Thyroid dysfunction in sea bass (Dicentrarchus labrax): Underlying mechanisms and effects of polychlorinated biphenyls on thyroid hormone physiology and metabolism

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ContentslistsavailableatScienceDirect

Aquatic

Toxicology

j o ur na l 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 / a q u a t o x

Thyroid

dysfunction

in

sea

bass

(Dicentrarchus

labrax):

Underlying

mechanisms

and

effects

of

polychlorinated

biphenyls

on

thyroid

hormone

physiology

and

metabolism

Joseph

G. Schnitzler

a,∗

_,

_Niko

_Celis

b

_,

_Peter

_H.M.

_Klaren

c

_,

_Ronny

_Blust

b

_,

_Alin

_C.

_Dirtu

d,e

_,

Adrian

Covaci

b,d

_{, Krishna}

_Das

a

a_Mare_Centre,_Laboratory_for_Oceanology_B6c,_Liège_University,_Liège,_Belgium

b_Laboratory_of_{Ecophysiology,}_Biochemistry_and_Toxicology,_Department_of_Biology_University_of_Antwerp,_B-2020_Antwerp,_Belgium c_Radboud_Univ_Nijmegen,_Inst_Water_Wetland_Res,_Fac_Sci,_Dept_Anim_Physiol,_{Heyendaalseweg}_135,_NL-6525_AJ_Nijmegen,_The_Netherlands d_{Toxicological}_Center,_University_of_Antwerp,_B-2610_{Wilrijk-Antwerp,}_Belgium

e_Department_of_Chemistry,_“Al._I._Cuza”_University_of_Iasi,₇₀₀₅₀₆_Iasi,_Romania

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received1June2011

Receivedinrevisedform26July2011 Accepted29July2011 Keywords: Dicentrarchuslabrax Polychlorinatedbiphenyls Thyroidhormones Deiodination Glucuronidation Sulfation Invivoexposure

a

b

s

t

r

a

c

t

ThecurrentstudyexaminestheeffectofsubchronicexposuretoamixtureofAroclorstandardsonthyroid hormonephysiologyandmetabolisminjuvenileseabass.Thecontaminantmixturewasformulatedto reﬂectthepersistentorganicpollutiontowhichtheEuropeanseabasspopulationcouldconceivablybe exposed(0.3,0.6and1.0␮g7PCBspergfoodpellets)andhigher(10␮g7PCBspergfoodpellets).After 120daysofexposure,histomorphometryofthyroidtissue,muscularthyroidhormoneconcentrationand activityofenzymesinvolvedinmetabolismofthyroidhormoneswereassessed.Meanconcentrationsof 8,86,142,214and2279ngg−1_ww₍₇_ICES_PCB_congeners)_were_determined_after₁₂₀_days_exposure.

TheresultsshowthattheeffectsofPCBexposuresonthethyroidsystemaredose-dependent.Exposureto environmentallyrelevantdosesofPCB(0.3–1.0␮g7PCBspergfoodpellets)inducedalargervariability ofthefolliclediameterandstimulatedhepaticT4outerringdeiodinase.Muscularthyroidhormonelevels

werepreservedthankstothePCBinducedchangesinT4dynamics.At10timeshigherconcentrations

(10␮g7PCBspergfoodpellets)animportantdepressionofT3andT4levelscouldbeobservedwhich

areapparentlycausedbydegenerativehistologicalchangesinthethyroidtissue.

1. Introduction

Inarecentﬁeldstudywehaveestablishedcorrelationsbetween

exposuretoorganochlorinecontaminantsandthyroidfunctionin

wildseabassfromEuropeancoasts(Schnitzleretal.,2011a).

Mul-tivariatestatisticalanalysisspeciﬁcallyrevealedtheinvolvement

ofhigherchlorinatedPCBsinthyroiddysfunction.Indeed,ﬁshes

withhigherPCBconcentrationsdisplayedalterationsinmetabolic

pathways,viz.deiodinationandsulfation,thataffectcirculatingand

tissuethyroidhormonelevels(Schnitzleretal.,2011a).

The mechanisms of how endocrine disruptors alter thyroid

function have been extensively investigated but are still not

fully understood. The regulatory pathways involvedin thyroid

∗ Correspondingauthor.

E-mailaddresses:joseph.schnitzler@ulg.ac.be(J.G.Schnitzler),

Niko.Celis@ua.ac.be(N.Celis),p.klaren@science.ru.nl(P.H.M.Klaren),

ronny.blust@ua.ac.be(R.Blust),alindirtu@yahoo.com(A.C.Dirtu),

adrian.covaci@ua.ac.be(A.Covaci),krishna.das@ulg.ac.be(K.Das).

homeostasisarenumerousandcomplex.Asaconsequence

envi-ronmentalchemicalscanactatmanylevelsinthethyroidsystem

(Ishiharaetal.,2003).Thereareatleastthree independent,but

possiblyinteracting,mechanismsthatmayexplaintheabilityof

PCBtoreducecirculatingand tissuelevelsofthyroidhormones.

First, PCBshave beenshowntochangethyroidglandstructure,

possiblydirectlyinterferingwiththyroidglandfunction(Collins

andCapen,1980b)anddisruptingdirectlythehormone

synthe-sisin thethyroidgland(Boasetal.,2006;Brown etal.,2004a;

Ishiharaet al.,2003).PCBsmaydirectlyinterferewiththe

abil-ityofthethyroidglandtosynthesizethyroidhormonesbyaltering

mechanismsinvolvedinactiveaccumulationofiodideand

prote-olysisofthyroglobulin.Second,PCBscantargetthyroidhormone

metabolism.Theymayaffectextrathyroidaliodothyronine

deiodi-nases,enzymesthatcontroltheconversionofthyroidhormones

and arethus essentialin theregulationoflevelsof biologically

active T3 locally and systemically (Ishihara et al.,2003; Zoeller

andTyl,2007).IthasbeenshownthatPCBexposureincreasesbile

ﬂowrateaswellasthebiliaryexcretionofT4(CollinsandCapen,

1980a).PCBexposurealsoinducestheexpressionandactivityof

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thephase-IIenzymesglucuronosyltransferaseandsulfotransferase

thatalsoutilizethyroidhormonesasconjugategroupacceptorsand

increaseT4 conjugation(KlaassenandHood,2001;Visseretal.,

1993).TheseactionsfacilitateT4clearancebyhepaticmetabolism,

reducingthebiologicalhalf-lifeofT4.Finally,PCBscompetitively

bindtothyroidhormonebindingproteinsliketransthyretin(TTR)

inblood(Boasetal.,2006;Ishiharaetal.,2003;Wadeetal.,2002)

andcanpotentiallydisplacethyroidhormonesfromtheircarrier

molecules.Moreover,thesemayinteracttoproducesummative

effects.Besidesthesedirecteffects,indirecteffectsviadisruption

ofthyroidhormonereceptorsandaccessoryproteinsthatdirectly

controlthegeneexpressionthroughthyroidhormoneresponsive

elementscanalsointerferewiththethyroidsystem(Blantonand

Specker,2007;Ishiharaetal.,2003).

