Mechanisms linked to differences in the mutagenic potential of 1,3-dinitropyrene and 1,8-dinitropyrene

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Toxicology Reports

j o u r n al ho me p ag e : w w w . e l s e v i e r . c o m / l o c a t e / t o x r e p

Mechanisms linked to differences in the mutagenic potential of 1,3-dinitropyrene and 1,8-dinitropyrene

J.A. Holme

^a,∗

, H.E. Nyvold

^a

, V. Tat

^b

, V.M. Arlt

^c

, A. Bhargava

^b

, K.B. Gutzkow

^a

, A. Solhaug

^d

, M. Låg

^a

, R. Becher

^a

, P.E. Schwarze

^a

, K. Ask

^b

, L. Ekeren

^a

,

J. Øvrevik

^a

aDivisionofEnvironmentalMedicine,NorwegianInstituteofPublicHealth,N-0403Oslo,Norway

bDepartmentofMedicine,McMasterUniversity,Hamilton,ON,Canada

cAnalyticalandEnvironmentalSciencesDivision,MRC-PHECentreforEnvironmentandHealth,King’sCollegeLondon,London, UnitedKingdom

dNorwegianVeterinaryInstitute,Oslo,Norway

a rt i c l e i n f o

Articlehistory:

Received26March2014

Receivedinrevisedform7July2014 Accepted8July2014

Availableonline27July2014

Keywords:

Nitro-PAHs 1,3-Dinitropyrene 1,8-Dinitropyrene DNAdamage Apoptosis

a b s t ra c t

This studyexploresandcharacterizesthe toxicityoftwoclosely related carcinogenic dinitro-pyrenes(DNPs), 1,3-DNPand1,8-DNP, inhuman bronchialepithelialBEAS-2B cellsand mouse hepatomaHepa1c1c7cells.Neither1,3-DNPnor1,8-DNP (3–30␮M) inducedcelldeathinBEAS-2Bcells.InHepa1c1c7cellsonly1,3-DNP(10–30␮M)induced amixture of apoptoticand necroticcelldeath after 24h. Both compoundsincreased thelevelofreactiveoxygenspecies (ROS)inBEAS-2BasmeasuredbyCM-H2DCFDA- fluorescence.AcorrespondingincreaseinoxidativedamagetoDNAwasrevealedbythe formamidopyrimidine-DNAglycosylase(fpg)-modifiedcometassay.Withoutfpg,DNP- inducedDNAdamagedetectedbythecometassaywasonlyfoundinHepa1c1c7cells.Only 1,8-DNPformedDNAadductmeasuredby³²P-postlabelling.InHepa1c1ccells,1,8-DNP inducedphosphorylationofH2AX(␥H2AX)andp53atalowerconcentrationthan1,3-DNP andtherewasnodirectcorrelationbetweenDNAdamage/DNAdamageresponse(DR) andinducedcytotoxicity.Ontheotherhand,1,3-DNP-inducedapoptosiswasinhibitedby pifithrin-␣,aninhibitorofp53transcriptionalactivity.Furthermore,1,3-DNPtriggeredan unfoldedproteinresponse(UPR),asmeasuredbyanincreasedexpressionofCHOP,ATF4 andXBP1.Thus,othertypesofdamagepossiblylinkedtoendoplasmicreticulum(ER)- stressand/orUPRcouldbeinvolvedintheinducedapoptosis.Ourresultssuggestthatthe strongercarcinogenicpotencyof1,8-DNPcomparedto1,3-DNPislinkedtoitshighergeno- toxiceffects.Thisincombinationwithitslowerpotencytoinducecelldeathmayincrease theprobabilityofcausingmutations.

Abbreviations: AhR,aromatichydrocarbonreceptor;B[a]P,benzo[a]pyrene;Chk,checkpointkinases;CYP,cytochromeP450;DMSO,dimethyl sulfoxide;DHE,dihydroethidium;1,3-DNP,1,3-dinitropyrene;1,8-DNP,1,8-dinitropyrene;DDR,DNAdamageresponse;ER,endoplasmicreticulum;

fpg,formamidopyrimidine-DNAglycosylase;Hoechst33342,2-(4-ethoxyphenyl)-2,5-bis-1H-benzimidazolehydrochloride);␥H2AX,phosphorylated H2AX; CM-H2DCFDA or H2DCFDA, 5-(and 6-)chloromethyl-2,7-dichlorodihydroﬂuorescein diacetate; Hoechst 33258, 2(2-(4-hydroxyphenyl)-6- benzimidazole-6-(1-methyl-4-piperazyl)benzimidazolehydrochloride);1-NP,1-nitropyrene;3-NBA,3-nitrobenzanthrone;RNS,reactivenitrogenspecies;

NR,nitro-reductasesnitro-PAHnitrosubstituted-polycyclicaromatichydrocarbon;PM,particularmatter;PAH,polycyclicaromatichydrocarbon;PARP, poly(ADP-ribose)polymerase;PI,propidiumiodide;PFT,piﬁthrin;ROS,reactiveoxygenspecies;SSB,singlestrandbreaks;UPR,unfoldedproteinresponse;

zVAD-FMK,benzyolcarbonayl-Val-Ala-Asp-ﬂuoromethylketone.

∗ Correspondingauthorat:DepartmentofAirPollutionandNoise,DivisionofEnvironmentalMedicine,NorwegianInstituteofPublicHealth,P.O.Box 4404,Nydalen,N-0403Oslo,Norway.Tel.:+4721076247;fax:+4721076686.

E-mailaddress:[email protected](J.A.Holme).

http://dx.doi.org/10.1016/j.toxrep.2014.07.009

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1. Introduction

Epidemiologicalstudieslinkexposuretourbanairpar- ticularmatter(PM)withanincreasedriskoflungcancer [1–3]andin October 2013theInternationalAgencyfor Research on Cancer (IARC) classiﬁed outdoor air pollu- tionascarcinogenictohumans(Group1).UrbanairPM isaheterogeneousmixtureofvarioustypeofPMinclud- ing combustion particles like diesel exhaust PM.These particlescontainpolycyclicaromatichydrocarbons(PAHs) andnitro-PAHs, formedduringtheincompletecombus- tion, which have been suggested to play an important roleinthePM-inducedcarcinogenesis[4].Experimental studieshaveshownthatmanyPAHsandnitro-PAHsare highlymutagenicandcancausetumorsinanimalmodels [5,6].

Nitro-PAHscanbemetabolizedintocorrespondingaryl- hydroxyamines both by cytosolic nitro-reductases (NR) andmicrosomalcytochromeP450(CYP)enzymes[7].CYP enzymescanactivatethePAH-ringtoepoxideinterme- diates that can be further converted to more reactive diol-epoxides[8].Arylhydroxyaminescanbefurtheracti- vatedbyN-acetylationorsulfonation[1,9].Reductionof nitro-PAHstothecorrespondingaminesmayalsoleadto theproductionofreactiveoxygen(ROS)ornitrogen(RNS) speciesdependingonthenitro-reductaseandtheavail- abilityofoxygen[1,10,11].Nitro-PAHscaninducecellular toxicityinmanywaysandbesidesDNAdamage,lysoso- malandmitochondrialdamagehavebeensuggestedtobe importanttriggeringsignals[12–15].

