A human relevant mixture of persistent organic pollutants (POPs) and perfluorooctane sulfonic acid (PFOS) enhance nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells

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A human relevant mixture of persistent organic pollutants (POPs) and per ﬂ uorooctane sulfonic acid (PFOS) enhance nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells

Ajay Yadav

^a,b,

*, Mazia Amber

^c

, Denis Zosen

^b

, Nils Anders Labba

^b

,

Eva Henriette Willemijn Huiberts

^b

, Johanna Samulin Erdem

^d

, Fred Haugen

^d

, Hanne Friis Berntsen

^a,d

, Shanbeh Zienolddiny

^d

, Ragnhild Elisabeth Paulsen

^b

, Erik Ropstad

^a

, Lisa Connolly

^c

, Steven Verhaegen

^a

aDepartmentofProductionAnimalClinicalSciences,NorwegianUniversityofLifeSciences,P.O.Box369Sentrum,NO-0102,Oslo,Norway

bSectionforPharmacologyandPharmaceuticalBiosciences,DepartmentofPharmacy,UniversityofOslo,P.O.Box1068,Blindern,NO-0316,Oslo,Norway

cInstituteforGlobalFoodSecurity,Queen’sUniversityBelfast,19ChlorineGardens,Belfast,BT95DL,NorthernIreland,UnitedKingdom

dNationalInstituteofOccupationalHealth,P.O.Box5330Majorstuen,NO-0304,Oslo,Norway

HIGHLIGHTS GRAPHICAL ABSTRACT

Nervegrowthfactor(NGF)induced neuritogenesisinPC12ratpheochro- mocytomacells.

Persistentorganicpollutants(POPs) enhancedNGF-inducedneuritogen- esis.

Perﬂuorooctanesulfonicacid(PFOS) contributed to 50 % of the POP mixtureeffect.

Nuclear and mitochondrial health wasunaffectedbythePOPmixture orPFOS.

ARTICLE INFO

Articlehistory:

Received23October2020

Receivedinrevisedform1December2020 Accepted6December2020

Availableonline9December2020

Keywords:

Glutathione Liveimaging Mitochondrialmass

Mitochondrialmembranepotential Ratpheochromocytoma

ABSTRACT

Disruptionofneuriteoutgrowthisamarkerforneurotoxicity.Persistentorganicpollutants(POPs)are potentialdevelopmentalneurotoxicants.WeinvestigatedtheireffectonneuriteoutgrowthinPC12rat pheochromocytomacells,inabsenceorpresenceofnervegrowthfactor(NGF),aninducerofneuronal differentiation.Cellswereexposedfor72htoadeﬁnedmixtureofPOPswithchemicalcompositionand concentrationsbasedonbloodlevelsintheScandinavianpopulation.Wealsoevaluatedperﬂuorooctane sulfonicacid(PFOS)alone,themostabundantcompoundinthePOPmixture.Onlyhigherconcentrations ofPOPmixturereducedtetrazoliumsalt(MTT)conversion.High-contentanalysisshowedadecreasein cell number, but no changes for nuclear and mitochondrial cellular health parameters. Robust glutathionelevelswereobservedinNGF-differentiatedcells.Liveimaging,usingtheIncuCyteZOOM platformindicatedongoingcellproliferationovertime,butslowerinpresenceofNGF.Thepollutantsdid not inhibitneuritogenesis, but ratherincreasedNGF-inducedneurite length.PFOSinduced neurite

* Correspondingauthorat:DepartmentofProductionAnimalClinicalSciences,NorwegianUniversityofLifeSciences,P.O.Box369Sentrum,NO-0102,Oslo,Norway.

E-mailaddress:ajay.yadav@nmbu.no(A.Yadav).

http://dx.doi.org/10.1016/j.toxlet.2020.12.007

ContentslistsavailableatScienceDirect

Toxicology Letters

j o u r n al h o m e p a g e :w w w . el s e v i e r . c o m / l o c a t e / t o x l e t

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outgrowthtoabout50%ofthelevelseenwiththePOPmixture.NeitherthePOPmixturenorPFOS affectedneuritelengthintheabsenceofNGF.Ourobservationsindicatethatrealisticcomplexmixtures ofenvironmentalpollutantscanaffectneuronalconnectivityviaNGF-inducedneuriteoutgrowth.

1.Introduction

Normaldevelopmentofthecentralnervoussystemrequiresthe coordinatedontogenyofproliferation,migration,differentiation, synaptogenesis, gliogenesis, myelination and apoptosis (Barone etal.,2000).Perturbationsoftheseprocessesthroughexposureto environmentaltoxicantsduringdevelopment,couldaffectstruc- turesandfunctionsofthebrain,leading tolifelongimpairment (GrandjeanandLandrigan,2006).Pregnancyandearlylifestages are particularly vulnerable to exposure to toxicants including persistentorganicpollutants(POPs).Thegrowthofdendritesand synapsesisfundamentalforthedevelopmentofneuronalcircuits that underlie human cognition and behaviour. Disruption of synaptic circuits are associated with intellectual disability, epilepsy, autism spectrum disorder (ASD), schizophrenia and bipolardisorder(Forrestetal.,2018).Thechemicalsthatdisrupt thegrowth oftheseaxonalanddendriticprocesses(collectively called“neurites”)havebeenlinkedwithdevelopmentalneurotox- icity (DNT) in vivo (Radio et al., 2008, 2010). Many of the neurodevelopmentalprocessesthatoccurinvivoforexamplecell differentiation, neurite outgrowth, and synaptogenesis, can be followed using in vitro neuronal systems. Among these, the measurement of neurite outgrowth has received the most attention (Radio and Mundy, 2008). Chemicals that can affect theneuriteoutgrowthincludePOPs.Variouscelllineshavebeen used to study the effects of chemicals on neurons (Radio and Mundy, 2008), including thepheochromocytomaPC12 cell line whichhasbeenwidelyusedasamodelforthestudyofneuronal differentiationandneuriteoutgrowth(Fujitaetal.,1989).PC12isa celllineestablishedfromatransplantableratadrenalpheochro- mocytoma(GreeneandTischler,1976).Serum-withdrawaltogeth- erwithstimulationwithnervegrowthfactor(NGF)resultsinthe cellsundergoingonedoublingandinductionofneuriteoutgrowth (Greene,1978).