Disruptionofthyroidfunctioncanhavesevereconsequences

asthyroidhormonesplayanimportantroleinthemaintenanceof

anormalphysiologicalstatusinvertebrates.Inadultﬁsh,thyroid

hormonesareofprimaryimportanceintheregulationofsuch

fun-damentalphysiologicalprocessesasgrowth,nutrientutilization,

andreproduction.Fishgrowfasterandarehealthierwhenthyroid

hormonelevelsareadequate(Poweretal.,2001;Yamano,2005),

providinganeconomicrationaletostudythyroiddisruptorsina

ﬁsheryandaquaculturecontext.Thisexplainsourchoiceofthe

testspecies,Europeanseabass(Dicentrarchuslabrax),asitisan

importantcommercialspecies,toppredatorofasimplefoodweb,

commonlyfoundinEuropeancoastalwaters,andwithawell

doc-umentedbiology(Loizeauetal.,2001;PickettandPawson,1994).

Polychlorinatedbiphenylshavebeenshowntoalterthyroid

hor-monelevelsinexperimentalanimals,includingﬁsh(Brouweretal.,

1989/7;Coimbra and Reis-Henriques,2007; Collins and Capen, 1980a,b;Fowlesetal.,1997;Hallgren,2001,2002;Iwanowiczet

al.,2009).Moststudiesonﬁshthyroidologyhaveinvolvedambient

concentrations ofxenobiotics deliveredatsublethal,

concentra-tionsthat, however, are still higher than those encountered in

theﬁeld(Blanton andSpecker, 2007;Brownet al.,2004a).Our

studyaimedtogainanintegratedinsightintotheeffectsofa

4-monthinvivoexposuretovariousenvironmentallyrelevantdoses

ofcommercialmixtures of polychlorinatedbiphenyls (PCBs)on

thethyroidsystemofD.labrax.Severalendpointswereanalyzed

simultaneously:thyroidglandhistology,hepatic5-deiodination

(orouterringdeiodination,ORD)activitiesthatconvertthe

thy-roidprohormoneT4 tothebioactivehormoneT3,andmuscular

T4andT3levels.Inaddition,twobiochemicalpathways,i.e.

sulfa-tionandglucuronidation,involvedinthyroidhormonemetabolism

andphase-2responsetotoxicants,wereassayed.Thisapproach

allowedustodetermineunderlyingmechanismsanddose

depen-dencyoftheeffectsofthesepollutantsonthethyroidsystemof

theseﬁsh,andtoexaminetheconsequencesofapotential

disrup-tionofthethyroidsystemongrowthperformanceandcondition

factorinthesecommerciallyimportantﬁshspecies.

2. Methods

2.1. Foodpreparation

Thecontaminantmixturewasformulatedtoreﬂectthe

persis-tentorganicpollutiontowhichtheEuropeanseabasspopulation

couldconceivablybeexposed.Ourpreviousﬁeldstudyonseabass

fromEuropeancoastalregionsrevealedPCBpatternsdominated

byalargecontributionfromthehepta-,hexa-and

pentachlori-natedPCBs(Schnitzleretal.,2011b).Thesecongenersarethemost

abundantduetotheirwidespreaduseincommercialmixturessuch

asAroclor1254and1260.Wethereforedecidedtoworkwitha

1:1mixtureofAroclor1254and1260.Loizeauetal.(2001)caught

suprabenthicspecies(gobies,shrimpsandmysidaceans)thatare

potentialpreyofseabassintheSeineestuary(Loizeau,2001).Based

onthereportedthePCBconcentrations(ondryweightbasiswith

standarddeviation),wecalculatedtheofthe7markerPCB

con-geners(IUPACnos.28,52,101,118,138,153,180)presentedin

Table1.Wecontaminatedthefoodwitha1:1mixtureofAroclor

1254and1260toreachconcentrationsofthe7ICESmarker

con-genersrangingfrom300to1000ngg−1 food,toreﬂectbestthe

observedconcentrationincommonseabassprey.

Aroclormixtureswereaddedto100mLhexaneand100-g

por-tionsofcommercialﬁshfood(T-2PClassic.Trouw.France)intoa

1000mLroundbottomﬂask.Themixturewasslowlystirredby

arotaryevaporator(waterbathat60◦C,refrigerationat5◦Cand

pressureat875Pa)tilldryness(ca.1h).Theresultingfoodspiked

withchemicalswasthenthoroughlydriedat40◦Covernight.The

foodpelletskepttheirinitialformandconsistency.Controlfood

waspreparedinthesamemannerasidefromaddingthetest

mix-ture.

The concentrations for the 7 ICES PCB congeners in

pel-lets designed for the ﬁve different exposure conditions were

measuredasdescribed below, andtheobtainedresultsare: (1)

27ngg−1 (assignedlabel: Control), (2) 329ngg−1 (assigned the

nominal valueof 300ngg−1dw),(3) 629ngg−1 (assigned value

600ngg−1dw), (4) 1021ngg−1 (assigned value 1000ngg−1dw)

and(5)11,395ngg−1 (assignedvalue10,000ngg−1dw).The

cor-relationfactor betweennominaland effectiveconcentrations is

0.9999(Table1).

2.2. Husbandry

ExperimentaltrialswereconductedintheBiologyDepartment

ofAntwerpUniversity,Belgium.Seventy-ﬁvejuvenileseabass(D.

labrax,L.)wereobtainedfromacommercialﬁshfarm(Ecloserie

marinedeGravelines,Gravelines,France).Theirbodymassranged

from7to20g(mean13.2±2.8g).Fishwerehousedin200-Ltanks

withanaturalphotoperiod.Thewatertemperaturewasmaintained

at15◦Cduringtheexperiment.Wateraerationwassettomaintain

100%airsaturation.Thewaterwascontinuouslyﬁlteredthrough

mechanical,charcoalandextensivebiologicalﬁltersbeforebeing

recycled.

Fish were randomly assigned to a control group and four

treatmentgroups (group size n=15in each case) thatreceived

contaminated foodat0.3, 0.6,1.0and10.0␮gg−1 (of[7ICES

PCBs] per gof food pellets),respectively. Fish were fedspiked

foodfor120days.Thedailyfeedingrationwas2.0%ofthemean

bodymassoftheﬁsh,adjustedaftereachsamplingperiodbased

onmeanweightofthesub-sampleﬁshthatweresacriﬁced.Feed

was presented by sprinkling at the surface of the water and

wasgenerallycompletelyconsumedbyeachgroupofﬁshwithin

1min.Fiveﬁshweresampledfromeachtankondays40,80and

120.Fish werealwayssampled24hafterthepreviousfeeding.