DNA damage triggers a complex protein kinase signaling cascade, the so-called DNA damage response (DDR),which canactivate cell cyclecheckpoint kinases (Chk1/2)therebypromotingDNArepairand/ortriggerapo- ptosis[16,17].TheresultingDDRdependsonthetypeof damage(DNAadductsaswellassingle-anddouble-strand breaks[SSBandDSB])aswellasamountofdamage.H2AX is a DDRprotein which is phosphorylated into ␥H2AX oftenfollowingDSB[18,19].TheproteinkinaseATM(ataxia telangiectasiamutated)andATR(ATMandRad3-related) regulatesChk1/2signaling,resultingin phosphorylation andactivationofp53[20–22].Increasedphosphorylation ofp53mayup-regulateexpressionofp21leadingtocell cyclearrestortheexpressionofcellsurfacereceptorslike Fasaswellasmitochondrial pro-apoptoticproteinslike Bax,Bak,PUMAandNOXA[23,24].Thusp53iscentralboth withregardtoregulatingcellcyclearrestgivingtimefor DNArepairortriggeringdeath/apoptoticsignalingpath- ways.

Endoplasmatic reticulum (ER) stress and the conse- quent unfolded protein response (UPR) are involved in variouspathological conditions [25,26]and canbetrig- gered by a variety of environmental factors including chemicalpollutants[27–29].ProlongedERstressissensed bytheUPRpathwaythroughthreeknownERmembrane boundtransducers:IRE1,ATF6andPERK[25].IRE1may be directly activated by unfolded proteins while ATF6 and PERK require the release of the chaperone GRP78 which dissociates when unfolded proteins accumulate in the ER [30]. Markers of the UPR response includes increased expression of GRP78, CHOP, ATF6,ATF4, and

Fig.1.Chemicalstructuresofthetestcompounds.

XBP1. These pathways attenuate ER stress by promot- ingproteinfolding, inhibitingmRNAtranslation, andby facilitatingdegradationofunfoldedproteins.However,if ER-stressisprolongedortooextensivetheUPRmayalso triggercelldeath.InparticularATF4anditsdownstream targetCHOP,apro-apoptotictranscriptionfactor,appears centralinUPR-mediatedcelldeath[30].

Celldeathhastraditionallybeenclassifiedintoapopto- sisornecrosis,butrecentscientificadvanceshaverevealed anumberofothermodesofcelldeathincludingvarious formsofprogrammednecrosis,senescence,autophagyand mitoticcatastrophe[31].Inpreviousstudieswehavesuc- cessfully usedthemousehepatomacelllineHepa1c1c7 as a model system to investigate various forms of cell deathinducedbyPAHsandnitro-PAHs[14,32,33].Thiscell linehasahigharylhydrocarbonreceptor(AhR)-inducible capacitywhichpromotesthemetabolicactivationofthese compounds.Oneofthemostimportantfindingswasthat reactivemetabolitesmaytriggerdifferentdeathsignaling pathwaysaswellasanti-apoptoticandpro-survivalsignals whichareimportantdeterminantsforthefinaloutcome.

Evencloselyrelatedchemicalswerefoundtotriggerdiffer- enttypesofcelldeath([12,13,34]).Thiswasalsothecase whenusinghumanbronchialepithelialBEAS-2Bcellsasan experimentalmodel[35].BEAS-2Bcellsareacommonly usedmodelforstudyingthecytotoxicityandgenotoxic- ity of air pollution components.In previousstudies we haveshownthaturbanairPMaswellasseveralindivid- ualnitro-PAHsresultedinAhR-dependentCYPexpression, DNAdamageandaDDRresultinginapoptosis,and/orthe releaseofcytokines[35–40].

Inthepresentstudywehavecomparedvariouscyto- toxicandgenotoxiceffects/responsesoftwocloselyrelated carcinogenicdinitro-pyrenes(DNPs)inthetwocellmod- els(Fig.1).Bothcelllineswereexposedto1,3-DNPandthe morepotentcarcinogen1,8-DNP[6,41,42].Overall,BEAS- 2BcellsrespondedwiththeinductionofROSandoxidative damagetoDNA,whileHepa1c1c7cellsseemedtobeamore sensitivecellularmodelwithregardstotheformationof DNAadducts,DDR(1,8-DNP)andcelldeath(1,3-DNP).Our resultssuggestthatthestrongercarcinogenicpotencyof 1,8-DNPcomparedto1,3-DNPislinkedtoitshighergeno- toxiceffects.Thisincombinationwithitslowerpotency toinducecelldeathincreasestheprobabilityof causing mutations.

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2. Materialsandmethods

2.1. Chemicals

LHC-9cellculturemedium,5-(and6-)chloromethyl- 2,7-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA in short H₂DCFDA), dihydroethidium (DHE) were from Life Technologies (Carlsbad, CA, USA). Sterile HBS and purified collagen,PureCol^TM,were fromInamed Bioma- terials(Freemont,CA,USA).1,3-Dinitropyrene(1,3-DNP), 1,8-dinitropyrene (1,8-DNP), benzo[a]pyrene (B[a]P), bovineserumalbumin(BSA),dimethylsulfoxide(DMSO), ethylenediamineteraacetic acid (EDTA), Hoechst 33258, Hoechst33342,aprotinin,Pifithrin-␮(PFT-␮),PonceauS, phenylmethylsulfonyl fluoride(PMSF),propidiumiodide (PI), polyoxyethylene octyl phenylether (TritonX-100) and zVAD-FMK were obtained from Sigma–Aldrich (St.

Louis,MO,USA).Piﬁthrin-␣(PFT-␣)andPepstatinAwere from Calbiochem (Cambridge,CA, USA). Leupeptinwas fromAmershamBiosciences(Uppsala,Sweden).Bio-Rad DC protein assay was from Bio-Rad Laboratories, Inc (Hercules,CA,USA). Fetalcalfserum (FCS), gentamycin, MEM alphamedium with l-glutamine, without ribonu- cleosidesanddeoxyribonucleosideswerefromGibcoBRL (Paisley, UK). UltraPure^TM Low Melting-Point Agarose was purchased from Invitrogen (Paisley, UK). All other chemicals wereof analytical gradeand purchased from commercialsources.

2.2. Antibodies

Antibodies against cleaved caspase 3, phospho-p53 (Ser15), p53,cleaved PARP (Asp214),␤-actin, phospho- H2AX(Ser139)wereobtainedfromCellSignaling(Beverly, MA, USA). NOXA was purchased from Santa Cruz Biotechnology, Inc,(Santa Cruz, CA,USA). As secondary antibodieshorseradishperoxidase-conjugated goat-anti- rabbit(Sigma),horseradishperoxidase-conjugatedrabbit anti-goatorrabbitanti-mouseIgGfromDako(Glostrup, Denmark)wereused.

