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ContentslistsavailableatScienceDirect

Toxicology Reports

jo u r n al h om ep a g 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

Omega-3 and alpha-tocopherol provide more protection against contaminants in novel feeds for Atlantic salmon (Salmo salar L.) than omega-6 and gamma tocopherol

Liv Søfteland

^a,∗

, Marc H.G. Berntssen

^a

, Jennifer A. Kirwan

^b

, Trond R. Størseth

^c

, Mark R. Viant

^b

, Bente E. Torstensen

^a

, Rune Waagbø

^a

, Pål A. Olsvik

^a

aNationalInstituteofNutritionandSeafoodResearch,Norway

bSchoolofBiosciences,UniversityofBirmingham,BirminghamB152TT,UK

cSINTEFFisheriesandAquaculture,Norway

a r t i c l e i n f o

Articlehistory:

Received6November2015 Receivedinrevisedform 23December2015 Accepted11January2016 Availableonline14January2016

Chemicalcompoundsstudiedinthisarticle:

Chlorpyrifos Endosulfan Phenanthrene Benzo(a)pyrene Arachidonicacid Eicosapentaenoicacid

␣-tocopherol

␥-tocopherol Keywords:

Interactions Nutrients PAH Pesticides Lipidomics Metabolomics RT-qPCRtranscriptomics

a b s t r a c t

ExtendeduseofplantingredientsinAtlanticsalmonfarminghasincreasedtheneedforknowledge ontheeffectsofnewnutrientsandcontaminantsinplantbasedfeedsonﬁshhealthandnutrient- contaminantinteractions.PrimaryAtlanticsalmonhepatocyteswereexposedtoamixtureofPAHsand pesticidesaloneorincombinationwiththenutrientsARA,EPA,␣-tocopherol,and␥-tocopherolaccord- ingtoafactorialdesign.CellswerescreenedforeffectsusingxCELLigencecytotoxicityscreening,NMR spectroscopymetabolomics,massspectrometrylipidomicsandRT-qPCRtranscriptomics.Thecytotoxi- cityresultssuggestthatadverseeffectsofthecontaminantscanbecounteractedbythenutrients.The lipidomicssuggestedeffectsoncellmembranestabilityandvitaminDmetabolismaftercontaminant andfattyacidexposure.Co-exposureofthecontaminantswithEPAor␣-tocopherolcontributedtoan antagonisticeffectinexposedcells,withreducedeffectsontheVTGandFABP4transcripts.ARAand

␥-tocopherolstrengthenedthecontaminant-inducedresponse,ARAbycontributingtoanadditiveand synergisticinductionofCYP1A,CYP3AandCPT2,and␥-tocopherolbysynergisticallyincreasingACOX1.

IndividuallyEPAand␣-tocopherolseemedmorebeneﬁcialthanARAand␥-tocopherolinpreventingthe adverseeffectsinducedbythecontaminantmixture,thoughacombinationofallnutrientsshowedthe greatestamelioratingeffect.

1. Introduction

Increaseduseofplantfeedingredientshasintroducedanew cocktailofplant-oilderivedcontaminants,suchaspolycyclicaro- matichydrocarbons(PAHs) andpesticides,formerlynot related withfarmingofsalmonids[5,23].Plantoilsintendedforanimalfeed productioncanbecontaminatedwithPAHlikephenanthreneand benzo(a)pyrene[81,12]duetoatmosphericdepositionofparticles oncropsbeforeharvestingorlaterduringthethermalprocessing

∗Correspondingauthor.

E-mailaddress:[email protected](L.Søfteland).

oftheoilseeds[20,64].Residuelevelsofpesticideslikeendosulfan andchlorpyrifoshavebeenreportedinproductsfromplantssuch assoyaormaize[33,49]whicharecommonlyusedasingredientsin salmonfeeds[4].BothPAHandpesticideshavebeenshowninsev- eralinvitroandinvivoexperimentstocauselipidandendocrine disturbancesandtoinducecytochromeP450enzymesinteleost ﬁsh[43,53,68,79,100,102].PAHsaregenotoxic[16,86,35]andexpo- surehasbeensuggestedtocausevitaminDsignallingdisruption [79]aswellasaneffectoncellmembraneintegrity[65,70].The pesticidesendosulfanandchlorpyrifoshavebeenshowntoinduce lipidaccumulationinAtlanticsalmonlivercellsbothinvitroand invivo[43,23,79].

http://dx.doi.org/10.1016/j.toxrep.2016.01.008

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Inadditiontoanalteredcontaminantprofile,theplant-based feedingredientsalsochangethenutrientprofileofthefish.Marine oils contain high levels of the n-3 polyunsaturated fatty acids (PUFAs)eicosapentaenoicacid(EPA,20:5n-3)anddocosahexaenoic acid(DHA,22:6n-3)[36]whilecommonplantoilscancontainhigh levelsofn-6PUFAlikelinoleicacid(LA,18:2n-6)[92].Sincethe fattyacidcompositionofoilyfishreflectsthefattyacidcomposi- tionoftheirfeed[85],replacingfishoilwithvegetableoilinfish feedtypicallyreducestheconcentrationinAtlanticsalmonfilletof then-3PUFAsEPAandDHAandincreasesconcentrationsofthe n-6PUFAsLAandarachidonicacid(ARA,20:4n-6)[25,62,84,85].

Theliverisacentralorganforlipidmetabolism[7]andthesyn- thesisofcholesterol and bile [42]. Immediatelyafter uptake in theliver,the PUFA canbe converted toenergy by ␤-oxidation [74],storedinadipocytesandinintracellularlipiddropletsindif- ferenttissues[55]orincorporatedintophospholipidmembranes [92].The n-3and n-6PUFAs canbe converted totheirrespec- tivegroupofeicosanoidsorlipidmediatorsbythelipoxygenase (LOX),cyclooxygenase(COX)andcytochromeP450(CYP)enzyme pathways[92].Then-6eicosanoidsare ageneralgroupofpro- inflammatoryeicosanoids[92].Bycontrast,then-3PUFAcanbe convertedton-3eicosanoidsthathaveanti-inflammatoryabilities [92].Severalstudieshavereportedincreaseinliverlipidwhenfish oilwasreplacedwithplantoilsindicatingthatnutrientsinfish oilsuchasEPA andDHA, n-6,saturatedfattyacids, cholesterol andphytosterolsplayarole(Torstensenetal.,2011)[45,47,62].

Highlevelsofthen-3PUFAsDHAandEPAinfishfeedcanprotect againstinductionofliversteatosisinAtlanticsalmon[45,47].␣- tocopherolisanessentialnutrientforfish[28]andisalsothemain formofvitaminEinfishfillet[76].Dietary␣-tocopherolistaken upmorerapidlythan␥-tocopherol[93],whichisthemainvita- minEcongenerinmostplantseeds[34]andmaize,rapeseedand soyaoils[77].␥-tocopherolseemstobeamoreeffectivetrapfor lipophilicelectrophilesthan␣-tocopherol[34]andincontrastto␣- tocopherol,␥-tocopherolpossessesanti-inflammatoryproperties [34].Similartothen-3PUFAs,highlevelsoftocopherolcaninhibit inductionofliversteatosis[91]andprotectorganismsagainstlipid oxidation[28].

Theaimofthisstudywastoexaminehowrelevantnutrientscan modulatethetoxicologicaloutcomeofacontaminantmixtureasso- ciatedwithplantfeedingredientsusingmetabolomic,lipidomic andtranscriptomicmethodstosearchfornovelbiomarkersand possibleinteractioneffects.ThestudyutilisedAtlanticsalmonpri- maryhepatocytesasabiologicalmodelsystem.