Weightand lengthweremeasuredandthespeciﬁcgrowthrate

(SGR=100%×(lnﬁnal weight−lninitialweight)/totaldays)and

conditionfactor(CF=weight×100/length3₎_were_calculated._The

subpharyngealareawasremovedandimmersedinformalin

ﬁx-ative (VWRInternationalBVBA). Approximately10gof skeletal

musclewasexcisedcaudallyofthehead,dorsaltothelateralline

andanteriortothedorsalﬁn.Muscleandliversampleswerefrozen

immediatelyondryiceandstoredat−80◦_C_until_analysis.

2.3. Organiccontaminantanalysis

2.3.1. Standardsandreagents

AllindividualPCBandpesticidestandardswereobtainedfrom

Dr. EhrenstorferLaboratories GmbH(Augsburg,Germany).

Ace-tone, n-hexane (Hex), dichloromethane, and isooctane were of

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Table1

Contaminationlevelsforthe7tracerPCBcongenersincurrentpreyofD.labraxandinartiﬁciallyArochlor1254and1260contaminatedfood.(Accordingto:Loizeau,V., Abarnou,A.,Ménesguen,A.,2001.ASteady-StateModelofPCBBioaccumulationintheSeaBass(Dicentrarchuslabrax)FoodWebfromtheSeineEstuary,FranceEstuaries Vil.24,No6B,p1074–1087.). 28 52 101 118 153 138 180 sum7PCB Preys N.integer 12.5±1.4 40.2±4.2 65.1±6.6 53.3±5.4 119.6±12.0 94.9±10.0 59.0±6.0 444.6±35.1 P.microps 9.3±1.0 36.5±3.3 74.6±7.2 71.5±6.9 146.5±15.0 121.5±12.8 44.0±4.6 503.9±48.3 P.longirostris 5.6±0.6 29.2±3.1 23.2±2.0 52.6±5.4 96.4±10.0 75.2±8.1 51.2±5.3 333.4±31.3 C.crangon 8.4±0.5 31.2±3.3 26.5±2.1 59.7±6.1 156.4±16.0 131.5±13.6 81.9±9.0 495.6±55.8 D.labrax 10.3±1.5 44.1±4.8 126.5±13.0 144.8±15.0 338.8±35.0 298.7±27.1 131.3±12.7 2397.7±502,4 Food Control 5.7±2.4 10.6±3.0 4.7±1.2 1.4±0.5 2.5±0.5 1.9±0.3 0.3±0.1 27.1±3.5 0.3␮gg−1 _5.7_±_0.1 _29.8_±_0.7 _55.6_±_0.8 _36.2_±_0.9 _87.3_±_3.5 _73.0_±_2.6 _41.2_±_1.6 _328.8_±_27.5 0.6␮gg−1 5.9±0.6 52.0±0.1 105.0±4.0 74.9±1.6 170.4±4.5 139.4±4.4 81.2±3.3 628.7±54.7 1.0␮gg−1 10.0±2.1 79.9±12.0 167.2±18.9 121.5±12.0 278.6±30.0 226.7±23.4 136.9±17.4 1020.8±89.5 10␮gg−1 _82.3_±_5.6 _825.9_±_110.7 _1907.6_±_219.1 _1342.7_±_141.2 _3106.4_±_312.3 _2579.8_±_263.4 _1549.9_±_1023.9 _11394.6_±_1023.9

basic aluminium oxide, and silica gel (Merck) were used after pre-washingwithHexandheatingovernightat120◦C.An acceler-atedSoxhletextractorB-811(Buchi,Switzerland)wasusedforthe extractionoftargetcompoundsfromﬁshtissuesandfeed.

2.3.2. Samplepreparationandanalysis

Afishfilletwasthawedatroomtemperature,andapproximately 1gwaspreciselyweighed,groundwith20ganhydroussodium sul-fateandplacedintoanextractionthimble.Afteradditionofinternal standards(PCB46andPCB143),themixturewasextractedfor2.5h byhotSoxhletwith80mLofhexane/acetone=3:1(v/v).Theextract wassubjectedtocleanupon5gofacidsilica(44%sulfuricacid, w/w).Hexane(20mL)wasusedforthecompleteelutionofPCBs. Thefinaleluatewasconcentratedundernitrogenuntil100␮Land transferredtoaGCvial.

One ␮Lwasinjected inpulsedsplitless modeona Hewlett-Packard6890GCconnectedviadirectinterfacetoaHP5973mass spectrometer.A50m×0.22mm×0.25mm,HT-8capillarycolumn (SGE,Zulte,Belgium)wasusedwithheliumascarriergasata con-stantflowof0.7mL/min.Injectorandinterfacetemperatureswere setat270and300◦C,respectively.Theoventemperatureprogram beganat90◦C,kept1min,andthenincreasedwith15◦C/minto 170◦C,heldfor3min,thenincreasedat4◦C/minto270◦C,heldfor 1min,andwasfurtherincreasedat10◦C/minto290◦Candheld for15min.Themassspectrometerwasoperatedinelectronimpact ionizationmode.Twomostabundantionsweremonitoredforeach levelofchlorinationforPCBs.Methodlimitsofdetection(LOD)for individualPCBcongenersrangedbetween0.1and0.5ngg−1lipid. Recoveriesoftargetcompoundsrangedbetween72%and80%.We investigated38PCBcongeners(IUPACnos.18,31,28,52,49,47,44, 74,95,101,99,87,110,118,105,151,149,146,132,153,138,128, 156,187,183,174,177,171,172,180,170,199,196/203,195,194, 205,206and209)inall75musclesamples,and21metabolites (IUPACnos.4-HO-CB119,4-HO-CB120,3HO-CB118,4HO-CB109, 3HO-CB153, 4HO-CB146, 4HO-CB127, 3HO-CB138, 4HO-CB130, 4HO-CB163,4HO-CB187,4-HO-CB162,4-HO-CB202,4-HO-CB177, 3HO-CB180,4HO-CB172,4HO-CB193,4-HO-CB198,4-HO-CB-199, 4-diHO-CB202and4-HO-CB208)infivemuscleandliversamples. The methodwassubmittedtoregular qualityassuranceand control procedures. Retention times, ion chromatograms, and intensityratios of themonitored ions wereused as identifica-tioncriteria. A deviationof the ion intensityratioswithin 20% ofthemeanvaluesof thecalibrationstandards wasconsidered acceptable.Themethodperformancewasassessedthrough rig-orous internal quality control,which includeda daily check of calibrationcurvesandregularanalysisofproceduralblanksand certifiedreferencematerialCRM350(PCBsinmackereloil).The methodwastestedbyregularparticipationtointerlaboratorytests organizedbytheUSNationalInstituteofStandardsand Technol-ogy(NIST)forthedeterminationofPCBsinbiologicalsamples.The

resultsoftheindividualPCBcongenersdeviatedlessthan20%from thetargetvalues.