2.3. Cellculture

BEAS-2B cells, immortalizedSV40-adenovirus-hybrid (Ad12SV40)transformedhumanbronchialepithelialcells, werepurchased fromtheAmericanTissueTypeCulture Collection(ATTCC,Rockville,MD,USA).Inthesecellsp53 ismutatedincodon47,asequencechangethatisreported nottochangeitsfunctionalproperties[43,44].Cellswere growninLHC-9mediumoncollagen(PureCol^TM)-coated culture flasks and dishes. Cells were grown in 5% CO₂ humidifiedairat37^◦Candkeptinalogarithmicgrowth phase(1–9×10⁶cells/75cm²flasks).Cellsweresplittwice aweek.

The mouse hepatoma Hepa1c1c7 cell line was purchased from the European Collection of Cell Culture (ECACC).Maintenanceofthecellswasdoneaccordingto ECACC’sguidelinesand theyweregrown inalphaMEM mediumwith2mMl-glutamine,withoutribonucleotides anddeoxyribonucleotides.Themediawassupplemented with 10% heat-inactivated FCS and 0.1mg/mL of the

fungicidegentamycin.Cellsweregrownin5%CO2humid- iﬁedairat37^◦Candkeptinalogarithmicgrowthphase (1–9×10⁹cells/75cm² ﬂasks). Cells were split twice a week.

2.4. Exposure

BEAS-2B cells were plated in 35mm 6-well dishes (8×10⁴or10×10⁴cells/well).Freshmediumwasadded thedayafterseedingandrightbeforeexposure.Hepa1c1c7 cellswereseededindishes(35mm6-wellculturedishes or90mmculturedishes)ortraysatadensityof70,000 cells per cm² the day before exposure. Fresh medium wasaddedbeforeexposure.Wheninhibitorswereused, cellswerepre-incubatedwiththeinhibitorfor1hbefore addingthetestsubstances.Cellsweretreatedwith1,3-DNP or1,8-DNP. Cells treatedwiththesolvent,DMSO, were usedascontrols.TheamountofDMSOaddedtothecul- turemedium wasalways less than0.5%. B[a]P(15␮M) wasincludedaspositivecontrol.Afterexposurecellswere alwaysbrieﬂyanalyzedbylightmicroscopytoverifyany toxicresponse.

2.5. Fluorescencemicroscopy

To characterize cytotoxicity, the cells were exposed totest substancefor 24hand analyzedby ﬂuorescence microscopyafterstainingwithHoechst33342(5␮g/mL) andPI (10␮g/mL), andprepared onamicroscopy slide.

Cell morphologywasevaluatedusing a NikonEclipse E 400fluorescentmicroscope,withanUV-2Aexcitationfilter 330–380nm(magnification1000×).Atleast300cellswere countedperslideandclassifiedaseitherviable,apoptotic or necrotic. Cells with clearly condensed and/or frag- mentednuclei(bothPI-negativeandPI-positive)aswell asPI-negativecells withpartialchromatincondensation werecountedasapoptoticanddeterminedasafraction ofthetotalnumberofcells.PI-stainedcellsexhibitinga roundedmorphologyandhomogenouslystainednucleus (typicalnecrotic)orpartiallycondensed chromatinwith lessfluorescentintensitiesweretermedPIpositive.Non apoptoticcells,excludingPI,wereconsideredasviablecells [12].

2.6. Flowcytometry 2.6.1. Cellcycleanalysis

Aftertreatmentfor24h,cellsweretrypsinatedandpre- paredforﬂowcytometry.CellularDNAwasstainedwith Hoechst33258(1.0␮g/mL)and TritonX-100(0.1%)and analyzedusingtheBDSLRIIﬂowcytometer.Percentages ofcellsinthedifferentphasesofcellcyclewereestimated usingtheMulticycleProgram(PhoenixFlowSystem,San Diego,CA,USA)[45,46].

2.6.2. ROSmeasurements

ROSproductionwasdetectedusingflowcytometryand theoxidation-sensitivefluorescentprobesCM-H₂DCFDA (1␮M) todetecthydrogenperoxideand DHE(5␮M) to detectsuperoxideanions[47].Briefly, cellswereloaded withCM-H₂DCFDAorDHEforthe2lasthofexposureto

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test-substancesfor2 or24h.Afterexposure, cells were washed twice with ice cold phosphate-buffered saline (PBS) and analyzed by ﬂow cytometry to exclude ROS excretedfromdeadcells,andeffectsmaskedbyanypossi- blyreductionofcellproliferation.Pro-oxidantsthatwere usedaspositivecontrolsincludedincubationwith1mM hydrogenperoxideforCM-H₂DCFDAand100␮Mmena- dioneforDHE.Thesubstanceshadsomeauto-ﬂuorescence, butthisdidnotaffecttheobtainedresults.

2.7. Singlecellgelelectrophoresis(cometassay)

Thecomet assay wasperformed as described previously[48].Briefly,cellswereexposedto1,3-DNP,1,8-DNP (1,3,10or30␮M)orB[a]P(10␮M)for24h.Mediawas removedandcellsweretrypsinatedandre-suspendedat 10⁶cells/mLin medium containing 10% FCS. Cells were dissolvedin 0.75% low melting point agarose dissolved in PBS with EDTA and molded as 48 (7␮L) gels onto GelBondfilmsattachedtoplasticframestofacilitatesub- sequent treatment steps. After lysis over night at 4^◦C (2.5Msodiumchloride,0.1MEDTA,10mMTrizmabase, 1%lauroylsarcosine sodiumsalt, pH10; with1%Triton X-100and10% DMSOfreshly added),filmswere rinsed once and then equilibrated/incubated in enzyme buffer for50minpriortoenzymetreatmentwiththebacterial formamidopyrimidine-DNA glycosylase (fpg; 1.0␮g/mL) orleftinthesamebufferbutwithoutthefpgenzyme(1h, 37^◦C).DNAunwindingwasperformedinelectrophoresis bufferinthedark(5+35min,4^◦C).Afterelectrophoresis at8–10^◦C(0.8V/cm,20min,pH13.2)andneutralization (0.4MTrizmabasebufferpH7.5for2×5min),filmswere fixedinethanolanddried.Rehydratedfilmswerestained withSyrbGold(10,000×dilutioninTris–EDTAbuffer,pH 8.0,20mininthedark)andscoredwiththeCometIVcap- turesystem(version 4.11)from PerceptiveInstruments (UK).Allsampleswereblindedand30nucleipergelwin- dowwerecounted.ThelevelofDNAdamagewasexpressed astailintensity,i.e.%fluorescenceinthecomettailrelative tothetotalfluorescenceofthecomet.