Glutathione(GSH)actsasanimportantantioxidant.Becauseof theextensiveoxidativemetabolisminthebrainfrommitochon- drial respiration,the cells of this organ continuously generate reactiveoxygenspecies. Asaresult,theantioxidantfunctionof GSHappearstobeespeciallyimportantforthebrain.Further,a compromisedGSHmetabolismcontributestotheprogressionof neurologicaldisorders(SchmidtandDringen,2012).

POPs are mostly halogenated compounds that are highly resistanttoenvironmental degradation,andthatbioaccumulate andbiomagnifyinlivingorganisms(UNEP,2008).SeveralPOPs, includingpolychlorinatedbiphenyls(PCBs),organochlorine pesticides(OCPs),polybrominateddiphenylethers(PBDEs),perﬂuor- ooctanesulfonicacid(PFOS),and perﬂuorooctanoicacid(PFOA) aresupposedtobedevelopmentalneurotoxicants(Grandjeanand Landrigan, 2014; Hoyer et al., 2015; Kodavanti, 2006; Schmidt etal.,2014).WhereasmanyofthestudiesonPOPsfocusonsingle compoundorasmallgroupof contaminants,weareinreallife exposedsimultaneouslytoacomplex mixtureof contaminants, which could have additive, synergistic or antagonistic effects (Boppetal.,2018;SCHER,2012;WHO,2017).

Previously,wedesignedanenvironmentallyrelevantmixture of POPs for use in animal and in vitro experimental studies, containing 29 different chlorinated, brominated, and perﬂuori- natedcompounds(Berntsenetal.,2017).Themixtureusedinthe presentstudycontainedPOPsconcentrationsbasedonmeasured

humanbloodconcentrationsinScandinaviaandaimedtoprovide adeﬁnedandrealisticmixtureofenvironmentalcontaminantsfor toxicity studiesthat couldreﬂect the relativelevelsof POPs to whichindividualsareexposed(Berntsenetal.,2017).

PFOS belongs to the chemical class of perﬂuorinated alkyl substances (PFAS),a familyofemergingpersistent pollutantsof increasing concern with regards to adverse health outcomes, includingcognitiveeffects (Spratlenetal.,2020).Inourunpub- lishedstudies(manuscriptunderreview)wefoundthatthePOP mixtureat500xfoldhumanbloodlevelsandPFOSat20

m

M,ina corresponding concentration, induced excitotoxicity via the involvement of N-methyl-D-aspartate receptor (NMDA-R) in culturesofchickencerebellargranuleneurons(CGNs).Asimilar resulthasbeenobservedbyourgroupusingthesamePOPmixture atconcentrationsfrom500x,andPFOSinrataswellasinchicken CGNs(Berntsenetal.,2018,2020).PFOS-inducedneurotoxicityhas alsopreviouslybeenstudiedinPC12cells(Lietal.,2017;Slotkin etal.,2008).AsPFOSis thecompound occurringatthehighest concentrationinthePOPmixture,wealsoevaluatedPFOSasan individualcompound.

High Content Analysis (HCA) is a novel, high-throughput, quantitative ﬂuorescence technique providing an important approach forthedetectionofcompound toxicityatthelevel of individualcells(O’Brienetal.,2006;Wilsonetal.,2016).Cellular healthmarkerslikecellnumber(CN),nucleararea(NA),nuclear intensity(NI), mitochondrialmembrane potential(MMP), mitochondrialmass(MM)covercellularmetabolicfunctions(O’Brien andHaskins,2007;Wilsonetal.,2016).Thedetection/investiga- tion of these multiple nuclear and mitochondrial parameters allowed us to evaluate cellular health at the end of the differentiationprocess.MeasurementsofintracellularGSHlevels wasusedasamarkerforpossibleeffectsofthePOPexposureson thecellulardefencemechanism.

Sinceneuritogenesisisa markerforneuronaldifferentiation, and neurite outgrowth is very sensitive to neurotoxicants we investigatedtheeffectsofPOPsonneuritedynamicsinthePC12 cellmodel.WeusedtheIncuCyteZOOMplatformwhichtakesnon- perturbing phase-contrast images of neuronal cultures under physiologicalconditionsoveranextendedperiodoftime(Garay et al., 2016), and hasbeen used to study neurite dynamics in differentneuronalcells(Mengeletal.,2019;Yagietal.,2015).This platform also allowed usto followcell proliferation over time, whichcomplementedtheendpointparametersdescribedabove.

2.Materialandmethods

2.1.Chemicals,PFOS,andthePOPmixture

Dulbecco’smodiﬁedEagle’smedium(DMEM)andL-glutamine wereobtainedfromLonzaBioWhittaker(Verviers,Belgium).Penicil- lin-streptomycin,sodiumpyruvateandhorseserumwerepurchased from Gibco, Life Technologies(Paisley, UK). Foetal bovine serum (South America) was from BioWest (Nuaille,France). Dimethyl sulfoxide (DMSO), monochlorobimane (mBCl), thiazolyl blue tetrazolium bromide (MTT), formalinand mouse neuralgrowth factor (NGF) wereallsuppliedbySigma-Aldrich(Poole,Dorset,UK). Hoechst33342 nuclearstainandmitochondrialmembranepotentialdye(MitoTrack- er^TM Red CMXRos) were provided by Thermo Scientiﬁc (UK).

Valinomycin,perﬂuorooctanesulfonicacidpotassiumsalt(PFOS98

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%)aswellasallotherreagentswerefromSigma-Aldrich(StLouis,MO, USA).

InthecelltoxicitytestPFOSconcentrationswerechoseninthe range of 10–100

m

M, based on previous studies in PC12 cells (Berntsenetal.,2018).Intheneuriteoutgrowthstudytherange wasbetween10–50

m

M,becausein thepreliminarystudiesthe concentration above 50

m

M showed no effect on neurite outgrowthandwasassociatedwithfewercells.

