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GENEREGULATIONINTELEOSTSBYESTRADIOLANDESTROGENMIMICS By CHRISTOPHERJAMESBOWMAN ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT OFTHEREQUIREMENTSFORTHEDEGREEOF DOCTOROFPHILOSOPHY UNIVERSITYOFFLORIDA 2001 ACKNOWLEDGMENTS Iwouldliketothankthemanypeoplewhocontributedtomyworkoverthepast fouryears. Theirhelpandadviceprovidedmewiththenecessarytrainingtoconduct thesestudies,andtheimportanceofpatienceandcollaborationwhileperformingsound scientificresearch. NancyD.Denslow,Ph.D.showedtremendouscompassionandincrediblesupport whileIwasinherlaboratory. Sheintroducedmetothefieldofendocrinedisruption,and togetherwelearnedmoreaboutenvironmentaltoxicology. Sheprovidedaplacetowork withseeminglyunlimitedresources. Shealsoofferedanenvironmentofcutting-edge biotechnologywithunlimitedpossibilities. Nancyfreelysharedideasandadvice,yet allowedmetodecidewhatprojectstoworkonandhowtocompletethem,this encouragedandfosteredmyindependenceasaresearcher. Ifeelliketogetherwewere abletowritefellowshipapplicationsthathavefinanciallysupportedmeforthepastfour years. Withhersupport,Ihadtheopportunitytosharemyworkwithmanydifferent scientificaudiences,teachingmethevalueofeffectivecommunication. Nancyembodies manydesirablequalitiesthatarerareinadoctoraladvisorandIfeelblessedtobeher firstdoctoralstudent. CollaborationwithLeroyFolmar,Ph.D.,MichaelHemmer,Ph.D.,andtheir colleaguesattheU.S.E.P.A.(UnitedStatesEnvironmentalProtectionAgency)was educationalandsuccessful. Theexperimentsanddiscussionswesharedledtomany 11 publicationsandanentirechapterofthisdissertation. Ithankthemallforthe contributionstheymadetomydoctoralresearch. Iwouldalsoliketothankmycommitteemembers:KathleenShiverick,Ph.D., StephenRoberts,Ph.D.,EvanGallagher,Ph.D.,andRosaliaSimmen,Ph.D. They providedoutstandingsupportandguidancethroughouttheresearchprocessandIthank themforreviewingthisdissertation. KevinKroll,MarjorieChow,MingChow,ScottMcClung,ScottMcMillan, AlfredChung,LiZhang,LuanLe,andBenO'NealoftheMolecularBiomarkersand ProteinChemistryCorelabsarethankedforallthetechnicalguidanceandsupportduring mytimeintheDenslowlaboratories. IespeciallyappreciateKevinandMarjorie'shard workandpatience. Withoutthemtheworkwouldhavetakenmuchlongerthanitdid. IwouldliketothankSandyGibbons,TeresaStevens,andRobertConeyinthe ICBR(InterdisciplinaryCenterforBiotechnologyResearch)forkeepingupwithmy never-endingrequests. RonFerguson,TammyFlagg,SteveLee,LindaGreen,Karen Kelley,ScottWhittaker,DavidMoraga,SharonNorton,KeithLowe,WilliamFarmerie, andSavitaShankerallprovidedvaluableassistancethroughtheirtechnicalexpertiseand advice. IwouldespeciallyliketothankRon,Sharon,andStevewhofirsttaughtmethe basicsofmolecularbiology. Iappreciatealltheirhelp. Withoutthemthisresearch wouldhavetakenmuchlongerthanitdid. Theinterdisciplinarynatureofmydissertationrequiredadministrativesupport fromseveraldepartments. IthankJudyAdamsandthestaffintheDepartmentof Pharmacologyfortheirunconditionalcareandunderstandingwhileexplainingthe Universitysystem. IwouldalsoliketothanktheadministrativestaffintheCenterfor in EnvironmentalandHumanToxicology,ICBR,andtheDepartmentofBiochemistryand MolecularBiologyforalltheirassistance. IextendmuchappreciationtoEvanGallagherandpeoplefromhislab(Karen Pastos,KristenHenson,andJamesGardner)whoprovidedsignificanttechnicaland