2.8. DNAadductanalysisby³²P-postlabelling

DNAwasisolatedusingastandardphenol/chloroform extraction protocol. For DNA adduct analysis by 1,3- DNPand 1.8-DNPthebutanol enrichmentprocedureof the ³²P-postlabelling method was used; B[a]P-derived DNAadductswereenrichedusingnucleaseP1digestion.

The procedures were essentially as described previously with minor modiﬁcation [49,50]. Brieﬂy, DNA (4␮g) wasdigested with 288mU micrococcal nuclease (Sigma, UK)and 1.2mU spleen phosphodiesterase (MP Biomedical,UK),enrichedandlabeledasreported.Chro- matographic conditions for thin-layer chromatography (TLC) on polyethyleneimine-cellulose (Macherey-Nagel, Düren,Germany)were:D1,1.0Msodiumphosphate,pH 6.0;D3,4Mlithiumformateand7Murea,pH3.5;andD4, 0.8Mlithiumchloride,0.5MTrisand8.5Murea,pH8.0.

Afterchromatography,TLCsheetswerescannedusinga PackardInstantImager(DowersGrove,IL),andDNAadduct levels(relativeadductlabeling[RAL])werecalculatedfrom

adductc.p.m.,thespeciﬁcactivityof[␥-³²P]ATPandthe amountofDNA(pmolofDNA-P)used.

2.9. GeneexpressionproﬁlingofgeneslinkedtotheUPR andinﬂammation

2.9.1. Microarrayanalysis

Hepa1c1c7cellsweregrownin90mmculturedishes (7×10⁵cells/dish)andexposedtothetestcompoundsfor 6h.Afterincubationandremovalofthecellsupernatant, cellswerestoredinRNAlateratroomtemperatureuntil processing.TotalRNA wasextracted usingtheRNAque- ousMicro Kit(Ambion)accordingtothemanufacturer’s instructions.RNAquantityandpuritywereassessedusing aNanodrop^®spectrophotometer.TheintegrityoftheRNA wasexaminedusingtheAgilentBioanalyzer2100toobtain anRNAintegrity number(RIN).Allsamplesusedinthe subsequentgeneexpressionanalysishadaRINofatleast 8.5.ThenCountersystem(NanostringTechnologies,Seat- tle,WA)wasusedtoquantifytheexpressionlevelsof67 pre-deﬁnedgenesofinterest.Eachhybridizationreaction contained100ngoftotalRNAina5␮Laliquot,reporterand captureprobes,sixpairsofpositivespike-inRNAhybridiza- tioncontrolsandsixpairsofnegativecontrolprobes.The hybridizationreactionproceededfor21hat65^◦C.

2.9.2. Real-timereverse-transcriptionpolymerasechain reaction(RT-PCR)

mRNA was reverse transcribed using Superscript II RT (Invitrogen, Carlsbad, CA) to obtain cDNA for gene expressionanalysis.Usinga7500Real-TimePCRMachine (AppliedBiosystems, FosterCity,CA) withTaqMan Uni- versal PCR MasterMix (Applied Biosystems) the PCR protocol involved: 20s initiation at 50^◦C followed by 10min at 95^◦C; 40 cycles of 15sec ampliﬁcation at 95^◦C; 1min at 60^◦C. Grp78 (Mm00517690g1), CHOP (Mm01135937g1), ATF6 (Mm01295317m1), spliced XBP1(Mm03464496m1),totalXBP1(Mm00457357m1), Nrf2(Mm00477784m1),PGK1(Mm00435617m1),RPLP2 (Mm00782638s1).CT wascalculated usingtheSDS softwarev2.2asdescribedbythemanufacturer(Applied Biosystems; Invitrogen, Life Technologies, Burlington, Ontario,Canada).

2.10. Westernblottingimmunoassay

Hepa1c1c7andBEAS-2Bcellsweregrownin90mmcul- turedishes(7×10⁵cells/dish),exposedtotestsubstances for24handthenwashedwithPBS.Cellswerethenfrozen, thawedand lysedin20mMTrisbuffer, pH7.5,150mM NaCl,1mMEDTA,1mMEGTA,1%TritonX-100,2.5mM sodiumpyrophosphate,1mM␤-glycerophosphate,1mM Na₃VO₄, 1mM NaF, 10mg/mL leupeptin, 1mM PMSF, 10mg/mLaprotininand10mg/mLpepstatinA.Aftercell lysis,solutionsweresonicated,centrifuged(290×g), and thesupernatantcontainingthecellularproteinswascol- lected. Protein concentrationwas measured by using a Bio-RadDCproteinassaykit.Asampleof12.5␮gproteinin eachwellwassubjectedto12%or15%sodiumdodecylsul- fatepolyacrylamidegelelectrophoresis(SDS-PAGE).Blots

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wereincubatedwithprimaryantibodiesovernightaccord- ingtothemanufacturer’srecommendations,washedand next incubated withhorseradish peroxidase-conjugated secondary anti-rabbit, anti-mouse or anti-goat antibodies (1:5000).The Westernblots where developed using the SuperSignal^® West Dura Extended Duration Sub- stratesystem(Thermo Scientiﬁc,Rockford, IL,USA) and aChemiDox^TMXRS+molecularimagerwithImageLab^TM software (Bio-Rad LaboratoriesInc., Hercules, CA, USA).

Typicalresultsarefromoneoutofthreeseparateexpo- sures/experiments.

InexperimentslinkedtoUPR,theproteinsampleswere loadedonto10%SDS-PAGE.Proteinsweretransferredusing awettransferapparatusontoaPVDFmembrane(Bio-rad) andimmunoblottedusingantibodiesfromSantaCruzBio- thecnologyagainstGrp78,Calnexin,andPDI.␤-Actinwas usedinallWesternblotsashousekeepingprotein.Anti- bodiesweredilutedinOdysseyBlockingBuffer(1:1000).

A twocolorWestern blotinfrared fluorescentdetection methodwas usedtovisualize theproteins usingIRDye fluorescent secondary antibodies and Odyssey^® Imager (LI-CORBiosciences).QuantificationoffluorescentWest- ernblotwasachievedwithImageJprogramdesignedby theNIH.

2.11. Statisticalanalysis

For the statistical analysis of Nanostring data, the nSolverAnalysisSoftwarev1.1(NanostringTechnologies) wasusedtonormalizetheraw geneexpressiondatato thepositivecontrolsand12referencegenes:ACTB,B2M, GAPDH,GUSB,HPRT1,IPO8,PGK1,POLR2A,RPLP2,TBP,UBC and YWHAZ. One-way ANOVA analysis was conducted using GraphPadPrism 5.0 (GraphPadSoftware, Inc,San Diego,CA,USA).Unsupervisedhierarchicalclusteringwas performedwithMultiexperimentViewer4.9usingEuclid- ian distance withaverage linkageclustering. The other resultswererepresentativesofthreeormoreindependent experimentswithidenticalconditions.Datawaspresented asmean±SEM.Statisticalsigniﬁcancewasevaluatedusing analysisofvariance(ANOVA)withtheDunnetpost-test.To analyzetheeffectoftreatmentoncytotoxicity,ROS,comet assayandRT-PCRresponsesstatisticswasperformedon log-transformeddata.P<0.05wasconsideredsigniﬁcant.

