BPAF-INDUCEDADIPOGENESIS:ROLEOFPERIPHERAL5-HTSIGNALING
BPAF-INDUCEDADIPOGENESISIN3T3-L1CELLS:ROLEOFPERIPHERALSEROTONINSIGNALING
ByKRISTINAD.WIGGERS,B.MSc.
AThesisSubmittedtotheSchoolofGraduateStudiesinPartialFulfilmentoftheRequirementsfortheDegreeMasterofScience
McMasterUniversity©CopyrightbyKristinaD.Wiggers,August2017
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McMasterUniversityMASTEROFSCIENCE(2017)Hamilton,Ontario(MedicalSciences)TITLE:BPAF-InducedAdipogenesisin3T3-L1Cells:RoleofPeripheralSerotoninSignalingAUTHOR:KristinaD.Wiggers,B.MSc.(UniversityofWesternOntario)SUPERVISOR:Dr.AlisonHollowaySUPERVISORYCOMMITTEE:Dr.WarrenFoster Dr.GregorySteinberg NUMBEROFPAGES:xi,80DESCRIPTIVENOTES
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ABSTRACT
There is evidence that synthetic chemicals inourenvironment called,obesogens,may
playan important role in theglobalobesityepidemic.BisphenolA (BPA) isoneof the
most studiedobesogens.Due to concerns surrounding theuseof BPA, BPA structural
analogues are being used as substitutes in consumer products. However, there is
currently little informationavailable regarding theireffectsonpathways important for
the development of obesity. Interestingly, serotonin (5-HT) signaling in peripheral
tissuesplaysanimportantroleinregulatingmetabolichomeostasis.Moreover,BPAhas
been shown to alter 5-HT signaling in the central nervous system. Therefore, the
objectiveofthisstudywastodeterminetheeffectsofBPAanaloguesonadipogenesis
andelucidatewhethertheirobesogeniceffectsinvolveperipheral5-HTsignaling.
3T3-L1 preadipocytes were treated with 10 μM BPA or four commonly used BPA
analoguesduringdifferentiation.LipidaccumulationwasassessedbyOilRedOstaining.
Adipogenic markers and genes important for 5-HT synthesis/metabolism were
subsequently examined following treatment with BPA and the structural analogue
bisphenol AF (BPAF). The roles of tryptophan hydroxylase 1 (TPH1) and monoamine
oxidase(MAO),inadditiontoglucocorticoidreceptor(GR)signalingwerealsoevaluated.
Treatment with 10 μM BPAF and Bisphenol S (BPS) significantly increased lipid
accumulation in differentiated 3T3-L1 cells. 10 μM BPAF significantly increased
adipogenic gene markers, as well as GR gene transcripts. Moreover, BPAF induced
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adipogenesis in the absence of the synthetic glucocorticoid dexamethasone. Further,
althoughBPAFsignificantlyincreasedTph1mRNAlevels,blockingitsactivitywithpara-
chlorophenylalanine(pCPA),didnotblocktheadipogeniceffectsofBPAF.However,co-
treatment with the MAO inhibitor, phenelzine, significantly decreased lipid
accumulation and peroxisome proliferator activated receptor gamma (Pparg)
expression.
Therefore,thesedatademonstratethatBPAFmayactasanobesogenandsuggeststhat
itsactionmightbemediated,inpart,byaltered5-HTsignaling.
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ACKNOWLEDGMENTS
Firstly, to my supervisor, Dr. Alison Holloway, thank you for your mentorship and
supportover the last twoyears.Thankyouforchallengingmetothinkcriticallyabout
myresearchprojectandgivingmetheopportunitytoexploremyownideas.Aboveall,
thankyou foralwaysbelieving inme.Youhavegivenmeastrong foundation,both in
research and in professional and leadership skills, that will forever follow me as I
progress inmycareer.Iwouldalso like to thankmycommitteemembers,Dr.Warren
FosterandDr.GregorySteinberg.Yoursuggestionsandinsightsthathavehelpedshape
myresearchprojecthavebeengreatlyappreciated.
Tothemembersof theHollowayLab,especiallyAndreaLIanos,AhmedAyyash,Sergio
RaezVillanueva,SarahKleiboer,NicoleLatchminarine, JuliaMartinko,MaddyRudolph,
PeterGariscsak,NicoleDeLong,OnnaWuandMichelleLo.Thankyounotonlyforyour
continuedhelpinthelaboratorybutalsoforthelastingfriendshipsthathavedeveloped.
Coming into the lab everyday would not have been the same without the countless
laughswehavesharedandtheobstaclesthatwereovercometogether.
Thankyoutomyfamilyforyourlove,understandingandrelentlessencouragementasI
completed this new and exciting chapter inmy life. Finally, thank you tomy partner,
Adam,foralwaysbeingthereformefromtheverystarttofinish.Iamforevergrateful!
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TABLESOFCONTENTS
DESCRIPTIVENOTES.....................................................................................................ii
ABSTRACT...................................................................................................................iii
ACKNOWLEDGMENTS..................................................................................................v
LISTOFFIGURES........................................................................................................viii
LISTOFTABLES............................................................................................................ix
LISTOFABBREVIATIONS...............................................................................................x
CHAPTER1:INTRODUCTION.........................................................................................11.1AdipogenesisandWhiteAdiposeTissueFunction(WAT)........................................11.2EndocrineDisruptingChemicalsandBisphenolA....................................................31.2.1HumanExposure................................................................................................41.2.2EpidemiologicalStudies.....................................................................................61.2.3Invitroandinvivostudies.................................................................................7
1.3AreBisphenolAAnaloguesSafeAlternatives?.......................................................101.4RoleofPeripheralSerotoninonAdipogenesisandWhiteAdiposeTissueFunction......................................................................................................................................131.5BPAandSerotoninSignaling..................................................................................161.6BPAandGlucocorticoidSignaling...........................................................................16
CHAPTER2:HYPOTHESISANDOBJECTIVES.................................................................192.1RationaleandHypothesis.......................................................................................192.2Objectives...............................................................................................................20
CHAPTER3:MATERIALSANDMETHODS.....................................................................213.1Cellculture,murineadipocytedifferentiationandtreatments.............................213.2LipidStainingandQuantification...........................................................................223.3RNAExtractionandQuantification........................................................................233.4ComplimentaryDNA(cDNA)Synthesis..................................................................233.5PrimerDesign.........................................................................................................243.6Real-TimeQuantitativePolymeraseChainReaction(RT-qPCR).............................243.7StatisticalAnalysis..................................................................................................25
CHAPTER4:RESULTS..................................................................................................274.1BPAFandBPSsignificantlyinducelipidaccumulationin3T3-L1cells....................274.2BPAFsignificantlyincreasesmRNAexpressionlevelsofadipogenicgenemarkers274.3Dose-dependenteffectsofBPAF-inducedadipogenesis........................................274.424hrtreatmentwith10μMBPAorBPAFdoesnotincreaselipidaccumulationinmatureadipocytes.......................................................................................................284.510μMBPAFsignificantlyincreasesmRNAexpressionlevelsofNr3c1(GR)..........28
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4.6Nr3c1(GR)andTph1genetranscriptsarepositivelycorrelatedwith10μMBPAFtreatment.....................................................................................................................284.7BPAFcaninduceadipogenesisintheabsenceofthesyntheticglucocorticoiddexamethasone............................................................................................................284.8BPAFincreases5-HTsyntheticenzymegenetranscripts.......................................294.9TPH1inhibitiondoesnotinhibitBPAF-inducedadipogenesis................................294.10BPAFsignificantlyincreasesMaoageneexpression............................................304.11MaoaandPparggenetranscriptsarepositivelycorrelatedwith10μMBPAFtreatment.....................................................................................................................304.12MAOinhibitionblocksBPAF-inducedadipogenesis.............................................30
CHAPTER5:DISCUSSION............................................................................................415.1TheeffectsofBPAFonlipidaccumulationduringadipogenesis............................415.2TheeffectsofBPAFonlipidaccumulationinmatureadipocytes..........................435.3ThepotentialroleofglucocorticoidsignalinginBPAF-inducedadipogenesis.......495.4TheeffectsofTPH1inhibitiononBPAF-inducedadipogenesis..............................525.5TheeffectsofMAOinhibitiononBPAF-inducedadipogenesis..............................555.6Implications............................................................................................................58
CHAPTER6:FUTUREDIRECTIONS...............................................................................61
CHAPTER7:CONCLUSIONS........................................................................................62
CHAPTER8:REFERENCES............................................................................................63
CHAPTER9:APPENDIX................................................................................................78
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LISTOFFIGURES
Figure1.Summaryofstagesofadipogenesisfrommesenchymalprecursortoamatureadipocyte.............................................................................................................................2Figure2.BPAchemicalstructure........................................................................................4Figure3.ChemicalstructuresofBPAstructuralanalogues..............................................11Figure4.Modelofadipogenesisinvitro...........................................................................26
Figure5.BPAstructuralanaloguesinducelipidaccumulationin3T3-L1cells.......................31
Figure6.mRNAexpressionlevelsofadipogenicgenemarkersin3T3-L1cellstreatedwith10μMBPAFandBPA...............................................................................................................32Figure7.Dose-dependenteffectsofBPAFandBPAonlevelsofadipogenicgenemarkersin3T3-L1cellsbyRT-qPCR.....................................................................................................33Figure8.24hr treatmentwith10μMBPAorBPAFdoesnotsignificantly increase lipidaccumulationinmatureadipocytes..................................................................................34Figure 9. BPAF is able to induce adipogenesis in the absence of the syntheticglucocorticoiddexamethasone(Dex)................................................................................35Figure10.mRNAexpressionof5-HTsyntheticenzyme,Tph1,issignificantlyincreasedwith10μMBPAFtreatment.......................................................................................................36Figure11.Co-treatmentwith10μMBPAForBPAwith50μMpCPAdoesnotsignificantlyalterlipidaccumulationin3T3-L1cells................................................................................37Figure12.Effectsof co-treatmentwith10μMBPAForBPA±50μMpCPAonadipogenicgenemarkersin3T3-L1cells...............................................................................................38Figure13.mRNAexpressionofMaoaissignificantlyincreasedwith10μMBPAFtreatment.
...........................................................................................................................................39
Figure 14. Co-treatment with 10 μM BPAF + phenelzine (PZ) significantly decreases lipidaccumulationandPparggeneexpressionin3T3-L1cells.....................................................40
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LISTOFTABLES
AppendixTable1.ComparisonofUrinaryConcentrations(ng/ml)ofTotalBPAandBPAStructuralAnaloguesAcrossHumanStudies....................................................................78AppendixTable2.SummaryofBPAConcentrationsInducingSignificantLipidAccumulationInVitro........................................................................................................79AppendixTable3.PrimerSequencesforRT-qPCR...........................................................80
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LISTOFABBREVIATIONS
5-HT–serotoninAADC–aromaticaminedecarboxylaseADIPOQ–adiponectinANOVA–AnalysisofVarianceATCC–AmericanTypeCultureConditionBMI–bodymassindexBPA–BisphenolABPA-G–BisphenolAglucuronideBPAF–BisphenolFBPB–BisphenolBBPC–BisphenolCBPF–BisphenolFBPS–BisphenolSBPZ–BisphenolZC/EBP;Cebp–CCAAT-enhancerbindingproteinCCL20–chemokine(C-Cmotif)ligand20cDNA–complimentaryDNACfd–complementfactorD;adipsinCHAMACOS–CenterfortheHealthAssessmentofMothersandChildrenofSalinasCNS–centralnervoussystemCt–comparativecycletimesDDC–dopadecarboxylaseddH20–doubledistilledwaterDex–dexamethasoneDHC–dehydrocorticosteroneDMEM–Dulbecco'sModificationofEagle'sMediumDMSO–dimethylsulfoxideDOHaD-DevelopmentalOriginsofHealthandDiseaseEDC–endocrinedisruptingchemicalEFSA–EuropeanFoodSafetyAuthorityEPA–EnvironmentalProtectionAgencyFABP4–fattyacidbindingprotein4FAS–fattyacidsynthaseFBS–fetalbovineserumFDA–FoodandDrugAdministrationFMI–fatmassindexGD–gestationaldayGR;Nr3c1–glucocorticoidreceptor;nuclearreceptorsubfamily3groupcmember1HIAA–5-hydroxyin-doleaceticacid
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HSL–hormonesensitivelipaseHtr–serotoninreceptorIBMX–3-isobutyl-1-methylxanthineIFN-γ–interferongammaIL-6–interleukin6iWAT-inguinalwhiteadiposetissueLPL–lipoproteinlipaseMAO–monoamineoxidaseMCP-1–monocytechemoattractantprotein1MIA–5-methoxy-indolacetateMSC-mesenchymalstemcellNHANES–NationalHealthandNutritionExaminationSurveyNOAEL–noobservedadverseeffectlevelpCPA–para-chlorophenylalaninePEPCK;Pck1–phosphoenolpyruvatekinasePGC-1α; Ppargc1a – peroxisome proliferator-activated receptor gamma coactivator 1alphaPlin–perilipinPND–postnataldayPPARγ;Pparg–peroxisomeproliferatoractivatedreceptorgammapWAT-parametrialwhiteadiposetissuePZ–phenelzineROSI–rosiglitazoneRT-qPCR–Real-TimeQuantitativePolymeraseChainReactionS.E.M–standarderrorofthemeanSERT–serotonintransporterSREBP1c–sterolregulatoryelementbindingprotein1cSSAO/PrAO–primaryamineoxidaseTBBPA–tetrabromobisphenolATCBPA–tetrachlorobisphenolATNF-α–tumornecrosisfactoralphaTPH–tryptophanhydroxylasetTDI–temporarytolerabledailyintakeWC–waistcircumferenceWHO–WorldHealthOrganization
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CHAPTER1:INTRODUCTION
1.1AdipogenesisandWhiteAdiposeTissueFunction(WAT)
Adipogenesisistheconversionoffibroblastprecursorstolipid-ladenadipocytes1.New
adipocytesarise fromadiposestromalmesenchymalstemcells (MSCs),arecommitted
topreadipocytesandundergodifferentiationintomatureadipocytesoverthecourseof
six distinct stages1,2 (Figure 1). Adipose mass is expanded through adipocyte
hypertrophy (increase in cell size) and hyperplasia (increase in cell number)3. The
formationofadipocytes inhumansbeginsduring the14thweekofdevelopment4. It is
postulated that themajority of new adipocytes are recruited early in childhood, and
thus, adipocyte number remains relatively stable in adulthood, with 10% of cells
renewedeveryyear5.
