环境毒理.docx

环境毒理.docx

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环境毒理

Thyroidendocrinedisruptingactivityofpentachlorophenol:

aninvitroandinvivoassa

Abstract

Theobjectiveofthepresentstudywastoevaluatethyroidendocrinedisruptingactivityofpentachlorophenol（PCP）byusinganinvitroandinvivoassay.Intheinvitroassay,ratpituitaryGH3cellswereexposedto0,0.1,0.3,and1.0μMPCP.Weselecteddeiodinase1（Dio1）genetranscriptionasthesensitiveandspecificthyroidhormone-responsivegenetoindicatethyroidendocrinedisruption.TheresultsshowedthatPCPexposurealonecouldsignificantlydown-regulatedDio1genetranscriptionsandthushasthepotentialthyroidendocrinedisruptingactivity;whileco-exposurePCPwithphysiologicallevelsoftriiodothyronine（T3）,theDio1genetranscriptionsinducedbyT3weremarkedlydown-regulatedbyPCP,indicatingantagnosticactivityofPCPinvitro.Intheinvivoassay,zebrafishembryoswereexposedto0,1,3,and10μg/LofPCPuntil14dayspost-fertilization（dpf）.PCPexposureresultedindecreasedofthyroxine（T4）levels,butelevatedcontentsofwhole-bodyT3.Genetranscriptionswerefurthermeasuredinthehypothalamic-pituitary-thyroid（HPT）axis.PCPexposuresignificantlyup-regulatedgenetranscriptions,includingthyroid-stimulatinghormone（TSHβ）,sodium/iodidesymporter（NIS）,thyroglobulin（TG）,Dio1andDio2,thyroidhormonereceptors（TRαandTRβ）,anduridinediphosphate-glucuronosyl-transferase（UGT1ab）.PCPexposuredidnotinfluencethegenetranscriptionoftransthyretin（TTR）.TheresultsindicatethatPCPhasthepotentialasthyroidendocrinedisruptorbothinvitroandinvivoandfurtherconfirmedthereliabilityofthedevelopingzebrafishlarvaeasmodelsforassessmentofthethyroidendocrinedisruptionofchemicals.

Keywords:

Pentachlorophenol;T-Screenassay;Zebrafishlarvae;Thyroidendocrinedisruption;Hypothalamic-pituitary-thyroid（HPT）axis

1.Introduction

Pentachlorophenol（PCP）wasmainlyusedaspesticideandwoodpreservativeworldwide.Duetoitswidespreaduseforthesepurposes,PCPhasbecomeaubiquitousenvironmentalcontaminant（reviewedbyZhengetal.,2012）.AlthoughsomecountrieshavebannedorcontroltheuseofPCP,itisstillusedasawoodpreservative,anddetectedintheaquaticenvironment,wildanimalsandhumansamples（Farhadietal.,2009;Zhengetal.,2011;Zhengetal.,2012）.ChinahasrestrictedtousePCPin1997,however,PCPwasusedasamolluscidetocontrolthere-emergenceofschistosomiasisinsomeareas（TanandZhang,2008）.BecauseofthelargeamountofuseofPCP,thereiswidespreadenvironmentalcontamination.PCPhasbeendetectedinsurfacewatersinChina,uptomicromolar（μg/L）concentrations（Zhengetal.,2000;Gaoetal.,2008;Hanetal.,2009）.ThesedataindicatedthatPCPusageinducedobviousPCPpollutioninthelocalwaterenvironmentandmaycauseadverseeffectsonaquaticorganismsandpublichealth.

Theimpactofenvironmentalchemicalsonthyroidendocrinesystemhavereceivedgreatattentioninrecentyears,especiallybecausethyroidhormones（THs）areofspecialimportanceinfetaldevelopment,asdevelopmentofthebrainisdependentonnormallevelsofthyroidhormones（reviewedbyBoasetal.,2012）.Endocrinedisruptingchemicalscanhaveadirectimpactonthyroidhormone（TH）synthesis,transport,binding,catabolismandclearanceofcirculatingTHs（KloasandLutz,2006）.LimitedinformationshowedthatPCPmayhavepotentialtodisruptthyroidendocrinefunctions.Forexample,PCPhas3,3′,5-triiodothyronine（T3）-antagonistactivityinvitroandinvivoassaysinXenopuslaevis（Sugiyamaetal.,2005）.DevelopmentalexposureofPCPtoratscausedlowertotalthyroxine（T4）concentrationsinplasma（Kawaguchietal.,2008）.Inhumans,thenegativeassociationbetweenmaternalplasmaPCPlevelsandcordplasmafreethyroxine（fT4）concentrationsinneonateswasreported（Dallaireetal.,2009）.GiventheimportantrolesofTHsinthegrowthanddevelopmentoffetuses,thepotentialthyroidendocrinedisruptionofPCPhasraisedgreatconcernoveritsadverseenvironmentalhealthrisks.