AllcalculationswereexecutedwithGraphPadPrismsoft- ware.

3. Results

3.1. Celldeath

BEAS-2BcellsandHepa1c1c7cellsweretreatedwith 1,3-DNP and1,8-DNP for24 and72h,or DMSOonlyas control.Toxicitywasexaminedbylightmicroscopy(data notshown)andquantiﬁedafterthestainingofcellswith Hoechst33342 and PI.No major cytotoxiceffects were observedinBEAS-2Bcellsafterexposureto1,3-DNPand 1,8-DNP(Fig.2).InHepa1c1c7cells(Fig.2),however,1,3- DNP caused a concentration-dependent increase in cell deathstartingat3␮Mafter24hexposure.Theinducedcell deathwasamixtureofapoptosisandnecrosis.Incontrast,

1,8-DNPdidnotinduceanysigniﬁcantcelldeathafter24h;

after72hincreasedcelldeathwasobservedatthehighest concentration(30␮M;Fig.2).

3.2. Characterizationofapoptosis

Wefurthercharacterizedthe1,3-and1,8-DNP-induced celldeathinHepa1c1c7cells. Afterexposuretovarious concentrationsofDNPsfor24h,Hepa1c1c7cellsweresam- pledandanalyzedbyWesternblotting.Thisrevealedan increasedcleavageofpro-caspase3and PARPat10and 30␮M1,3-DNP (Fig.3A),while1,8-DNP had noeffects.

Further,thepan-caspaseinhibitorzVAD-FMKreduced1,3- DNP-inducedapoptosisfrom31%tolessthan15%(Fig.3B), butthenecrosiswassomewhatincreased.

Effectsoncellcyclewereanalyzedbyﬂowcytometry after24hofexposure(Fig.3C).1,3-and1,8-DNP(10␮M) bothdecreasedthenumberofcellsinG1,andincreasedthe numberofcellsinSphase.1,3-DNPseemedtobeslightly morepotentthan1,8-DNP,andalsogaveasigniﬁcantG2 increaseevenat3␮M.

3.3. Cellularmechanismsinvolvedinthecytotoxicity ROS may be formed during nitro-PAHs metabolism as well as during the cell death process as a result of mitochondrial damage. As it has been suggested that ROS is an important determinant both with regard to inducedcytotoxicityandgenotoxicity,wemeasuredROS formation in BEAS-2B and Hepa1c1c7 cells after exposure tovarious concentrations of 1,3- and 1,8-DNP for 2 and 24h (Fig. 4; Supplementary Fig. 1). Both compounds increased the level of ROS as determined by CM-H2DCFDA-ﬂuorescencedetectinghydrogenperoxide.

In BEAS-2B cells, both compounds showed signiﬁcant responsesafterboth 2and24hexposure, with1,8-DNP givingaslightlylargerresponse.InHepa1c1c7cells,1,8- DNPwastheonlycompoundthatincreasedROSafter2h;

andclearlygivingamorerobustresponsethan1,3-DNP.

ROSformation/mitochondriafunctioncanalsobedeter- minedbyDHE-ﬂuorescencemeasuringsuperoxideanions^. Asshown in Fig.5 and SupplementaryFig. 2, theonly positiveresponsewasseenat10␮M1,8-DNPinBEAS-2B cells.

3.4. DNAdamage

Toexamineifthecytotoxicresponseobservedby1,3- DNPinHepa1c1c7cellsisduetoDNAdamageand/orto testiftheincreasedROSresultedinoxidativedamageto DNA,thecometassaywasused.Thealkalinecometassay measuresSSBsandalkali-labilesites,however,theuseof thefpg-modiﬁedcometassayalsoallowsthedetectionof oxidizedbasessuchas7,8-dihydro-8-oxoguanine(8-oxoG) andvariousring-openedpurines[48,51].InBEAS-2Bcells bothDNPsshowednoresponseinthecometassaywith- outfpg(Fig.6A),whiletheassaywaspositiveinHepa1c1c7 cells with1,8-DNP beingslightly morepotent (Fig.6B).

InBEAS-2Bcellsboth1,3-DNPand1,8-DNPincreasedthe levelofoxidativedamagetoDNAat3␮M,whileamarked responseinHepa1c1c7cellswasﬁrstseenatsomewhat

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Fig.2.Celldeathdeterminedbyfluorescencemicroscopy.BEAS-2BorHepa1c1c7cellswereexposedtovariousconcentrationsof1,3-DNP,1,8-DNPor DMSO(control)forupto72h.CellswerestainedwithHoechst33342andpropidiumiodide(PI),andsubsequentlyanalyzedforapoptosis(Ap)(including apoptoticnecrotic)andnecrosis(Nec)usingfluorescencemicroscopy.Thethirdcolumnsinthegraphsarethesumsofthetwofirst.Datapresentsthe mean±SEMofatleast3independentexperiments.*SignificantlydifferentfromDMSO-treatedcontrols(p<0.05).

higherconcentrations(Fig.6AandB).Theseresultscorre- spondedroughlytomeasuredROSproduction(seeabove).

Furthermore, we foundthat in BEAS-2B cells only 1,8- DNPresultedin DNA adductformation asmeasuredby the³²P-postlabellingassay(Fig.6CandD).Adductlevels werelowerthanthosereportedinapreviousstudyusing Hepa1c1c7cells.Inthatstudyalso1,8-DNPbutnot1,3-DNP inducedDNAadducts[14].

3.5. DNAdamageresponse

To investigate if thedifferences in toxicity between thesetwo strongly related DNPscouldbe explainedby differences in their DDR, we looked at the phosphorylation of H2AX and p53 by Western blotting. H2AX is phosphorylatedatserine139(␥H2AX)uponinductionof DSB. It is often used as an indicator of DSB formation

[52] which is considered to be a particular lethal DNA damage. InBEAS-2B cellsDNP treatmentonlylead toa slight increasein the␥H2AX formation(Fig.7A); while markedresponseswereobtainedinHepa1c1c7cellsafter exposure to low concentrations of both DNPs. 1,8-DNP inducedstronger␥H2AXresponsescomparedto1,3-DNP, and1,8-DNPalsoseemedtoinducetheseeffectsatlower concentrations (Fig.7B).Similarly, littleeffectsin BEAS- 2BandastrongereffectofDNPsinHepa1c1c7cellswere seenwithregardstop53phosphorylation,animportant transcriptionfactorparticularlyforgenescontrollingcell cyclearrest and/orapoptosis[37,53–56].Afterexposure theHepa1c1c7cellsto1,8-DNP,amarkedconcentration- dependent increase in phosphorylated p53was already seen at 1␮M. In contrast, only slight increases were observedafter1,3-DNPexposureathigherconcentrations (Fig.7).