2. Materialsandmethods

2.1. Chemicals

Endosulfan (6,7,8,9,10-hexachloro-1,5,5a,6,9,9,a-hexahydro- 6,9-metano-2,4,3-benza-dioxathiepin-3-oxide, ␣+␤–2+1;

PESTANAL^®, analytical standard), chlorpyrifos (O,O-diethyl-O- 3,5,6-trichlor-2-pyridylphosphorothioate,PESTANAL^®,analytical standard), phenanthrene (≥98% pure), benzo(a) pyrene (≥96%

pure),arachidonicacid(ARA≥99%),eicosapentaenoicacid(EPA,

≥99% pure), potassium hydroxide, ␣-tocopherol (␣T, >95.5%

pure) and ␥-tocopherol (␥T, ≥96% pure) were all purchased fromSigma–Aldrich(Oslo,Norway).Dimethylstocksolutionwas purchasedfromScientiﬁcandChemicalSuppliesLtd.(Bilston,UK), chloroform (HPLC grade) was purchased from Fisher Scientiﬁc (Loughborough,UK)andammoniumacetatewaspurchasedfrom Sigma–Aldrich Co., Ltd. (Dorset, UK). The fatty acid free-BSA (FAF–BSA)waspurchasedfromPAA(Parching,Austria).

2.1.1. BovineSerumAlbumin(BSA)couplingoffattyacids

Bindingoffattyacids(FA)tofattyacidfree-BSA(FAF–BSA)was performedasperGhionietal.[22].Tosummarize,FAdissolvedin 0.04mlchloroformpermgFAwasaddedtoaglasssampletube andN2wasusedtoevaporatethechloroform.Potassiumhydrox- ide(KOH)wasappliedtotheFAina1:3ratioandthesolutionwas shakenfor10minusingavortexmixer.FAF–BSAwasemployed ina2.5:1relationshiptotheFAandthesolutionwasmixedfor 45minbeforeit wassterile-ﬁlteredand preserved at−80^◦C in anoxicconditions.

2.2. Isolationofprimaryculturesofhepatocytes

JuvenileAtlanticsalmonwereobtainedandkeptattheanimal holdingfacilityatIlab,UniversityofBergen(UiB),Bergen,Norway.

Theﬁshwerefedoncedailywithaspecialfeedproducedwithout additionofsyntheticantioxidantsandwithlowlevelsofcontam- inants,suppliedbyEWOS,Norway(HarmonyNatureTransfer75).

Allglassware,instruments and solutionswere autoclaved prior toliverperfusion.HepatocyteswereisolatedfromeightAtlantic salmon(278–381g)withatwo-stepperfusionmethodpreviously describedinRef.[78].TheﬁnalcellpelletwasresuspendedinL- 15mediumcontaining10%ﬁshserum(FS)fromsalmon(Nordic BioSite, Oslo, Norway), 1% glutamax (Invitrogen, Norway) and 1%penicillin–streptomycin–amphotericin(10,000units/mlpotas- siumpenicillin,10000␮g/mlstreptomycinsulphateand25␮g/ml amphotericinB)(Lonzo,Medprobe,Oslo,Norway).TheTrypanBlue exclusion method,performedin accordance withthe manufacturer’s protocol (Lonzo,Medprobe, Oslo, Norway),was usedto determinecellviability.Thedifferentcellsuspensionsusedinthis studyhadcellviabilitybetween85–90%.Thecellsuspensionswere platedon5␮g/cm²laminin(Sigma–Aldrich,Oslo,Norway)coated cultureplates(TPP,Trasadingen,Switzerland),andthehepatocytes werekeptat10^◦CinasterileincubatorwithoutadditionalO2/CO2

(Sanyo,CFCFREE,EttenLeur,TheNetherlands).Thefollowingcell concentrationswereused;7.2×10⁶cellsperwellin6-wellplates (in3mlcompleteL-15medium),2.6×10⁶cellsperwellin12-well plates(in2mlcompleteL-15medium),0.2×10⁶cellsperwellin xCELLigence96-wellplates(in0.2mlcompleteL-15medium).

2.3. Chemicalandnutrientexposure

Theprimarycellswereculturedfor36–40hpriortochemical exposurewithonechangeofmedium(containing 10%FS)after 18–20h. Thecells wereexposed for 48hto singlenutrientsto establishcytotoxicdose-responsecurves andtonutrientsand a contaminantmixtureaccordingtoafactorialexperimentaldesign forinteractionevaluation.Cytotoxicitydose-responsecurveswere establishedfor␣-tocopherol(1,10,100,1000and10,000␮M)and EPA(100,200,4000and600␮M)andcellsfromthreeﬁsh(n=3) wereusedtomakethedose-responsecurves.Basedonindividual cytotoxicitydose-responsecurvesforthenutrients,non-cytotoxic concentrationsofthe␣-tocopherol(100␮M)andthefattyacidEPA (200␮M)wereused(datanotshown).Tobeabletoassesstheeffect ofbothmarineandplantderivednutrientsonthetoxicityofamix- tureofPAHsandpesticides,comparableconcentrationswereused fortheplantderived nutrients␥-tocopherol(100␮M)and ARA (200␮M)inafactorialdesign.Thecontaminantmixtureusedhere wasselectedbasedonapreviousstudybySøftelandetal.[79]and wascomposedof100␮MofthePAHsbenzo(a)pyreneandphenan- threneand1␮Mofthetwopesticideschlorpyrifosandendosulfan.

Theseconcentrationswerechosenafterearlierindividualassess- mentofthefourcontaminantsaimingfor levelsjustbelowthe onset ofcytotoxicity,and thecontaminantmixture usedinthe presentstudywasapotentcombination[79].Afullfactorialdesign wasusedwithtwolevels(lowandhighconcentrations),azero

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Table1

Overviewofthedifferentconcentration(␮M)combinationsusedforthevarious nutrients(eicosapentaenoicacid(EPA),arachidonicacid(ARA),␣-tocopherol(␣T) and␥-tocopherol(␥T))andcontaminantmixture(CM)usedinthefactorialdesign forlipidomicandRT-qPCRevaluation.CMcontained100␮Mofbenzo(a)pyreneand phenanthreneand1␮Mofchlorpyrifosandendosulfan.

Exp.no. EPA ARA ␣T ␥T CM

1 0 0 0 0 0

2 200 0 0 0 0

3 0 200 0 0 0

4 200 200 0 0 0

5 0 0 100 0 0

6 200 0 100 0 0

7 0 200 100 0 0

8 200 200 100 0 0

9 0 0 0 100 0

10 200 0 0 100 0

11 0 200 0 100 0

12 200 200 0 100 0

13 0 0 100 100 0

14 200 0 100 100 0

15 0 200 100 100 0

16 200 200 100 100 0

17 0 0 0 0 100

18 200 0 0 0 100

19 0 200 0 0 100

20 200 200 0 0 100

21 0 0 100 0 100

22 200 0 100 0 100

23 0 200 100 0 100

24 200 200 100 0 100

25 0 0 0 100 100

26 200 0 0 100 100

27 0 200 0 100 100

28 200 200 0 100 100

29 0 0 100 100 100

30 200 0 100 100 100

31 0 200 100 100 100

32 200 200 100 100 100

33 100 100 50 50 50

point(control,0.4%DMSO),andonecentrepointinordertoevalu- atelinearity(Table1).InadditionaBSAcontrolwasincluded.Cells fromﬁveﬁshwereemployedforthefactorialdesignexperiment.

Theexposuremedium contained1%FS.The chemical exposure mediumwassubstitutedwithnewmediumafter18–20h.