Adipogenesis is a highly regulated process consisting of a cascade of transcription
factors.AsreviewedbyRosenetal.(2000)andFarmer(2006),CCAAT-enhancerbinding
proteins (C/EBPβ, C/EBP𝛿) activate peroxisomeproliferator activated receptor gamma
(PPARγ), the master regulator of adipogenesis6,7. This results in activation of C/EBPα
resultinginapositivefeedbackloopbetweenPPARγandC/EBPα6–8.Anotherimportant
regulator is sterol regulatory element binding protein 1c (SREBP1c) which has been
shown toactivatePPARγ6,9 and induceexpressionof theSREBP1c targetsof amature
adipocyte,fattyacidsynthase(FAS)andlipoproteinlipase(LPL)9,10.Othermarkershighly
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expressed in mature adipocytes include fatty acid binding protein 4 (FABP4) and the
adipokines,adiponectin(ADIPOQ)andadipsin11.
Figure1.Summaryofstagesofadipogenesisfrommesenchymalprecursortoamature
adipocyte.Figureadaptedfrom1.
Adipocytesplayacriticalrole inregulatingglobalenergyhomeostasis12.Whenthere is
caloricexcess,adipocytesexpandandstoreenergyastriglyceridesprotectingcellsfrom
ectopiclipidaccumulationandlipotoxicity(Reviewedin:12).Whenthereisashortageof
nutrients, free fatty acids and glycerol are subsequently released from the stored
triglyceridepool12. Free fattyacidsare incorporated intoother tissueswhere theyare
re-esterifiedtotriglyceridesorundergoβ-oxidation12.Freefattyacidsmayalsoremain
withinadipocytestoundergoβ-oxidation12.
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While it was once thought that adipose tissue was merely a storage depot, adipose
tissuealsoactsasanendocrineorgancapableofsecretingfactorstermedadipokines13.
These biologically active factors include leptin, ADIPOQ, resistin and inflammatory
mediatorssuchastumornecrosisfactoralpha(TNF-α),interleukin6(IL-6)andmonocyte
chemoattractantprotein1(MCP-1)13.Thereisconsiderableevidencedemonstratingthe
effectsoftheseadipokineson insulinsensitivity, lipidmetabolism,energyhomeostasis
and inflammatoryprocesses13.Therefore,perturbations inadiposetissuedevelopment
and/orfunctionareanessentialcomponentintheetiologyofobesity.
1.2EndocrineDisruptingChemicalsandBisphenolA
It has been postulated that endocrine disrupting chemicals (EDCs) play a key role in
mediating the obesity epidemic14. An endocrine disruptor has been defined by the
WorldHealthOrganization (WHO) as: “anexogenous substanceormixture that alters
the function(s) of the endocrine system and consequently causes adverse effects in
intact organism, or its progeny or (sub) population”15. The “obesogen hypothesis”
proposes that EDCs found in our environment and food play a key role inmediating
adipose tissue development and function16. This can be through increasing the
formation of new adipocytes, promoting lipid accumulation in existing fat cells or
through alterations in food intake and satiety17. Exposure to potential endocrine
disruptors is widespread. Phthalates, pesticides, herbicides, pharmaceuticals, flame
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retardants,industrialwasteandfoodadditiveshavebeenextensivelyevaluatedinorder
todeterminetheireffectsonadipogenesisandadiposetissuefunction(Reviewedin:14).
Oneof themostwidelystudiedEDCs,BisphenolA (BPA),hasalsobeen identifiedasa
potentialobesogen18.
Figure2.BPAchemicalstructure(https://pubchem.ncbi.nlm.nih.gov).
1.2.1HumanExposure
BPA is predominantly used is themanufacturing of polycarbonate plastics, and epoxy
resins19. Additionally, BPA is found in thermal paper products such as cash register
receipts20.Human exposure is ubiquitous; BPAhas been detected in urine and serum
samples from infant toadultpopulationsaround theworld21–23.Biomonitoring studies
have reported BPA concentrations in urine and serum in the ng/ml range (urine 1–5
ng/ml;serum0.5–2ng/ml)24.Acomparisonbetweentwolargescalestudies intheU.S
(NationalHealth andNutrition Examination Survey (NHANES) 2007–2008) andCanada
(Canadian HealthMeasures Survey (CHMS) 2007–2009) revealed urinary levels of 2.1
ng/ml(95%CI1.9,2.3)and1.2ng/ml(95%CI1.1,1.2)respectively25.Thiscorresponded
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toanestimatedintakeof36.9ng/kgbodyweight/day(95%CI34.1,40.0)intheU.Sand
21.4ng/kgbodyweight/day(95%CI20.0,22.9)inCanada.
BPA exposure through dietary sources is regarded as the primary route of human
exposure, although exposure can also occur via dermal, sublingual and inhalation19,21.
Onceingested,BPAisrapidlymetabolizedintheliverandguttoBPAglucuronide(BPA-
G)andsulfatemetabolites26.Closeto100%ofBPAtakenorallyisexcretedinurineinits
conjugatedformwithin24hrs26.Initsconjugatedform,BPA-G,hasbeenthoughttobe
biologicallyinactive,however,arecentstudyhasdemonstratedapossibleactiverolefor
thismetabolite27. The no observed adverse effect level (NOAEL) set by the Food and
DrugAdministration(FDA)forBPAis5mg/kgbodyweight/dayandtheEuropeanFood
Safety Authority (EFSA) has set the temporary tolerable daily intake (tTDI) at 4 μg/kg
bodyweight(EFSA2015).Recently,Huangetal.(2017)usedurinarylevelsofBPAfrom
studiesworldwidetoestimateglobaldaily intakeofBPA29.Theypredictedtheaverage
global daily intake as 30.76 ng/kg body weight /day for adults, 60.08 ng/kg body
weight/day for children and 42.03 ng/kg body weight/day for pregnant women. This
corresponds to levels 2 and 1.4 times greater in children and pregnant women
comparedtoexposures innon-pregnantadults, suggestingthat thesetwogroupsmay
haveincreasedsusceptibilitytothedeleteriouseffectsofBPA.Becausetheseestimated
intakesarebelowthetTDIandNOAEL,somecontroversyremainsastowhetherthese
thresholdsshouldbeloweredfurther.
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1.2.2EpidemiologicalStudies
AlthoughthereremainssomequestionabouttheimportanceofBPAexposureinoverall
obesity rates, cross-sectional human studies in children, adolescents and adults have
shown primarily positive associations between higher urinary BPA levels and odds of
obesity.Indeed,arecentmeta-analysisof12studiesreportedthatwhencomparingthe
highestversusthelowesturinaryBPAconcentrations,thepooledoddsratios(ORs)were
1.67 (95% CI 1.41, 1.98) for obesity and 1.48 (95% CI 1.25, 1.76) for increased waist
circumference (WC)30. A cross sectional study of 3390 adults determined BPA was
positively associated with obesity and insulin resistance in individuals in the highest
quartileofurinaryBPAexposure31.Similarly,aprospectivestudyfollowing888adultsfor
4years foundthatparticipantswithhigherurinaryBPA levelshadan increasedriskof
centralobesity32.
In child/adolescent populations, a recent study using data from NHANES 2003–2008
foundapositiveassociationbetweenincreasingBPAurinaryconcentrationsandobesity
aftertakingintoaccountconfoundingfactors,withanORof2.57(95%CI1.72,3.83)for
obesity in the highest urinary quartile33. Using data from the Center for the Health
AssessmentofMothersandChildrenofSalinas(CHAMACOS)longitudinalstudy,Harley
et al. (2013) found higher urinary BPA levels during pregnancy were associated with
decreased BMI-z score, percent body fat and obesity in females at age 934. However,
whenurinaryBPAlevelsweremeasuredat9yearsofageinbothmalesandfemales,
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levels were positively associated with BMI, WC, body fat percentage and obesity34.
Similarly, a recent study evaluating prenatal BPA exposure in 500 mother-child pairs
from Crete, Greece evaluating BMI, WC and skinfold thickness found a negative and
positiveassociationingirlsandboysrespectivelyatage435.WhenurinarylevelsofBPA
were measured at 4 years, a positive association was determined for all the above
parameters35. Conversely, another study evaluating BPA exposure during pregnancy
found no change in BMI but positive associations with percent body fat,WC and fat
massindex(FMI)atage7.Whensexwastakenintoaccount,apositiveassociationwas
onlyobservedwithFMIingirls36.
Taken together, based on current epidemiological studies higher levels of BPA in
childhood and adolescence havemost consistently been associatedwith an increased
oddsofobesity37. Importantly, this is aperiodofextensiveadiposetissuegrowthand
development5,38–40.
1.2.3Invitroandinvivostudies
Inboth invitroand invivomodels,BPAhasbeenshowntopromoteadipogenesisand
altergenesessentialtolipidmetabolism41–44.
AppendixTable1providesasummaryofstudiesevaluatingtheeffectsofBPAon lipid
accumulation invitro,demonstratingthatBPAhasadipogeniccapacityatbothlowand
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high doses.Moreover, in addition to enhanced lipid accumulation, BPAhas also been
shown to alter adipokine expression/secretion45–48, glucose uptake/insulin stimulated
glucose utilization49–51 and inflammatory markers50,51 in adipocytes in vitro. Taken
together,thesestudiesdemonstratethatBPAnotonlyhaseffectsonlipidaccumulation,
butalsoonkeymetabolicprocessesregulatingadiposetissuefunction.
Studies evaluating the effects of in vivo BPA exposure in rodents have not shown
alterations in body weight52,53; however, disruptions of other metabolic parameters,
suchasglucosehomeostasis,insulinsensitivity,cholesterolbiosynthesisaswellasgenes
involved indenovo lipogenesishavebeenshowntobealteredbyBPAtreatment.For
example,inmiceBatistaetal.(2012)didnotobservechangesinbodyweightfollowing
exposure to 100μg/kg BPA for 8 days53. However, the BPA-treated animals displayed
dysregulatedglucose/insulinhomeostasis.Similarly,Marmugietal. (2014)didnot find
anyalterationsinbodyweightwhenmaleCD1miceweretreatedfrom6weeksofage
for 8 months with BPA (5–5000 μg/kg/day)52. The authors did observe increased
parametrialwhite adipose tissue (pWAT)weight, glucose intolerance, hyperglycaemia,
hypercholesterolemia and increased cholesterol biosynthesis in the liver. Although
exposuretoBPAinadulthoodhasbeenassociatedwithmetabolicdisruption,itseffects
on body weight may be more profound when the exposure occurs during critical
windowsofdevelopment(i.e.,duringpregnancy,earlychildhood,duringpuberty),when
formationofnewfatcellsisrapidlyoccurringandadipocytenumberisestablished5,38.
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InsupportoftheDevelopmentalOriginsofHealthandDisease(DOHaD)hypothesis,that
perturbations during critical periods of development may lead to adverse metabolic
outcomes later in life (Reviewed in: 54,55), BPA has been extensively evaluated for its
effectsonobesityriskwhenexposuresoccurduringearlylife.StudiesofBPAexposure
duringpregnancyandlactationhavebeenshowntoincreasebodyweightandadiposity
by adulthood with clear sex specific effects42,43,56–60. However, the literature is
inconsistentwithsomestudiesreportingdecreasedbodyweightonly infemales56,61,62,
while others have found no effect of BPA exposure in utero through lactation63–65.