Thedeﬁned POPmixturewas designed and preparedat the Norwegian University of Life Sciences (NMBU), Oslo, Norway (Berntsen et al., 2017). The mixture contained 29 different compounds(SupplementaryTable1),includingsixperﬂuorinated (PFAA)(i.e.,PFHxS,PFOS,PFOA,PFNA,PFDA,andPFUnDA),seven brominated(Br)(i.e.,PBDE47,PBDE99,PBDE-100,PBDE153,PBDE 154,PBDE209, andHBCD),andsixteen chlorinatedcompounds (Cl)compounds(i.e.,PCB28,PCB52,PCB101,PCB118,PCB138,PCB 153,PCB 180,p,p'-DDE, HCB,α-chlordane, oxychlordane, trans- Nonachlor, α-HCH, β-HCH,

g

-HCH and Dieldrin). The relative concentrations of thechemicals in the mixturewere based on Scandinavianhumanblood levelsand thechoiceofcompounds were based on compounds found at high levels in blood, breastmilk and/or food. The stocks used in the present study hadaconcentrationof10⁶timesmeasuredhumanbloodlevelsand stockswerecreatedusingDMSOasasolvent.WeusedthePOP mixtureconcentrationfrom10to500x(foldhumanbloodlevels), as previously exposure with this mixture showed biological activityintherangeof1/10 500x(foldhumanbloodlevels)in awidevarietyofcellsystems(Doanetal.,2019;McCombetal., 2019;Shannonetal.,2019).

2.2.Cellcultureandtreatment

The ratpheochromocytoma cell linePC12 was cultivatedin growthmediumconsistingofDMEMwithpenicillin-streptomycin, sodiumpyruvate,andsupplementedwith5%horseserumand10% foetal bovine serum as described previously (Rakkestad et al., 2014).Cellswereseededoutin96wellsplates(100

m

L/well)and 35mmdiameterdishes(1mL/dish)inaserumfreemediumand thenincubated for4 6hforattachmentand serum starvation.

Withoutremovingthemedium,anequalvolumeoffreshmedium containinghorseserum(ﬁnal2%),NGF(ﬁnal50ng/mL)andtest compounds/controls,was thereafter added to each well. Plates wereincubatedfurtherfor72hpriortotheassay.Theexposure scenarioisoverviewedinFig.1.

2.3.MTTconversionassay

Cell viability was measured by MTT colorimetric assay, as describedpreviously(McCombetal.,2019).Thisassayisbasedon theconversionofMTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, a pale yellow substrate to a dark blue formazan product by the mitochondrial enzyme succinate dehydrogenaseinlivingcells(Mosmann,1983).Cellswereseeded inclearﬂat-bottomed96-wellplates(Nunc,Roskilde,Denmark)at a density of 110⁴ cells/cm² and exposed to test compounds eitherinthepresenceorabsenceofNGF.Following72hexposure withtestcompounds, thesupernatant wasdiscarded, and cells were incubated further with 2mg/mL MTT (in PBS) solution diluted1:6inassaymedia.Again,thesupernatantwasremoved, and 200

m

L/well of DMSOwas added todissolve theformazan crystals. Subsequently, the plate was incubated at 37C with agitationfor10min.Opticaldensitywasmeasuredat570nmwith a referenceﬁlterat 630nm usinga Sunrise spectrophotometer (TECAN,Switzerland).Cellviabilitywasmeasuredasthepercent- ageabsorbanceofthesamplecomparedwiththeabsorbanceofthe solventcontrol.

Valinomycin(Vln)was usedasa positivecontrolintheMTT conversion assay and HCA assay. Exposure to Vln induces a dissipationofthemitochondrialtransmembranepotential,leading tomitochondrialdysfunction(Abrahametal.,2008).

2.4.CellularandmitochondrialhealthassessedbyHighContent Analysis(HCA)

The experimentalprocedure was followedas detailedprevi- ously(Shannonetal.,2017;Wilsonetal.,2016).TheCellomics¹ HCS reagent series multiparameter cytotoxicity assay was per- formed following the manufacturer’s instructions. Cells were seeded into collagenbio-coat BD Falcon 96 well ﬂat bottomed microtitreplates(BDBiosciences,Bedford,MA,US)atadensityof 110⁴ cells/cm² and exposed totest compounds either in the presence or absence of NGF. Brieﬂy, mitochondrial membrane potentialdyewaspreparedbyadding117

m

LofanhydrousDMSO tomakea1mMstock.Following72hincubation,50

m

Loflivecell stain(ﬁnal0.1

m

M)wasaddedtoeachwellfor30minat37Cand protectedfromlight.Thelivestainwasremoved,andcellswere then ﬁxed with a 10 % formalin solution for 20min at room temperature(RT)andwashedwithPBS.Hoechst33342dye(ﬁnal 1.6

m

M)wasaddedtoeachwellandincubatedfor10minatRT, afterwhichcellswerewashedwithPBS.Thewellswerethenﬁlled with 200

m

L PBS, sealed withplate sealer and evaluated ona CellInsight^TM NXT High Content Screening Platform (Thermo FisherScientiﬁc,UK).Thisinstrumentanalysesepiﬂuorescenceof individual cell events using an automated micro-plate reader analyzerinterfacedwithaDellprecision136T5600workstation.

HoechstdyewasusedtomeasureCN,andnuclearmorphology including NI and NA. Mitochondrial membrane potential dye (MitoTracker^TMRedCMXRos)wasusedtomeasuremitochondrial health, specificallyMMPand MM.Data werecapturedforeach plateusinga10xobjectivemagnificationintheselectedexcitation andemissionwavelengthsofHoechstdye(Ex/Em350/461nm)and mitochondrialmembranepotentialdye(Ex/Em554/576nm).For each well,ninefieldsofviewimageswereacquiredtoexamine eachparameter.