2.4. Cytotoxicitytestingofchemicals

For the cytotoxicity assessment of the nutrients and contaminant mixtures, real time impedance data obtained by the xCELLigence systems (ACEA Biosciences, Inc. (ACEA), Aarhus, Denmark)wasused.ThexCELLigencesystemquantiﬁeselectrical impedanceacrosselectrodesin96-wellcellcultureE-Plates.The impedancemeasurementgivesquantitativeinformationaboutthe cells’healthstatusincludingmorphology,cellnumberandviability andisindicatedwiththeparameterCellindex(CI)orthenormal- izedCI(NCI).ThexCELLigenceinstrumentwasusedtoestablish dose-response relationship for EPA and ␣-tocopherol exposed singlytotheprimaryhepatocytesandinteractionevaluationusing cellsexposedaccordingtoafactorialdesign,asmentionedabove.

Therealtimecellmonitoringwasconductedat10^◦Cinanincubator withoutadditionalO₂/CO₂(Sanyo,CFCFREE,EttenLeur,Nether- land),usingtheRTCAsingleplatexCELLigenceplatform.Thedata wascollectedaccordingtoSøftelandetal.[79].Brieﬂy,thedata wascollectedwithintervalsof2minaftercontaminantexposure for12h,andthenevery15minfor120h.Thelasttimepointbefore compoundexposurewasusedforthenormalization,allowinga moreprecisecomparisonoftheeffectofthedifferentcontaminant concentrationstested.TheCIvaluespresentedherewerecalcu- latedfromthreetoﬁvereplicatevalues.Determinationofcytotoxic

effectswasdoneaccordingtotheInternationalstandardisedtest forinvitrocytotoxicity,ISO10993-5:2009[32].Contaminantswill bedeemedcytotoxicwhencellsviabilityexceeds30%reduction comparedtothecontrol.

2.5. Metabolomicsandlipidomics

Based onthefactorialdesign cytotoxicityscreening,a selec- tionofmixtureswereanalysedwithlipidomicandmetabolomic methodstodeterminehownutrientsmodifythetoxiceffectofthe contaminantmixtures.TheexposuregroupsanalysedwereEPA, ARA,␣T,␥T,CM,C-EPA,C-ARA,C-␣T,C-␥T,C-Allhigh,andC-All low,andthesegroupscorrespondedtofollowingmixtures2,3,5, 9,17,18,19,21,25,32and33,respectively,inTable1.

2.5.1. Metaboliteextraction

Lyophilizedsampleswereextractedusinga1145␮lmixtureof chloroform:methanol:water(2:2:1.8)and vortexedin2mlglass vials.Thepolarandnon-polarphasesofthisbi-phasicmixturewere separated,andthepolarphase(500␮l)wasvacuumcentrifuged (30minat300K),frozenandfreezedriedfornuclearmagneticreso- nancespectroscopy(NMR)analysis.Forthenon-polarphase,300␮l wereevaporatedunderN₂andstoredat−80^◦CbeforeMSanalysis.

2.5.2. FT-ICRmassspectrometrybasedlipidomics

All dried lipid samples were resuspended in an equal vol- umeof2:1methanol:chloroformwith5mMammoniumacetate.

Lipidomicanalyseswereconductedinnegativeion modeusing a hybrid7-T FT-ICR mass spectrometer (LTQ FT Ultra, Thermo Fisher Scientiﬁc, Bremen, Germany) with a chip-based direct infusionnanoelectrospray ionisationassembly (Triversa,Advion Biosciences,Ithaca,NY).Nanoelectrosprayconditionscomprisedof a200nl/minﬂowrate,0.4psibackingpressureand-1.2kVelec- trosprayvoltagecontrolledbyChipSoftsoftware(version8.1.0).

Massspectrometryconditionsincludedanautomaticgaincontrol settingof5×10⁵andamassresolutionof100,000.Analysistime was4.25min(pertechnicalreplicate),controlledusingXcalibur software(version2.0,ThermoFisherScientiﬁc).Spectrawerecol- lectedusingthe“SIMstitching”method,i.e.,fourteenoverlapping selectedionmonitoring(SIM)massranges,rangingfromm/z70 to2000,wereacquiredandsubsequentlyfusedtogether,[72,95].

Duplicateanalyseswereaveragedforeachsample.Aqualitycon- trol(QC)sampleconsistingofapooledaliquotofthesampleswas analysedbeforeduringandaftertheanalysis.

2.5.3. NMRspectroscopybasedmetabolomics

Subsequently thedried polarmetabolite fractionwasresus- pendedin200␮lD₂Owith1mMTMSPandtransferredtoNMR tubes.Allsamplesweremaintainedat277Kandanalysedwithin 48hofresuspension.NMRwasperformedonaBrukerDRU600 NMRspectrometer(600.23MHzfor¹H)ﬁttedwitha5mmCPQCI cryogenicprobe(BrukerCorporation).ThreemmNMRtubeswere usedwiththeBrukerSampletrackautosamplerinwhichthesam- pleswere keptat279Kbefore(andafter) analysis.Thespectra wererecordedat300Kwithsuppressionoftheresidualwaterreso- nanceusingthenoesygppr1dpulsesequencefromtheBrukerpulse sequencelibrary.Apulsewidthof7.91␮swasusedtocollect128 freeinductiondecayswith32Kdatapointswithaspectralwin- dowof12,019Hz(20ppm).Theacquisitiontime was2.73sand theinterscandelaywas3s.Thenoesymixingtimewas10ms,the datawerezeroﬁlledto64Kandexponentiallinebroadeningof 0.3HzappliedbeforeFouriertransformationandthespectrawere phasedandbaselinecorrected.

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Table2

PCRprimers,GenBankaccessionnumbers,ampliconsizesandefﬁciency.

Gene Accessionno. Forwardprimer(5-3) Reverseprimer(5-3) Productsize(bp) Efﬁciency

CYP1A AF364076 TGGAGATCTTCCGGCACTCT CAGGTGTCCTTGGGAATGGA 101 1.93

PPARA NM001123560 TCTCCAGCCTGGACCTGAAC GCCTCGTAGACGCCGTACTT 58 2.00

CYP3A DQ361036 ACTAGAGAGGGTCGCCAAGA TACTGAACCGCTCTGGTTTG 146 1.90

ACOX1 DY733173 CACTGCCAGGTGTGGTGGTA GGAATTCGTACGTTCTCCAATTTCA 94 2.04

FBP4 BT125322 CCGCCGACGACAGAAAAA TTTTGCACAAGGTTGCCATTT 61 1.99

CPT2 BG934647 TGCTCAGCTAGCGTTCCATATG AGTGCTGCAGGACTCGTATGTG 49 2.07

VTG AY049952 GACTTCGCCATCAGCCTTTC GCCACGGTCTCCAAGAAGTCT 110 2.11

EF1AB AF321836 TGCCCCTCCAGGATGTCTAC CACGGCCCACAGGTACT 59 2.04

UBA52 GO050814 TCAAGGCCAAGATCCAGGAT CGCAGCACAAGATGCAGAGT 139 2.01

B-ACTIN BG933897 CCAAAGCCAACAGGGAGAA AGGGACAACACTGCCTGGAT 92 1.96

2.6. Quantitativereal-timeRT-PCR 2.6.1. RNAextraction

The RNeasy Plus mini kit (Qiagen, Crawley, UK) was used to extract total RNA according to the manufacturer’s protocol.

RNA was eluted in 30␮l RNase-free MilliQ H2O and stored at

−80^◦C. The RNA quantity and quality were assessed with the NanoDrop^®ND-1000UV–visSpectrophotometer(NanoDropTech- nologies,Wilmington,DE,USA)andtheAgilent2100Bioanalyzer (AgilentTechnologies,PaloAlto,CA,USA)pursuanttothemanufac- turer’sinstructions.TheintegrityoftheRNAwasevaluatedwiththe RNA6000NanoLabChip^®kit(AgilentTechnologies).Thesamples usedinthisexperimenthad260/280nmabsorbanceratiosanda 260/230nmratiosabove2andRNAintegritynumber(RIN)values above9,whichindicatepureRNAandintactsamples[66].