Further, a study evaluating BPA exposure during adolescence found increased body
weightandadiposityinbothmalesandfemales66.
Tworecentstudies,usingenvironmentallyrelevantdosesbelowtheNOEL,determined
dose and sex specific differences in BPA exposed offspring. Lejonklou et al. (2017)
exposedratsinuteroto0.5μg/kgbodyweight/dayor50μg/kgbodyweight/dayBPA63.
Whilenodifferences inweightgainwereobserved, femaleshad significantlyelevated
plasma triglyceride levels (50 μg/kg body weight/day) and significantly increased
adipocyte density with exposure at the lower dose. Males, however, only displayed
significantincreasesinplasmatriglyceridelevelsatthelowerdose63.Rubinetal.(2017)
treated mice perinatally, or perinatally in addition to peripubertally, to determine
whetheraspecificexposurewindowalteredbodyweightandbodycompositioninCD-1
mice60.Theychosedosesrangingfrom0–250μgBPA/kgbodyweight/day,withnodose
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exceedingtheNOELof5mg/kgbodyweight/day.Whileperinatallyexposedmaleshad
anincreaseinweightgain,whencombinedwithperipubertalexposure,thedifferences
inweightwithcontrolanimalsdeclined.However,femaleshadincreasedbodyweight,
elevated triglyceride levels, dysregulated glucose homeostasis and evidence of insulin
resistancewiththeadditionofperipubertalexposure.Thesestudieshighlightthatmany
factors can lead to variations among findings including type of species, animal strain,
sex, dose, mode of administration, diet and window of exposure60,63. Moreover,
althoughdata surroundingBPA exposure in vivo is not consistent,mounting evidence
fromhuman,animalandinvitrostudiessuggestBPAcanaffectpathwaysimportantfor
thedevelopmentofobesity.
1.3AreBisphenolAAnaloguesSafeAlternatives?
WiththerisingconcernofhumanexposuretoBPA,theuseofBPAstructuralanalogues
as replacements has become widespread67,68. This is alarming as very limited
toxicologicaldataon theseanalogues is available. Structural analoguesofBPA include
BisphenolS(BPS),BisphenolF(BPF),BisphenolB(BPB)andBisphenolAF(BPAF)among
others67.
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Figure3.ChemicalstructuresofBPAstructuralanaloguesfromlefttoright:BPB,BPAF,
BPS,BPF(https://pubchem.ncbi.nlm.nih.gov).
While these BPA-replacement compounds should ideally exhibit less harmful effects
than BPA, a growing number of studies have demonstrated that these structural
analoguesalsopossessdeleteriouseffects,comparabletoorgreaterthanBPAitself68–72.
Of the structural analogues, BPShasbeen themost extensively studied. BPS is often
usedasanalternative in“BPA-free” thermalpaper73 aswellas inbabybottles74.BPS
hasalsobeen found to leach from foodcans75. Héliès-Toussaintetal. (2014) showed
thatatlowdoses(i.e.,ƒMrange)bothBPAandBPSincreasedtriglyceridelevelsin3T3-
L1 cells. Additionally, lipolysis was decreased significantly in both BPA and BPS
treatments, with a greater reduction observed with BPS at lower concentrations76.
Additionally, BPS was shown to act through a PPARγmediatedmechanism in 3T3-L1
cells, significantly increasing lipid accumulation (10–50 μM)77. Of concern, BPS was
showntoactasastrongerinducerofadipogenesisthanBPA.Further,anotherstudyhas
alsoreportedtheobesogeniceffectsofBPSinvivo78.Inanearlierstudy,80μMofBPB,
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BPE,BPFandBPSwasabletoincreasetriglyceridelevelsin3T3-L1cells,tolevelssimilar
to that seen with BPA41. Recently, the adipogenic effects of BPAF were evaluated in
AmericanTypeCultureCondition(ATCC)3T3-L1cellsatdosesrangingfrom1nMto10
μM.BPAFtreatmentresultedinsignificantlipidaccumulation79,aneffectwhichmaybe
duetoitspotentialinteractionwiththePPARγligandbindingdomain80.
WiththeuseofBPAdecliningandtheuseofitsstructuralanaloguesontherise,itisnot
surprisingthatthisparallelstheincreaseindetectionintheenvironmentandinhuman
exposure levels. BPAF and BPF have been detected in high levels in surfacewater in
Taihu Lake, China81. Recently, BPAF, Bisphenol C (BPC) and Bisphenol Z (BPZ) were
detected in the highest quantities in aquatic organisms, displaying higher
bioaccumulativeabilitythanBPA82.AnotherstudymeasuredurinesamplesofAmerican
adultsduring2000-2014ateighttimepointstodetectconcentrationsofBPAandthree
analogues,BPS,BPFandBPAF83 .BPAwasdetectedmostfrequentlybutdisplayedthe
greatestdecline,(99–74%)whereasBPSshowedthegreatestriseindetection(19–74%).
BPFwasthehighestdetectedanalogue(42–88%),howeverBPAFwasonlydetected in
<3% of samples. Appendix Table 2 shows urinary concentrations of BPA and BPA
structuralanaloguesacrosshumanstudies.
Recently, data from NHANES 2013–2014 was used to see if BPA, BPF and BPS were
positively associated with obesity in adults84. While BPA was shown to be positively
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associatedwithbothgeneralandabdominalobesity,noassociationswereobservedfor
BPSandBPF.However,forBPS,theauthorshighlightedthatasignificantassociationwas
foundwithBMI,butonlyinthethirdurinaryquartile84.Moreover,themetabolictoxicity
ofthesecompoundsislargelyunknowndespitethefactthattheyarestructurallysimilar
toBPA.Thus,theymaytargetmanyofthesamepathwaysthatareimportantforenergy
homeostasisincludingserotoninsignaling,whichisdisruptedbyBPAinthebrain.
1.4RoleofPeripheralSerotoninonAdipogenesisandWhiteAdiposeTissueFunction
Serotonin (5-hydroxytryptamine, 5-HT), a monoamine formed from the amino acid
tryptophan, has been shown to play an important role in both central andperipheral
nervous system functions85,86. Tryptophan hydroxylase (TPH; Tph), the rate limiting
enzyme in the synthesis of 5-HT, has two isoforms87. TPH1 is responsible for the
production of 5-HT in peripheral tissueswhereas TPH2 is predominantly found in the
centralnervoussystem(CNS)87.As5-HT isunabletocrossthebloodbrainbarrier, it is
important to note that two distinct functional pools of 5-HT exist87. Serotonin is
producedintwoenzymaticsteps85.Firstly,L-tryptophanbecomes5-hydroxytryptophan
through the activity of TPH1. Secondly, aromatic amine decarboxylase (AADC), also
knownasdopadecarboxylase(DDC;Ddc),actstoconvert5-hydroxytryptophanto5-HT.
IntheCNS,5-HTactsasaneurotransmittercontrollingphysiologicalprocessesincluding
feeding, behavior and energy expenditure88,89. In peripheral tissues, 5-HT has been
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shown to regulate metabolic homeostasis in adipocytes, liver, pancreas, muscle and
macrophages(Reviewedin:85).Mostofthe5-HTfoundintheperipheryarisesfromthe
enterochromaffin cells located in the gut85. Serotonin produced by the gut is either
stored in platelets or circulates in the blood85. Serotonin signaling in the periphery is
mediated throughbinding tooneof fourteen receptors foundon target tissueswhich
havebeenseparatedintosevendistinctclasses(Htr1toHtr7)85.Serotoninreceptorsare
G-protein coupled receptors, with the exception of Htr3, the only ligand-gated ion
channel85,90.
Serotoninhasbeenshowntopromoteadipogenesisandaffectadiposetissuefunction.
In rodentsonahigh-fatdiet,an increase in5-HT levelsbeenshown tobeassociated
withobesity91,92whereasTph1deficientmicehaveloweradiposityandgainlessweight
whileonahigh fatdiet93,94. Indifferentiatingprimary ratadipocytes,Tph1 andmRNA
levels of 5-HT receptors,Htr2a,Htr2b andHtr2c,were significantly upregulatedwhen
compared to undifferentiated control cells95. Moreover, primary rat adipocytes were
abletoindependentlyproduceandsecrete5-HT95.Kinoshitaetal.(2010)reportedthat
TPH1 is essential for adipogenesis in 3T3-L1 cells96 and 5-HT has been shown to be
producedoverthecourseofdifferentiation94.Inthesecells,the5-HTreceptorsubtypes,
Htr1a,Htr1b,Htr1d,Htr1f,Htr2a,Htr2c,Htr5a,Htr5b,Htr6,Htr7havebeenidentified96.
Additionally,antagonistsforbothHtr2candHtr2adecreasedadipogenesis,suggestinga
role for these receptors during the differentiation process96. Another group showed
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inhibitionoftheHtr2areceptorresultedinsignificantlyreducedexpressionoflipogenic
genes94. Therefore, Htr2a receptor activation in WAT may promote lipogenesis94.
Conversely, stimulation of Htr2b seems to suppress lipogenesis97 while stimulating
lipolysis through itseffectsonhormonesensitive lipase (HSL) inWAT98.Stimulationof
Htr2ahasalsobeenshowntoblockadiponectinexpressioninadipocytes99,whichcould
leadtoelevatedgluconeogenesisintheliverandimpairedinsulinsensitivity(Reviewed
in: 100). Taken together, these studies suggest a functional role for 5-HT signaling in
regulatingkeyprocessesofadipogenesisandlipidmetabolisminWAT.
Following5-HTuptakeby5-HTtransporters(SERT),5-HTismetabolizedto5-hydroxyin-
doleaceticacid(HIAA)and5-methoxy-indolacetate(MIA)bymonoamineoxidase(MAO;
Mao)101.While adipocyteshavebothMAOAandMAOB,MAOA is expressed inhigher
amounts in human adipocytes102 and has been shown to be present in 3T3-L1 cells
duringdifferentiation96.Moreover, themetabolitesof5-HT,HIAAandMIA,havebeen
implicated in the adipogenic process. MIA is believed to act as a PPARγ agonist
stimulatingadipogenesisin3T3-L1cells101.Additionally,MIAandHIAAhavebeenshown
todirectlybindtotheactivationfunction2pocketofPPARγ101.Tofurthersupportthis
hypothesis,Grèsetal.(2013)showedthatoxidationof5-HTinadipocytesincreaseslipid
accumulation and stimulates the expression of downstream PPARγ genes103.
Pharmacologically inhibiting MAO with an irreversible non-selective MAO inhibitor,
phenelzine,significantlydecreasestriglyceridecontentin3T3-L1cells104.Therefore,the
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role of 5-HT in promoting adipogenesis may be two-fold. Serotonin can promote
adipogenesisbyactingthrough5-HTreceptorssuchasHtr2aorHtr2cor ina receptor
independentpathwaythrough5-HTcatabolism.
1.5BPAandSerotoninSignaling
ThereisevidencelinkingBPAexposureand5-HTsynthesisandmetabolismintheCNS.
Previousreports inrodentshaveshownBPAsignificantly increases5-HTlevels105–107.A
studyevaluatingperinatalexposuretoBPA inmicereported increasedmRNAlevelsof
Tph2 and Htr1a in the hippocampus, Htr2a in the midbrain and decreased Htr2c
expression in the striatum108. Additionally, the effects of BPA, BPF and BPS were
evaluated on the 5-HT system in the prefrontal cortex of female rats109. BPA
increased5-HTreceptorsHtr3aandHtr7whilebothBPFandBPS increasedexpression
ofHtr4109.Tph1wasdecreasedinallthreetreatmentgroups109.Furthersupportforthe
associationbetweenBPAandanaloguesandserotonergicsignalingcomesfromtheU.S
Environmental Protection Agency’s (EPA) ToxCast database
(http://www.epa.gov/ncct/toxcast/).BPA,BPAFandBPBhaveallbeenpredictedactive
against5-HTreceptors(Htr1a,Htr2a,Htr2c,Htr3a,Htr4,Htr5a,Htr6,Htr7).Thiscoupled
with the effects of BPA on5-HT signaling in the brain demonstrates a plausible
mechanismfortheactionofBPAanditsstructuralanaloguesinperipheraltissues.
1.6BPAandGlucocorticoidSignaling
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Glucocorticoidsignalingplaysanimportantroleduringadipogenesis8,110,111andthusany
alterations in signaling may impact the adipogenic process. In adipogenesis, GR
activation leads to the induction of C/EBP𝛿, a transcription factor critical during early
adipogenesis112;bothC/EBP𝛿andC/EBPβhavebeenshowntoactivateC/EBPαin3T3-L1
cells113. GR knockdown in 3T3-L1 cells, significantly impairs lipid accumulation8.
