Repeated neonatal exposure to sucralose does not alter the

expression of GAP-43, CaMKII, synaptophysin and tau proteins

in cortex and hippocampus of mice.

Kiara Aiello

Degree project in biology, Bachelor of science, 2009Examensarbete i biologi 15 hp till kandidatexamen, 2009Biology Education Centre and Department of Environmental Toxicology, Uppsala UniversitySupervisor: Henrik Viberg

Contents

ACKNOWLEDGEMENT.............................................................................................................. 2ABSTRACT............................................................................................................................... 3INTRODUCTION........................................................................................................................ 4Sucralose .........................................................................................................................................4

BrainDevelopment .........................................................................................................................6

GAP‐43(growthassociatedprotein43) ..........................................................................................7

CaMKII(Ca2+Calmodulin‐dependentproteinkinaseII)...................................................................8

Synaptophysin .................................................................................................................................8

Tau...................................................................................................................................................9

AIMS ....................................................................................................................................... 9MATERIALS AND METHODS..................................................................................................... 9Animals............................................................................................................................................9

Chemicals ......................................................................................................................................10

Treatments ....................................................................................................................................10

Samplecollection ..........................................................................................................................10

Samplepreparation.......................................................................................................................10

Slot‐blotanalysis ...........................................................................................................................11

Statisticalanalysis..........................................................................................................................11

RESULTS................................................................................................................................ 11EffectsofsucraloseonGAP‐43,CaMKII,synaptophysinandtauproteinlevelsinneonatalhippocampusandcortex...............................................................................................................11

DISCUSSION AND CONCLUSION.............................................................................................. 13REFERENCES ......................................................................................................................... 15

2

Acknowledgement

IwouldliketothankSimonBolivarUniversityinCaracas,UppsalaUniversityandallthepeoplethat

mademyexchangethisyearpossible.Ithasbeenagreatexperiance,somanymanythanks!

OfcourseIwouldlovetothankmytutorwhohastoughtmeallthetechnicquesandmadethis

projectpossible:Ph.D.HenrikViberg,itis100%truethatyouareawesome!!!

ThankstoJanÖrbegandandPerErikssonforteachingmeandshowingmetheworldoftoxicology

andecotoxicology.Withsuchinterestingandchallengingcoursetheyhaveopenedthedoortowhat

islikelytobemyfuturefieldofwork.

TackJanÖ.alsoforintroducingmetothiswonderfullaboratory,whereIwasprivilagedtoworknext

toreallyinspiringandencouragingprofessionals!

IwouldalsoliketothankAndersFredrikssonforthehelpinthelaboratorywork,myfamily(los

amo!)andeverybodythatmadethispossible.

Ihavereallyenjoyeditandlearnedalot,

Tacksåmycket!

KiaraAiello

3

Abstract

Sucraloseisanartificialsweetener,whichusehavebeenincreasingasawayofmanaginghealthand

as anoption to improve thequalityof life to thediabetic population. Several toxicological studies

haveshownthatsucralosedonotpresentanydangertolaboratoryanimals(mice,rats,rabbit,dogs).

Studies regarding potential for carcinogenesis,mutagenesis, reproductive effects, between others,

havebeendone,butnostudiesregardingneurotoxicity,suchaschangesinlevelsofbrainproteins,

behavior,learningandmemoryafterexposuretosucraloseduringtheperiodofbraingrowthspurt

havebeenrealized.

Mammalshaveamarkedperiodofrapidbraingrowthanddevelopment(BGS),whichispostnatalin

miceandrats,spanningthefirst3‐4weeksoflifeandreachingitspeakaroundpostnatalday10.The

presentstudywasundertakentoexplorethepossibleeffectsofrepeatedexposuretosucraloseon

levelsoffourproteinsimportantfornormalbraindevelopment,duringtheBGS.

InthepresentstudyneonatalmaleNMRImiceweregiven125mgsucralose/kgbodyweight,when8,

9,10,11and12daysold,andeuthanizedonday13.Theagent(sucraloseorNaCl)wasadministered

as a singleoraldose via ametal gastric tube. Proteins assayswereperformedmeasuring levelsof

GAP‐43,CaMKII,synaptophysinandtauinhippocampusandcortex.Thestudyshowednosignificant

effects on protein levels in hippocampus or cortex. Therefore sucralose seems not likely to cause

developmentalneurotoxiceffectsinneonatalbrainafterrepeatedexposure.