2.6.2. Quantitativereal-timeRT-PCR

Thetranscriptionallevelsofselectedtargetgeneswerequan- tiﬁedwithatwo-stepreal-timeRT-PCRprotocol.Aserialdilution curveoftotalRNAwithsixpointsintriplicatesbetween1000–31ng weremadeforPCRefﬁciencycalculations.500ngoftotalRNAwas addedtothereactionforeachsample,andreversetranscription (RT)reactionswereruninduplicatesusing96-wellreactionplates.

No-templatecontrol(ntc)andno-ampliﬁcationcontrol(nac)reac- tionswererunfor qualityassessment foreverygeneassay.The 50␮lRTreactionswereperformed at48^◦Cfor 60minutilizing aGeneAmpPCR9700thermocycler(AppliedBiosystems, Foster City,CA,USA).IndividualRTreactionscontained1XTaqManRT buffer(10×), 5.5mM MgCl₂, 500mM dNTP (of each), oligo dT primers(2.5␮M), 0.4U/␮lRNase inhibitorand 1.67U/␮lMulti- scribeReverseTranscriptase(AppliedBiosystems)andRNase-free water.

Foreverygeneanalysed,real-timeqPCRwasrunin10␮lreac- tionsonaLightCycler^®480Real-TimePCRSystem(RocheApplied Sciences, Basel, Switzerland) containing 2.0␮l cDNA (diluted twofold).Thereal-timeqPCRwascarriedoutintwo384-wellreac- tionplatesusingSYBRGreen MasterMix(LightCycler480SYBR Greenmastermixkit,RocheAppliedSciences,Basel,Switzerland) containinggene-speciﬁcprimersandFastStartDNApolymerase.

PCRrunswereperformedwitha5minactivationanddenaturing stepat95^◦C,followedby45cycleswitheachcycleconsistingofa 10sdenaturingstepat95^◦C,a10sannealingstep(60^◦C)andfinally a10sextensionstepat72^◦C.Theprimerpairshadanannealing temperatureof60^◦C;seeTable2forprimersequences,amplicon sizesandGenBankaccessionnumbers.Finalprimerconcentrations of500nMwereused.Forconfirmationofamplificationofgene- specificproducts, a melting curveanalysiswascarried out and thesecondderivativemaximummethod[82]wasusedtodeter- minecrossingpoint(CT)valuesusingtheLightcycler480Software.

Tocalculatethemeannormalizedexpression(MNE)of thetar- getgenes,thegeNormVBAappletforMicrosoftExcelversion3.4 wasusedtocalculateanormalizationfactorbasedonthreeref-

erencegenes.Byusinggene-speciﬁcefﬁcienciescalculatedfrom thestandardcurves,theCTvaluesareconvertedintoquantities [89].Elongationfactor1AB(EF1AB),acidicribosomalprotein(ARP) and␤-actinweretheselectedreferencegenesforthisexperiment.

Thereferencegeneswerestablewithgeneexpressionstability(M) valuesof0.304.

2.7. Dataanalysis 2.7.1. xCELLigence

GraphPadPrism6.0software(GraphPadSoftwareInc.,PaloAlto, CA,USA)wasusedforthestatisticalanalysesofthexCELLigence dose-responseevaluation using one-way ANOVAfollowed by a Dunnett’sposthoctest(p<0.05)todetecttreatmentvariationin nutrientexposedhepatocytes.Mean±SEwerecalculatedforthree replicates.

2.7.2. Metabolomics

TheprocessedNMRspectrawereimported intoMatlab (The Mathworks,Inc.)usingProMetabv3.3software[90].Theregion from10to0.5ppmwasimportedwitha resolutionof0.02ppm whichresultedin4750datapoints,andtransformedusingagener- alizedlogtransformation.PCAandPLS-DAanalysiswasperformed inPLS-toolboxv7.0.1(EigenvectorResearch,Inc.)onnormalized and mean centred data prior to multivariate statistical analy- ses.ThequantitativedatafromNMRwereﬁrstsubjectedtothe Shapiro–Wilk testfor normalityand themetabolitesweresub- jected to the Kruskal–Wallis analysis of variance followed by Games–Howellposthoctesting[21]inordertoassessthecompar- isonsofstatisticallysigniﬁcantchangesbetweengroupscompared tothecontrol.

2.7.3. Lipidomics

AllanalyseswereperformedinMatlab7.8.0withPCAandPLS- DAanalysisperformedinPLS-toolboxv.6.7.1.Massspectrawere processedusingathree-stageﬁlteringalgorithmasdescribedin Ref.[59].Samplesweresubsequentlynormalisedusingprobabilis- ticquotientnormalization[14].Processingtherawmassspectra yielded a datasetthat wasfurtheroptimisedusing a QC based methodas described in Ref. [39]. Mass features withover 20%

missingvaluesacrossallsampleswereremovedandtheresulting intensitymatrix wassubmitted for univariate statistical analy- sisasdescribed below.Ak-nearestneighborapproach[31] was appliedtoimputemissingvaluestothesamedatasetanditwas transformedusingageneralizedlogtransformation(glog)priorto multivariatestatisticalanalysis[14].Thefinallipidomicsdataset wascomprisedof901massfeaturesuponwhichstatisticalanal- yseswereconducted.PCAandPLSDAwereperformedtoassess theoveralleffect(threeoutlierswhichwereoutsidetheHotellings t2plotwerefirstremoved).Allsupervisedmodelswerevalidated using cross validation and permutation testing to avoid over- fitting.Univariatestatisticalanalyseswerealsoconductedonthe

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Fig.1.ASimplifiedscaledandcenteredPLSregressioncoefficientswith95%confidenceintervalsforNormalizedcellindex(NCI)levelsmeasuredinprimaryAtlantic salmonhepatocytesexposedtoeicosapentaenoicacid(EPA),arachidonicacid(ARA)and␣-tocopherol(␣T),␥-tocopherol(␥T)andcontaminantmixture(CM)accordingly tothefactorialdesign(N=5).TheCMwascomposedof100␮Mofbenzo(a)pyreneandphenanthreneand1␮Mofchlorpyrifosandendosulfan.Themodelisbasedon 33experimentalobjects,andhadonePLScomponent.Themodelcontainingfivelineartermsandeightinteractionterms(R²=0.85andQ²=0.55).Onlylinear(CM)and interactiontermsrepresentingcontaminant-nutrientinteractions(C-EPA,C-ARA,C-␥T)andimportantnutrient-nutrientinteraction(EPA-ARA,␣T-␥T)wereincludedin thefigure(confidencelevel=0.95).SignificantPLSregressioncoefficientsareindicatedwitha*(p<0.05).ThecompletePLSregressionmodelequationisdescribedinthe supplementaryA1.TheregressioncoefficientsreflectingtheimpactofthefactorsonthePLSmodel.B4DcontourplotofxCELLigencecytotoxicityNCIlevelsasafunctionof EPAandARAwithincreasinglevelsofCMand␥TontheX-andY-axis,respectively,keeping␣Tconstantat100␮M.ThehighlightedvaluesintheplotrepresentNCIlevels forthedifferentstratificationbeddings(isoboles).

lipidomicsdataset.AKruskal–Wallisanalysisofvariancefollowed byGames–Howellposthoctesting[21]wasconductedinorderto assessthecomparisonsofstatisticallysigniﬁcantchangesbetween groupscomparedtothecontrol.ABenjamini–Hochbergcorrection of10%wasappliedtotheKWresultstocontrolforfalsediscovery [3].TheeigenvaluesforthedifferentgroupsinthedifferentPCA plotsarepresentedintheSupplementaryTableA3.1-A3.5.