2.5.Glutathionemeasurements

TotalreducedGSHlevelsinCGNsweremeasuredwithmBClas describedpreviously(Sørviketal.,2018).Inbrief,cellswereseeded in black 96 wells plates at a density of 110⁴ cells/cm² and

Fig.1.Exposureoverviewofexperiments.

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exposed with test compounds for 72h. Then medium was removedandreplacedwithnewmediumcontaining40

m

Mof mBCl and incubated further in the dark at 37C for 30min.

Subsequentlythemediumwasremoved,andplateswerewashed with freshly prepared experimental buffer containing 140mM NaCl,3.5mMKCl,15mMTrisHCl(pH7.4),5mMglucose,1.2mM Na2HPO4(pH7.4),and2mMCaCl2.100

m

lofthebufferwasadded to each well, and mBCl ﬂuorescence was measured at the excitation wavelength of 380nm (15nm bandwidth) and emission wavelength of 478nm (21nm bandwidth) using a CLARIOstar¹platereader.Further,inthesameexposure,wealso quantiﬁedcellnumberusingnucleistainingwithHoechst33342.

Buffers were replaced with Hoechst 33342 (0.4

m

g/mL) and incubatedindarkfor1min.Subsequently,theplatewasreadat excitation wavelengths of 350nm (22nm bandwidth) and emission wavelengths of 461nm (36nm bandwidths). This measurement was used to correct for cell number in the calculationsforGSHlevelsaftersubtractionofblankvalues.

2.6.TransfectionandluciferaseassayforGCLCpromoteractivity assessment

Theprocedurehasbeendetailedpreviously(Sørviketal.,2018).

CellswereseededinØ35mmdishesatadensityof1.2510⁴cells/

cm²andallowedtoattachfor24h.Oncultureday1,cellswere transfected with GCLC (catalytic subunit) promoter coupled to luciferase (GCLC-luc; 0.8

m

g; a kind gift from R. Blomhoff, DepartmentofNutrition,UniversityofOslo)andinternalcontrol

vectorpRL-TK(0.2

m

g;purchasedfromPromega,WI,US)toatotal of 1

m

g DNA/mL culture medium. Transfection medium was replaced with fresh medium after 4h. On culture day 2, cells wereexposed with testcompounds dissolved in serum free or serumfreeplusNGFmedium.After48hluciferasewasmeasured withaluminometer(LumatLB9507;BertholdTechnologiesGmbH, Bad Wildbad, Germany) as described previously (Strom et al., 2010). Renilla luciferase was measured using the kit Dual- Luciferase¹ Reporter Assay System in accordance with the manufacturer’sinstructions(Promega,WI).

2.7.LivecellimagingusingtheIncuCyteneuriteanalysis

TheIncuCyteZOOMlive-cellimagingsystem(EssenBioScience, UK)wasusedtoanalyseneuritedynamics.Differentparameters werequantiﬁedovertimeusinganautomatedIncuCyte¹Neuro- tracksoftwaremodule,asdetailedpreviously(Garayetal.,2016).

PC12cellswereseededin96-wellTPP(TechnoPlasticProductsAG, Switzerland)platesandexposedtotestcompoundseitherinthe presenceorabsenceofNGF,asdescribedabove.Cellswereseeded at a density of 0.810⁴ cells/cm² in the presence of NGF and 1.610⁴ cells/cm² in theabsence of NGF. Plates werescanned every60minovera72hperiodusinga10xobjective.Fourimages per well were captured and images were analysed for neurite length, branchpoints, cell-bodyclusters number and area. The masks/ﬁlters adjustment for Neurotrack phase contrast image analysiswereasfollows:Segmentationmode:Texture;Holeﬁll:0;

Adjustsize: 5

m

m;Mincellwidth:8

m

m;Neuriteﬁltering:Best;

Fig.2.AssessmentofcytotoxiceffectsinPC12usingthetetrazoliumMTTconversionassayorhighcontentanalysis(HCA).Cellswereexposedinthepresenceorabsenceof NGF(50ng/mL)withsolventcontrol(SC,DMSO0.1%),POPmixture(xhumanbloodlevels)orPFOS(mM).Valinomycin(Vln,15mM)wasincludedasapositivecontrol.

Following72hexposuretothecompounds,cellviabilitywasmeasuredbyconventionalMTTconversionassayforA)POPmixture,andB)PFOS.Cellnumbermeasuredby multi-parameterHCAisshownforC)POPmixture,andD)PFOS.Dataaremean,n=3-4independentexperiments,withreplicate>3.Allvalueswerepresentedrelativetothe correspondingsolventcontrol(SC,0.1%DMSO)deﬁnedas100%,*p<0.05,comparedtoSCwithNGFand⁺p<0.05comparedtoSCwithoutNGF,w=withNGFand wo=withoutNGF.

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Neuritesensitivity:0.35

m

m;andNeuritewidth:1

m

m,detailedin (NeuroTrack^TM,2020).Thefollowingparameterswerequantiﬁed:

1Cell-BodyClusters(CBC)=totalnumberofcellbodyclusters/

areaofimageﬁeld;

2Cell-BodyCluster Area(CBCA)=sumofareasofallcell-body clusterspooled/areaofimage;

3NeuriteLength(NL)=sumoflengthsofallneuritespooled/area ofimageﬁeld;

4NeuriteBranchPoints(NBP)=totalnumberofbranchpoints/

areaofimageﬁeld.

2.8.Statisticalanalysis

Resultsarepresentedasmeanstandarderrorofthemean (SEM),n=3–4independentexperiments,withreplicates>4per group. IncuCyte data were analysed using the mixed model personality in JMP Pro 14¹ (SAS, Cary, USA). Dependent variables were CBC, CBCA, NL and NBP. For the statistical analysislogtransformeddependentvariablesgaveasatisfacto- ry ﬁt to the normal distribution. Independent ﬁxed effect variables were: exposure group, time in culture and their interaction. Experiment and the nested effect of time within experiment were entered as random effects allowing for repeated measurements along the time scale. Dunnett’s test wasused tocomparedifferencesbetween groupsexposed to chemicalsandcontrol(solventcontrol,0.1%DMSO).Theeffect

ofNGFonlogtransformeddependentvariables(CBC,CBCA,NL andNBP)wasexploredinaseparatemixedmodelusingasubset ofdataincludingonlycontrols.Independentvariablesincluded asﬁxedeffectsweretimeinculture,NGFandtheirinteraction.

