermore,thesameholds trueforthechemicalreactivityofthesesubstances(Wangetal., 2012). Differences between SM and CEES induced toxicity also became apparent, when comparing apoptosis and necrosis Fig.7.ExcisionofCEES-inducedN7-ETE-guanineadductsisnotdependentoncellularPARylation.A.Cellswerepre-incubatedABT888(1mM)for30min,treatedwith 1mMCEESfor1h,thenincubateduptotimepointsasindicated,andsubjectedtoimmunochemicalstainingusinganN7-(H)ETE-G-specificantibody2F8asdescribedinKehe etal.,2008.Scalebarindicates50mm.B.Densitometricanalysisfromthreeindependentexperiments,meansSD.Statisticalevaluationwasperformedusingtwo-way ANOVAtestingfollowedbySidak’smultiplecomparisontesting.Foradose-responseanalysisofCEES-inducedN7-ETE-guanineadductsrefertoFig.S2.