Forputativeannotationofthedetectedmassfeatures,theKEGG databaseand MI-Pack software (‘single peak search’ approach) wereﬁrstusedtoassignoneormoreempiricalformula(e),e.g., C_cH_hN_nO_oP_pS_s,aswellasputativemetabolitenamestoeachmass feature[94],assuminga maximum masserrorof ±2ppm.This assignmentequatestoMSIlevel2[75].

2.7.4. RT-qPCR

RegressionwasperformedwithPLS[98]tocorrelatethedesign matrixtotheresponsesofdifferenttranscripts.MODDE9.0(Umet-

rics, Umeå,Sweden)wasused fortheexperimentaldesign and thePLS analysis.Before thePLS analysisthe blend matrix was augmentedwithinteractionterms,thedatawerescaledtounit varianceandmeancentred.ThePLSmodelswerevalidatedwith respecttoexplainedvarianceandgoodnessofprediction(shownas Q²),obtainedaftercrossvalidation[97].Inaddition,thePLSmodels wereevaluatedwithrespecttogoodnessofﬁt(R²).

3. Results

3.1. xCELLigencecytotoxicityscreening

Amultivariatemodelwasusedtoanalysethecytotoxicitydata.

ThePLSmodel,basedonthexCELLigencenormalizedcellindex (NCI),containedthreesigniﬁcantpositivenutrient–contaminant interactions.Ofthenutrient–contaminantinteractions,theinterac- tionofthecontaminantmixture(CM)and␥T,theC-␥T(p=0.005),

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displayed increasedcell viability (Fig. 1A). In addition, thePLS plotcontainedtwosigniﬁcantnutrient–nutrientinteractionsofthe lipids(EPA–ARA)andthetocopherols(␣T–␥T)withtheEPA–ARA (p=0.000078)interactionshowingthestrongestpositiveeffecton theresponse,increasingthecellviability.Thecontourplotanalysis ofallthenutrientsandtheCMshowedthatthecellviabilityreduc- tioncausedbytheCMwasalmostcompletelycounteractedwhen cellswereexposedtotheCMincombinationwithallthenutrients duetoasynergisticeffect(Fig.1B).Theindividualnutrientsonly moderatelyreducedcellviability.Onlyco-exposuretoallnutrients providedsufﬁcientprotectionagainstCMinducedcytotoxicity.

3.2. Metabolomicsandlipidomics

3.2.1. FT-ICRmassspectrometrylipidomics

A selection of the different mixtures were analysed with lipidomicsandmetabolomicstoexaminethemechanismofhow nutrientsaffectthetoxiceffectsoftheCM.Theexposuregroups analysedwere;cellsexposedtoasinglenutrient(EPA,ARAand␣T and␥T)orCM,onenutrientincombinationwiththeCM(C-EPA, C-ARA,C-␣TandC-␥T)andeitheraloworhighconcentrationof theCMandallnutrients(C-AlllowandC-Allhigh).Initiallyeachof thefournutrientsalongwiththeCMwasinvestigatedwithPCA andPLSDAtorevealtheindividualeffectsof thetreatments. In thelipidomicsPCAplots,thecellstreatedwiththeCM,ARAand EPAshowedclearseparationfromeachotherandthecontrolalong PC1(Fig.2AandB).ARAwasthenutrientthatshowedthegreatest globaleffectonthelipidome.ThePLSDAanalysisdisplayedsimilar effectswithp≤0.05(frompermutationtesting)andclassiﬁcation errorrate≤5%fortheseparticularcompounds(datanotshown).

Cellstreatedwiththeothernutrients(tocopherols)couldnotbe reliablyseparatedfromthecontrolcells(Fig.2C(p=0.104,Table A4.4)and2D(p=0.004,TableA4.5)).Incontrastto␣T,the␥Twas signiﬁcantdifferentfromtheothergroups,however,duetosmall samplesizeandlargenumberoflatentvariables,␥Tweretherefore togetherwith␣Texcludedfromfurtheranalysis.

PCAscoresplotsforcellstreatedwitheithercontrolDMSO,CM orafattyacidplusCMshowedthattheadditionofthefattyacids totheCM(Fig.2AandB)shiftedthelipidomebacktowardsthe normal(untreated)state.Asimilarbutdoserelatedeffectonthe lipidomewasobservedwhenallnutrientswerecombinedtogether andadministeredalongsidetheCMateitherahighoralowdose (Fig.2E).

Toassess ifthefattyacidshadanamelioratingeffectonthe CMtoxicity,massfeaturesthathadbeenshowntobesignificantly changingbetweenCMtreatedcellsandthecontrolDMSOtreated cellswereusedasaproxyindicatoroftoxicity.Thelistofsignifi- cantlydifferentmassfeatureswascheckedtoseewhetherthese features remained significantly differentfrom the control class inthecombinednutrientandCMtreatmentgroup.Analysiswas conductedusingKruskal–Wallisanalysisofvariance(levelofsignificancesetatq<0.1)followedbyGamesHowellposthoctesting (levelofsignificancedefinedasp<0.05).Intotal,88massfeatures weredeemed significant across thedifferent treatment groups (TablesA2.1andA2.2inSupplementary),however,only13metabo- liteswereannotatedwithindividualputativeidentitiesand are presentedinTable3.TheC-EPAgrouphadthelargestnumberof41 affectedmetabolites,e.g.,fourdifferentadductsofdihydroxynorvi- taminD3(hydroxypropyl) withameanfold changeofbetween 6.84and11.34inadditiontoxeniasterol,phosphatidylinositol(PI 42:5)and cholesterol sulphatewithmeanfold changesof 2.26, 1.96,and−1.52,respectively.TheCMwasthesecondmostaffected groupwith31 individualmetabolitesthat changedsignificantly comparedtothecontrol.Fourfeatures,allputativelyannotatedas differentadductformsofdihydroxynorvitaminD3,hadameanfold changeofbetween10.11and15.39andthephosphatidylglycerol

(P-33:2)andphosphatidylserine(P-32:1)levelswerebothsignifi- cantlyreducedintheCM.IntheARAexposedgroup,27metabolites wereaffectedoverall,andoftheindividualputativelyannotated metabolites, three metabolites were elevated compared tothe control; twophosphatidylglycerols (PG 22:4)withfold changes of 13.44 and 13.42, and phosphatidylethanolamine (PE 42:10) withafoldchangeof2.60.Eighteenmetaboliteswereaffectedin C-ARAgroup,e.g.,phosphatidylethanolamine(PE42:10)andphos- phatidylethanolamine(PE42:9)werebothelevatedcomparedto thecontrolwithafoldchangeof2.43and1.52,respectively.Inthe EPAgroup,6metaboliteschangedsignificantlycomparedtothe control,howevernoputativeidentificationscouldbeascribedto these.BothEPAandARAeffectedtheCMlipidomicresponseincells, howevercellsexposedtoallnutrientsshowedgloballyastronger protectiveeffectagainstthelipidomicdisturbancescausedbythe CM.