IntheevaluatingTHendocrinedisruption,aninvitromodelusingtheratpituitarytumorcelllineGH3（T-Screen）hasbeendeveloped.GH3cellscansynthesizeandsecretgrowthhormone（GH）andprolactin（PRL）andT3caninducethetwohormonesproductionaswellasgenetranscriptions（Spindleretal.,1982;Stanley,1988）.ThisT-ScreenisbasedontheT3dependentcellgrowth,mediatedbyspecific,high-affinitythyroidreceptor（TR）,bindingTHstothyroidhormoneresponsiveelements（TREs）andultimatelyleadingtogeneexpression.ThustheT-ScreenassaycanbeusedforinvitrodetectionofagonisticandantagonisticpropertiesofcompoundsattheleveloftheTRintheabsenceandinthepresenceofT3（Gutlebetal.,2005;Schriksetal.,2006）.Inaddition,genetranscriptionscanbemeasuredbasedonmodulationofbasalGHandPRLsecretioninGH3cellsisrelatedtotheregulationofGHandPRLgenetranscription（Tamuraetal.,2000）.Furthermore,GH3cellspossessbothdeiodinase1（Dio1）and2（Dio2）activity（St.Germain,1985;Morietal.,2007）,andareresponsivetoT3（Baueretal.,1997）.BoththedeiodinasesareimportantroleinmaintaininglocalT3levelsinthebrainandpituitaryinvivo.T-Screenassayhasproventosuccessfullypredicttheeffectsofsomethyroidhormonedisruptingchemicals,suchasplasticizers,alkylphenols,pesticides,polychlorinatedbiphenyls（PCBs）,andbrominatedflame-retardants,personalcareproducts（GhisariandBonefeld-Jorgensen,2005;Hamersetal.,2006;Schriksetal.,2006;Hansenetal.,2009;Taxvigetal.,2011;Hintheretal.,2011）.

Infish,thethyroidendocrinesystemiscontrolledprimarilybythehypothalamic–pituitary–thyroid（HPT）axis,whichisresponsibleforregulatingthyroidhormonedynamicsbycoordinatingtheirsynthesis,secretion,transportandmetabolism（reviewedbyCarrandPatiño,2011）.Recently,aninvivomodelfortestingendocrinedisruptionofTHswasdevelopedindevelopingzebrafishlarvae（Yuetal.,2010）.ThisassayindicatedthattheHPTaxiscanbeevaluatedtodeterminethyroidendocrinedisruptionofTHendocrinedisruptors,e.g.,PBDEsandalsotosomeextentonpotentialmechanismsofaction（Yuetal.,2010;Chenetal.,2012）.However,thethyroidendocrinedisruptionofPCPandenvironmentalriskinfishisnotwell-known.

TheobjectivesofthepresentstudyweretoinvestigatethethyroidendocrinedisruptingactivityofPCPbymeansofinvitroandinvivoassay.Intheinvitrostudy,theTH-endocrinedisruptingactivitywasassessedusingtheT-Screenassay.TH-responsivegenetranscriptionswereexamineduponPCPexposure;WefurtherexaminedtheimpactofPCPonTHslevelsandgenetranscriptionsintheHPTaxisandthepotentialmechanismsofthyroidendocrinedisruptioninthedevelopingzebrafishlarvae.

2.Materialsandmethods

2.1.Chemicals

Pentachlorophenol（PCP）（>99%,CASNo.87-86-5）waspurchasedfromSigma-Aldrich（St.Louis,MO,USA）.Itwasdissolvedindimethylsulfoxide（DMSO）,andstoredat4°C.[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide（MTT）waspurchasedfromDuchefa（theNetherlands）.Allotherchemicalsusedinthepresentstudywereofanalyticalgrade.

2.2.Cellculture

TheratpituitaryGH3cellswereobtainedfromtheCellCenteroftheInstituteofBasicMedicalSciences,ChineseAcademyofMedicalSciences（Beijing,China）.Thecellswereculturedinphenol-redfreeDMEM/F12medium（Sigma,St.Louis,MO,USA）,supplementedwith10%fetalcalfserum（FCS,Gibco）,2.5%100U/mLpenicillin,and100μg/mLofstreptomycin.Thecellsweremaintainedat37 °Cinanatmosphereof5%CO2.

2.3.Cellviabilityassayandchemicalsexposure

CellviabilitywasmeasuredbythecolorimetricmonitoringconversionofMTTtoformazan.Briefly,theGH3cellswereseededinto96-wellcultureplates（Falcon,FranklinLakes,NJ,USA）atadensityof5×104cells/well.After24hofculture,theculturedmediumwasremovedandserum-starvedculturemediumwasaddedforanother24h.PCPwasdilutedwithserum-freeculturemedium（0,0.1,0.3,and1.0M）andwasaddedtothewellsfor44h.Thena20μLofMTTsolution（5mg/mLMTTinPBS）wasaddedtoeachwellandtheplateswereincubatedforadditional4h.Theculturemediumwasremovedand150μlofDMSOaddedtoeachwell.Afterincubationforanother20min,theextenttowhichtheMTThadreducedtoaformazanproductwasdeterminedusingamicroplatereader（M2,MolecularDevice,UnionCity,CA,USA）at490nm.Thecellviabilitywasexpressedasapercentageofthecellsurvivalratecomparedwiththecontrol（Yuetal.,2008）.Thereweresixreplicatesandeachtreatmentcontainedablankandsolventcontrol（0.1%DMSO）.

Forthyroidendocrinedisruptionassay,theGH3cellswithadensityof2×105perwellwereseededinto12-wellplatesfor24h.Thentheculturemediumwasremovedandthecellswererinsedwithserum-freemedium.ThecellswerethenexposedtoPCP（0.1,0.3,and1.0μM）inserum-andphenolred-freeDMEM/F12for48h.Therewerethreereplicateforeachtreatmentandcontrol.

2.4.Zebrafishmaintenanceandembryoexposure

Adultzebrafish（Daniorerio）（ABstrain）maintenanceandembryoexposuretoPCPwerecarriedoutfollowingthemethoddescribedpreviously（Yuetal.,2010）.Briefly,embryosthatdevelopednormallyandreachedtheblastulastage（2hourspost-fertilization,hpf）wereselectedforexperiments.Approximately400embryoswererandomlydistributedintoglassbeakerscontaining500mLofPCPsolution（0,1,3,and10μg/Lorequalto0.0037,0.011,0.033μM）.Therewere3replicatesineachexposuregroupandcontrolgroupforgene