scientificadvicetothisresearch. IwouldalsoliketothankGregStaufferandKathy ChildressfortheirhelpintheAquaticToxicologylab. Iwillmissourjointlabmeetings. IthankTimothyGrossandhisstaffattheUnitedStatesGeologicalSurvey (ShaneRuessler,CarlaWieser,andJonWiebe)forprovidingfacilities,personnel,and fishforsomeoftheexperiments. Ialsogreatlyappreciatetheplasmahormoneanalysis conductedbyTim'slab. Thisdisserationwouldnothavebeenpossiblewithoutthefinancialsupportofthe SuperfundBasicResearchProgram(NationalInstitutesofEnvironmentalHealth Sciences)CentergrantandGraduateFellowship. Inaddition,theU.S.EPA-STAR (SciencetoAchieveResults)GraduateFellowshipIreceivedprovidedsupportformy salary,tuition,andlimitedexpensesfrom1999to2001. Finally,withspecialrecognitionandlove,Ithankmywife,RobinBowman,for herunconditionalsupportandpatience. OurmanyyearsspentinGainesvillewillnever beforgotten. Ialsothankclosefriendsandfamilyfortheirkindwordsand encouragement. Iwouldnothavebeenabletofinishthisdegreewithoutthem. IV TABLEOFCONTENTS page ACKNOWLEDGMENTS ii LISTOFTABLES viii LISTOFFIGURES ix ABSTRACT xii CHAPTERS 1 INTRODUCTIONANDLITERATUREREVIEW 1 Background 1 TheEndocrineDisruptionHypothesis 1 SupportingEnvironmentalEvidence 1 SupportingLaboratoryEvidence 3 XenobioticswithEstrogenicActivity 5 PharmaceuticalEstrogens 6 OrganochlorineCompounds 6 Phenolics 7 NaturallyOccurringCompounds 8 OtherCompounds 8 FishModels 9 Vitellogenin 11 ModelofHormonalRegulation 12 BiomarkerofEstrogenicity 12 EstrogenReceptorMediatedPathway 13 2 COORDINATEINVIVOGENEEXPRESSIONINLARGEMOUTHBASS (MICROPTERUSSALMOIDES)AFTERACUTEESTRADIOLEXPOSURE 20 Introduction 20 CoordinateGeneRegulation 21 LargemouthBass 23 ExperimentalObjectivesandHypothesis 24 MaterialsandMethods 24 FishCollectionandMaintenance 24 ExperimentalExposures 25 1 RNAIsolation 26 EstrogenReceptorandVitellogeninRT-PCR 27 CloningandSequencingofIsolatedcDNAs 27 mRNAQuantification 28 DifferentialDisplayRT-PCR 30 ProteinAnalysis 31 Results 33 EstrogenReceptorandVitellogeninSequencingandCharacterization 33 RegulationofmRNAsinAdultLargemouthBass48HoursPostInjection 34 PlasmaVitellogeninDoseResponse 36 mRNACharacterizationAfterAcuteExposureinJuvenileLargemouthBass 37 Discussion 38 LargemouthBassEstrogenReceptorandVitellogenin 39 ExposureofAdultLargemouthBasstoEstradiol 42 PlasmaSteroidandVitellogeninDoseResponsetoEstrogens 44 ExposureofJuvenileLargemouthBasstoEstradiol 48 DifferentialmRNARegulationbyEstradiol 53 3 DEVELOPMENTOFANINVITROMODEL: LARGEMOUTHBASSPRIMARY HEPATOCYTES 77 Introduction 77 Rationale 78 FishPrimaryHepatocytes 79 Objective 81 MaterialsandMethods 82 FishCollectionandMaintenance 82 ReagentsandEquipment 82 BuffersandMedia 84 HepatocyteIsolation 85 TheFish 86 ThePerfusion 86 TheIsolationofHepatocytes 88 HepatocyteCulture 89 CellViability 89 CultureMorphology 91 ExperimentalResults 91 Discussion 93 4 VITELLOGENININDUCTIONINSHEEPSHEADMINNOWASANINVIVO MODELFORESTROGENICITY 106 Introduction 106 VitellogeninmRNAandProteinasBiomarkersofExposure 107 EnvironmentalEstrogens 108 ExperimentalObjective 110 MaterialsandMethods 11 VI FishCollectionandMaintenance Ill ExperimentalExposures 112 RNAIsolation,IdentificationandVerificationofVitellogeninSequences 114 mRNAQuantification 115 DifferentialDisplayRT-PCR 117 ProteinAnalysis 