2.4. Thyroidparameters 2.4.1. Standardsandreagents

Thyroxine(T4),uridine5-diphosphateglucuronicacid(UDPGA)

and 3-phospho-adenosine-5-phosphosulfate (PAPS) were obtained from Sigma Chemical Co. (St. Louis, MO). Sephadex LH-20waspurchasedfromAmershamPharmaciaBiotechBenelux (Roosendaal, The Netherlands). Outer ring labeled [125_I]T

4

(23.3TBq/mmol) was obtained from Perkin-Elmer Life Science, Inc.(Boston,MA).Allotherchemicalswereanalyticalgradeand obtainedfromcommercialsuppliers.Radiolabelediodothyronines werepuriﬁedshortlybeforeusebySephadexLH-20column chro-matography.Radioactivitywasmeasuredina 1272-Clinigamma gammacounter(LKB/WallacOy,Turku,Finland).Protein concen-trationsweredeterminedusingaCoomassieBrilliantBlueG-250 kit(Bio-Rad,München,Germany)andbovineserumalbuminasa standard.

2.4.2. Muscularthyroidhormonedeterminations

MusculartotalT3andtotalT4concentrationsweremeasured

byradioimmunoassay(SiemensCoat-a-Count,Brussels,Belgium) according to themanufacturer’s instructions. Details of extrac-tion methods and the assay protocol are described elsewhere (Schnitzleretal.,2008).Theaccuracyoftheassaywasdetermined

byblindanalysisofqualitycontrolstandardsathigh,mediumand

lowconcentrations.Thesesampleswereinsertedinduplicateat

thefront,middleandendoftheassayandmeanmeasured

concen-trationswerethencomparedtoactualconcentrationstodetermine

assayreliability.Theassaywasacceptedwithreliabilitybetween

90and110%.Todeterminetheefﬁcacyoftheextractionprocess

inrecoveringthyroidhormonesaswellastransferofsamplesinto

differenttypesoftubes,tworecoverysystemswereused.

Unla-belledthyroidhormonewasaddedtothemincedﬁshmuscleprior

tohomogenization.Thesampleswerethensubjectedtothesame

homogenization,extraction,reconstitutionandthyroidhormone

assayproceduresastheunknownandstandardcurvesamples.The

percentageofthyroidhormonerecoveredfromeachspikedtube

wascalculatedandrevealedquantitativerecoveriesof92%T4and

93%T3.

2.4.3. Sulfotransferase

SulfotransferaseactivitywasmeasuredinduplicatewithT4as

theconjugategroupacceptor,whilePAPSwasusedasthesulfate

groupdonor.SulfotransferaseactivitytowardsT4wasmeasuredby

theincubationofapproximately50␮ghomogenateproteinat37◦C

for 120minin 200␮L100-mMNa-phosphate bufferand 2mM

EDTA(pH7.2),1␮M125_I-labeled_T

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wasterminatedwith800␮Lice-cold0.1MHCl,andthequenched

incubatewasappliedtoSephadexLH-20 minicolumns(2mLof

a10%,w/vsuspension)toresolveliberatediodide,water-soluble

conjugatesandnativeiodothyronines,respectively,asdescribedin

detailpreviously(vanderHeideetal.,2002).Radioiodideactivities

inthewater-solublefractionswereinterpretedtohaveoriginated

fromthepresenceofsulfatediodothyronines.Controlincubations

intheseassayswereintheabsenceofPAPS.Netsulfotransferase

activitiesareexpressedasapercentageofthetotalsumofall

frac-tionsoftheSephadexLH-20chromatograms.

2.4.4. UDPglucuronyltransferase(UGT)

UGTactivitywasmeasuredinduplicatewithT4 asthe

conju-gategroupacceptor.UDPGAwasusedastheglucuronosylgroup

donor.TheglucuronidationofT4wasmeasuredbytheincubation

of50␮ghomogenateproteinat37◦Cfor120minin200␮Lbuffer

containing100mMTris/HCl(pH7.4),5mMMgCl2and0.05%Brij56

(Polyethyleneglycolhexadecylether;acolorlessnonionic

deter-gent and emulsiﬁer; Sigma–Aldrich), supplemented with 1_␮M

125_I-labeled_T

4and5mMUDPGA.Thereactionwasquenchedwith

200␮Lice-cold methanol,andtheincubatewascentrifugedfor

10minat 1500×g.To 300␮Lof thesupernatantthus obtained

700␮L 0.1M HCl was added, and the mixture was subjected

toSephadexLH-20columnchromatographyasdescribedabove.

Radioiodide activities in the water-soluble fractions were here

interpretedtohaveoriginatedfromthepresenceofglucuronidated

iodothyronines.ControlincubationswereintheabsenceofUDPGA.

2.4.5. Outerringdeiodinase(ORD)

5-Deiodinase activities were measured in duplicate as

describedindetailelsewhere(Klarenetal.,2005).Brieﬂy,50␮g

homogenateproteinwasincubatedfor1hundersaturating

sub-strateconditionsof20␮MT4in200␮Lof100mMNa-phosphate

buffer(pH7.2).Outerringlabeled[125_I]T

4wasusedasatracer,and

waspuriﬁedona10%(w/v)SephadexLH-20mini-columnshortly

beforeuse.Thereactionwasquenchedbytheadditionof100␮L

ice-cold5%BSA,followedby500␮Lice-cold 10%TCA,and

cen-trifugedat1400×g(15min,4◦C).To500␮Lofthedeproteinised

supernatantthusobtainedanequalvolumeof1.0MHClwasadded,

andliberatediodidewasseparatedfromthenativeiodothyronine

usingSephadex LH-20columnchromatography. Non-enzymatic

outerringdeiodinationwasdeterminedintheabsenceofa

prepa-ration.

2.4.6. Thyroidhistomorphometricanalysis

Thethyroidtissuesenclosedinthesubpharyngealareawere

storedinformalinﬁxative.Thetissuewasthendecalciﬁedin5%

formicacidand5%formaldehydeforadayandtransferredintoa

sodiumsulfatesolutionforanotherday.Thetissueswere

dehy-dratedin agraded seriesof ethanol beforebeingembeddedin

parafﬁnwax. Theparafﬁn blocks werelongitudinally sectioned

(8␮m)throughallthethyroidtissuesandstainedconventionally

withhaematoxylin-eosin.

Sections were analyzed with a light microscope. Images of

50 randomlyselectedfollicles at100times magniﬁcation were

observed. Thyroid histomorphometry was measured using the

Macniﬁcation® _software_(version_1.6.1_Orbicule_Enhanced_Labs).

Thedifferentmeasurementsinthethyroidtissueweredetermined

bymanuallyselectingthecontoursofthefolliclesinthetissue.The

folliclearea,perimeter,diameter,lengthandwidthofevery

fol-liclecrosssectionweremeasured.Theshapeofthefollicleswas

describedwiththreedimensionlessshapedescriptors:roundness,

formfactorandaspectratiothatwerecalculatedasfollows.

• Roundness=4Area(␮m2_)/_Diameter2 ₍_␮m)._A_follicle_with_a

maximumroundnessvalueof1perfectlyresemblesacircle.