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Fig.3. Effectsof1,3-DNPand1,8-DNPapoptosisoncellcycledistribution.Hepa1c1c7cellswereexposedtovariousconcentrationsof1,3-DNP,1,8-DNP,or DMSO(control)for24h.(A)LevelsofPARPandcaspase3wereanalyzedbyWesternblotting(shownisonerepresentativeexperimentoutofthreeseparate incubations).(B)Hepa1c1c7cellswerepre-treatedfor1hwithzVAD-FMK(20␮M)followedbyco-exposurewith1,3-DNP(30␮M)orDMSO(control)for 24h.Percentageofcelldeathwasestimatedbyfluorescencemicroscopycounts.Datapresentsthemean±SEMof3independentexperiments.*Significantly differentfromDMSO-treatedcontrols(p<0.05).^#SignificantlydifferentfromtreatmentswithoutzVAD-FMK(p<0.05).(C)Hepa1c1c7cellswereexposedto variousconcentrationsof1,3-DNP,1,8-DNPorDMSO(control)for24h.CellswerestainedwithHoechst33258andthecellcycledistributionwasmeasured byflowcytometer.Dataispresentedastherelativeproportionsofcells(%)inthedifferentcellcyclephases.Eachbarrepresentsthemean±SEMof3 independentexperiments.*SignificantlydifferentfromDMSO-treatedcontrols(p<0.05).

Pifithrin-␣(PFT-␣)suppressesp53-mediatedtranscrip- tion,whilepifithrin-␮(PFT-␮)inhibitsp53locatedtothe mitochondriabyreducingitsaffinitytotheanti-apoptotic proteinsBcl-xl and Bcl-2 [57]. Fluorescence microscopy analysesafterHoechst33342andPIstainingshowedthat PFT-␣almostcompletelyinhibited1,3-DNP-inducedcell death (Fig. 8A), while PFT-␮ was clearly less effective

suggestingthatp53-mediatedtranscriptionisrequiredfor thisapoptoticprocess.

3.6. EffectsonER-stressandUPR

Although 1,8-DNP induced considerably more ROS and DNA damage aswellas stronger DNA-DR and p53

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Fig.4.ROSasdeterminedbyH2DCFDA-fluorescence.BEAS-2BorHepa1c1c7cellswereexposedtovariousconcentrationsof1,3-DNP,1,8-DNP,orDMSO (control)for2or24h.Duringthelast2hCM-H2DCFDA(H2Dinfigure)wasadded.Afterexposurecellswereanalyzedbyflowcytometer(seealso SupplementaryFig.1).Eachbarrepresentsthemean±SEMof3independentexperiments.*SignificantlydifferentfromDMSO-treatedcontrols(p<0.05).

activation,1,8-DNPinducedlittleapoptosiscomparedto 1,3-DNP.Thus,additionalpathwaysarelikelyinvolvedin theapoptotic process inducedby 1,3-DNP. Oneof such pathwayscouldbeERstressandsubsequentUPR,which hasrecentlybeenfoundtobetriggeredbyvariousenvi- ronmentalfactors[27].Inordertoexplorethispossibility weexposedthecells totheDNPsfor 6hand examined theireffectongene expressionlinkedto UPR.First, we used Nanostring’snCounter technologyto analyzeRNA expressionprofilingfor67primarygeneslinkedtoUPRand inflammation.AsshowninFig.9AandB,1,3-DNPbutnot 1,8-DNPinducedaincreaseinAFT4,Grp78andXBP1gene expression,allofwhicharelinkedtotheUPRresponse.In linewithastrongerUPRalsotheincreaseofAFT6,CHOPwas markedhigher for 1,3-DNP than1,8-DNP. Interestingly, 1,3-DNP also increased the MAPK-related gene TAOK3, andgeneslinkedtoinflammation(e.g.IL-6).However,no releaseof IL-6 wasseenin BEAS-2BorHepa1c1c7 cells (datanotshown).Unsupervisedhierarchicalclusteringwas usedtoidentifysubgroupsofgenesandsampleswithsim- ilarexpressionpatterns.Theninesamplesinourdataset weresuccessfullygroupedintotheirrespectivetreatment groupsbasedsolelyontheirgeneexpressionprofiles.1,3- DNP-treatedcellswereidentifiedasadistinctclusterfrom thecontrolandthe1,8-DNP-treatedcells.Theaforemen- tionedUPRgenes and inflammatory markers were also sortedintoauniquegenecluster.Theclusterwaschar- acterizedbyincreasedexpressionofitscomponentgenes

inthe1,3-DNPsamplescomparedtothecontrols.Asimilar trendof1,3-DNP-inducedUPRactivationwasveriﬁedin independentexperimentsusingRT-PCR(datanotshown).

4. Discussion

1,3-DNP and 1,8-DNP are both reportedto begeno- toxicandinducetumorsinrats,but1,8-DNPisconsidered tobethemost potent[6,41,42].In previousstudieswe havefoundthathumanlungepithelialBEAS-2Bcellsare more sensitiveto3-nitrobenzanthrone (3-NBA)-induced DNAdamageandcelldeaththanHepa1c1c7cells,while B[a]Pand1-nitropyrene(1-NP)appeartobemoretoxicin Hepa1c1c7cells[35,37,58].Inthepresentstudywefound thatBEAS-2BcellswerelessresponsivethanHepa1c1c7 cells to 1,3- and 1,8-DNP with regards to cytotoxicity, DNAadductformationandDDR.However,thetwoDNPs induced at least as much ROS and even more oxida- tivedamagetoDNAinBEAS-2BcellsthaninHepa1c1c7 cells. It is noteworthy that 1,3-DNP induced an apoptosis inHepa1c1c7 cellswhich wasdependingonp53 transcriptionalactivity.In contrast,1,8-DNP inducedno apoptosis despitemore DNAadducts, a largerDDRand a considerably stronger p53 activation implying a role of additional mechanism(s) for the resulting apoptosis. In line with this, we observed that 1,3-DNP but not1,8-DNP,inducedactivationofseveralUPR-response genes, includingtheapoptosis-related ATF4 gene and it

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Fig.5. ROSasdeterminedbyDHE-fluorescence.BEAS-2BorHepa1c1c7cellswereexposedtovariousconcentrationsof1,3-DNPor1,8-DNP,orDMSO (control)for2or24h.Duringthelast2hDHEwasadded.Afterexposurecellswereanalyzedbyflowcytometer(seealsoSupplementaryFig.2).Eachbar representsthemean±SEMof3independentexperiments.*SignificantlydifferentfromDMSO-treatedcontrols(p<0.05).

downstream mediator CHOP.Thus, we suggest that the strongermutagenic/carcinogenicpotencyof1,8-DNPcom- pared to 1,3-DNP is linked to its greater DNA damage properties,whichincombinationwithitslowerpotency toinducecelldeathincreasestheprobabilityofinducing mutations.