Additionally, upon hormonal induction, both GR and C/EBPβ co-localize resulting in
histoneH3acetylationwhichinturnpromotesexpressionofadipogenicmarkerssuchas
PPARγ8. Therefore, as PPARγ and C/EBPα are key regulators in adipogenesis, it is not
surprisingthatglucocorticoidsareusedtostimulate3T3-L1differentiation111.
A number of EDC’s, including BPA, have been evaluated for their ability tomodulate
glucocorticoid signaling114,115. Througha luciferase reporterassay in3T3-L1 cells,BPA
was reported to significantly activateGR activity115.While BPA alonewas not able to
induce adipogenesis in the absence of the synthetic glucocorticoid, dexamethasone,
under submaximal conditions with the addition of dehydrocorticosterone (DHC),
significant increases inadipocytedifferentiationwereobserved115.Additionally,others
have investigated the potential link between BPA and GR signaling as a potential
mechanism of BPA-induced adipogenesis44,116. Interestingly, in contrast to the above
study115, BPAwas shown to promote adipogenesis in the absence of dexamethasone
(0.01–10nM),howevertheauthorsfoundnotranscriptionalactivationoftheGRusing
luciferasegeneassays116. Inprimaryhumanpreadipocytes, theGRantagonist,RU-486
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didnotblockBPA-inducedadipogenesissuggestingthattheactionsofBPAmaynotbe
mediatedsolelyviaGRsignaling44.Nonetheless,recentlythebindingaffinitiesofseveral
bisphenolcompoundswithGRweredeterminedusingacompetitivebindingassay.Of
theelevencompoundscompared,BPAandBPSdisplayed intermediatebindingaffinity
withGR,whileBPAFhadtheleastpotentbindingaffinity117.Interestingly,arelationship
between glucocorticoid and 5-HT signaling has been suggested in a previous study.
Moreover, treatment with a glucocorticoid in rats has been linked to significantly
increasedmRNA and protein expression ofTph1 andDdc, in addition to significantly
higher 5-HT levels in intra-abdominal adipose tissue118. Taken together, these reports
suggest thepotential role forBPA inmodulatingGRsignalingduringadipogenesisand
highlightsapossiblemodeofactionforotherbisphenolcompounds.
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CHAPTER2:HYPOTHESISANDOBJECTIVES
2.1RationaleandHypothesis
Rationale:
Concerns have been raised about the impact of human exposure to environmental
chemicals. The“obesogenhypothesis”postulatesthatEDCsfoundinourenvironment
and food play a key role in mediating adipose tissue development and function16.
AlthoughanumberofEDCshavebeenshowntocausedysregulatedlipidmetabolism14,
the mechanistic pathways have yet to be fully elucidated. BPA, one of the highest
volumechemicalsproducedglobally,isusedfortheproductionofpolycarbonateplastic
and epoxy resins19. Based on human, animal and cell culture studies, BPA has been
identified as a potential obesogenic compound37,41,42.With the increasing healthrisks
associatedwithBPAexposure,BPAanalogueshavecommonlybeenusedassubstitutes
in consumer products. More alarming, the associated risks of BPAanalogues on
metabolic diseases are poorly defined. There is now substantial evidence that 5-HT
signaling in peripheral tissues plays an important role in regulating metabolic
homeostasis, including effects on adipose tissue deposition and lipid homeostasis85.
Interestingly,BPAhasbeenshowntoalter5-HTsignalingintheCNS106,108.Therefore,it
is plausible that BPA and its structural analoguesmay disrupt peripheral serotonergic
networksleadingtotheincreasedfatdepositionandalteredlipidhomeostasisobserved
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inobesity.
Hypothesis: Structural analogues of BPA (BPAF, BPS, BPF and BPB) will potentiate
adipogenesis and cause dysregulated lipid homeostasis. I postulate these metabolic
perturbationswillbeduetoadisruptioninperipheral5-HTsignaling.
2.2Objectives
ThespecificaimsofmyM.Sc.projectare:
1) ToexaminetheeffectsofBPAanaloguesonadipogenesisandlipidhomeostasis
in3T3-L1cells.
2) To determine whether glucocorticoid signaling plays a role in BPAF-induced
adipogenesis.
3) Todetermine the roleof5-HTsignaling inmediating theeffectsofBPAand its
structuralanaloguesonadiposetissuedevelopmentandfunction.
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CHAPTER3:MATERIALSANDMETHODS
3.1Cellculture,murineadipocytedifferentiationandtreatments
The3T3-L1cell line, fibroblasticcellswhichareprogrammedto theadipocyte lineage,
has been widely used to investigate adipocyte differentiation in vitro111,119. A
differentiation cocktail consisting of insulin, 3-isobutyl-1-methylxanthine (IBMX), fetal
bovine serum (FBS) and glucocorticoids is frequently used to stimulate adipogenesis
110,111,120. This model system has been essential in evaluating preadipocyte-adipocyte
differentiationinvitro.
3T3-L1preadipocytes(ATCC®CL-173™)weregrowninDulbecco'sModificationofEagle's
Medium (DMEM) (Corning, 10-014-CV, Manassas, VA, USA) with 1 g/L glucose
supplementedwith 10% FBS (Gibco, 12483-020, Grand Island, NY, USA), 1% Penicillin
Streptomycin(P/S)(Gibco,15140-122)and1%L-Glutamine(L-Glut)(Gibco,25030-081)
at37°Cinahumidifiedatmosphereof95%airand5%CO2.Cellsweregrownin100mm
x20mmpolystyrene tissue-culture treatedplates (Corning, 353003,Corning,NY,USA)
andpassagedonce60-70%confluencewasreached.Forexperiments,cellswereplated
in6-wellplates (Corning,353046)ormaintained in100mmx20mmplates togrowto
confluence.At1daypost-confluence,designatedasday0,cellsweredifferentiatedinto
adipocytesinDMEM(Corning,10-013-CV)with4.5g/Lglucosecontaining10%FBSusing
an inductioncocktailconsistingof2µg/ml insulin(Sigma-Aldrich, I0516,St.Louis,MO,
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USA), 0.5 mM of IBMX (Sigma-Aldrich, I5879), and 0.25 µM dexamethasone (Dex)
(Sigma-Aldrich,D4902), unless otherwise indicated (51).After the initial 72hours, the
differentiationmediawasreplacedwithmaintenancemediacontainingDMEMwith4.5
g/Land2µg/mlinsulin.Mediawasreplacedevery48hrsuntilday9(Figure4).
CellsweretreatedwithBPA,BPS,BPB,BPFandBPAF(TorontoResearchChemicalsInc.,
Toronto,ON,Canada)ata finalconcentrationof10μMbyadding indicatedbisphenol
compoundsdissolvedindimethylsulfoxide(DMSO)startingatday0.Fordoseresponse
experiments,cellsweretreatedwithBPAandBPAFatvaryingconcentrations(0.01–10
μM). Cells were treated with DMSO at or below 0.02% as a vehicle control. For
antagonistexperiments,cellswereco-treatedwith50μMpara-chlorophenylalanine(4-
Chloro-DL-phenylalanine; pCPA) (Sigma-Aldrich, C6506) or 100 μM Phenelzine sulfate
salt(Sigma-Aldrich,P6777)dissolvedindoubledistilledwater(ddH20)asindicatedfrom
day0 today9. ForAim1,2µMrosiglitazone (ROSI) (Sigma-Aldrich,R2408)wasalso
addedtotheinductioncocktailfor72hrs.
3.2LipidStainingandQuantification
Atday9,cellswerestainedwithOilRedO(Sigma-Aldrich,00625)solution.Briefly,cells
werefixedwith10%formaldehydefor60minatroomtemperature.Cellswerewashed
withDulbecco’s Phosphate Buffered Saline (DPBS) (Corning, 21-031-CV) followedwith
60%isopropanolfor5minatroomtemperature.FilteredOilRedOsolution(300mgof
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OilRedOaddedto100ml99%isopropanol)wasaddedtoeachwell(1ml/well)for20
min.ThewellswerethenwashedwithddH2Ountilclearandimmediatelyfollowedwith
60%isopropanol.After10min,100µlofeachextractedsamplewastransferredtoa96-
wellplateintriplicate.Theplateswerereadat510nminaplatereader(Synergy™H4
Hybrid Microplate Reader, BioTek Instruments, Winooski, VT, USA) to measure
absorbance.
3.3RNAExtractionandQuantification
At day 9, cells from differentiated adipocytes were harvested using TRIzol® Reagent
(Ambion,15596018,CarlsbanCA,USA)andstoredat-80°Cuntilfurtheruse.Cellswere
lysedwith20GneedleandRNAwasextractedbyadding0.2mLchlorophorm/1mltrizol.
Following centrifugation, the upper aqueous phase was removed and 0.5 ml
isopropanol/1mLtrizolwasadded.Aftercentrifuging,thepelletwaswashedwith75%
ethanol2–3times.Thepelletwasthenairdriedandre-suspendedin30μLnucleasefree
water(Qiagen,129115,Hilden,Germany).RNAconcentrationinng/µlwasdetermined
byNanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific USA) and stored at -
80°Cuntilfurtheruse.
3.4ComplimentaryDNA(cDNA)Synthesis
Atotalof4μgofcDNAwasmadeusingtheHighCapacitycDNAReverseTranscription
Kit (Applied Biosystems, 4368814, Foster City, CA, USA) as per manufacturer’s
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instructions.40μLof cDNAwasmadeusing the followingconditionsusing the iCycler
thermocycler (BioRad Laboratories,Hercules, CA,USA): 25°C for 10min; 37°C for 120
min;85°Cfor5min.cDNAwasstoredat-20°Cuntilfurtheruse.
3.5PrimerDesign
Primers were designed using Primer-BLAST (National Center for Biotechnology
Information, Bethesda,MD,USA). If transcript varianceswere present, ClustalOmega
(EMBL-EBI,Hinxton,Cambridgeshire,UK)wasusedto findoverlappingvariantregions.
PCR product size was set to 50–150 base pairs. The following melting temperature
conditions were followed: Minimum: 58°C; Optimal: 60°C; Maximum: 60°C; Max
difference: 2°C. Each primer sequencewas run through OligoAnalyzer 3.1 (Integrated
DNA Technologies, Coralville, IA, USA) to check for hairpin, self-dimer and hetero-
dimerization. Sequences selected had a ∆G (kcal/mol) > than -9 for all parameters.
Primer sequences were synthesized by MOBIX Lab (Sanger Sequencing and Oligo
Synthesis Facility, Hamilton, ON, Canada) and validated by examining melting point
curves.
3.6Real-TimeQuantitativePolymeraseChainReaction(RT-qPCR)
RT-qPCRwasperformedusingtheQuantabioPerfecta®SYBR®GreenFastMix®(95072-
05K,Beverly,MA,USA) todeterminerelative foldchangeofeachgenetranscript.The
plates(LightCycler480384-MultiwellPlates,0472974900,Roche,Mannheim,Germany)
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were analyzed by Light Cycler 480 IIMachine (Roche) under the following conditions:
polymerase activation (95°C for 10 min); 50 cycles of denaturing (95°C for 10 sec);
annealing(60°Cfor10sec);elongation(72°Cfor15sec).Sampleswereranintriplicate
and the expression of each gene was normalized to the expression of β-actin. The
relativefoldchangesweredeterminedusingthecomparativecycletimes(Ct)method,
usingthe formula2∆∆CT.AppendixTable3showsthe listofprimersequencesused for
RT-qPCR.
3.7StatisticalAnalysis
StatisticalAnalysiswasconductedbySigmaPlotv11(SystatSoftwareInc.,SanJose,CA,
USA)andgraphswerecreatedusingGraphPadPrism7.0c,(GraphPadSoftwareInc.,San
Diego, CA). Images were taken using the Moticam X2 camera (National Optical &
Scientific Instruments Inc, Schertz,TX,USA).Datawereassessed foroutliersusing the
Grubb’s test (GraphPadQuickCalcs,GraphPadSoftware Inc.).Comparisonsamong two
groupswereanalyzedusingStudent’sT-tests.Comparisonsamongmultiplegroupswere
analyzed using One-Way Analysis of Variance (ANOVA) followed by the appropriate
multiplecomparisonstest.Whennormalityorequalvariancefailed,theMann-Whitney
Rank Sum Test or Kruskal-Wallis One-Way ANOVA on Ranks were used to determine
significance.Thelevelofsignificancewassetatp=0.05.Alldataarepresentedasmean±
standarderrorofthemean(S.E.M).
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Figure4.Modelofadipogenesisinvitro.
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CHAPTER4:RESULTS
4.1BPAFandBPSsignificantlyinducelipidaccumulationin3T3-L1cells.
ToidentifyadipogenicBPAstructuralanalogues,theeffectsofBPA,BPAF,BPS,BPBand
BPF on lipid accumulationwere evaluated by Oil Red O staining. Both BPAF and BPS
significantly increased lipidaccumulation indifferentiated3T3-L1cells (P<0.05) (Figure
5).