• Form factor=4 Area (␮m2_)/Perimeter2 ₍_␮m)._The _form

fac-torexpressestheevennessofthefolliclesoutline;asitsvalue

approaches1,theoutlineresemblesacircle.

• Aspectratio=maximumlength(␮m)/maximumwith(␮m).The

largertheaspectratio,themoreelongatedthefollicleis;aratio

of1correspondstoaperfectlycircularfollicle.

Aselectionofthyroidtissuewasglutaraldehyde-ﬁxedandthen

embeddedinepoxyresin.Ultra-thinsectionswereobtainedusing

adiamondknifeonaReichert–Jungultra-microtome(UltracutE),

contrastedwithuranylacetate(alcoholicsolution)andleadcitrate,

andobservedinaJeolJEM100-SXelectronmicroscopeat80kVof

acceleratingvoltage.

2.5. Calculationsandstatistics

Meanvalues±standarddeviation,(median)andmin–maxare

presented, unlessindicated otherwise.Statisticalanalysisofthe

datawasperformedusingSPSSforMac®_software_(SPSS_Inc.,

ver-sion16.0.2).TheKolmogorov–Smirnofftestwasusedtotestfor

normalityofthestatisticallytreated variables.Treatmentgroup

comparisonsofthethyroidparametersweredonebyanalysisof

variance(ANOVA)tocomparemeans.Therelationshipsbetween

thyroidparameters(folliclehistomorphometry,thyroidhormone

concentrations and metabolicpathways) and toxicological data

wereanalyzedbycorrelationtests.Growthcurveswerecompared

byadistribution-freestatisticalmethodology.Resultswere

consid-eredsigniﬁcantwhenp<0.05.

3. Results

Nodifferencesinlength,weightandconditionfactorwerefound

betweenPCBexposedandcontrolseabassandnootherexternal

signofadverseeffectsofPCBexposurecouldbeobserved(Fig.3A).

Allﬁshwereinexcellentconditionwithaconditionfactoraround

1.18±0.06.

TheinitialaverageconcentrationsofPCBsmeasuredinseabass

muscleusedinaccumulationexperimentswereclosetoorlower

than themethodLOQ(meanconcentrationof 10ngg−1ww 7

markerPCBcongeners).ThisinitialaverageconcentrationofPCBs

didnotchangesigniﬁcantlyinthemusclesofseabassfromthe

controltankduringtheexperimentalperiod(p=0.81).Also,

mus-cularPCBsconcentrationsincreasednotsigniﬁcantlywithexposure

timeinallaccumulationexperiments(p>0.05)(Fig.1).The

penta-,hexa-andhepta-CBsrepresented88%ofthePCBcongenersand

thesetoptencongenerstendedtobefoundindecreasing

abun-dance,asfollows:CB153>CB180>CB138>CB149>CB101>CB

110>CB95>CB118>CB 187(Fig. 2).The muscular

concentra-tionsofthedifferentcongenerscorrelatedstronglyandwiththe

PCBs (R=0.65–0.99, p<0.001), as the contamination mixture

wasformulatedfromthesamestocksolution.Asaconsequence,

congener-speciﬁcanalysisofeffectsonthyroidsystemcameout

withidenticalresults.Wethereforepresentinthefollowingeffect

analysis,thecorrelationbetweeneffectsandthePCBs.

Mean concentrations of 8, 86, 142,214 and 2279ngg−1ww

(7ICESPCBcongeners)weredeterminedafter120days

expo-sure(Fig.1).AlthoughPCBsarenotreadilymetabolizedbyﬁsh

(Letcheret al., 2000),we investigated the presence of

hydrox-ylated PCB metabolites in both muscle and liver ﬁsh samples

collected from highest treatment group. Only a few HO-PCB

congeners wereidentiﬁed in liver, but nonecould bedetected

in muscle samples. With a mean±standard deviation (based

(5)

Fig.1.Bioaccumulationof[7ICESPCB]inthemuscleasafunctionoftimeinthe differentexposuretanks.

consisted of approximately 0.4% from thesum PCBs measured

in the same tissues (data not shown). According to theirrank

orderof concentration,themostabundant metabolites(mainly

high-chlorinated ones) were: 4HO-CB187>4HO-CB163>

4-HO-CB-199>4HO-CB172>4HO-CB193>4HO-CB146>4-HO-CB177.

LowerchlorinatedPCBs(penta-hexa),despitebeingdominantin

thediet,didnotformHO-PCBmetabolitesatdetectablelevelsin

skeletalmuscletissue.

The tested thyroid parameters did not vary signiﬁcantly

betweentime-points(40, 80 and 120days)indicating thatthe

exposures exert already their effect after 40 days (Fig. 3A–F).

Thedataarethereforepooledbyexposuregroups,without

tak-ingthedifferent time-pointsintoaccount in Table2. Muscular

T4 concentrations did not change signiﬁcantly following

expo-sure to contaminated food containing 0.3 up to 1.0␮gg−1dw

[7 ICES PCB]. Muscular T3 levels had increased 1.5 fold from

0.52±0.17to0.76±0.22ngg−1(F3,56=3.68,p=0.017,n=60)inﬁsh

fedcontroland contaminatedfood(1.0␮gg−1dw[7ICESPCB]),

respectively(t=−3.26,p=0.003).Inthecaseofexposure

exceed-ingtheenvironmentallyrelevantrange(10␮gg−1dw[7ICESPCB]),

lowermuscularthyroidhormoneconcentrationscouldbeobserved

(t=6.58,p<0.001andt=2.03,p=0.045forT4andT3respectively)

comparedtocontrol.TheT4andT3concentrationwerereducedto

75%and30%comparedtocontrols(Fig.3B,C,DandTable1).

The thyroid parameters were not directly related to each

other.Correlationtestcouldnotidentifysigniﬁcantrelationships

betweenT4, T3 and hepatic deiodinase,sulfotransferaseor

glu-curonyltransferaseactivity(p>0.05).

A1.9-foldincreaseinORDactivitycouldbeobservedinﬁshes

with higher contamination levels (Fig. 3E).This observationis

supportedbythepositive correlationbetweenthehepaticORD

activity and the effective PCB concentration measured in the

muscle of these ﬁsh (R=0.414, p<0.001, n=75). The activities

of conjugatingenzymes in liverresponded differentially tothe

PCBexposure.Whereastheglucuronyltransferase(UGT)activity

remainedunchanged(R=_−0.026,p=0.906,n=55),thehepatic

sul-fotransferase(SULT)activitywasmaximallyreducedto47%ofits

controlvalue.Thisobservationwasalsosupportedbyanegative

correlationbetweenthehepaticsulfotransferaseactivityandthe

effectivePCBconcentrationmeasuredinthemuscleoftheseﬁsh

(R=−0.512,p=0.009,n=75)(Fig.3FandTable1).

Histologicalexaminationrevealedchangesinthethyroid

fol-licles of ﬁshes exposed to 0.3–10␮gg−1dw [7 ICES PCB]). The

epithelialcellheightrangedfrom18to31␮mand didnotvary

signiﬁcantlybetweentheexposuregroups(F4,21=1.94,p=0.157).