AllotherdatawereanalysedusingGraphpad8.2(SanDiego,CA, US). Outlier valueswere tested for by the built-in feature in GraphPad (Robust regression and Outlier removal, Q=1%). A one-way analysis of variance (ANOVA) followedby Dunnett’s multiple comparisons test was usedto determine signiﬁcant differences between treatments and solvent control. An unpaired t-test was used for comparison when appropriate between two groups. A p-value of < 0.05 was considered signiﬁcant.

3.Results

3.1.EffectsofPOPmixtureorPFOSoncytotoxicityasmeasuredbyMTT conversionassay

Inaﬁrstapproach,cytotoxiceffectsofpollutantsonPC12cells viabilitywereevaluatedusingtheMTTconversionassay.Bothin thepresenceand absenceofNGF,exposure tothePOPmixture reduced MTT activity only at the highest concentration tested (Fig.2A).PFOSalonereducedMTTactivitysigniﬁcantlyat50and 100

m

M,whichwere2.5and5timeshigherthanthecorresponding concentrationinthePOPmixtureat500x(Fig.2B).Intheabsence of NGF, reduced MTT activity was only observed at 100

m

M (Fig.2B).

Fig.3.AssessmentofchangesinnuclearhealthinPC12usinghighcontentanalysis(HCA).CellswereexposedinthepresenceorabsenceofNGF(50ng/mL)withsolvent control(SC,DMSO0.1%),POPmixture(xhumanbloodlevels)orPFOS(mM).Valinomycin(Vln,15mM)wasincludedasapositivecontrol.MultiparameterHCAendpoints weremeasuredfollowing72hexposuretothetestcompounds.ResultsfornuclearareaisshownforA)POPmixture,andB)PFOS.NuclearintensityisshownforC)POP mixture,andD)PFOS.Dataaremean,n=3-4independentexperiments,withreplicate>3.Allvalueswerepresentedrelativetothecorrespondingsolventcontrol(SC,0.1% DMSO)deﬁnedas100%.*p<0.05,comparedtoSCwithNGFand⁺p<0.05comparedtoSCwithoutNGF,w=withNGFandwo=withoutNGF.

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3.2.EffectofPOPmixtureorPFOSoncellnumbermeasuredbyHCA ToinvestigatewhetherdecreasesinMTTactivitywasduetoa reductionincellnumberweevaluatedcellnumberindependently byHCA.ForthePOPmixtureinthepresenceofNGF,reductionsin cellnumberasmeasuredbyHCAwerealreadysigniﬁcantat10x (foldhumanbloodlevels,Fig.2C).However,intheabsenceofNGF thecellnumberremainedunaffectedatanyoftheconcentrations tested (Fig. 2C). For PFOS alone no signiﬁcant concentration- responsewasobservedforcellnumberinthepresenceorabsence ofNGF(Fig.2D).

3.3.EffectsofPOPmixtureorPFOSoncytotoxicityasmeasuredby nuclearandmitochondrialhealthusingHCA

Alternatively, MTT activity can be affected by diminished mitochondrialhealthresultingincelllossviacelldeath.Thus,we investigatedadditionalHCAtoxicityparametersfornuclearand mitochondrial health. Both in thepresence or absence of NGF, exposure toeither thePOP mixtureor PFOSdidnot affect the nuclear(Fig.3A–D)ormitochondrialparameters(Fig.4A–D).

HCA images using Hoechst dye (nuclear staining) and mitochondrialpotentialdye(mitochondrialstaining)areillustrat- edinSupplementaryFig.1.

3.4.EffectsofPOPmixtureorPFOSonglutathionelevelsandGCLC promoteractivity

SinceintracellularGSHlevelsareimportantforcellsurvival,we measuredtheeffectsofpollutantsontotallevelsofGSHbymBCl probe,aswellastheproductionofGSHbyGCLCpromoteractivity.

In thepresenceof NGFthePOP mixturesigniﬁcantlyincreased glutathione levels at 10x blood levels, whereas at higher concentrations the glutathione levels were comparable with control levels. In the absence of NGF, glutathione levels were reduced ina concentration-dependent mannertohalf thelevel seeninthesolventcontrol(SC,0.1%DMSO;Fig.5A).Bothinthe presenceorabsenceofNGFtheGCLCpromoteractivityfollowing 48h exposure to POP mixture 500x remainedunaffected. This suggeststhatthesynthesisofGSHdidnotchangefollowingthe exposuretoPOPmixture(Fig.5B).

3.5.EffectsofPOPmixtureorPFOSoncellbodyclustersandareain PC12cellswithandwithoutNGF

Wequantiﬁedthenumberofcellbodyclusters(CBC)andarea (CBCA)usingIncuCytelivecellimagingwhichcanbeinterpretedas a measure of proliferationand cellbody size, respectively.CBC numberandCBCAincreasedwithtimeincultureforallconditions Fig.4.AssessmentofchangesinmitochondrialhealthinPC12usinghighcontentassay(HCA).CellswereexposedinthepresenceorabsenceofNGF(50ng/mL)withsolvent control(SC,DMSO0.1%),POPmixture(xhumanbloodlevels)orPFOS(mM).Valinomycin(Vln,15mM)wasincludedasapositivecontrol.MultiparameterHCAendpoints weremeasuredfollowing72hexposuretothetestcompounds.ResultsformitochondrialmassisshownforA)POPmixture,andB)PFOS.Mitochondrialmembranepotential isshownforC)POPmixture,andD)PFOS.Dataaremean,n=3-4independentexperiments,withreplicate>3.Allvalueswerepresentedrelativetothecorrespondingsolvent control(SC,0.1%DMSO)deﬁnedas100%.*p<0.05,comparedtoSCwithNGFand⁺p<0.05comparedtoSCwithoutNGF,w=withNGFandwo=withoutNGF.

