Gene regulation in teleosts by estradiol and estrogen mimics

212 Pages·2001·9.1 MB·English

by  Bowman, Christopher James.

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