bpaf-induced adipogenesis: role of peripheral 5-ht … · 2017-10-18 · m.sc. thesis – k,...

BPAF-INDUCEDADIPOGENESIS:ROLEOFPERIPHERAL5-HTSIGNALING

BPAF-INDUCEDADIPOGENESISIN3T3-L1CELLS:ROLEOFPERIPHERALSEROTONINSIGNALING

ByKRISTINAD.WIGGERS,B.MSc.

AThesisSubmittedtotheSchoolofGraduateStudiesinPartialFulfilmentoftheRequirementsfortheDegreeMasterofScience

McMasterUniversity©CopyrightbyKristinaD.Wiggers,August2017

M.Sc.Thesis–K,Wiggers;McMasterUniversity–MedicalSciences

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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


13

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


14

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


15

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


16

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


17

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


18

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.


19

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


20

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.


21

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,


22

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


23

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


24

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)


25

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).


26

Figure4.Modelofadipogenesisinvitro.


27

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


28

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


29

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-


30

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).


31

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.


32

Figure6.mRNAexpressionlevelsofadipogenicgenemarkersin3T3-L1cellstreatedwith10μMBPAFandBPA.mRNAlevelsweredeterminedbyRT-qPCRinmurine3T3-L1preadipocytestreatedwithcontrol(DMSO)or10μMBPAFandBPAfor9days.Dataarenormalizedtoβ-actin.Datarepresentmean±SEM.*P<0.05(n=4–15)relativetocontrolcalculatedbyOneWayANOVAfollowedbyHolm-Sidak.


33

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.


34

Figure8.24hrtreatmentwith10μMBPAorBPAFdoesnotsignificantlyincreaselipidaccumulationinmatureadipocytes.Differentiated3T3-L1cellsatday9weretreatedwithcontrol(DMSO),10μMBPAor10μMBPAFfor24hrs.a)Representativeimagesofcontrol(DMSO),10μMBPAand10μMBPAFstainedwithOilRedO(10x).b)LipidaccumulationwasdeterminedbyOilRedOstaining.Datarepresentmean±SEM.*P<0.05relativetocontrol(n=3)calculatedbyOne-WayANOVA.


35

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).


36

Figure10.mRNAexpressionof5-HTsyntheticenzyme,Tph1,issignificantlyincreasedwith10μMBPAFtreatment.mRNAlevelsweredeterminedbyRT-qPCRinmurine3T3-L1preadipocytestreatedwithcontrol(DMSO)or10μMBPAFfor9days.Dataarenormalizedtoβ-actin.Datarepresentmean±S.E.M.*P<0.05(n=4–11)calculatedbyMann-WhitneyRankSumTest.


37

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.


38

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).


39

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).


40

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.


41

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


42

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.


43

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


44

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.


45

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


46

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


47

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


48

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


49

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


50

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


51

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


52

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


53

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.


54

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


55

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.


56

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


57

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


58

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


59

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


60

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.


61

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.


62

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.


63

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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


80

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

bpaf-induced adipogenesis: role of peripheral 5-ht … · 2017-10-18 · m.sc. thesis – k,...

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