4.2BPAFsignificantly increasesmRNAexpression levelsofadipogenicgenemarkers.
mRNA levels of key markers of adipogenesis (PPARγ; Pparg, C/EBPα; Cebpa) and of
mature adipocytes (FABP4; Fabp4, ADIPOQ; Adipoq) were evaluated by RT-qPCR.
Treatmentofcellswith10μMBPAFsignificantlyincreasedmRNAlevelsofPparg,Cebpa,
Fabp4 andAdipoq (P<0.05) (Figure6).BPAat anequimolardose (i.e., 10μM)didnot
haveanyeffectontheexpressionofPparg,CebpaorAdipoq,butresultedindecreased
(P<0.05)Fabp4mRNAlevels(Figure6).
4.3Dose-dependenteffectsofBPAF-inducedadipogenesis.
Because the effects of BPAF on adipogenic gene markers had not previously been
evaluated, I determined the effects of this compound over a range of concentrations
(0.01–10μM)ontheexpressionofkeymarkersofadipogenesis(PPARγ,C/EBPα)andof
matureadipocytes (FABP4,ADIPOQ).BPAF significantly increasedmRNAexpressionof
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thekeyadipogenicmarkerPpargatdosesrangingfrom0.1to10μM(P<0.05).However,
markersofmatureadipocytes (Fabp4,Adipoq)wereonly significantly increasedat the
10μMdose(P<0.05)(Figure7).NochangesingeneexpressionwithBPAwereobserved
atanydosetested.
4.424hr treatmentwith10μMBPAorBPAFdoesnot increase lipidaccumulation in
matureadipocytes.
TodeterminewhetherBPAorBPAFincreaseslipidaccumulationinmatureadipocytes,
fullydifferentiated3T3-L1cellsatday9weretreatedwithcontrol(DMSO),10μMBPA
or 10 μM BPAF for 24hrs. Lipid accumulation was not significantly different among
treatmentgroups(P<0.05)(Figure8).
4.510μMBPAFsignificantlyincreasesmRNAexpressionlevelsofNr3c1(GR).
Treatment with 10 μM BPAF during differentiation significantly increased nuclear
receptor subfamily 3 group c member 1 (Nr3c1; GR) gene transcripts in 3T3-L1 cells
(P<0.05)(Figure9a).
4.6Nr3c1 (GR)andTph1gene transcriptsarepositively correlatedwith10μMBPAF
treatment(R2=0.829,P<0.0001)(Figure9b).
4.7 BPAF can induce adipogenesis in the absence of the synthetic glucocorticoid
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dexamethasone.
To determine whether glucocorticoid signaling plays a role in BPAF-induced
adipogenesis,dexamethasonewasomittedfromtheinductioncocktail.Treatmentwith
10 μM BPAF in the absence of dexamethasone from Day 0 to Day 3 significantly
increasedlipidaccumulationcomparedto(-)Dex(DMSOcontrol)treatedgroup(P<0.05)
(Figure9d),butthisdidnotreachthesamelevelascellswhichreceiveddexamethasone
intheinductionmedia.TreatmentwithBPAFfromDay0toDay9elicitedanincreasein
lipidaccumulation,howeverthiswasnotsignificantlydifferentfromthe(-)Dex(DMSO
control)treatedgroup(Figure9e).
4.8BPAFincreases5-HTsyntheticenzymegenetranscripts.
To determine whether BPA and its structural analogues affect 5-HT synthesis, the
expression levelsofTph1andDdcwereevaluatedbyRT-qPCR indifferentiated3T3-L1
cells.Steady-statemRNAexpressionofthe5-HTsyntheticenzymeTph1wassignificantly
increased by 10μMBPAF (P<0.05) (Figure 6a).DdcmRNA levelswere also increased,
howeverthisdidnotreachsignificance(p=0.103;Figure10b).
4.9TPH1inhibitiondoesnotinhibitBPAF-inducedadipogenesis.
TodeterminewhetherBPAF-inducedadipogenesis ismediatedthrough increased5-HT
synthesis, cellswere co-treatedwithpCPA, a TPH inhibitor, duringdifferentiation.Co-
treatmentwith10μMBPAorBPAF±50μMpCPAdidnotimpairdifferentiationin3T3-
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L1 cells; pCPA administration did not reduce BPAF-induced lipid accumulation (Figure
11b) or the BPAF-induced increase in mRNA expression of adipogenic gene markers
(AdipoqandFabp4)(P<0.05)(Figure12).Interestingly,pCPA+10μMBPAresultedina
significant increase inPpargexpression;aneffectnotseenwithBPAalone (Figure8e)
anditdidnotblocktheBPA-inducedincreaseofAdipoq(Figure12h).
4.10BPAFsignificantlyincreasesMaoageneexpression.
To determine whether BPAF affects 5-HT catabolism, the expression levels of MAOA
were evaluated by RT-qPCR in differentiated 3T3-L1 cells. mRNA expression ofMaoa
wassignificantlyincreasedby10μMBPAF(P<0.05)(Figure13a).
4.11 Maoa and Pparg gene transcripts are positively correlated with 10 μM BPAF
treatment(R2=0.5312,P=0.0021)(Figure13b).
4.12MAOinhibitionblocksBPAF-inducedadipogenesis.
Co-treatmentwith100μMphenelzine(PZ),anon-selectiveirreversibleMAOinhibitor,in
thisstudywasabletoblocktheadipogeniceffectsofBPAF,significantlydecreasinglipid
accumulation (Figure 14b). Moreover, mRNA expression levels of Pparg were
significantly decreased in the BPAF + PZ treatment group (Figure 14d). Lipid
accumulationwasalsosignificantlydecreased inthe10μMBPA+PZtreatmentgroup
(Figure14c).
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Figure5.BPAstructuralanaloguesinducelipidaccumulationin3T3-L1cells.a)Murine3T3-L1preadipocytesweretreatedwithcontrol(DMSO)or10μMBPA,BPAF,BPS,BPBandBPFfor9days.LipidaccumulationwasdeterminedusingOilRedOstaining.b)Representativeimagesofcontrol,10μMBPAand10μMBPAFstainedwithOilRedO(10x).Datarepresentmean±S.E.M.*P<0.05relativetocontrol(n=3–20)calculatedbyOne-WayANOVAfollowedbyHolm-Sidak.
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Figure6.mRNAexpressionlevelsofadipogenicgenemarkersin3T3-L1cellstreatedwith10μMBPAFandBPA.mRNAlevelsweredeterminedbyRT-qPCRinmurine3T3-L1preadipocytestreatedwithcontrol(DMSO)or10μMBPAFandBPAfor9days.Dataarenormalizedtoβ-actin.Datarepresentmean±SEM.*P<0.05(n=4–15)relativetocontrolcalculatedbyOneWayANOVAfollowedbyHolm-Sidak.
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Figure7.Dose-dependenteffectsofBPAFandBPAonlevelsofadipogenicgenemarkersin3T3-L1cellsbyRT-qPCR.Cellsweretreatedwithcontrol(DMSO),BPAF(0.01–10μM)andBPA(0.01–10μM)for9days.Dataarenormalizedtoβ-actin.Datarepresentmean±S.E.M.*P<0.05(n=3–12)relativetocontrolcalculatedbyOne-WayANOVAfollowedbyHolm-SidakorKruskal-WallisOne-WayANOVAonRanksfollowedbyDunn’sMethod.
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Figure8.24hrtreatmentwith10μMBPAorBPAFdoesnotsignificantlyincreaselipidaccumulationinmatureadipocytes.Differentiated3T3-L1cellsatday9weretreatedwithcontrol(DMSO),10μMBPAor10μMBPAFfor24hrs.a)Representativeimagesofcontrol(DMSO),10μMBPAand10μMBPAFstainedwithOilRedO(10x).b)LipidaccumulationwasdeterminedbyOilRedOstaining.Datarepresentmean±SEM.*P<0.05relativetocontrol(n=3)calculatedbyOne-WayANOVA.
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Figure9.BPAFisabletoinduceadipogenesisintheabsenceofthesyntheticglucocorticoiddexamethasone(Dex).3T3-L1cellsweretreatedwith0.25μMDex(Day0–Day3),0.25μMDex(Day0–Day9),10μMBPAF(Day0–Day3),10μMBPAF(Day0–Day9)and(-)Dex(DMSOcontrol).a)mRNAexpressionlevelsofNr3c1(GR)in3T3-L1cellstreatedwith10μMBPAF.Dataarenormalizedtoβ-actin.Datarepresentmean±S.E.M.*P<0.05(n=4–11)calculatedbyStudent’sT-test.b)Nr3c1(GR)andTph1genetranscriptsarepositivelycorrelated(R2=0.829,P<0.0001).c)RepresentativeimagesoflipidaccumulationasdeterminedwithOilRedOstaining(10x).d)Lipidaccumulationin3T3-L1cellstreated0.25μMDex(Day0–Day3),10μMBPAF(Day0–Day3)or(-)Dex(DMSOcontrol)byOilRedOstaining.e)Lipidaccumulationin3T3-L1cellstreatedwith0.25μMDex(Day0–Day9),10μMBPAF(Day0–Day9)or(-)Dex(DMSOcontrol)byOilRedOstaining.Datarepresentmean±S.E.M.*P<0.05(n=4)calculatedbyStudent’sT-test(d)orMann-WhitneyRankSumtest(e)versus(-)Dex(DMSOcontrol).
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Figure10.mRNAexpressionof5-HTsyntheticenzyme,Tph1,issignificantlyincreasedwith10μMBPAFtreatment.mRNAlevelsweredeterminedbyRT-qPCRinmurine3T3-L1preadipocytestreatedwithcontrol(DMSO)or10μMBPAFfor9days.Dataarenormalizedtoβ-actin.Datarepresentmean±S.E.M.*P<0.05(n=4–11)calculatedbyMann-WhitneyRankSumTest.
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Figure11.Co-treatmentwith10μMBPAForBPAwith50μMpCPAdoesnotsignificantlyalterlipidaccumulationin3T3-L1cells.a)Representativeimagesofcontrol(DMSO),10μMBPAF,10μMBPAF+50μMpCPA,10μMBPAand10μMBPA+50μMpCPAfollowingOilRedOstaining(10x).b)LipidaccumulationwasdeterminedbyOilRedOstainingincellstreatedwithcontrol(DMSO),10μMBPAFand10μMBPAF+50μMpCPAfor9days.c)LipidaccumulationwasdeterminedbyOilRedOstainingincellstreatedwithcontrol(DMSO),10μMBPAand10μMBPA+50μMpCPAfor9days.Datarepresentmean±S.E.M.*P<0.05relativetocontrol(n=5–6)calculatedbyOne-WayANOVAfollowedbyHolm-Sidak.
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Figure12.Effectsofco-treatmentwith10μMBPAForBPA±50μMpCPAonadipogenicgenemarkersin3T3-L1cells.mRNAlevelsweredeterminedbyRT-qPCRinmurine3T3-L1preadipocytestreatedwithvehicle(DMSO),10μMBPAForBPA±50μMpCPAfor9days.Dataarenormalizedtoβ-actin.Datarepresentmean±S.E.M.*P<0.05(n=4–5)relativetocontrolcalculatedbyOneWayANOVAfollowedbyHolm-Sidak(a–d,f–h)orKruskal-WallisOne-WayANOVAonRanksfollowedbyDunnett’sMethod(e).
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Figure13.mRNAexpressionofMaoaissignificantlyincreasedwith10μMBPAFtreatment.a)mRNAlevelsweredeterminedbyRT-qPCRinmurine3T3-L1preadipocytestreatedwithcontrol(DMSO)or10μMBPAFfor9days.Dataarenormalizedtoβ-actin.Datarepresentmean±S.E.M.*P<0.05(n=4–11)calculatedbyMann-WhitneyRankSumTest.b)PpargandMaoagenetranscriptsarepositivelycorrelated(R2=0.5312,P=0.0021).
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Figure14.Co-treatmentwith10μMBPAF+phenelzine(PZ)significantlydecreaseslipidaccumulationandPparggeneexpressionin3T3-L1cells.a)Representativeimagesofcontrol,10μMBPAF,10μMBPAF+PZ,10μMBPAand10μMBPA+PZin3T3-L1cellsstainedwithOilRedO(10x).b)3T3-L1cellsweretreatedwithcontrol(DMSO)or10μMBPAF±100μMPZfor9days.LipidaccumulationwasdeterminedbyOilRedOstaining(n=3).c)3T3-L1cellsweretreatedwithcontrol(DMSO)or10μMBPA±100μMPZfor9days.LipidaccumulationwasdeterminedbyOilRedOstaining(n=3).d)mRNAexpressionofadipogenicgenemarkerPpargbyRT-qPCR(n=4–5).DataarenormalizedtoRplp0.Datarepresentmean±S.E.M.Groupswithdifferentlettersaresignificantlydifferentfromeachother(P<0.05)calculatedbyOne-WayANOVAfollowedbyHolm-Sidak.