BothDNPsinducedcelldeathtovariousextentsinthe twodifferentcelllines.InBEAS-2Bcellsneither1,3-DNP nor1,8-DNPexposureresultedinincreasedcelldeath.We havepreviouslyshownthatexposureofdieselexhaustpar- ticlesresultinanincreasedreleaseofcytokinesinBEAS-2B, andrelatedthisresponsetochemicalpollutantsboundto theparticles[40,59].Onemajorchemicalcompoundlinked tourbanairPMwas1-NP,whichincreasedproductionof thecytokinesIL-6andIL-8inBEAS-2Bcells[37].ROShas oftenbeenregardedtobeanimportanttriggeringfactor forincreasedcytokineproduction/releasebyambientair particles aswellasorganicpollutantsattachedtothem [40].However,in thepresentstudywedidnotobserve anyeffectonthereleaseofIL-6andIL-8after1,3-DNPor 1,8-DNPexposure(datanotshown).Interestingly,thiswas despitethefactthatbothcompoundsmarkedlyinduced ROSandoxidativedamagetoDNAinBEAS-2Bcells,without anincreaseincytotoxicity.Thisobservationisinlinewith previousﬁndingsfromourgroupsuggestingthatoxida- tivestressaloneis insufﬁcientforcytokineresponsesin theBEAS-2Bcellline[37,60].ThisimpliesthatwhileROS

maybeanimportantdeterminantforactivationofcytokine responses,additionalsignalsarerequired.

InBEAS-2Bcells1,3-DNPand1,8-DNPrapidlyenhanced ROSformationtoalevelwhichwassustainedfor24hafter exposure,suggestingtherateofROSformationforboth DNPswaslong-lasting.IncreasedROSlevelswerefoundto beaccompaniedbyamarkedincreaseinoxidativedamage toDNAasmeasuredbythecometassay.However,little, ifany,increaseinSSB-levelswasobserved(cometassay withoutfpg),indicatingthattheoxidativedamagetoDNA mayhavebeenrepairedinabalancedwayinthesecells.

Furtherstudiese.g.withDNArepairinhibitorsareneeded toassesstheactualrateofformationoftheoxidativedam- agetoDNAanddamageremovalinthesecells.

WehavepreviouslyshownthatwinterurbanairPM_2.5 fromMilancontaining a highlevelof PAH, couldcause mitotic delay, mitotic catastrophe and/or apoptosis in BEAS-2B cells [36]. So far tested neither various diesel exhaustPMs,B[a]P,1-NP,3-NBAnorvariousquinoneshave beenfoundtoinducesucheffectsinBEAS-2B[35,40,61].

However, 3-NBA which is activated by the nitroreduc- taseNAD(P)H:quinoneoxidoreductase(NQO1)wasfound to be a potent inducer of DNA adducts and apoptosis [35].In thepresent studywe foundthat althoughboth DNPsinduced ROSand oxidative damage toDNA, little DDR,nocelldeathandnomarkedeffectsonproliferation (visualexamination;datanotshown)wereobservedinthe

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Fig.6.DNAdamagemeasuredbythecometassayandDNAadductformationassessedby³²P-postlabelling.BEAS-2B(A,C)andHepa1c1c7(B)cellswere exposedto1,3,10or30␮Mof1,3-DNP,1,8-DNP,B[a]P(15␮M)orDMSO(control)for24h.DNAstrandbreaksweremeasuredbythecometassay(without fpg),andoxidativedamagetoDNAwasdeterminedusingthefpg-modiﬁedcometassay(AandB).Data(%tailDNA)representsthemean±SEMof3 independentexperiments.(CandD)DNAadductformation(RAL,relativeadductlabeling)inBEAS-2Bwasmeasuredby³²P-postlabellingassay.ND,not detectable.

Fig.7. TheeffectonDNAdamageresponse.BEAS-2B(A)andHepa1c1c7cells(B)wereexposedtovariousconcentrationsof1,3-DNP,1,8-DNPorDMSO for24handlevelsofH2AX,phosphorylatedH2AX(␥H2AX)andp53wereanalyzedbyWesternblotting(semiquantiﬁcationaregivenasnumbersabove theblots).Resultsfromonerepresentativeofthreeseparateexperimentsarepresented.

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Fig.8.Effectsofp53inhibitorson1,3-DNPinducedcelldeath.Hepa1c1c7cellswereexposedto10␮M1,3-DNPfor24hwithorwithoutpre-incubation for1hwith20␮Mpifithrin-␣(PFT-␣,A)orPFT-␮(B).ControlcellsweretreatedwithDMSOonly.CellswerestainedwithHoechst33342andpropidium iodide(PI),andsubsequentlyanalyzedforapoptosis(Ap)(includingapoptoticnecrotic)andnecrosis(Nec)usingfluorescencemicroscopy.*Significantly differentfromDMSO-treatedcontrols.^#Significantlydifferentfromtreatmentwithoutinhibitor(p<0.05).

BEAS-2Bcells.Thus,apparentlysomeother/strongersig- nalsareneededtoinducetheseeffects.Thelackofresponse inBEAS-2BcellswhencomparedtoHepa1c1c7cellsmaybe linkedtotheirlowerCYPenzymelevelsresultinginlower PAHmetabolism[62,63].ThehighROSresponsesobserved suggest that nitro-reduction reactions resulting in ROS dominateinBEAS-2Bcells.Furthermore,theloweramount of 1,8-DNP-DNA adductsformedin BEAS-2B relative to Hepa1c1c7 cells[14] maysuggest metabolicdifferences notonlywithregardstotheexpressionofCYPenzymes.

These metabolicdifferences couldhave impacton DNA adductformation.

WhilebothDNPswerenotcytotoxicinBEAS-2Bcells, 1,3-DNP induceda concentration-dependent increase in celldeathinHepa1c1c7cellsafter24h,startingatcon- centrationsof≥3␮M.Incontrast,1,8-DNPonlyinduced celldeathafterprolongedexposureatmuchhighercon- centrations(30␮Mat72h).Inpreviousstudieswehave foundthatvariousPAHsandnitro-PAHshaveresultedin differentformsofcelldeath.Forexample,exposuretoB[a]P waslinkedtoamixtureofapoptosisandnecrosis,1-NP toapoptosisandanon-apoptoticprogrammedcelldeath [13], and 3-nitroﬂuoranthrene to apoptosis and a pro- grammedcelldeathwithnecroptoticfeatures[34].Judged bymicroscopicexaminationandWesternblotanalysis,the 1,3-DNP-inducedcelldeathwasamixtureofapoptosisand necrosis.SimilarresultswerepreviouslyreportedforB[a]P [64].Becausethepan-caspase inhibitor zVAD-FMKonly partiallyinhibited1,3-DNP-inducedcelldeath,wecannot excludethatthereisanalternativecaspase-independent pathwayasreportedpreviouslyfor1-NP[65].Thismixed picturewhichisoftenseenafterchemicalexposureisprob- ablyduetoanincompleteapoptoticprocessin manyof thecellsduetotheapoptoticprocessbeingswitchedto necrosis,possiblyasaresultofmitochondrialdamageand lowATPlevels,and/orinactivationofcaspasesassuggested previously[64,66,67].