118 Results 120 VitellogeninI&IICloning,SequencingandCharacterization 120 TimeCourseofVitellogeninInductionPostInjection 122 VitellogeninDoseandTimeResponsewithConstantAqueousExposure 124 Generegulationbyestradiol,diethylstilbestrol,andethinylestradiol 125 Vitellogenininductionbynonylphenol,methoxychlor,andendosulfan 126 Decreasedvitellogeninlevelsfollowingtransfertocleanwater 128 Discussion 129 AcuteEstradiolExposures 132 AqueousDoseResponseExposures 135 DecreasedVitellogeninLevelsAfterExposure 140 5 CONCLUSIONS 160 LISTOFREFERENCES 172 BIOGRAPHICALSKETCH 196 vn . LISTOFTABLES Table Page 2-1. Plasmavitellogenininlargemouthbass48hourspostestradiolinjection 56 3-1 Compositionofbufferedperfusionsolutionsusedtoisolateprimaryhepatocytes..97 3-2. Mediaandsupplementsusedtocultureprimaryhepatocytes 97 4-1. Nominalandactualmeasuredwaterconcentrationsofchemicalsevaluatedin sheepsheadminnow 144 4-2. VitellogeninmRNAandproteininductionaftersevendaysaqueousexposure....145 Vlll LISTOFFIGURES Figure Page 1-1. Representativechemicalstructuresofdifferentfamiliesofsuspected xenoestrogens 17 1-2. Relationshipbetweentheliverandovaryduringvitellogenesisinfemales 18 1-3. Simplifiedschemeoftheestrogenreceptormediatedpathway 19 2-1. AcutemRNAresponsehypothesis 57 2-2. Largemouthbass{Micropterussalmoides) 57 2-3. Largemouthbasssamplingbylabpersonnel 58 2-4. Histologyoflargemouthbassgonads 59 2-5. EstrogenreceptorandvitellogeninRT-PCRproducts 60 2-6. CloningstrategyforestrogenreceptorandvitellogeninPCRproducts 61 2-7. LargemouthbassvitellogenincRNAstandardcurve 62 2-8. Multiplesequencealignmentofclonedlargemouthbassestrogenreceptor fragment 63 2-9. Multiplesequencealignmentofclonedlargemouthbassvitellogeninfragment..64 2-10. LargemouthbasslivermRNAcharacterization 65 2-11. Plasmasteroidmeasurementsoutto48hourspostinjection 66 2-12. EstradiolinducedmRNAsover48hours 67 2-13. Plasmavitellogenininductionover48hours 68 2-14. LargemouthbassdifferentialdisplayprimerpairG-23 69 2-15. LargemouthbassdifferentialdisplayusingprimerpairsG-10andC-l 70 IX . 2-16. VerificationoflargemouthbassERp72 71 2-17. Plasmasteroids48hoursfollowingacuteexposure 72 2-18. Doseresponseinductionofplasmavitellogenininlargemouthbass 73 2-19. TimecourseofestradiolinducedmRNAsover21days 74 2-20. Timecourseofplasmavitellogenininduction 75 2-21 LargemouthbassestrogenreceptorandvitellogeninmRNAcomparison 76 3-1. Liverperfusionsetup 98 3-2. Hepatocyteisolationcytology 99 3-3. Electronmicroscopyofisolatedhepatocytes 100 3-4. Trypanblueofisolatedhepatocytes 101 3-5. Hepatocyteviabilityovertime 102 3-6. Osmolalityofhepatocytereagents 103 3-7. Lightmicroscopyofculturedhepatocytes 104 3-8. Primaryhepatocytegeneexpression 105 4-1. Sheepsheadminnow{Cyprinidonvariegatus) 145 4-2. RT-PCRonestrogentreatedsheepsheadminnowRNA 146 4-3. MultiplesequencealignmentofsheepsheadminnowVIT1andVIT2 146 4-4. NorthernblotanalysisofthetwosheepsheadminnowvitellogeninmRNAs 147 4-5. TimecourseofvitellogeninmRNAinductioninsheepsheadminnowfollowing acuteestradiolexposure 148 4-6. Timecourseofsheepsheadminnowplasmavitellogenininductionfollowing acuteestradiolexposure 149 4-7. InductionofsheepsheadminnowvitellogeninmRNAandproteinaftersevendays constantexposure 150 4-8. InductionofsheepsheadminnowvitellogeninmRNAfollowingconstantaqueous exposure 151