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CHAPTER5:DISCUSSION
5.1TheeffectsofBPAFonlipidaccumulationduringadipogenesis
As a known endocrine disruptor, BPA has been extensively studied for its role in the
developmentofobesity. There isnowconsiderableevidence ranging fromcell culture
and animal experiments to human epidemiological studies to support the hypothesis
thatBPAisanobesogenaffectingadipogenesisandlipidhomeostasis. Duetogrowing
concernssurroundingtheuseofBPA,thereisincreasinguseofBPAstructuralanalogues
as replacements for BPA in consumer products. However, there is currently little
informationavailableregardingtheirtoxicity;thisisevenmorenotableinthecontextof
theireffectsonobesity.Theresultsofmyworkdemonstratedthatatthedoseusedin
this study (i.e., 10 μM), BPAF is a stronger inducer of adipogenesis than BPA. BPAF
significantly increased lipid accumulation, as well as mRNA expression levels of
adipogenicgenemarkers(PpargandCebpa)andmarkersofmatureadipocytes(Fabp4
andAdipoq).Althougharangeofdoseswasevaluated,theseeffectswereonlyseenat
the highest (i.e., 10 μM) dose tested. The results of this study are in agreementwith
recent findings showing that BPAF, at the same dose, stimulated triglyceride
accumulation in ATCC 3T3-L1 cells; the effect of BPAF to induce triglyceride
accumulation was approximately 45–50% of the response seen relative to ROSI79, a
known inducerof triglycerideaccumulation.Theauthorsobservedminimal changes in
triglyceridelevelsinthelowernanomolarrangeswhichisalsoconsistentwiththelackof
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effectsonadipogenicgeneexpressioninthelowerdoserangesinthisstudy.Although
BPAF has recently been shown to increase lipid accumulation in 3T3-L1 cells79, the
results of this study, tomy knowledge, are the first to evaluate its effects onmRNA
expressionofadipogenicgenemarkers.
TheresultsofthisstudyalsosupportrecentfindingsdemonstratingtheabilityofBPSto
increase murine adipogenesis in vitro76,77. Ahmed and Atlas (2016) showed that BPS
significantlyincreasedlipidaccumulation(10–50μM)in3T3-L1cells77.Moreover,25µM
BPS significantly increasedadipogenic genemarkers (ie.,Fabp4,Pparg, perilipin (Plin),
Lpl,adipsin(complementfactorD;cfd)andCebpa)andlipidaccumulationtoagreater
extentthanBPAatanequivalentdose.Halogenatedbisphenols,tetrachlorobisphenolA
(TCBPA)andtetrabromobisphenolA (TBBPA),havebeenshowntoactivatePPARγand
inducelipidaccumulationin3T3-L1cells121.Thefactthatstructurallysimilarderivatives
of BPA have stronger ability to activate PPARγ121,122, the master regulator of
adipogenesis,supportsthenotionthatthesealternativesmaystimulateadipogenesisto
agreaterextentthanBPA.Additionally,BPAwasfoundtobetheleastpotentchemical
whencomparedtoTCBPA,TBBPAandBPAFatinducingtriglycerideaccumulation(23%
vs 45–50%) relative to cells maximally stimulated with ROSI79. Taken together, the
results of this study add to the growing body of literature supporting the fact that
structuralanaloguesofBPAmaybemoreobesogenicthanBPAitself.
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While BPA has been shown to stimulate in vitro differentiation (i.e., increase
adipogenesis)at10µM122,123,other studiesusing10μMBPAdidnot report increased
adipogenesissimilartowhatisseeninmywork124,125.However,BPAhasbeenshownto
affect adipogeneis at lower doses (fentomolar- nanomolar range)50,76,115 as well as in
higher micromolar ranges (20–80 μM)41,44,48,77,124. Inconsistencies between studies,
however, may be attributed to varied differentiation models (e.g., murine, human
preadipocytes) differentiation protocols (e.g., in the absence/presence of
dexamethasone;differinginductionreagents)andtiming/lengthofexposure.Therefore,
comparisons across studies can be challenging when attempting to draw definitive
conclusionsofthemostpotentadipogenicdoseofBPA.Regardless, inmystudy,BPAF
andnotBPAattheequimolarconcentrationsinducedadipogenesissuggestingthatBPAF
maybeamorepotentobesogenthanBPA.
5.2TheeffectsofBPAFonlipidaccumulationinmatureadipocytes
Todeterminewhether theobesogenicactionsofBPAFwerespecific to theprocessof
adipogenesis,IevaluatedtheeffectsofBPAFonfullydifferentiatedadipocytes.Ididnot
observechangesinlipidaccumulationinmatureadipocyteswhentreatedfor24hrswith
10μMBPAF.ThissuggeststhatalthoughBPAFmayactduringadipogenesistoincrease
differentiationofpreadipocytes, itmaynotplayarole inregulating lipogenesis infully
differentiated fat cells. A previous study evaluating the effects of BPA in mature
adipocytes isolated from subcutaneous adipose tissue, determined that 1 and 10 nM
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BPA significantly increasedmean lipid area after 24hr treatment126. Triglyceride levels
were also increased at 1 and 10 nM, though only the 10 nM dosewas significant126.
Treatment with 10 μM BPA in this study exhibited no effects on mature murine
adipocytes. However, othermetabolic parameters have been shown to be altered by
BPA in mature adipocytes, such as adipokine secretion, insulin sensitivity and
inflammatorymarkers45,46,51,126.Adiponectinplaysarole in improving insulinsensitivity
andits levelhasbeenshowntobereducedinobesity127.Moreover,BPAexposurehas
beenassociatedwithdecreasedadiponectin levels.Hugoetal. (2008) isolatedmature
adipocytes from subcutaneous tissue in women and observed significantly decreased
adiponectinreleasewith0.1and10nMBPAtreatment45.Anotherstudydiscoveredthat
BPA,inadditiontoBPF,BPEandBPBat80μMsignificantlydecreasedbothadiponectin
productionandsecretion in3T3-L1adipocytes46.Additionally,10nMand100nMBPA
hasbeenfoundtodecreaseinsulinstimulatedglucoseutilizationindifferentiated3T3-L1
cells and differentiated human adipocytes51. A role for inflammation has also been
determinedwithdysregulationofIL-1B,IL18,IL-6,CCL20(chemokine(C-Cmotif)ligand
20)and interferongamma (IFN-γ) cytokines inBPA treatedmatureadipocytes51,126.As
obesity is multifactorial, with dysregulation of multiple metabolic parameters,
inflammation, insulin sensitivity in addition to lipid homeostasismayplay a role in an
EDC’sdeleteriouseffects.Therefore,althoughBPAFdidnotincreaselipidaccumulation
inmatureadipocytes,otherdeleteriousmetaboliceffectscannotberuledoutsuchasits
effectsoninsulinsensitivityandinflammatorymarkers.
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ResultsfrommystudyalsosuggestthatexposuretoBPAFinuteroorinearlylifemaybe
amorecriticalwindowofexposure.Thishypothesisissupportedbyanumberoflinesof
evidence. Firstly, based on current epidemiological studies higher levels of BPA in
childhood and adolescence havemost consistently been associatedwith an increased
odds of obesity37. Secondly, from my study, BPAF appears to have more profound
effectsonadipogenesis;adipogenesisbeginsduringthe14thweekofdevelopmentand
formation of majority of new adipocytes occurs during childhood/ adolescence. This
occurs predominately before the first two years of life and during puberty from
approximatelyage8 to185,38,40.Thirdly, ithasbeensuggested fromworkonBPAthat
theBPAmetaboliteBPA-Gmaybeunabletocrosstheplacentaoncemetabolizedbythe
fetus and that in fact BPA-G can be deconjugated back to free BPA by the fetus128.
Importantly, BPA-G itself has been shown to have adipogenic capacity129. These data
suggestthatwithincreasingexposuretoBPAduringpregnancy,thefetusisexposedto
thesepotentadipogenicstimuli.
There have been a number of animal studies which have evaluated the effects of
prenatal exposure to BPA on the development of obesity in the offspring during
postnatallife.Sommetal.(2009)foundelevatedbodyweightandanincreaseingenes
regulatinglipogenesis,lipolysisandhypertrophicadipocytesatpostnatalday21(PND21)
infemaleSpragueDawleyratsexposedto1mg/ml(70μg/kgbodyweight)BPAinutero
and lactation42. Another recent study in rats had similar finding (increased adipocyte
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densityinfemaleoffspringandalterationsinmetabolicmarkers(triglyceridelevels,lipid
homeostasis,adipokinesecretion)usingamuchlowerdose(0.5μg/kgBPA)63,whichwas
belowtherecentlyloweredtTDIsetbytheEFSAof4μg/kg/day.vanEsteriketal.(2014)
found dose dependent increases in body weight in male mice (0–3000 ug/kg body
weight) fromweek 6 toweek 21; however, in females a dose dependent decrease in
body weight and adipocyte size was observed (week 8–week 21). Similarly, another
study with male offspring by Angle et al. (2013) reported significant weight gain in
addition to adipocyte number/volume at 19 weeks of age58. Conversely, Alonso-
Magdalena et al. (2010) foundmalemice exposed in utero to 10 or 100 μg/kg body
weight/daydisplayednochangesinbodyweightat6monthsofage;femaleshowever
hadsignificantlyreducedbodyweight(10μg/kgbodyweight)beginningat3monthsold
whichpersisteduntil6months61.StudiesevaluatingtheeffectsofinuteroBPAexposure
furtherintoadulthoodhavereportedanincreaseinweightgaininOF-1malemiceat28
weeksofage(10μg/kg)59.
ExposuretoBPAinconjunctionwithahighfatdietmayalsoexacerbateBPA’seffects.
When rats were exposed to 50 μg/kg bodyweight from gestational day (GD) 0 until
PND21,bothmaleand femaleoffspringhad significantly increasedweightwhen feda
normal diet; an effect which began at week 19 and week 17 for males and females
respectively43.Thisincreasewasmoreprofoundandappearedearlierinageinanimals
onahighfatdiet.Inaddition,bodyfatpercentageandadipocytesizeweresignificantly
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increasedinmalesat27weeks.However,asignificantincreaseinbodyfatpercentage
andadipocytesizewasonlyobservedinfemaleswhenonahighfatdietwhencompared
to control high-fat diet fed animals. This was also evident in a study by Somm et al.
(2009) who only observed significant weight gain in males exposed to BPA in
utero/lactationwhentheywerefedahighfatdiet42.Conversely,anotherstudydidnot
finddifferencesinBPAexposedanimalswhentheywereplacedonhighfatdiet64.
Additionally, BPA exposure during adolescence may represent a period of increased
vulnerability as both male and female mice exposed to BPA (5–5000 ug/kg body
weight/day)at5weeksofagefor30daysdisplayedsignificantweightgainandadiposity
66.Moreover, inmales, increasedadipogenesiswasevidentwithelevatedmRNAlevels
ofPparg,CebpaandFabp4ininguinalwhiteadiposetissue(iWAT),aswellasincreased
adipogenic capacityof stromal vascular fraction (SVF) cells. Similarly, females treated
perinatally (GD8–PND16) and during puberty from PND21–35 displayed augmented
weight gain and dysregulated glucose homeostasis when compared to females only
treated during pregnancy/lactation60. Therefore, sex, timing and dose dependent
differencesacrossinvivostudiesareapparent.
Finally,exposuretoBPSinuterohasalsobeenassociatedwithincreasedweightgain.In
vivo,malemiceexposedto1.5and50μg/kgbodyweight/dayinuteroupto22weeks
ofagehadsignificantly increasedfatmassandbodyweight inconjunctionwithahigh
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fat diet78. Therefore, this demonstrated that a commonly used structural analogue of
BPAisabletoexertitseffectsduringacriticalwindowofexposure.
In human prospective studies, the association between in utero BPA exposure and
obesity havemixed findings, however consistent with other cross-sectional studies in
children33,37, BPA concentrations in childhood have mostly been associated with
elevated BMI in some34,35 but not all36 studies. In the CHAMACOS longitudinal study,
urinary BPA concentrations during pregnancy were associated with decreased BMI-z,
percentbodyfatandreducedoddsofoverweight/obesity(OR=0.37;95%CI0.16,0.91);
an effect only observed among girls at 9 years of age34. Interestingly, urinary BPA
concentrationsinbothgirlsandboysmeasuredatage9werepositivelyassociatedwith
BMI, WC, percent body fat and overweight/obesity (OR = 4.20; 95% CI 1.60, 11.02;
urinaryconcentrations>median)34.Asimilarfindinginfemaleswasalsoobservedina
studybyVafeiadietal.(2016)whichfoundanegativeassociationbetweenBMI-zscore
atages1through4andBPAconcentrationsinthe1sttrimesterofpregnancy;apositive
associationwasobserved forboysat all at ages1 through435. ChildhoodurinaryBPA
levels at age 4 were cross-sectionally associated with BMI-z score, WC and skinfold
thickness. Hoepner et al. (2016) found fat mass index, percent body fat and waist
circumference were positively associated with urinary BPA concentration during
pregnancy,withnoassociationwithBMI-zscoreforbothsexes36.Aftertakingsexinto
account, FMIwas the only parameter positively associatedwith prenatal urinary BPA
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concentrationsandthisassociationwasonlyobservedforfemales.Further,Valvietal.