DifferencesinDNAadductformationafter1,3-and1,8- DNPexposuremaybedue todifferencesinmetabolism [68]. The activation/detoxiﬁcation pathways for both nitro-PAHsarenotpreciselyknown.Differencesintheir

nitro-reduction have been reported. While 1,3-DNP is reducedbyanitroreductasetransferringasingleelectron, 1,8-DNP isreduced byanenzymethat transferringtwo electrons[69]. However,no clearrelationship hasbeen foundfor 1,8-DNP betweenDNA adduct formation and NQO1expression ina panelof mouseembryonic fibro- blastcelllines[49].Othernitroreductasessuchasxanthine oxidasehavealsobeenshowntoactivatenitro-PAHslike 3-NBA[1,70].Inactivationof1,3-and1,8-DNPsbyhuman livermicrosomalP450shasbeenreported[9].Otherstud- iesindicatethat1,6-DNP,and1,8-DNPcanbeactivatedby humanP4501B1[68].Collectively,thesefindingssuggest thatmechanismsinvolvedintheactivationanddetoxifica- tionofNPiscomplexandmaydependonthechemicalas wellastheexperimentalsystemused.

Inthisstudyweobservedthat1,8-DNPinducedmore DNAdamage,astrongerDNA-DRresponse,andastronger activationofp53than1,3-DNP.Nevertheless,p53inhibi- tionwithpifithin-␣(PFT-␣)markedlyreducedcelldeath inducedby1,3-DNPintheHepa1c1c7cells,whilePFT-␮ had noeffect.Thissuggeststhat transcriptionalactivity ofp53isrequiredtoexecutethe1,3-DNP-inducedapop- toticprocess.Inlinewiththisfindingwehavepreviously observedthatB[a]P-inducedDNAdamagewasassociated withap53-dependentcelldeathinthesecells[64].Itis noteworthy that in a previous study we observed that 1,8-DNPinducedp53phosphorylationinHepa1c1c7cell, but this was not accompanied by nucleartranslocation [14].Therefore,wesuggested thattheinabilityofphos- phorylatedp53toenterthenucleusin1,8-DNP-exposed Hepa1c1c7cells,couldexplainthelackofcelldeath.How- ever,inthepresentstudy,p53didappeartotranslocate tothenucleusfollowing1,8-DNPexposureinHepa1c1c7 cells(datanotshown).Itremainstobeclarifiedwhether thisdiscrepancy mayberelatedtobatch/passagediffer- encesoftheHepa1c1c7cells.Nevertheless,1,8-DNPfailed toinduceapoptosisinthepresentstudy,despitetheappar- entlyfunctionalp53-translocation.Thusitispossiblethat the1,8-DNP-inducedp53-activationalone isinsufficient totriggerapoptosis,andthatadditionalp53-independent cellularsignalsareneededforacytotoxicresponse.

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Fig.9.Effectsonproteinunfoldingresponse(UPR).Hepa1c1c7cellswereexposedto10␮M1,3-DNPor1,8-DNPfor6handgeneexpressionwasmeasured byNanoString’snCountertechnology.Aheatmapshowingfoldchangesforthe1,3-DNPand1,8-DNPcells(A).Eachcolumnrepresentsanindividualcell culturesample.Changesingeneexpressionwerecalculatedusingthelog2-transformedfoldratiosbetweeneachexperimentalsampleandthemeanofthe controls.HierarchicalclusteringwasperformedusingEuclidiandistanceandaveragelinkage.Ahistogramdemonstratingthelog2-foldratiosforselected UPRgenesandIL-6(B).Eachbarrepresentsthemeanfoldchange±SEMofthreereplicatesforeachtreatmentcondition.*p<0.05,**p<0.01,***p<0.001 vs.DMSO-treatedcontrols.

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Fig.9.(Continued)

Incontrastto1,8-DNP,1,3-DNPinducedER-stressand UPR as judged by increased expression of several UPR response genes. Of particular interest is that 1,3-DNP inducedincreasedexpressionofATF4anditsdownstream target CHOP, which are both considered to be among themaineffectorsofUPR-inducedapoptosis(30).Recent ﬁndingsshowedthatover-expression ofATF4and CHOP induced apoptosis, throughincreased protein synthesis, ATPdepletionandROS-formation[71].Thissuggeststhat the increase in ATF4 and CHOP expression may have contributedto1,3-DNP-inducedapoptosisinthepresent study,andthatER-stressandUPRplaysacentralroleinthe inducedcelldeath.Thus,despiteinducingonlymoderate DNAdamageandDDR,1,3-DNPappearstoinducemore cytosolic damagethan 1,8-DNPthereby triggeringaddi- tionaldeathsignals.Thissuggeststhatcross-talkbetween DDRandcytotoxicsignalingfromthecytosolmaybecen- tralindeterminingwhetherDNAdamageresultsincell cyclearrestandDNArepair,ortriggersapoptosisin1,3- DNPexposedHepa1c1c7cells.Inconcordancewiththis, reactive metabolitesknowntopreferentially reactwith DNA comparedtoothermacro-molecules areknownto haveahighermutagenicpotential[72].Furtherstudiesare neededtoexplorethesehypotheses,whichmaybevery importantwhenevaluatingvariouschemicalsfortheircar- cinogenicproperties.

5. Conclusion

This study showed that a small change in molecu- lar structure, such as changing the position of a nitro groupfrom3rdpositionto8thpositioncanhavemarked impactonthecellularresponse.Theobservedeffectswere markedlydifferentdependingonthecellularmodelused.

BEAS-2B cells respondedwith more ROS and oxidative damage toDNA,while Hepa1c1c7 cells seemedtobea moresensitivecellularmodelwithregardstoDNAadduct formation,DDR(1,8-DNP),andcelldeath(1,3-DNP).Our resultsindicatethatbothDDRandUPRplaycentralroles in1,3-DNP-inducedapoptosisinHepa1c1c7cells.Ourdata suggests that thestronger carcinogenic potencyof 1,8- DNPcomparedto1,3-DNPislinkedtoitshighergenotoxic effects, whichin combinationwithitslowerpotencyto

inducecelldeathmayincreasetheprobabilityofcausing mutations.

Conﬂictofintereststatement

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

WethankHans-JørgenDahlmanforassistancewiththe ﬂowcytometer.TheworkwassupportedbytheResearch CouncilofNorway,throughtheEnvironment,Geneticsand Health-program(grantsno.165386and160863).Volker M.ArltissupportedbyCancerResearchUK.KjetilAskis supportedbytheCanadianLungAssociationandOntario ThoracicSociety.

AppendixA. Supplementarydata

Supplementary material related to this article can be found, in the online version, at doi:10.1016/

j.toxrep.2014.07.009.

Transparencydocument

TheTransparencydocumentassociatedwiththisarticle canbefoundintheonlineversion.

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