4

Introduction

SucraloseSucralose (1,6 dichloro‐1,6dideoxy‐ß‐D‐fructofuranosyl‐4‐chloro‐4‐deoxy‐α‐D‐galactopryranoside), a

high‐intensity sweetener derived from sucrose (Fig. 1), was first approved by EU in 1998 after

several studies concluded that sucralosehavenoacute, sub‐chronic, carcinogenic, reproductiveor

genotoxicity effects (FDA, 1998). Later in 1999, the FDA allowed sucralose as a general‐purpose

sweetenerinallfood(FDA,2006).ThebrandnameofsucraloseisSplenda.

The increase intheuseofsucralose isdueto itsexcellenttasteprofile. Inaddition, thehighwater

solubilityandphysicochemicalstabilityofsucraloseallowittobeusedinacidicbeveragesandbaked

goods without loss of sweetness, during processing and storage. Furthermore, sucralose is also

considered away ofmanaging health and an option to improve the quality of life of the diabetic

population(Grotz,2008).Atpresentsucraloseisusedinasmanyas15foodcategories(Kinghornet

al.,1998),includingasatabletopsweetenerandforuseinproductssuchasbeverages,bakegoods,

chewing gum, frozendesserts, gelatins,milk products, dry‐mixproducts, saladdressing, processed

fruitandfruitspread.(FDA,2006;GriceandGoldsmith,2000).

Sucraloseisconsideredtobehydrophilicratherthanlipophilic,havingconsiderablewatersolubility

(28.3 g/100ml at 20°C) (Jenner and Smithson, 1989). The molecular weight is 397.63346 g/mol

(Convertsunit.com, 2009) and the octanol/water partition coefficient is low (0.3) confirming that

sucraloseispoorlysolubleinlipids(GriceandGoldsmith,2000;JennerandSmithson,1989).

Figure 1. Sucralose is derived from sucrose by selective replacement of three hydroxyl groups by chlorine

atoms.

Themetabolicfateofsucralosehasbeenstudiedinseveraldifferentspeciesofexperimentalanimals

andhumantoevaluatethesafetyofsucralose.5‐10%ofanoraldose(10‐1000mg/kg)isexcretedin

5

theurineandapproximately90% isexcreted inthefecesasunchangedparentcompound(Simset

al.,2000).Biliaryexcretionwasshowntobelessthan10%oftheorallyadministereddose(Griceand

Goldsmith,2000;Mannetal.,2000;Simsetal.,2000)meaningthatsucraloseispoorlyabsorbedby

experimentalanimals (Johnetal.,2000a,b;Woodetal.,2000).Studiesrealized inhumansshowed

sucralose as a safety compound concerningmetabolic, biochemical toxicological and clinical data,

includingdiabetic individuals.Afterexposuretosucralose,humansshowed lowblood levels,which

attainamaximumwithin2‐3hoursandtheeffectivehalf‐lifeofsucraloseis13hours.Also,humans

showedno accumulation of sucralose after exposure to doses 10 times higher than the projected

meandailyhumanintake(GriceandGoldsmith,2000).

Twominorurinarymetabolitesaccountingforonly0.15%‐0.25%oftheoraldosewerefoundinthe

rat (Sims et al., 2000). These twourinary components have similar chromatographic properties to

metabolites found in dog urine, and two metabolites found in human urine, which have been

identifiedbymassspectrometryasglucuronideconjugatesthatdonotpresentanythreattoneither

oftheexperimentalanimals(Mannetal.,2000;Robertsetal.,2000;Woodetal.,2000).

Several studies have searched for adverse effects of sucralose but only a few has presented any

effects. First, sucralose suppressed beneficial bacteria and directly affected the expression of the

effluxtransporterP‐glycoproteinandcytochromeP‐450isozymes,whichareknowntointerferewith

thebioavailabilityofdrugsandnutrientsandmayexplainwhy65‐95%ofthesucraloseadministrated

orally is reportedly not absorbed from the gastrointestinal tract (Abu‐Donia et al., 2008; Federal

Register, 1998;). Furthermore, these effects on decreased intestinal bacteria in rats occur at

sucralose levels approved by the FDA for use in the food supply (from 1.1 till 11mg/kg bw/ day

sucraloseduring12‐wkstudy)(Abou‐Doniaetal.,2008).Severalinvestigationshaveshowndecreases

in food consumption, bodyweight gain, and selected organweights (thymus, spleen and caecum)

afterfeedingsucralosetoratsatdietary levelsof0.3,1.0,3.0%.Theseeffectswereassumedtobe

secondary toadecrease in foodconsumptionor to theconsumptionof largeamountsof thenon‐

nutritive,poorlyabsorbed,osmoticallyactivesubstance(Abou‐Doniaetal.;Goldsmith,2000).