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tested(Figs.6and7).CBCnumberforDMSOcontroltripledfor PC12 cells stimulated with NGF alone (Fig. 6A, C) and nearly quadrupledwithoutNGF(Fig.6B,D).NGFexposurewasassociated withasigniﬁcantdecreaseintheoverallleastsquares(LS)mean logCBC and logCBCA (Figs. 6 and 7; Supplementary Table 3).

Exposuretohighconcentrations ofPOP mixtureand PFOSwas associatedwitha signiﬁcantdecrease inlogCBC and logCBCA whereaslowandintermediateconcentrationshadvariableeffects (SupplementaryTable2;Figs.6and7).

Phase contrast IncuCyte images and images after applying Neuro Track masks for CBCs and neurites are illustrated in SupplementaryFigs.2and3.

3.6.EffectsofPOPmixtureorPFOSonneuritelengthandbranchpoints inPC12cellswithandwithoutNGF

We quantiﬁed neurite length and neurite branch points as measuresofneuriteoutgrowthusingIncuCytelivecellimaging.NL Fig.5. Assessmentofglutathione(GSH)levelsandGSHproductionlevels.CellswereexposedinthepresenceorabsenceofNGF(50ng/mL)withsolventcontrol(SC,DMSO0.1

%)orPOPmixture(xhumanbloodlevels)orPFOS(mM).A)GSHlevelusingmBClwasmeasuredfollowing72hexposure,andB)GCLCpromoteractivityfollowing48exposure.

Dataaremean,n=3-4independentexperiments,withreplicate>3.Allvalueswerepresentedrelativetothecorrespondingsolventcontrol(SC,0.1%DMSO)deﬁnedas1for GSH,*p<0.05,comparedtoSCwithNGFand⁺p<0.05comparedtoSCwithoutNGF,w=withNGFandwo=withoutNGF.

Fig.6.Assessmentofchangesincellbodyclusters(CBC)inPC12cellsafterexposuretoPOPmixtureorPFOSusinglive-cellimaging.Effectofthecompoundsinpresenceof NGF(50ng/mL)(AandC)oritsabsence(BandD)wereevaluated.CellwereexposedtothePOPmixture(AandB),ortoPFOS(CandD).SolventcontrolwasDMSO0.1%.

ConcentrationofthePOPmixtureisindicatedasPOPsxhumanbloodlevels.PFOSconcentrationsareinmM.AverageCBC(totalnumberofcellbodyclusters/areaofimage ﬁeld)wereautomaticallymeasuredbyNeuroTracksoftware,every60min,followedfor72h.Forclarity,thegraphispresentedonlywithmeanvalueatdifferenttimepoints.

Dataaremean,n=3-4independentexperiments,withreplicate>3pergroup.Statisticaldifferencebetweentheoverallaverageoftheexposedgroupsandthesolventcontrol (0.1%DMSO)isindicatedontheleft-handsideoftheﬁgurelegends(*p<0.05),w=withNGFandwo=withoutNGF.

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andNBPincreasedwithtimeincultureforallconditionsinthe presence of NGF (Figs. 8A, C, and 9 A, C, respectively). Only a marginalincreasewasobservedwithtimeinculturewhenNGF wasnotpresent(Figs.8B,Dand9B,D,respectively).

InPC12cellsstimulatedwithNGF,thePOPmixturesigniﬁcantly increasedtheneuritelengthinaconcentration-dependentmanner compared to the effect of NGF alone (Fig. 8A; Supplementary Table2).PFOShadasimilar,butlesspronouncedeffect.PFOSat concentrations of 20

m

Mand abovesigniﬁcantlyincreased the neuritelengthcomparedtoNGFaloneoverthe72hperiod(Fig.8C;

SupplementaryTable2).IntheabsenceofNGF,theeffectsofPOPs andPFOSonNLweremarginal(Fig.8B,D;SupplementaryTable2).

Similarpatternswereobservedforthenumberofbranchpoints (Fig.9A–D;SupplementaryTable2).

4.Discussion

WeinvestigatedifaPOPmixturebasedonhumanbloodlevels fromtheScandinavianpopulationcanaffectcellularhealthand neuritedynamics,usingPC12 cells asa model. Outcomeswere comparedtoPFOSalone,themostabundantcompoundinthePOP mixture.

CytotoxicityofthePOPmixtureexposureafter72hevaluated by MTT conversion was only seen at higher concentrations.

Reduced conversion of MTT to formazan by PC12 cells could indicateareductionofmitochondrialactivity,ratherthanreduced proliferationorlossofcells.However,adecreaseincellnumber wasmeasured byHCA.Yet,theHCAnuclearandmitochondrial parametersremainedunchangedandwereindicativeofhealthy cells.ThissuggeststhatthereductionseeninMTTwasmainlydue toareducedproliferation.TheIncuCyteexperimentsallowedusto follow cell proliferation in a dynamic way over the full 72h

exposure.Indeed,thenumberofcellbodyclusters(CBC)canbe interpreted as a measurement of cell proliferation, although it givesnoinformationaboutthenumberofcellsineachindividual cluster.Thiscanbegatheredfromthecellclusterarea(CBCA),a measurementofthetotalareaofcellclustersperimageﬁeld.Inall exposures, both CBC number and CBCAincreased linearly over time,indicativeofcontinuedcellproliferation.However,therise wasslowerinthepresenceofNGF,indicatinganegativeeffectof NGF on cell proliferation. This is in accordance with the observations that NGF-induced differentiation in PC12 cells is linkedwithreducedcellproliferation(GreeneandTischler,1976).

Duringthe72hperiodthemostpotentPOPmixtureconcentration (500x)sloweddowntherateofCBCnumberincreasecomparedto solvent control. However, in the absence of NGF, the same concentrationhadmoreoutspokeneffectontherateofreduction inCBCnumberincrease.Thiscouldberelatedtotherelativelower levelofGSHasdiscussedbelow.