Themeanfolliclediameterintheexposuregroupsrangedbetween

92and121␮mandnosigniﬁcantdifferencecouldbeidentiﬁed

(F4,21=0.34,p=0.897).Alargerheterogeneityoffolliclesizewas

observed in thyroidsof the1.0 and 10␮gg−1dw [7ICES PCB]

groups,thestandarddeviationwas4–6timeshigherthanin

con-trolgroupandtwiceashighthanintheotherexposuregroups

(0.3and0.6␮gg−1dw[7ICESPCB]).Thyroidsofhighlyexposed

ﬁsh (1.0 and 10_␮gg−1dw [7 ICES PCB]) contained small

folli-cles(Ø≈80␮m)andvery bigfollicles (Ø≈180␮m).Onlyslight

differences inroundness, formfactor andaspect ratiocouldbe

identiﬁedamongthedifferenttreatmentgroups(Table2).Electron

microscopyrevealedthatcellsformingthelargerfolliclesobserved

inthethyroidsofhighexposedﬁsh(1.0␮gg−1dw[7ICESPCB]),

containedextensivelamellararraysofroughendoplasmic

reticu-lum.Therewerenumerouslargeelectrondensecolloiddroplets

(6)

Table2

Histomorphometricanalysis,muscularthyroidhormonelevels,meanhepaticmetabolicactivityandcontaminationlevelsinwhitemuscleofEuropeanseabassThe concentrationsaregiveninngg−1wetweight(mean±standarddeviation,(median)andmin–max)*_signiﬁcant_ANOVA;a_signiﬁcant_after_Dunnett’s_multiple_comparisons.

Food[7ICESPCB] Control 0.3␮gg−1dw 0.6␮gg−1dw 1␮gg−1dw 10␮gg−1dw ANOVA

n 15 15 15 15 15 SumICESPCB(ngg−1₎ 9.9±10.2 60.4±30.5 104.8±76.8 175.9±99.5 1497.0±797.3 F(4,71)=46.5 (8.3) (51.4) (75.2) (137.2) (1234.5) p<0.001* 3.8–46.9 23.4–118.6 46.2–338.5 79.3–443.1 652.6–3222.3 SumPCB(ngg−1₎ 31.3±23.3 156.2±76.8 268.9±187.1 441.4±240.5 3641.1±1924.4 F(4,71)=46.1 (27.1) (133.4) (194.3) (348.4) (3003.7) p<0.001* 17.9–117.0 63.4–301.7 130.0–835.9 211.0–1084.1 1615.2–7840.7 T4(ngg−1) 9.3±3.6 5.5±2.7a _10.0_±_2.2 _9.2_±_3.1 _2.9_±_1.1a _F(4,71)₌_19.7 (9.9) (5.4) (10.5) (10.3) (3.1) p<0.001* 1.1–13.9 1.6–11.3 4.8–13.0 1.3–13.7 0.8–4.6 T3(ngg−1) 0.52±0.17 0.56±0.26 0.66±0.19 0.76±0.23a _0.40_±_0.14a _F(4,71)₌_6.7 (0.51) (0.54) (0.71) (0.83) (0.37) p<0.001* 0.20–0.80 0.10–0.90 0.30–1.01 0.30–1.10 0.20–0.70 deiodinaseactivity (fmolmin−1_␮g−1₎ 3.8±2.8 2.8±2.0 5.4±3.8 6.4±2.5a _8.2_±_1.3a _F(4,71)₌_7.0 (2.8) (2.2) (4.4) (5.3) (7.8) p<0.001* 0.3–9.3 0.3–7.0 0.6–13.0 3.1–11.5 0.8–17.1 sulfatationactivity (fmolmin−1_␮g−1₎ 3.2±0.4 3.2±1.9 2.4±1.3 1.7±1.0 1.5±1.3a _F(4,21)₌_1.9 (3.2) (3.5) (2.7) (1.7) (2.1) p=0.154 2.5–3.5 0.5–5.7 0.6–3.9 0.7–2.8 0.1–2.5 glucuronidationactivity (fmolmin−1_␮g−1₎ 7.7±5.2 7.2±5.7 7.1±3.1 6.6±3.2 5.8±3.2 F(4,21)=1.5 (7.4) (6.2) (7.9) (6.8) (6.3) p=0.960 1.2–15.0 1.3–15.1 3.4–10.0 1.4–9.6 1.4–9.1 folliclediameter(␮m) 92±5 110±14 116±14 121±21 116±29 F(4,21)=0.3 (92) (108) (117) (120) (110) p=0.897 88–94 98–126 103–118 92–169 80–180 cellheight(␮m) 21±5 21±4 22±3 29±3 22±4 F(4,21)=1.9 (21) (19) (22) (30) (23) p=0.157 18–25 19–26 20–25 25–31 18–25 roundness 0.81±0.03 0.73±0.01 0.77±0.05 0.74±0.02 0.75±0.02 F(4,21)=2.4 (0.81) (0.73) (0.76) (0.74) (0.74) p=0.081 0.79–0.83 0.73–0.75 0.73–0.83 0.72–0.75 0.74–0.77 formfactor 0.83±0.02 0.75±0.02 0.79±0.04 0.77±0.01 0.76±0.03 F(4,21)=3.2 (0.83) (0.75) (0.78) (0.78) (0.76) p=0.034* 0.82–0.85 0.72–0.76 0.76–0.84 0.76–0.78 0.74–0.79 aspectratio 0.98±0.07 0.94±0.18 1.01±0.05 0.99±0.07 1.23±0.35 F(4,21)=1.0 (0.98) (1.03) (1.00) (0.97) (1.06) p=0.433 0.93–1.03 0.74–1.06 0.98–1.06 0.94–1.08 1.00–1.65

similartothatofluminalcolloidwithinfollicularcells.Large lyso-somalbodieswithaheterogeneousinternalstructurewerepresent inlargernumbersthaninsmallerfollicles.TheGolgiapparatusand mitochondriaarewelldevelopedandcompressedbythenumerous colloiddroplets.Longprojectionsoffollicularcellcytoplasmoften extendedfromtheapicalsurfaceintothecolloidallumen(Fig.4).

Inthecaseof exposureexceedingtheenvironmentallyrelevant

range(10␮gg−1dw[7ICESPCB]),enlargementofinterstitialtissue

betweenfolliclesanddegeneratedcolloidwereobserved(Fig.5).

4. Discussion

Ourstudydemonstratesthatsubchronicexposuretoamixture

ofPCBsaffectsthyroidhormonephysiologyinjuvenileseabass.

Clearly,effectsofexposuretoenvironmentallyrelevant

concen-trationsdifferedineffectsandpathwaysfromthoseobservedat

higherlevels.Atlowerexposuredoses(0.3–0.6_␮gg−1dw[7ICES

PCB]infoodpellets),thyroidhormonehomeostasisappeared

unaf-fectedwhileasigniﬁcantdiminutionofthyroidhormonelevelswas

observedathigherconcentrations.