The“AcceptableDailyIntake”values(ADI)refertotheamountofthecompoundstudied,whichcan

be ingesteddailyovera lifetimewithoutappreciablehealthrisk (ADA,2004;WHO,1987).TheADI

definedbytheJointFAO/WHOExpertCommitteeonFood(JECFA)andtheScientificCommitteeon

Food of the European Union (SCF), for sucralose is 0‐15 mg/ kg bw (Renwick, 2006), and the

correspondingADI definedby theU.S. Food andDrugAdministration (FDA) is 5mg/kg/day. (ADA,

6

2004;FDA,1999;GriceandGoldsmith,2000).Theestimatedmeandaily intakeof sucralose forall

ageswouldbe1.1mg/kgbw,basedonMarketResearchCorporationofAmericaintakedata(Grice

andGoldsmith,2000).

BrainDevelopment

Everybrain regionhas itsownvulnerableperiodbutonecan roughlydivide themammalianbrain

development intotwocriticalperiods.Thefirstperiodincludesearlyembryonicbraindevelopment

wheretheshapeofthebraintakesformandtheprecursorstogliacellsandneuronsmultiply.The

secondcriticalperiodiscalledthebraingrowthspurt(BGS),andduringthisperiodthebraingrowsat

anacceleratedrate(DavisonandDobbing,1968).

Figure2.Ratecurvesofbraingrowthinrelationtobirthindifferentspecies.Valuesarecalculatedatdifferent

timeintervalsforeachspecies.FromDavisonandDobbing(1968)andEriksson(unpublished),withpermission.

IllustrationbyYlvaStenlund.

The BGS is characterized for expansion and accommodation of new synaptic connections of the

newly differentiated neurons, and includes axonal and dendritic outgrowth, synaptogenesis, and

proliferation of glia cells with accompanyingmyelinization (Davison and Dobbing, 1968; Kolb and

Whishaw, 1989).During this period animals acquiremany newmotor and sensory abilities (Bolles

andWoods,1964)andspontaneousmotorbehaviorpeaks(Campbelletal.,1969).

Thebraingrowthspurtoccursindifferentmammalianspeciesatdifferenttimesrelativetobirth,see

Fig. 2. For example in rodents (as rats and mice), the brain growth spurt occurs in the neonate,

spanning the first 3–4 weeks of life and reaching its peak around postnatal day (PND) 10, but in

7

human,itbeginsduringthethirdtrimesterofpregnancyandcontinuesthroughoutthefirst2years

oflife,coincidingwiththelactationperiod(DavinsonandDobbing,1986).

Severalstudieshaveshownthatthisperiodofrapidbraindevelopmentisvulnerabletoinsultsfrom

xenobiotics and that the presence of the compound in the brain during a defined period of

maturational processes is a critical factor (Ahlbom et al., 1995; Eriksson et al. 1992, 2000, 2002;

Fredrikssonet al., 2000;Viberg, 2009c;Viberget al., 2003b,2008b). In several studies it hasbeen

shown that neonatal exposure to neurotoxic and environmental agents can lead to widespread

neurodegeneration and disruption in brain function of adult mice. Among the neurotoxic and

environmental agents testedarebioallethrin,DDT,nicotine, ketamine,PCBs,PFOS,PFOA,PBDE99,

PBDE209 and PBD206. (Ahlbom et al., 1994; Ericksson, 1992, 1997, 1998; Eriksson et al., 2000;

Jevtovic‐Todorovic et al., 2003; Johansson,2009: Johansson et al., 2008; 2009a,b; Rice et al, 2007;

Vibergetal.,2004a,b,2007,2008a,b,c;2009b).

Neurotypicproteinscanserveassensitive indicatorsofeffectsofchemicalsonthedevelopingCNS

(O'Callaghan,1988a,b).Severalstudieshaveshownthat theeffectsof toxiccompoundsoncritical

developmentalprocesseswouldbereflectedbychangesinbiochemicalsubstratesunderlyingthem.

Thus, we examined levels of four proteins important for normal brain development involved in

neuronalsurvival,growthandsynaptogenesis.Alteredlevelsoftheseproteinsduringacriticalperiod

of the brain growth spurt (BGS) are good indicators of futuremalfunctions in the brain, and have

been reflected in dose response changes in behavioral tests of adult mice (Johansson, 2009;

Johanssonetal.,2009a,b;Viberg,2004a;2009a;Vibergetal.,2007,2008c).