3.2.2. NMRspectroscopymetabolomics

ThePCAandPLSDAanalysesofthewater-solublemetabolites showedalargereffectgloballythanattheindividualmetabolite level.ThevariationsintheentiremetabolicfingerprintsfromNMR representedbyPCAandPLSscoresplotsshowedshiftsin some ofthegroupscomparedtothecontrol.TheCMgroupwassepa- ratedfromthecontrolalongPC2(Fig.3A),thusindicatingmetabolic shiftsduetotheexposure.Theadditionofthetwofattyacidsin combinationwiththeCMtreatment(C-EPAandC-ARA)shiftedthe metabolicstatus observedbyNMRtowardsthecontrolsamples comparedtotheCMgroupalone(Fig.3BandC).Ascanbeobserved, thedirectionofchangeinthescoresplotswasthesameforthe C-EPAand C-ARA,indicating similarshifts in the NMR observ- ablemetabolome,butwithdifferentmagnitudes.Thealleviating effectwasmoreexpressedforARAwheretheC-ARAgroupwas betterseparatedfromtheCMgroup(Fig.3C).FortheEPAandC- EPAgroups,bothclusteredbetweenthecontrolandtheCMgroup (Fig.3B).Noapparentclusteringwasobservedtosupportanyalle- viatingeffectfromtheC-␣TandC-␥Tgroups(Fig.3DandE).The controlandbothC-AllgroupswereseparatedfromtheCMgroup along PC2,but therewasnoobservabledifferences betweenC- AllhighandC-Alllowgroups (Fig.3F).Thelow andhighlevels oftheCM incombination withallnutrientsreduced thetoxic- itymorethanindividualfattyacidsexposedincombinationwith CM,byshiftingthemetabolicresponsetoalmostoverlappingthe metabolicresponseobservedinthecontroltreatment.Attheindi- vidualmetabolitelevel,statisticalanalysiswithaKruskal–Wallis testfollowedbytheGamesHowellposthoctestresultedinnosig- nificantmetabolites(datanotpresented)whichmaybereflective ofthesmallsamplesizecombinedwiththelowerstatisticalpower inherentinallnon-parametrictests.Cellsexposedtoallnutrients incombinationwiththeCMshowedastrongeramelioratingeffect thanindividualfattyacidsexposedincombinationwithCM.

3.3. RT-qPCR

PLS interaction evaluation was performed on candidate biomarkers that belong to well-known toxicologicalpathways;

cytochromeP4501A(CYP1A,Fig.4A),CYP3A(Fig.4B),vitellogenin (VTG,Fig.4G),andfourlipidmetabolismcandidatemarkers;fatty acid-bindingprotein4(FABP4,Fig.4F);peroxisomeproliferator- activatedreceptors(PPARa,Fig.4C),carnitinepalmitoyltransferase 2 (CPT2, Fig. 4D)and peroxisomal acyl-coenzyme A oxidase 1 (ACOX1,Fig.4E).PLSmodelswerebasedonthemeannormalized expressionlevels(MNE)obtainedfromcellsexposedtonutrients andcontaminantmixtureusingafactorialdesigninordertodeter- minepossiblecontaminant-nutrientinteractions.

InallPLSmodels,exceptforACOX1,theCMhadthestrongest effectonthetranscriptionallevelscomparedtotheothertreat-

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Fig.2. PCAscoresplotsfromlipidomicsdataofsalmonhepatocytestreatedwithdifferentnutrientscombinedwithacontaminantmixture(CM)versuscontrolDMSO andtheCMalone.AEffectofeicosapentaenoicacid(EPA)comparedtotheC-EPA,Barachidonicacid(ARA)comparedtoC-ARA,C␣-tocopherol(␣T)comparedtoC-␣T,D

␥-tocopherol(␥T)comparedtoC-␥T.EThedoserelatedeffectonthelipidomicproﬁlewhenalowdose(C-Alllow)orahighdose(C-Allhigh)ofcombinationofCMandall nutrientswereused.

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Fig.3. MultivariateanalysesofNMRmetabolicﬁngerprintsforAtlanticsalmonhepatocyteresponsestoexposureshownbyAPCAanalysisoftheresponseofthecontaminant mixture(CM)exposureversuscontrol(DMSO).BTheeffectsofeicosapentaenoicacid(EPA)andC-EPA,Carachidonicacid(ARA)andC-ARA,D␣-tocopherol(␣T)andC-␣T, E␥-tocopherol(␥T)andC-␥T,andFC-AlllowandC-Allhigh(loworhighconcentrationoftheCMandallnutrients)comparedtotheeffectofCM.

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Fig.4. SimplifiedscaledandcenteredPLSregressioncoefficientmodelsfordifferenttranscriptsmeasuredinprimaryAtlanticsalmonhepatocytesexposedtoeicosapentaenoic acid(EPA),arachidonicacid(ARA),␣-tocopherol(␣T),␥-tocopherol(␥T)andcontaminantmixture(CM)usingmeannormalizedexpression(MNE)andafactorialdesign (N=5).CMcontained100␮Mofbenzo(a)pyreneandphenanthreneand1␮Mofchlorpyrifosandendosulfan.Thecombinedeffectsidentifiedwithcontourplotanalysis likeadditivity,synergismorantagonismarepresentedinthedifferentPLSregressioncoefficientmodels(confidencelevel=0.95).SignificantPLSregressioncoefficientsare indicatedwitha*(p<0.05).ACytochromeP4501A(CYP1A),R²=0.90,Q²=0.76.BCytochromeP4503A(CYP3A),R²=0.84,Q²=0.62.CPeroxisomeproliferator-activated receptors(PPAR␣),R²=0.76,Q²=0.53.DCarnitinepalmitoyltransferase2(CPT2),R²=0.78,Q²=0.52.EPeroxisomalacyl-coenzymeAoxidase1(ACOX1),R²=0.79,Q²=0.55.

FFattyacid-bindingprotein4(FABP4),R²=0.74,Q²=0.47.GVitellogenin(VTG),R²=0.77,Q²=0.58.ThecompletePLSregressionmodelequationsaredescribedinthe supplementaryA1.Onlyimportantlinerandinteractiontermsrepresentingcontaminant-nutrientandnutrient–nutrientinteractionswerepresentedinthefigures.The regressioncoefficientsreflectingtheimpactofthefactorsonthePLSmodel.

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Table3

TableofputativelyannotatedmassfeaturessignificantlydifferentbetweenhepatocytestreatedwithcontrolDMSOandthosetreatedwitheitheracontaminantmixture (CM),eicosapentaenoicacid(EPA),arachidonicacid(ARA),orCMandARA(C-ARA)orCMandEPA(C-EPA).Foldchangesinboldindicateresultsthatweresignificantly differentwithrespecttothecontrol.Massfeaturesarerepresentedmorethanonceiftheyweresignificantlydifferentwithrespecttothecontrolformorethanoneclass.

AnalysiswasconductedusingKruskalWallisanalysisofvariance(levelofsignificancesetatq<0.1)followedbyGamesHowellposthoctesting(levelofsignificancedefined asp<0.05).

Meanfoldchange(comparedtothecontrol)

m/z EPA C-EPA CM ARA C-ARA Empiricalformula PutativeAnnotation Adducts

461.3636 1.39 6.91 10.11 1.26 8.95 C29H50O4 (Hydroxypropyl)DihydroxynorvitaminD3 [M−H]- 462.3671 1.38 6.84 10.12 1.29 8.86 C29H50O4 (Hydroxypropyl)DihydroxynorvitaminD3 [M−H]-C13 521.3846 1.51 11.24 15.26 1.08 12.35 C29H50O4 (Hydroxypropyl)DihydroxynorvitaminD3 [M+Hac-H]- 522.3882 1.51 11.34 15.39 1.04 12.40 C29H50O4 (Hydroxypropyl)dihydroxynorvitaminD3 [M+Hac-H]-C13

559.3044 – – -1.89 13.42 7.40 C28H49O9P PG(22:4) [M−H]-

560.3077 – – -1.61 13.44 7.68 C28H49O9P PG(22:4) [M−H]-

810.5082 – – 1.01 2.60 2.43 C47H74NO8P PE(42:10) [M−H]-

812.5241 – – 1.02 1.66 1.52 C47H76NO8P PE(42:9) [M−H]-

501.28166 −1.53 −1.52 1.2 – – C27H46O4S Cholesterolsulphate [M+Cl]-

551.39556 1.32 2.26 2.36 – – C30H52O5 XeniasterolB [M+Hac-H]-

775.51214 −1.04 −1.18 −1.27 – – C39H73O9P PG(P-33:2) [M+Hac-H]-

776.50978 −1.05 −1.18 −1.26 – – C38H72NO9P PS(P-32:1) [M+Hac-H]-

939.59786 2.25 1.96 1.20 – – C51H89O13P PI(42:5) [M−H]-

ments.ThePLSanalysisofthetranscriptsCYP1AandPPAR␣showed thatARAwastheonlynutrient thathad asigniﬁcantcontribu- tiontothetranscriptionlevels;havinganadditivecombinedeffect togetherwiththeCM.ForCYP1A(Fig.4A),theadditiveresponse (p=0.044)causedincreasedtranscriptionwhilethePPAR␣addi- tivity(p=0.002)gaveareducedtranscription(Fig.4C).Theother nutrients,EPA,␣Tand␥Tdidnothaveanysigniﬁcantcontribution onCYP1AandPPARatranscription.