Environmentally relevant exposures to PCBs affected

thy-roid hormone metabolism and thyroid hormone synthesis. The

histomorphometricalanalysisshoweda largervariabilityof the

folliclediameter and especially increasedepithelialcell heights

withhigherPCBexposure.Theﬁnestructureofﬁshthyroidgland

ismoreheterogeneousthanitsmammaliancounterpart,

contain-ingfolliclesandcellsofdifferentsizesandfunctionalstates(Eales,

1979).Themechanismsthatgeneratethemarkedheterogeneity

infolliclearchitectureinresponsetocontaminantsareunknown.

Eales(1979)recognizedtheimportanceofthisheterogeneityof

ﬁshthyroidcontainingbothfolliclesandcellsofdifferentsizesand

functionalstatesthatarehypothesizedtogothrougha

histophys-iologicalcycleofgeneration,maturationanddecay(Eales,1979).

Therefore, anyminorproliferation ofthyroidtissueis fairly

dif-ﬁculttodetermine.Thesizeofthefolliclesand theformofthe

follicularcellsgivesanindicationofthesecretoryactivityofthe

gland.Thyroidsdominatedbysmallfollicleslinedbycuboidand

columnarcellscanbeclassiﬁedashighlyactive.Relatively

inac-tivethyroidsshowlargefollicleslinedbyloworﬂattenedepithelial

cells(Hallgren,2002).Ourobservationssupportthehypothesisthat

thecontaminationbyPCBmixturesinducesahyperactivityofthe

thyroid tissueindicatedbythehypertrophyoffollicular

epithe-lial cells. Theseobservations are in accordance withpreviously

reportedchangesinthyroidhistologicalappearance(Leatherland,

1993;Leatherlandand Sonstegard,1978,1980;Schnitzleretal., 2008).

(7)

Fig.3.(A)Meanspeciﬁcgrowthrate(%/day),(B)meanmuscular[T3]/[T4]ratio,(C)meanmuscularT4concentration(ngg−1),(D)meanmuscularT3concentration(ngg−1),

(E)meandeiodinaseactivity(fmol/min/␮g)andF:meansulfotransferaseactivity(fmol/min/␮g)asafunctionofPCBconcentrationinfoodandexposuretime.Thedataare giveninmean±standarddeviation;a_{signiﬁcantly}_different_from_control_after_Dunnett’s_multiple_comparisons_of_exposure_groups;_n_per_time_point_is_5.

Thesimultaneouspresenceofsmallandbigfolliclesinhighly

exposedﬁsh(1.0and10␮gg−1dw[7ICESPCB])indicatedan

asyn-chronyofcellularactivityin thethyroid gland.Thehistological

ultrastructureofepithelialcellssurroundingthesebiggerfollicles

wasdifferentcomparedtothesmallerfollicles,indicatingdifferent

secretoryactivities.Theorganellesinthecytoplasmofthesecells

weremoredeveloped,especiallytheroughendoplasmicreticulum,

probablyrelatedtoanincreasedsynthesisactivity.Withregardto

theprojectionsoffollicularcellcytoplasmfromtheapicalsurface

intothecolloidallumen,thefollicularcellstakeupcolloiddroplets

byendocytosis.A highnumber of colloiddroplets accumulated

noticeablyinthecytoplasmoffollicularcells.Comparable

obser-vationshavebeenmadeinPCB-fedrats(Capenetal.,1977;Collins

andCapen,1980a,b).CollinsandCapensuggestedthatthe

lysoso-malbodieswereunabletointeractwithcolloiddropletsinanormal

manner,leadingtotheinhibitionofthyroglobulinproteolysisand

thyroidhormonerelease(CollinsandCapen,1980b).

Themainmetabolicpathwaysforthyroidhormonesare

deio-dination,glucuronidation and sulfation (Brouwer et al., 1998b;

Ealesand Brown,1993).Iodothyroninedeiodinaseareenzymes

involvedlikewise inthe activationof thyroid hormone.In ﬁsh,

apparentlymorethaninothervertebrates,theseimportant

thy-roidhormonetransformationsarecontrolledoutsidethethyroid

andoccurmainlyinperipheraltissues(liver,brain,kidney,gill).

FishliverexpressesthehighestT4ORDactivity,whichsupportsthe

notionthatlivercouldplayadualrole,contributingtobothlocal

andsystemicsupplyofT3(BlantonandSpecker,2007).Exposureof

ratstoPCBsresultedinaninhibitionofhepaticdeiodinaseactivity

(Brouweretal.,1998a;Morseetal.,1996;Visseretal.,1993).

Enzy-maticouterringdeiodinationactivitiesweresigniﬁcantlyhigherin

animalsexposedto1and10␮gg−1dw[7ICESPCB]infoodpellets.

ThisincreasewasaccompaniedbyanincreaseinmuscularT3in

ﬁshexposedtoenvironmentalrelevantdosesofPCB.Adamsetal.

(2000)examinedthyroidhormonedeiodinationinplaice

follow-ingshort-termexposuretoPCB77and126(Adamsetal.,2000).

TheyfoundthatPCB77increasesthedeiodinationenzymeactivity

whereasPCB126didnotalterT4ORDinplaice.Thesedifferencesin

effectmaybeduetothegreaterpotentialforPCB77tobe

hydroxy-lated(Brouweretal.,1998b).Inmammals,co-planarPCBsgenerally

depressedhepaticT4ORD(Brouweretal.,1989/7).Unfortunately,

duetothecross-correlationofthedifferentPCBs,itwasnot

(8)

Fig.4. Thyroidfollicularcellsofseabassexposedto1␮gg−1[7PCBs]infood (×2000):

(A)betweentwosmallerfollicles,wecanseefewapicalcytoplasmicprocesses extendingintofollicularlumen,welldevelopedroughendoplasmicreticulum(RE) andlargeGolgiapparatuses(G)andfewcolloiddroplets(C)andlysosomalbodies (L)

(B)oflargefollicle,wecanseeapicalcytoplasmicprocessesextendinginto follicu-larlumen(Arrow),dilatedproﬁlesofroughendoplasmicreticulum(RE),numerous largecolloiddroplets(C)andlysosomalbodies(L).

thePCB-inducedchangesindeiodinatingactivitylikelyrepresents

compensatoryresponsestodisrupting effectsthat might

other-wisehavedepressedtheplasmaT3levels.Thedifferentresponses

betweenmammalianandﬁshspeciesrestsonthefactthatinﬁsh

theT3levelsareprimarilyunderperipheralcontrol.