ForPFOSinthepresenceofNGF,noreductioninMTTwasseen at lower concentrations (10 and 25

m

M), although higher concentrations(50and 100

m

M)showedareduction compared to control. No signiﬁcant changes in cell number at any concentration couldbeobserved. Aswith thePOP mixture,no effectswereseenonnuclearandmitochondrialparameters.PFOS at lower concentration (10 and 20

m

M) seemed to have a stimulatingeffectontherateofCBCnumberincreasecompared tocontrol,whereashigherconcentrations(30and50

m

M)ledto lowerrate.Thus,wecanconcludePFOSconcentrations(20and25

m

M)relevanttothosepresentin thetotalPOPmixturedidnot adverselyaffectneithercellproliferationnorcellulartoxicity.In theabsenceofNGF,onlyat100

m

MareductioninMTTactivitywas observed,butthisPFOSconcentrationisabout5timeshigherthan relevanttothePOPmixture.Again,nosigniﬁcanteffectsoncell Fig.7.AssessmentofchangesinCell-BodyClusterArea(CBCA)inPC12cellsafterexposuretoPOPmixtureorPFOSusinglive-cellimaging.Effectofthecompoundsin presenceofNGF(50ng/mL)(AandC)oritsabsence(BandD)wereevaluated.CellwereexposedtothePOPmixture(AandB),ortoPFOS(CandD).SolventcontrolwasDMSO 0.1%.ConcentrationofthePOPmixtureisindicatedasPOPsxhumanbloodlevels.PFOSconcentrationsareinmM.AverageCBCA(sumofareasofallcell-bodyclusterspooled/

areaofimage)wereautomaticallymeasuredbyNeuroTracksoftware,every60min,followedfor72h.Forclarity,thegraphispresentedonlywithmeanvalueatdifferent timepoints.Dataaremean,n=3-4independentexperiments,withreplicate>3pergroup.Statisticaldifferencebetweentheoverallaverageoftheexposedgroupsandthe solventcontrol(0.1%DMSO)isindicatedontheleft-handsideoftheﬁgurelegends(*p<0.05),w=withNGFandwo=withoutNGF.

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number and cellularhealth parameters in HCAwereobserved.

However,concentrationsof30and50

m

MleadtodecreaseinCBC number.

Intracellular GSH levels are important for cell survival and protectthecellsagainstinjurycausedbyROS.Wehavepreviously observedthatthePOPmixtureandPFOSnegativelyaffectedGSH levels in chicken cerebellar granule cells (manuscript under review).Thus, we testedif PC12 cells wereequallyaffected.In thepresenceofNGF,lowconcentrationsofPOPmixtureraisedGSH level, which was absent for the higher concentrations. In the absenceofNGF,POPmixtureexposureledtoadecreaseinGSH, suggestingthatNGF-differentiatedcellshaveamorerobustGSH defencemechanism.Indeed,treatmentwithNGFhasbeenshown toincreasethelevelofGSHinPC12cells(Kamataetal.,1996).We foundnoevidencethateitherinthepresenceorabsenceofNGF, the POP mixture at 500x affected GCLC promotor activity, suggestingitdidnotaffectproductionratesofGSH.

NGFcanstimulateneuriteoutgrowthinPC12cells,andneurite outgrowthcanbeusedasa markerforneuronal differentiation (RadioandMundy,2008).Indeed,inoursystemNGFalonewasa sufﬁcient driver for altering the cellularand neuritedynamics compared to DMSO control. Chemicals, including POPs, could potentiallyaffectneuronaloutgrowth(Addaeetal.,2013;Angus and Contreras, 1995; Radio et al., 2008). However, it seems exposure to the POP mixture cannot mimic the NGF-receptor signalonitsown.WefoundthatexposuretothePOPmixturedid enhanceNGF-inducedneuritelengthand branchednetworksof neurites in a concentration-and time-dependent manner. This suggeststhatthePOPmixturemighthavepotentiallyinﬂuenced theNGFneurotrophicsystem.Similartoourresults,Angus and Contreras(1995)foundthatAroclor1254(atechnicalmixtureof

PCBs)enhancedNGF-stimulatedneuriteoutgrowthinPC12cells andcausedanincreaseintheaffinityofbindingofNGFtothehigh- affinity NGF receptors. However, Aroclor 1254 is an industrial mixturewitha lot-to-lot variationof ortho-and non-ortho-PCB composition(Kodavantietal.,2001).Incontrast,ourmixtureis chemically defined and contains mainly non-dioxin like PCBs.

Thus,ourobservationsindicatethatnon-dioxin-likePCBsmight contributetoneuritogenesis.TheeffectsofPFOSonbothneurite lengthand branch pointparameters in absolutenumberswere about50%ofthefullmixture.TheeffectofPFOSseemedtoplateau from20

m

Monwards.Thisalsorepresentstheconcentrationof PFOSpresentinthePOPmixtureat500.Thus,PFOSalonecan explainpartoftheeffectseenwiththePOPmixture,butadditional compounds contributetotheoverall effect.Atpresent, mecha- nismsarenotknown.

Studiesinvitrohaveshownthedifferentialeffectsofindividual POPsonneuriteoutgrowthusingdifferentneuronalcells.PCB153 orPCB180at100nMfacilitatedtheneuriteoutgrowthassociated with a decrease in cell proliferation, indicating an increase in neuronal differentiation in primary rat embryonic neural stem cells(Toﬁghietal.,2011).InthePOPmixtureat500x,PCB153is presentat349nMandPCB180at170nM.Generally,OCPshave beenshowntoreduceneuriteoutgrowth.p,p`-DDTando,p`-DDT decreasedtheneuriteoutgrowthdosedependently(1–50

m

M)and induced apoptosisin NGF-stimulated PC12 cells. p,p`-DDE also revealed a similareffect but toa lesserextent (Shinomiya and Shinomiya,2003).InthePOPmixtureat500x,p,p`-DDEispresent at 0.5

m

M. Christen et al. (2017) reported that the different organochlorine pesticides including dieldrin at 10 or 100

m

M suppressed theneuriteoutgrowthinPC12cells co-treatedwith NGF, but this compound is only present at 28nM in the POP Fig.8.AssessmentofchangesinneuritelengthovertimeinPC12cellsafterexposuretoPOPmixtureorPFOSusinglive-cellimaging.Effectofthecompoundsinpresenceof NGF(50ng/mL)(AandC)oritsabsence(BandD)wereevaluated.CellwereexposedtothePOPmixture(AandB),ortoPFOS(CandD).SolventcontrolwasDMSO0.1%.