(2013)foundurinaryBPAlevelsinpregnantwomenwereweaklyassociatedwithhigher
BMI andWC in their childrenwhen theywere4 yearsold130. Conversely, Braunet al.
(2014) found no association for BMI or WC between BPA urinary levels in pregnant
women and their children at 2–5 years of age131. However, studies examining this
association into adulthood have not been reported in the literature. Therefore, as
pregnant women and children have detectable amounts of BPA, it remains unknown
whetherdetrimentaleffectsassociatedwithBPAexposureextendintoadulthood,when
obesity andmetabolic syndrome aremost likely tomanifest. The results ofmy study
demonstrate that BPAFmay specifically act during adipogenesis, not on already fully
differentiated adipocytes, therefore early life exposures to this BPA replacement
compound may have the most profound effects on disruption of lipid homeostasis.
BecauseIshowedthatBPAFprimarilyaffectsadipogenesis,andbothglucocorticoidand
5-HT signaling play an important role during this process, I wanted to determine
whetherthesesignalingpathwaysmediateBPAF-inducedadipogenesis.
5.3ThepotentialroleofglucocorticoidsignalinginBPAF-inducedadipogenesis
IthasbeensuggestedthatBPA-inducedadipogenesismaybemediatedviaactivationof
GR signaling. Therefore, I evaluated whether BPAF may also be acting through this
pathway. Treatmentwith dexamethasone in 3T3-L1 cells has been shown to increase
bothCEBP𝛿(Cebpd)andPpargexpressionlevels112,132.Additionally,C/EBP𝛿andC/EBPβ
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stimulates C/EBPα in 3T3-L1 cells113.Moreover, inNIH 3T3 fibroblasts, overexpressing
C/EBPβ and C/EBP𝛿 provides maximal induction of PpargmRNA levels, however this
response is only observedwith the addition of glucocorticoids132. Steger et al. (2010)
found that in 3T3-L1 cells GR and C/EBPβ binding results in an epigenomic transition
state involving histone H3 acetylation, PPARγ activation (which results in a positive
feedbackloopwithC/EBPα)andsubsequentinductionofadipogenicgenes8.Therefore,
glucocorticoid signaling plays an important role in regulating adipogenesis through its
associationwithC/EBPsandsubsequentPPARγactivation.
Many studies evaluating adipogenesis in vitro use an induction cocktail with
dexamethasone (a synthetic glucocorticoid) to activate the GR and stimulate
glucocorticoidsignalingpathways44,111. ToevaluatewhetherBPAFalonecanstimulate
adipogenesisviaGRsignaling,dexamethasonewasomittedfromtheinductioncocktail.
Intheabsenceofdexamethasone,BPAFsignificantlyincreasedlipidaccumulation(Day0
–Day 3) when compared to control treated cells. This suggests that BPAF is able to
induceadipogenesisintheabsenceofdexamethasoneandisalsoabletopotentiallyact
in the early phases of adipogenesis (Day 0–Day 3) to accelerate differentiation. This
suggests thatBPAFmaybeacting likeaGRagonist,althoughtheresponsewasnotas
strong as seen with dexamethasone, a potent GR agonist, alone. Using a luciferase
bindingassay,Sargisetal.(2010)reportedthatBPAcoulddirectlyactivateGR,however
BPAwas only able to induce adipogenesis under submaximal conditions with DHC in
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3T3-L1cells115.Conversely,theauthorsofanotherstudydidnotobserveGRstimulation
with BPA treatment using luciferase assays, however BPAwas able induce significant
lipidaccumulationintheabsenceofdexamethasone116.However,itwasshownthat25
μMBPAupregulatedtranscriptionalactivityofGRandC/EBP𝛿specificallyattheFABP4
promoter116. Therefore, the adipogenic effects of BPA may be mediated through co-
activators/co-repressorswith promoter specificity116,133. Additionally, BPAmay also be
actingsynergisticallywithGRtoincreaseadipogenesis,asevidentwithsubmaximalGR
activationorlowdosedexamethasoneexposure115,116.Ofnote,itisinterestingthatGR
activation has been found to accelerate adipogenesis, however cells are able to
accumulatelipidtothesameextentwhenadipogenesis iscontinuedtothreeweeksin
GR knockdown cells134. The authors concluded that although GR accelerates
preadipocyte differentiation, it is not absolutely necessary to stimulate lipid
accumulationin3T3-L1cells.
Inthisstudy,mRNAsteady-statelevelofGRwassignificantlyincreasedinBPAF-treated
cells. While the results of this study point to a possible link between BPAF and GR
signaling, further studies areneeded todeterminewhetherBPAF is actingdirectly via
theGRtoincreaseadipogenesis.Recently,BPAFhasbeenshowntobindtoGRhowever,
it only displayed weak binding activity, whereas BPA showed more potent binding
activity117.Similarly, inanotherstudybothBPAFandBPAwerenotshowntostimulate
GRactivity in luciferaseassays135. Sargisetal.(2010)reportedthatBPAtreatmentdid
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not affect GR protein expression despite the fact that there was an increase in GR
transcriptionalactivity115.Therefore, it isunknownwhether thechanges inNr3c1 (GR)
mRNA expression levels in this study are indicative of activation ofGR transcriptional
activity.Although Iwould liketo investigatetheroleofGRsignaling inBPAF-mediated
adipogenesis,therearesometechnicallimitationsassociatedwithusing3T3-L1cellsto
addressthisquestion.IthasbeenreportedthatusingGRantagonistsin3T3-L1cellsis
challenging. For example, RU486, a GR antagonist, has been reported to act as an
inducerofadipogenesis136,137,thereforealternativemethodssuchasluciferaseassaysor
knockdown cellmodel should be used. Finally, it is also possible that the association
betweenBPAFexposure,GRexpressionandincreasedadipogenesismaybeindirectand
involve5-HTsignalingpathways.
In rats, dexamethasone treatment significantly increased Tph1 and Ddc mRNA and
proteinexpressioninintra-abdominaladiposetissue,aswellas5-HTlevelsinbothintra-
abdominal adipose tissue and serum118, suggesting that activation of the GR may
transcriptionally regulate TPH1andDDC. Interestingly, inmy study I foundapositive
correlation betweenNr3c1 (GR) and Tph1 mRNA levels. These data suggest that the
effectofBPAFtoincreaseadipogenesismaybemediated,inpart,viaalterationsin5-HT
pathways.
5.4TheeffectsofTPH1inhibitiononBPAF-inducedadipogenesis
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Invitro,5-HTlevelshavebeenshowntobeproducedduringadipocytedifferentiation94
and5-HT stimulates lipid accumulationwhenadded to3T3-L1 cells96. In TPH1mutant
cells, importantadipogenicgeneregulators,Ppary,CebpaandFabp4weresignificantly
reduced96. Blocking Htr2a has also been shown to inhibit lipid accumulation in
differentiated3T3-L1cellsandtreatmentwithanHtr2aagoniststimulatesexpressionof
genesinvolvedinlipogenesis94.Thesestudiessuggestthatboth5-HTandTPH1playan
integralroleinregulatingadipogenesisandlipogenesisin3T3-L1cells.However,Htr2B
signalinginadipocyteshasbeenshowntoincreaselipolysis98andmicroRNA-448located
on Htr2c plays a role in suppressing kruppel-like factor 5 (KLF5) expression96, an
important regulator of adipogenesis. Therefore, 5-HT seems to act differentially in
adiposetissuedependingonitactionsonaspecificreceptorsubtype.
Additionally, although inhibiting peripheral 5-HT synthesis has been shown to play a
protective role against obesity, in contrast, another study has pointed to beneficial
effect of 5-HT inmice fed a high fat diet138.Mice injected intraperitoneallywith 5-HT
gained less weight, had lower intra-abdominal lipid accumulation and improved
insulin/glucosetolerance,howeverthiseffectwasonlyseenwhencombinedwithahigh
fatdiet138.Theauthorspostulatedthatan increase inoxidativemetabolism inskeletal
muscle,aswellasanupregulationinmRNAlevelsofperoxisomeproliferator-activated
receptorgammacoactivator1-alpha-b,c(Ppargc1a-b,-c;PGC-1α)mayplayarole.
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Inthecurrentstudy,althoughtreatmentwith10μMBPAFsignificantlyincreasedTph1
levels in 3T3-L1 cells, blocking its activity with pCPA, a TPH inhibitor, did not impair
adipogenesis as hypothesized. This was evident as there were no alterations in lipid
accumulationwithOilRedOstaining,aswellasnosignificantchangesinmRNAlevelsof
adipogenic gene markers in cells treated with BPAF and pCPA. These results do not
supportthehypothesisthatBPAF-inducedadipogenesis ismediatedviaincreased5-HT
production.
OnelimitationofthisstudyisthatonlyTph1mRNAlevelswerereportedand5-HTlevels
werenotmeasured.Therefore,itisunknownwhetherthe50μMpCPAdosechosenin
this study sufficiently blocks TPH1at a level to effectively reduce5-HT levels in these
cells. Previous studies in 3T3-L1 cells have shown significant reductions in lipid
accumulationwithpCPAatdosesrangingfrom8–200μM96.Therefore,thedoseusedin
thisstudyof50μMwaswithintherangereportedtoinhibitlipidaccumulationin3T3-L1
cells.Additionally,a30μMdosewasusedtodeterminetheeffectsof5-HTinbuffalorat
liver 3A cells (BRL-3A)118.However, pCPA seems tobeamuch lesspotent inhibitorof
TPH1thanotherTPHinhibitorssuchasLP-533401.Inonestudymeasuringinhibitionof
humanTPH1 invitro,theIC50ofpCPAwas250μMwhereastheIC50forLP-533401was
reportedtobe0.7μM139.Additionally,inhibitionof5-HTlevelsdisplayedIC50valuesof
43μMand0.4μMforpCPAandLP-533401respectively.Interestingly,inthebrain,Tph
gene transcripts and 5-HT levels were shown to differ with pCPA treatment, with an
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increase in Tph mRNA levels corresponding with reduced 5-HT levels 140. Therefore,
these results presented in the current study may not reflect alterations in enzyme
activity and interpretation of data should be made with careful consideration.
Moreover, it ispossiblethatpCPAmayhaveashorthalf-life incultureconditions,and
adding it in more frequent intervals may block TPH1 activity to a fuller extent.
Furthermore,experimentsevaluatingtheefficacyofpCPAtoinhibitTPH1activityand5-
HTproductionin3T3-L1adipocytesareneeded.
5.5TheeffectsofMAOinhibitiononBPAF-inducedadipogenesis
While5-HThasbeenshowntoactonreceptors,mostnotablyHtr2a,Htr2bandHtr2cin
adipocytes94,96,98,apossibleroleforitsmetaboliteshasalsobeenreported.Wakuetal.
(2010)discoveredthatMIA,ametaboliteof5-HT,isabletoincreaselipidaccumulation
in 3T3-L1 cells.Moreover, both HIAA andMIA, indole-acetatemolecules can activate
PPARγviabindingtoactivationfunction2helixH12.When5-HTisoxidizedin3T3-F442A
cells by MAO, there is enhanced lipid accumulation and stimulation of
phosphoenolpyruvate kinase (Pck1; PEPCK) and Fabp4 expression, both PPARγ
responsive genes103. Interestingly, indole-acetate can be produced from tryptophan
itself, a pathway independent of 5-HT metabolism141. Therefore, a role for indole-
acetate molecules, which have been shown to directly bind to PPARγ and stimulate
adipogenesisislikely.
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Inthisstudy,10uMBPAFsignificantlyincreasedmRNAexpressionofMaoa,theenzyme
responsibleforthebreakdownof5-HTtoitsmetabolites.TodeterminewhetherBPAF-
induced adipogenesis occurs via altered 5-HTmetabolism, I investigatedwhether the
adipogeniceffectsofBPAFweredue inpartto itsactionsonMAO.Phenelzine,anon-
selectiveirreversibleMAOinhibitor, isusedfortreatingdepression142. Treatmentwith
phenelzinehasbeenreportedtobeassociatedwithincreasedweightgaininhumans143,
however the mechanisms underlying this effect are poorly understood. Interestingly,
pharmacologicallyblockingMAOinadipocyteswith100μMphenelzinehasbeenshown
to significantly decrease triglyceride accumulation during adipogenesis104 and reduce
weight gain inobese rats144. Moreover, inmy study I observed apositive correlation
betweenMaoa andPparg gene transcripts. Co-treatmentwith 100 μM phenelzine in
thisstudysignificantlydecreasedlipidaccumulationin3T3-L1cellsandwasabletoblock
theadipogeniceffectsofBPAF.Adoseof100μMwasused in thepresent studywas
chosen as it has been reported to sufficiently inhibit amine oxidase activity in rat
adipocytes144. Therefore, these results suggest a role for increasedMAOAactivity and
potentially 5-HT metabolites in mediating the adipogenic effects of BPAF. However,
there still remain some questions regarding the role of 5-HT metabolites in the
adipogenicactionsofBPAFseeninthisstudy.