AsynergisticinteractioneffectbetweenARAandtheCMwas predictedbythePLSmodelsforCYP3A(Fig.4B,p=0.013)andCPT2 (Fig.4D,p=0.001), resultinginincreasedtranscription. ThePLS modelforACOX1(Fig.4E)showedasimilarsynergisticinteraction effectbetweenyTandtheCM(p=0.0008).ForFABP4(Fig.4F),the negativeinteractiontermforC-␣Tpredictedanantagonisticeffect between␣TandtheCM(p=0.044),reducingthetranscriptionlev- els.Lastly,theVTGPLSmodel(Fig.4G)revealedanantagonistic effectbetweenEPAandtheCM(p=0.014).Basedontheeffects ontoxicologicalimportantmarkers,EPAand␣-tocopherolseemed morebeneﬁcialthanARAand␥-Tinpreventingtheadverseeffects inducedbytheCM.

4. Discussion

Today’sAtlanticsalmonfeedwithhighinclusionlevelsofplant ingredientscontainareducedamountofEPA,␣Tandcontaminants suchaspersistentorganicpollutants(PCBs,dioxinsetc.)andheavy metals(Cd,Hg).Atthesametime,higherlevelsofplant-derived contaminantsarefoundinplantbasedfeedslikePAHandpesti- cides,andnutrientssuchas␥TandARAderivedfromlinoleicacid (LA).Thelevelsoffeednutrientsandcontaminantswillvarywith whichtypeoffeedingredientsusedandactualfeedcomposition ofthefeedingredients[5,25,28,34,62,77,84,85].Toassurethatthe feedcontainsufﬁcientlevelsoftocopherol,synthetic␣Tissupple- mentedasa-tocopherylacetatetoAtlanticsalmonfeeds[28].These tocopherolsandfattyacidsEPAandARAhavedifferentkinetics andbiologicalrolesinculturedsalmon[2,28,60],withthepossi- bilitytoaffectthetoxicityofcontaminantsdifferently.Thisstudy showthatthenutrientsEPA,ARAand␣T,␥Taffect,individually andtogether,thetoxicityofaCMcomposedoftwoPAHsandtwo pesticides.Theadditionofnutrientstothecontaminantmix(CM) signiﬁcantlyincreasedtheviabilityofAtlanticsalmonhepatocyte cellscompared toexposuretoCMalone.PAHs andparticularly lighterPAHs,likephenanthrene,areabletoreducecellviability [65,79]inteleostinvitrosystems,used atthesameconcentra- tionrangeasinthisstudy[79].Ofthepesticides,onlyendosulfan

haspreviously beenshown to negatively affectcell viabilityof Atlanticsalmonhepatocytes,howeverthereductionwasobserved athigher exposureconcentration[79] thanusedin thepresent study.Therefore,itismostlikelythatitisthePAHsthataredriv- ingthenegativeeffectobservedincellviability.Thecytotoxicity ofPAHshasbeensuggestedtobecausedbytheirabilitytoembed inanddisruptcellularmembranes[65]byincreasingtheirﬂuidity [46].Thecytotoxicitydataobtainedfromthecurrentcontaminant- nutrientinteractionevaluationsuggestthatthenegativeeffectsof theCMmightbecounteractedbythenutrients.Oftheindividual nutrients,␥Thadthestrongestpositiveeffectonthecellviabil- ity.EvaluationoftheC-␥Tinteractiontermshowedthat␥Tseems toamelioratethetoxicityoftheCM.␣Tisknowntobethemost importantantioxidantthatresideswithincellmembranes,protect- ingthelipidsfromperoxidation[1],andmaintainingtheintegrity ofmembranesbypreventingtheinitiationofcelllysis[28].Mam- malianendothelialcellsenrichedwiththen-6PUFALAshoweda weakenedendothelialbarrierforPCBowingtoadiscrepancyincel- lularstatusofantioxidant/oxidativestress[101].Inaddition,PCB causedanincreaseduptakeofLA.However,thedysfunctionofthe endothelialbarriercausedbythePCBwascompletelyrestoredby

␣Towingtoreducedoxidativestressandproductionofinﬂam- matorycytokines[101].In astudywhere ratswereexposedto iron-dextranincombinationwith␣Tor␥T,itwasshownthat␥T similarto␣Tisabletoinhibitlipidoxidation[29].Thissuggests that␥Ts,bypreventinglipidoxidation,areabletoamelioratethe effectonthecellviabilitycausedbytheCM.

Togeneratehypotheses aboutthepotentialmodesof action ofthestudiedCM,nutrientsandnutrient–contaminantcombina- tions,lipidomicandmetabolomicprofilingwereemployed.ARA wasthenutrient that showedthe greatestglobal effectonthe lipidome, having a stronger effect than the CM and EPA. Both forms of vitamin E had the least effect on the lipidomic and metabolomic responses,eventhough previousresearchsuggest vitaminEhasloweffectonhepatocytescellviability[50].Intotal, 13putativeannotatedmetabolitesweresignificantlyaffected.The C-EPAcombinationinducedthemostsignificantperturbedmass feature changes followed by the CM, ARA and C-ARA.EPA did notinduceanysignificantmassfeaturechanges.BothC-EPAand theCMsignificantlyalteredfourmassfeatures,identifiedasthe vitaminDanalogue19nor-2␣-(3-hydroxypropyl)-1␣,25(OH)2D3 (dihydroxynorvitaminD3).Thelipidomicdatashowedthat19nor- 2␣-(3-hydroxypropyl)-1␣,25(OH)2D3 was significantincreased 10.11–15.39foldintheCMgroupand6.84–11.34foldintheC-EPA group compared to the control. 19 nor-2␣-(3-hydroxypropyl)-

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1␣,25(OH)2D3isananalogoftheactivevitaminDform1␣,25(OH) 2D3[9].Thisconﬁrmspreviousresultswherebenzo(a)pyreneand asimilarcontaminantmixtureofPAHandpesticidesinducedan effectonthesteroidsynthesispathwayandvitaminD3metabolism inprimaryAtlanticsalmonhepatocytes[79].The19nor-2␣-(3- hydroxypropyl)-1␣,25(OH)2D3hasbeenshowntobeeffectivein inhibitingcellproliferationinHepG2cells[9]andinpreventingcell growthinMCF-7cells,andBxPC-3tumordevelopmentinmouse [8].Theincreasedsynthesisofthe19nor-2␣-(3-hydroxypropyl)- 1␣,25(OH) 2D3 in exposed Atlantic salmon hepatocytes might thereforepreventpotentialgenotoxiceffectsinducedbyPAHs,like benzo(a)pyren[16,86,35].