Thyroidhormoneconjugationwithglucuronicacidinactivates

thethyroidhormones,increasestheirsolubilityandfacilitatestheir

excretioninbileandurine(Brouweretal.,1998b).Inour

exper-iment noeffect of PCB exposureon T4UGTcould beobserved,

whereasotherstudieshavereportedmarkedinductioninrats

fol-lowingexposuretoindividualPCBcongeners(Adamsetal.,2000;

Brownetal.,2004b;Morseetal.,1993;Spearetal.,1990;Visser

etal.,1993)andmixtures(Hood,1999;KlaassenandHood,2001;

Morseetal.,1996).InmostofthesereportsreducedT4levelsinthe

sameanimalaccompaniedanincreasedT4UGTactivityand

nega-tivecorrelationsbetweenthosetwoparameterssuggestedacausal

relationship.Generally,phenolicPCBsundergodetoxiﬁcationby

glucuronidationand inducehepaticUGTs tofacilitateexcretion

ofPCBs(KlaassenandHood,2001;Visseretal.,1993).Although

therewasnoincreaseinhepaticT4-UGTactivityinourexperiment.

ThismaybebecausetheT4-glucuronidationenzymeassaydoesnot

evaluateactivityforspeciﬁcUGTisoforms.UGTactivityinduction

canbedependentonarylhydrocarbonreceptor(AhR)activation

(Richardsonetal.,2008).AntagonisticinteractionsbetweenAhR

agonistssuchasPCB126andotherAhR-inactivePCBshavebeen

demonstrated(Safeetal.,1998).

Anotheressentialstepinmetabolismofiodothyroninesisthe

sulfationbysulfotransferases(Schuuretal.,1999).Theyalso

inac-tivatethethyroidhormones,increasetheirsolubilityandfacilitate

theirexcretioninbileandurinelikeUGT.FurthermoreTHsulfates

donotbindtoT3receptorsandarethusunabletomimicthyroid

hormoneactivityandarerapidlydegradedbyinnerring

deiodi-nases(Brouweretal.,1998b;Schuuretal.,1999).Inourexperiment

weobservedageneraldecreaseofSULTactivity.Thisisin

accor-dancewithinvitrostudiesusingratandhumanhepatomacelllines

thatrelatedastronginhibitionofthyroidhormonesulfationwith

hydroxylated metabolitesofPCB(Brouweretal.,1998b;Schuur

etal.,1999).ThisreducedT4SULTactivityisaccompaniedbyan

increaseofmuscularT4levelsinanimalsexposedto

environmen-tallyrelevantdosesofPCB.

Theheterogeneityofﬁshthyroidsystemsandtheirresilienceto

perturbationsmakeitdifﬁculttointerpretthesechangesinactivity.

ItiswellacceptedthatsomePCBcongeners,duetotheirstructural

similarity,areabletoserveasbindingligandsforT4-binding

pro-teins(Darnerudetal.,2001).Bythesemeanstheyreducethyroid

hormone levelsbydisplacing themfromtransportproteinsand

increasingtheirexcretion.BothT4 andT3 havea negative

feed-backeffectonTSHsecretionbythepituitaryinteleostﬁshspecies

(Yoshiuraetal.,1999).Thatmayhavestimulatedtheproductionof

T4,revealedbythyroidhistomorphometry.Thedeiodinase

activ-ity wasincreased,thus more conversionof T4 toT3, and there

isless excretionofthyroid hormonesthroughthehepatic

path-wayasthesulfationactivitydecreasedwithraisingPCBexposure.

Thesemodiﬁcationsin thyroidhormonedynamicscontributeto

maintainthyroidhormonelevelsinanacceptablerange.ThePCB

induceddisruptionofthyroidsystemiscounteredbyanextensive

self-regulatoryfeedbackcontrol.

Nevertheless,weobserveanimportantloweringofmuscularT3

andT4levelsinanimalsexposedto10␮gg−1dw[7ICESPCBs]in

foodpellets.Thisobservationindicatesthatatthoseexposure

lev-elsothercausesthanthemetabolicpathwaysareinvolved.Inthese

ﬁshesthehistologicalexaminationrevealedlymphoidcell

inﬁltra-tionandenlargementoftheinterstitialtissuebetweenfolliclesand

degeneratedcolloid.Thefolliclesappearedinlowernumberand

thetissueseemsdisorganized.Thesedegenerativechangesmight

havecausedtheobservedhypothyroidismintheseﬁsh.Probably,

thepollutantsatthisdoseinterferewiththesynthesisandsecretion

ofthyroidhormones.

(9)

Thethyroidstatushaspronouncedeffectsongrowthand

devel-opment in ﬁsh (Blanton and Specker, 2007; Inui et al., 1995;

Klarenetal.,2008;Poweretal.,2001;Shiaoand Hwang,2006; Yamano,2005).Dependingonthedosageused,T3supplementation

hasanabolicandcataboliceffectswhereashypothyroidismalways

resultsingrowthretardation(Theodorakis etal.,2006;Vander

Geytenetal.,2001).Inthisstudy,neithersizenorweight

differ-encescouldbefoundbetweenthetreatmentgroups,whichcan

beexplainedbythehighfeedingrateoftheseﬁshes.Thyroid

hor-monereserveshavenotbeendeterminedfullyinanyﬁshspecies

butbasedonhumanvaluesitwouldseemadvisabletocontinue

studiesforseveralmonthstodetermineatruemeasureofeffecton

thethyroidalstatus(Brownetal.,2004a).

5. Conclusions

ThepresentedresultsshowclearlythattheeffectsofPCB

expo-sures on the thyroid system are dose-dependent. Exposure to

environmentallyrelevantdosesofPCBmodiﬁeshepaticT4outer

ringdeiodinaseandinducedthehormonesynthesisandsecretion.

Ultrastructuralhistologicalinvestigationsshowedapotential

inhi-bitionofthyroglobulin proteolysisandthyroidhormone release

inthethyroidsofhighexposedﬁsh(1.0␮gg−1dw[7ICESPCB]).

Meanwhile, the thyroid hormone levels were preserved. The

presentedmechanismsare partof theextensiveautoregulatory

feedbackcontrol at both central and peripherallevels,and the

inducedchangesinthyroidhormonedynamicskeepthelevelsinan

acceptablerange.At10timeshigherconcentrations,animportant

depressionofmuscularT3andT4levelscouldbeobservedwhich

areapparentlycausedbyothermechanismsthanmetabolic

path-ways.Hereweobserveddegenerativehistologicalchangesinthe

thyroidtissuethatmighthavecausedthehypothyroidisminthese

ﬁsh.

Acknowledgements

Schnitzler,J.receivedgrantsfromFRIA(Fondspourlaformation

àlarecherchedansl’industrieetdansl’agriculture).Krishna,D.is

aF.R.S.–FNRSResearchAssociate(FondsdelaRecherche

Scien-tiﬁque).TheauthorsaregratefultoPr.Thomé,JP.,andLouvet,M.

fortheirlogisticalassistanceinfoodpelletpreparation.Covaci,A.

acknowledgesapostdoctoralfellowshipfromtheResearch

Scien-tiﬁcFoundationofFlanders(FWO).Dirtu,A.acknowledgesﬁnancial

supportfromUA.ThisisaMAREpublication213.

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