Chicheetal.(2009)questionedwhetherphenelzine’slipiddepletingeffectswereinfact
due to a direct inhibition of amine oxidase activity104. When other amine oxidase
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inhibitors were examined for their ability to regulate triglyceride levels during
adipogenesis,noapparent relationshipwasobserved in3T3-F442Acells;notallamine
oxidaseinhibitorsinhibitedtriglycerideaccumulationtothesamedegree.However,itis
apparent thatother inhibitorsblockamineoxidaseactivityatdifferingconcentrations,
some more potent than others144,145. Moreover, it is plausible that if phenelzine’s
antiadipogenic effects were due its amine oxidase inhibitory ability and 5-HT
metabolitesdirectlybindtoPPARγ,addingaPPARγagonistshouldrescuephenelzine’s
antiadipogenic effects. However, the authors concluded thatphenelzine’s actions are
notmediatedthroughPPARγsignalingas troglitazone,aPPARγagonist,wasunableto
rescue phenelzine’s inhibitory effects on adipogenesis in 3T3-F442A cells and in cells
constitutivelyexpressingPPARγ.Moreover,thesemetaboliteshavenotbeenshownto
alter PpargmRNA expression levels103 but they may act as PPARγ agonists altering
PPARγ receptor activity101. In my study, although mRNA expression of Pparg was
significantlydecreasedwhencellsweretreatedwithBPAFandphenelzine,phenelzine’s
effectsmaynotbemediatedthroughalterationsinPPARγactivity.
ItalsomustbenotedthatphenelzineisnotonlyaninhibitorofMAOAandMAOB,but
alsoofprimaryamineoxidase (SSAO/PrAO).SSAO/PrAO isalso involved inmodulating
adipose tissue function144,146,147. Grès et al. (2013) reported that oxidation of 5-HT in
adipocyteswaspredominantly throughaMAOdependentmechanism103. Therefore, if
phenelzine acts largely through SSAO/PrAO, this may represent an additional
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mechanism of phenelzine to inhibit adipogenesis which is independent of 5-HT
catabolism.
ItisalsopossiblethatMAOandGRmaybelinked.Boucheretal.(2014)foundMAOA
to be the third highest upregulated gene in their microarray analysis upon 48hr
treatment with 1 μM dexamethasone in human preadipocytes148. Therefore, if BPAF
doesplayaroleinglucocorticoidsignaling,asoutlinedabove,itmayalsoplayarolein
the increasedMaoa gene expression levels that were observed in the current study.
Nonetheless,thisstudydemonstratesthatBPAFisnotabletoovercometheinhibitory
actionsofphenelzine,pointingtoapossibleroleforMAOinBPAF-inducedadipogenesis.
5.6Implications
AlthoughmyworkdemonstratedBPAFasapotentialobesogen,alteringadipogensis,it
is only one of several known replacement chemicals (i.e., Bisphenol AP, Bisphenol E,
BPSIP, Bisphenol E, Bisphenol PH, Bisphenol Z) that has been implicated in obesity67.
These structurally similar substitutes have been detected in sediment, food, receipts,
resinandmunicipalsewagesludge(Reviewedin:67,68).BPAstructuralanaloguesarealso
increasingly being detected in urine samples in humans68,83,84. Though limited
toxicological data exists, these replacements have been shown to targetmany of the
sameendocrinepathwaysasBPAsuchasestrogen,androgen,pregnaneXreceptorand
glucocorticoidsignaling149.Additionally,severalanalogues(BPAF,BPB,BPFandBPS)are
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suspectedtodisplayequivalentorgreatertoxicitythanBPA68.Moreover,themetabolic
consequencesofthesereplacements,includingtheireffectsonadipocytenumber,lipid
storage and satiety/food intake are limited. This is especially concerning in regard to
theirpotentialeffectsduringcriticalwindowsofexposure(i.e.,duringpregnancy,early
childhood,adolescence)whenexposuretothesechemicalscanpermanentlypredispose
anindividualtoobesityandmetabolicdisruptionlaterinlife.
As previously mentioned, adipocyte number remains relatively stable in adulthood.
Therefore, increasing the number of fat cells through exposure to an EDC during
development may create changes to an individual’s metabolic set point17,40,150.
Ultimately,thisleadstoanincreaseinstemcellscommittedtobecomingadipocytesor
morefatcellsthathavetheabilitytoexpandandbefilledwithlipid.This,coupledwith
thefactthatadipocytenumbercannotbediminishedthroughdiet,physicalactivityor
surgery creates an environment favoring energy storage5,150. Additionally, a “second-
hit”,suchasahighfatdietmayfurtherexacerbatetheeffectsonanindividualwhohad
previouslybeenexposedtoanEDCearlyinlife150.
Secondly,while invivostudieshaveshownBPAstimulatesadipogenesisandadipocyte
hypertrophy42,63, its exposure has also been associated with dysregulation of glucose
homeostasis and impaired insulin sensitivity43,58,61. Therefore, obesogensmay also be
implicatedinalteringawiderangeofmetabolicfactorssotheireffectsonwholebody
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metabolic homeostasis need to be assessed. Thirdly, adipogenic EDCs have also been
shown to possess transgenerational effects151 which can permanently altermetabolic
outcomesinsubsequentgenerations.
Takentogethermyworkhasidentifiedanewpotentialobesogenwhichinmystudy,has
beenshowntobeevenmorepotentthanthechemicalitisreplacing(i.e.,BPA).Thefact
that more than one BPA replacement compound has been shown to have similar or
more profound effects than BPA on metabolic outcomes raises significant concerns
abouttheirincreasinguseinconsumerproducts.
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CHAPTER6:FUTUREDIRECTIONS
ForfutureinvestigationintotheroleBPAFplaysduringadipogenesis,proteinexpression
levelsofPPARy,C/EBPα,FABP4andADIPOQshouldbeassessed.Moreover,asgeneand
protein expression do not reflect activity, transcriptional binding assays to determine
whetherBPAF candirectly bind to and alter specific promoter regions ofGRor other
transcriptionalregulatorssuchasPPARyneedstobeexamined.Additionally,tofurther
determinetheroleof5-HTsignalinginBPAF-inducedadipogenesis,5-HTlevelsneedto
be measured. An alternate MAO inhibitor could be used to delineate whether the
antiadipogenic effects observed in this study are due to its actions on amine oxidase
activity.Additionally,asBPAplaysaroleinglucosehomeostasis,itwouldbeinteresting
to observe whether BPAF also alters glucose transport and insulin sensitivity in
adipocytes.
Finally,althoughthe3T3-L1modelhasbeenextensivelyusedforstudyingadipogenesis
invitro,usinghumanpreadipocytesmaymorecloselyresembleahumanmodel.AsBPA
has been studied for its effects during development, the effects of BPAF in rodents
exposedperinatallycouldalsobeexamined.
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CHAPTER7:CONCLUSIONS
Dueto thegrowingconcern fromscientists, regulatorsandthepublicsurroundingthe
use of BPA, increasing attention has been placed on its structural analogues as
replacements.However,littleiscurrentlyknownabouthowthesesubstitutesmayaffect
human health. The data from this study suggest that both BPAF and BPS, common
replacements forBPA,mayalsocontribute tometabolicperturbationsassociatedwith
the development of obesity.Moreover, the results of this study add to themounting
evidencethatEDCshavetheabilitytomodulateadiposetissuephysiology.Thefindings
of this study also identify peripheral 5-HT signaling as a potential novel mechanism
throughwhichEDCsmayexerttheirobesogeniceffects.WhileexposuretoBPAhasbeen
showntoalterkeycomponentsofthecentralserotonergicsignalingpathway,nostudy
has attributed this pathway as a casual linkage between exposure to BPA and its
structural analogues andmetabolic dysfunction. Therefore, further studies evaluating
the link between how EDCs might modulate their obesogenic effects through the
peripheralserotonergicnetworkareneeded.Asobesityisaseriousglobalpublichealth
challengerepresentingoneoftheleadingcausesofmorbidity,mortalityandhealthcare
expenditureworldwide,thereisanurgentneedtodeterminewhichchemicalsposethe
greatesthealthrisktosociety.
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CHAPTER9:APPENDIX
Table1.SummaryofBPAConcentrationsInducingSignificantLipidAccumulationInVitro.Author Model Windowof
Exposure[]Tested Effect[]Lipid
AccumulationSargisetal.115 3T3-L1 Day0–Day3 100nM 100nMMasunoetal.152 3T3-L1 Day2–Day8 4–80μM 20μM,40μM,80μMMasunoetal.41 3T3-L1 Day0–Day2
Day0–Day1120μg/ml(87.6μM)20μg/ml(87.6μM)
20μg/ml(87.6μM)20μg/ml(87.6μM)
Wadaetal.123 3T3-L1 Day0–Day5/6 10μM 10μMAtlasetal.116 3T3-L1 Day0–Day8 0.01–10nM 0.01–10nMChamorro-Garcíaetal.122
3T3-L1mouseMSCs
humanMSCs
Day2–Day7Day0–Day14
100pM–10μM1nM–100μM
100nM,1μM,10μM(↔)
Héliès-Toussaintetal.76
3T3-L1 Day2–Day10 1ƒM–1μM 1ƒM,1pM,1nM
Taxvigetal.124 3T3-L1 Day3–Day6 5–20μM 20μMAriemmaetal.50 3T3-L1 3weeks
(preadipocytes)&Day0–Day8
1nM 1nM
Wangetal.153 humanvisceralpreadipocytes
Day4–Day18 10nM,1μM,80μM 10nM,1μM,80μM
Ohlsteinetal.125 humanadiposestromalcells
Day0–21Day0–14
100pM–10μM 100nM,1μM10nM–1μM
Boucheretal.44 primaryhumanpreadipocytes
Day2–Day14 25μM,50μM 25μM,50μM
Biemannetal.154
MSCs
Day0–Day12
10nM,10μM
10nM(↔)10μM(↓)
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Table2.ComparisonofUrinaryConcentrations(ng/ml)ofTotalBPAandBPAStructuralAnaloguesAcrossHumanStudies.
Study Author Location N Urine(ng/ml)GM*BPA NHANES2003–2004 LakindandNaiman155 USA 2517(age6–60+) 2.4CMHS2007–2009 Lakindetal.25 Canada 5476(age6–79) 1.2NHANES2005–2006 LakindandNaiman22 USA 2548(age6–60+) 2.0NHANES2007–2008 Lakindetal.25 USA 2489(age6–79) 2.1NHANES2011–2012 LakindandNaiman156 USA 2489(age6–60+) 1.5NHANES2013–2014 Liuetal.84 USA 1521(age20+) 1.3a Yeetal.83 USA 141 0.67 42 0.36 Yangetal.157 China 94b 0.886BPS Yeetal.83 USA 141 0.22(2013) 42 0.25(2014) Liaoetal.158 USA 31 0.299 Yangetal.157 China 94b 0.028NHANES2013–2014 Liuetal.84 USA 1521(age20+) 0.4a
BPAF Yeetal.83 USA 141 N/C(2013) 42 N/C(2014) Yangetal.157 China 94b 0.018BPF
Yeetal.83 USA 141 0.18(2013) 42 0.41(2014) Yangetal.157 China 94b 0.228NHANES2013–2014 Liuetal.84 USA 1521(age20+) 0.3a
*GM–geometricmeanaurinarymedianbresidentsnearBPAFmanufacturingplant
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Table3.PrimerSequencesforRT-qPCR.
Gene Forward(5'-3') Reverse(5'-3')Pparg GCGGAAGAAGAGACCTGGG GTGACTTCTCCTCAGCCCGCebpa CCGTGGTGGTTTCTCCTTGA TTTTTGCTCCCCCTACTCGGAdipoq ATCTGGAGGTGGGAGACCAA TGGGCTATGGGTAGTTGCAGFabp4 ATTTCCTTCAAACTGGGCGTG CTTTCCATCCCACTTCTGCACTph1 GACCATCTTCCGAGAGCTAAACAA AGCAAAGGGAGGTTTCTGAGGTADdc TCTTCGCTTACTTCCCCACG AGGAGAAACCAATGCAGCCAMaoa GGCTGTCATCAAGTGCATGG CATGATGGCAGGCATTGACCNr3c1(GR) GGACCACCTCCCAAACTCTG ATTGTGCTGTCCTTCCACTGβ-actin GCAAGCAGGAGTACGATGAG GTGTAAAACGCAGCTCAGTAACARplp0 CCAGCAGGTGTTTGACAACG TCCAGAAAGCGAGAGTGCAG