ConcentrationofthePOPmixtureisindicatedasPOPsxhumanbloodlevels.PFOSconcentrationsareinmM.Averageneuritelengths(sumoflengthsofallneuritespooled/

areaofimageﬁeldinmm/mm²)wereautomaticallymeasuredbyNeuroTracksoftware,every60min,followedfor72h.Forclarity,thegraphispresentedonlywithmean valueatdifferenttimepoints.Dataaremean,n=3-4independentexperiments,withreplicate>3pergroup.Statisticaldifferencebetweentheoverallaverageoftheexposed groupsandthesolventcontrol(0.1%DMSO)isindicatedontheleft-handsideofﬁgurelegends(*p<0.05),w=withNGFandwo=withoutNGF.

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mixtureat500.TreatmentwithHCBat0.5nMrepressedneurite outgrowthbut didnotaffect cellviability inmouse embryonic stem(ES)cells(Addaeetal.,2013).Thiscompoundispresentinthe POPmixture(500x)at114nM.CommercialPBDEsdecabromodi- phenylether(deca-BDE)at15

m

M,inhibitedtheneuritegrowthin cultured rat hippocampal neuron (Liao et al., 2008a). In our mixture,individualBFRsarepresentatnanomolarrange.Nano- molar concentrations of PFOS, have been shown to facilitate neuriteoutgrowthassociatedwithdecreasedcellproliferationin ratprimaryembryonicneuronalcells(WanIbrahimetal.,2013).

However, micromolar concentrations of PFAAs, comparable to ours,inhibitedtheneuriteoutgrowthinculturedrathippocampal neuronscells (Liao et al.,2009,2008b).These discrepancies in outcomesmightbeexplainedbymanyfactors,suchasconcentra- tion ranges, differences in different types of neurons, or the experimental conditions used. The effects of POPs on neurite outgrowth in PC12 could also be species specific or cell type specific effect. In contrast to other neurons from the central nervoussystem,PC12is lackingfunctionalexpressionofNMDA receptors.Thisreceptormediatesglutamateinducedexcitotoxicity andcanresultinexcessivecalciuminfluxleadingtocelldeathin many neurological disorders (Edwards et al., 2007). Another possible reason could be an ontogenetic effect, linked to the developmentalstageofthecellsused.Inthepresent,weexamined theeffectsofPOPsinPC12cellsthatwerenotexposedpreviously toNGF(i.e.notdifferentiated,unprimed).Althoughnotexamined inthepresentstudy,theotherpossiblewaywouldbetodetermine theeffectsofPOPsinPC12thathavebeenexposedpreviouslyto NGF(primed).Parranetal.(2003)observedthatinunprimedPC12 cells, mercuric chloride exposure increased NGF-stimulated neuriteoutgrowth.However,inprimedPC12cells,thiscompound

inhibitedNGF-stimulatedneuriteoutgrowth.Itshouldbenoted that our POP mixture contains 29 different compounds which couldhaveadditive,synergisticorantagonisticeffects.

Itshouldbestressedthatneuritelengthnumberreturnedby theIncuCyteimageanalysissoftwarerelatestothesummationof theindividuallengthsofoutgrowthsperimageﬁelds,ratherthan a mean length of individualneurites.Similarly, thenumber of branchpointsreturnsa countperimageﬁeld.Also,itdoesnot differentiatebetweentruebranchpoints(oneextensionsplitting in 2 or more)or crossings formed byoverlapping outgrowths.

Nevertheless, wefound both parametersare highly correlated, and their increases suggest that cells are investing in the formationofmoreneuritematerial,andthatthecomplexityof theoutgrowthsincreases.

OurPOPmixturedidnotinduceimmediatecelldeath.Rather, the inhibition of neurite outgrowth has been considered an important mode of action for speciﬁc developmental neurotoxicants(Radioetal.,2008).However,inourstudyPOPmixture andPFOSleadtoenhancedneuriteoutgrowthindicatinglackof cytotoxicity. Someeffects onthe rate ofcell proliferationwere observed, but nuclear and mitochondrial health parameters remainedunaffected.

The neurite outgrowth occurs in a strictly controlled way.

Duringdevelopmentofthenervoussystem,manymoreneurons aregeneratedthanultimatelyintegrateintoneuronalcircuitsand survive(Mattson,2008).Althoughpollutantsshowednocytotoxic effects in these circumstances,overactive neuritogenesisat the wrongtimingmightalsobedetrimental.Forexample,studieshave foundthatspinedensityisincreasedinASD,whichcouldsuggesta developmentalalterationinspinemorphogenesis(Forrestetal., 2018).

Fig.9. AssessmentofchangesinneuritebranchpointsinPC12cellsafterexposuretoPOPmixtureorPFOSusinglive-cellimaging.EffectofthecompoundsinpresenceofNGF (50ng/mL)(AandC)oritsabsence(BandD)wereevaluated.CellwereexposedtothePOPmixture(AandB),ortoPFOS(CandD).SolventcontrolwasDMSO0.1%.

ConcentrationofthePOPmixtureisindicatedasPOPsxhumanbloodlevels.PFOSconcentrationsareinmM.Averageneuritebranchpoints(totalnumberofbranchpoints/

areaofimageﬁeld)wereautomaticallymeasuredbyNeuroTracksoftware,every60min,followedfor72h.Forclarity,thegraphispresentedonlywithmeanvalueatdifferent timepoints.Dataaremeann=3-4independentexperiments,withreplicate>3pergroup.Statisticaldifferencebetweentheoverallaverageoftheexposedgroupsandthe solventcontrol(0.1%DMSO)isindicatedontheleft-handsideofﬁgurelegends(*p<0.05),w=withNGFandwo=withoutNGF.