Perturbed lipid and cholesterol homeostasis by contami- nantshasbeendemonstratedinnumerousmammalian[19]and teleostean[19,79]studies.Contaminantsaffectingthelipidcom- position of membranes can signiﬁcantly inﬂuence membrane transport,bioenergeticsandcellsignalling,aswellasmembrane function and integrity [19]. ARA and C-ARA treatments gave the strongest effect oncell membrane lipids in exposed cells;

phosphatidylglycerol(PG22:4)was13-foldupregulated inARA exposedcellandphosphatidylethanolamine,(PE42:10)was2.6- and2.43-fold upregulatedin theARAandC-ARAexposed cells, respectively.Thelipidphosphatidylglycerolissynthesizedinthe mitochondriaand isusedfor production ofcardiolipin,a mito- chondrialinnermembrane lipidstabilizingtheelectrontransfer complex[51,88,87].Phosphatidylethanolamine,phosphatidylser- ineand phosphatidylinositol,key structurallipidsin eukaryotic membranes[88]andparticularlyenrichedintheinnercytoplas- micleaﬂet[38],wereallfoundtobeaffectedinthis study.The physicalpropertiesofthemembranedependsonthen-3andn-6 PUFAacylchainswhichaffectthelateralorganization,curvature etc.[69].Bilayersbecomesmoredeformableanddisorderedwith ARAthanthosecontainingmoredoublebondslikeDHA.InPAH andPCBcontaminatedmussels,anincreaseinthenon-polar/polar lipidratiointhemusselshasbeenobserved,suggestingthatthe contaminantsaffectedthelipidhomoestasisbyreducingthecon- versionofstoragelipidtomembranelipids[19].Asimilarreasoning canbeusedtoexplainthedownregulationofthemembranelipids phosphatidylglycerolandphosphatidylserinebytheCMtreatment inthepresentstudy.Bothexcessiveanddeﬁcientamountoffatty acidscaninducemitochondrialandER-stressresponses,produc- tionoffreeradicalsandsteatosisinhepatocytes[11,24,99].Only hepatocytestreatedwithC-EPAshowedaneffectonthecholesterol analoguecholesterolsulphatethatwas−1.52-folddownregulated.

Thehomeostasisofcholesterolistightlyregulatedinanimals,i.e., theamountabsorbedviathediet,producedviadenovocholesterol synthesis,andexcretedasbilesaltsorbiliarycholesterol[10,37,48].

Inmammals,cholesterolsulphateisanimportantcomponentof cellmembraneswhereitfunctionasastabilizingagentinasimi- larwayascholesterol[73].ThissuggeststhattheC-EPAtreatment reducedthestabilityofthecellmembrane,thoughthiseffectwas minor.

To furtheridentifynutrient–contaminant interactions, genes encoding selected targeted biotransformation enzymes, lipid metabolismandendocrineeffectmarkerswereanalysedwithqPCR andPLS.Similartothelipidomicsdata,ARAgavethestrongesteffect ongenetranscription.CellsexposedtotheCMincombinationwith ARAshowedanadditiveandsynergisticinductionoftheCYP1A andCYP3Atranscripts,respectively.Theothernutrients,EPA,␣T and␥TdidnotsigniﬁcantlyaltertheCM-inducedresponse.Sev- eralCYPtranscriptslikeCYP1AandCYP3Aarenotonlyinvolvedin biotransformationofcontaminantslikebenzo(a)pyreneandchlor- pyrifos[79]butalsoplayacriticalroleindegradationandsynthesis ofendogenouscompoundslikelipids[6].SeveralCYPenzymeslike CYP1AandCYP3Ahavepreviouslybeenfoundtobeinvolvedin theCYP-dependentmetabolising of ARA orLA into eicosanoids

inmammalsbothinvivoandinvitro[6,13,41]CYP1A,whichhas beenseenastheprimarilybiotransformationenzymeandactiva- toroftoxicandcarcinogeniccontaminants[56],isimportantin thebioactivationof benzo(a)pyrene,into theultimatecarcino- gen,thediolepoxide[40].Inmammals,CYP1AandCYP3Ahave been shown to bioactivate chlorpyrifos toa chlorpyrifos–oxon, whichisapotentanticholinesterase[80],andCYP3Atoconvert␤- endosulfantoendoulfansulphatewhichisastoxicasthemother compound[44].CYP1Abiotransformationofpoorlymetabolised inducerscancauseproductionofROS,lipidperoxidationandulti- matelycanchangethemembranefunction[19].Thus,theadditive andsynergisticincreasedexpressionofCYP1AandCYP3AbyARA canincreasethetoxicityoftheCMwhenexposedincombination.

Peroxisomeproliferator-activatedreceptor␣(PPAR␣)regulates the expression of several target genes linked to mitochondrial and peroxisomal ␤-oxidationof lipids[61].ARA exposuregave anadditiveeffectontheCMinduceddownregulationofPPAR␣. Though,despitethedownregulationofPPAR␣,theC-ARAcaused a weaksynergistic increase of carnitine palmitoyltransferase 2 (CPT2).TheCPT2transcriptisaninnermitochondrialenzymethat takespartintheoxidising oflong-chainfattyacids[21],andis usedasamarkerformitochondrial␤-oxidation.␥Tcontributed synergisticly totheCM inducedincreaseof thetranscriptionof peroxisomal ␤-oxidationenzyme peroxisomal acyl-coenzymeA oxidase1(ACOX1).ACOX1istheﬁrstenzymeinperoxisomal␤- oxidation and is in charge of thedesaturation of acyl-CoAs to 2-trans-enoyl-CoAs[21].Thisﬁndingisincontrasttoastudywith ratswherethemitochondriawerefoundtohavethemainrolein

␤-oxidationof␣T[54].Thesynergisticeffectoftheplantnutrients ARAand␥TontheCMeffectonmitochondrialandperoxisomal

␤-oxidationmightcausetheAtlanticsalmontoloseweight.Pes- ticideslikeendosulfanhavepreviouslybeenshowntocauselipid metabolismdisturbancessuchassteatosisinAtlanticsalmon,both inexposedhepatocytes[43]andininvivostudies[23].However, anunbalanceddietcanproduceasimilareffectinAtlanticsalmon feddietswithhighinclusionlevelsofplantingredientsinaddi- tion tolow levelsof EPAand DHA [45]. Thefatty acidbinding proteins(FABPs)areafamilyofproteinsinvolvedinlipidfluxin cells[71].Inhumanhepatocytesandtrophoblasts,increasedlipid accumulationhasbeenlinkedtoelevatedtranscriptionofFABP4 [17,67,96].AnorthologuetomammalianFABP4thatmayrepre- senttheadiposetissuetypeFABP(h6FABPorFABP11)infish[83], wasinducedbybothchlorpyrifos[58,79]andendosulfan[79]in previousAtlanticsalmonhepatocytesstudies.Inthepresentstudy, FABP4wassignificantlyupregulatedincells exposedtotheCM.

Incells co-treatedwith␣T,however,theFABP4transcriptlevel wasdownregulated, possiblydue toanantagonistic interaction betweentheCMand theantioxidant.A similardownregulation hasbeenobservedinAtlanticsalmonhepatocytesco-treatedwith chlorpyrifosand␣T[58].Thepresentﬁndingthusconﬁrmsthat

␣TcanreduceFABP4 transcriptionand possibleprotectagainst chemical-inducedsteatosisinAtlanticsalmon.

Further,anantagonisticeffectwasalsodetectedfortheVTG transcriptwhereEPAincombinationwiththeCMinducedaweak antagonisticeffectontheVTGexpressionlevel.Theeggyolkpre- cursorproteinVTGisgenerallyproducedinthefemalelivercells byestrogenicstimulation,originatingfromthedevelopingovarian follicles[18,30].AllfourcontaminantsintheCMhavetheabilityto interferewiththeestrogenreceptor(ER)pathwayinﬁsh[43,27,79].

InaccordancewithSøftelandetal.[79],exposuretotheCMsignif- icantlyincreasedVTGtranscription,however,incombinationwith EPA,VTGupregulationwassubstantiallyreducedduetoanantag- onisticinteraction.TheseresultssuggestthatEPAcanameliorate thenegativeeffectinducedbytheCMontheendocrinesystem.

ThemechanismbehindEPA’samelioratingeffectishowevernot known.