GAP­43(growthassociatedprotein43)

GAP‐43 is a phosphoprotein enriched at presynaptic nerve terminals; it is involved in axonal

outgrowthandplasticityinsynapticconnections,activitycommontoallormostneurons(Benowitz

andRouttenberg,1997;Yamanouchi,2005).GAP‐43playsakeyroleinguidingthegrowthofaxons

andmodulating the informationofnewconnections. The roleof this protein is consistentwith its

selectiveenhancementduringdevelopment,elevatingtheexpressionduringBGSdevelopmentand

regeneration.GAP‐43patternlevelspeakaroundPND10followedbyagradualdecreaseduringthe

remainingpartofthefirst4weeksoflife(Jacobsonetal.1986;Skene,1989;Viberg,2009c;Viberget

al,2008b).Duetoitscharacteristicsandpatternofexpression,GAP‐43isfrequentlyusedasamarker

foraxonalsproutingandgrowth(Oestreicheretal.1997).

8

CaMKII(Ca2+Calmodulin­dependentproteinkinaseII)

CaMKIIisanenzymewhichispresentinallcelltypes.Itisthoughttobethemostabundantprotein

kinaseinneuronaltissues,especiallythehippocampus,whereitaccountsfor1‐2%oftotalproteins

(EronduandKennedy,1985).

CaMKII plays a significant role in the cellularprocessof long‐termpotentiation (LTP) and vesicular

releaseofneurotransmitters.CaMKIIactivationinthepostsynapticterminaloccursfollowingcalcium

influx via NMDA receptors,which are critical to the induction of LTP (Braun and Schulman, 1995;

HeistandSchulman,1998).Furthermore,lackingofCaMKIIexhibitsdeficitsinmodelsoflearningand

memory(McGBlade‐McCullen1993).

CamKII expression increases continuously during the first 4 weeks of life in mice and rats. The

greatest rate of increase in the amount of CamKII occurred between PND 7 and PND 14, which

coincideswiththepeakinneonatalbraingrowth(Viberg,2009;Vibergetal,2008b).

Synaptophysin

Synaptophysin isa38kDaglycoproteinassociatedwiththemembraneofpresynapticvesicles,and

because of this, it is highly concentrated in the axonal terminals in the neuron (Sarnat and Born,

1999). The protein is involved in processes regarding the formation and cycling of the synaptic

vesicle, from which neurotransmitters are released in order to exchange information between

neurons(Ovtscharroffetal.,1993;Valtortaetal.,1989).

Long‐termpotentation(LTP)isbelievedtobefundamentalforinformationstorageinthebrain.In

order to reachLTP, synaptophysin,alongwithotherproteins involved inneurotransmitter release,

needs tobeactivated. Inorder toactivatesynaptophysin itneeds tobephosphorylated.CamKII is

theproteinwhichphosphorylatessynaptophysinandthereforeregulating itsactivity(Lynch,2004).

Studies by Fujita and coworkers, and Viberg, have described the ontogeny of synaptophysin as

increasingduringtheearlydevelopmentofthebrain,andduringthefirst4weeksof life. (Fujitaet

al.,1996;Viberg,2009;Vibergetal.,2008b).

Synaptophysinisausefulmarkerforsynapticdensity(Hamosetal.,1989;Masliahetal.,1990)and

widelyusedasan immunohistologicalmarkerfordeterminationofthedensityofsynapses inbrain

disorders(Valtortaetal.,2004).

9

TauTau, tubuline associated unit, is present in associationwith tubulin functioning as an activator for

tubulinpolymerizationandhenceamajorregulatorofmicrotubuleformationincells.Microtubuleis

involvedincellularmotionandthemaintenanceanddeterminationofthecellshape(Weingartenet

al. 1975). Tau has been implicated in the outgrowth of neuronal processes, the development of

neuronalpolarity,andthemaintenanceofnormalmorphologyoftheneurons(Brandt,1996;Wang

and Liu, 2008). It is additionally involved in the promotion of microtubule assembly and the

maintenanceofstabilityofaxonalmicrotubules(Vila‐Ortizetal.,2001;Weingartenetal.1975).

Tauontogenyinbraintissuefrommice,isaincreasingleveloftheproteinduringtheearlyneonatal

period, reaching peak levels between PND 3 and 10, and a subsequently decrease during the

remainingpartofthe28daysperiod(Viberg,2009;Vibergetal.,2008a;Vila‐Ortizetal.,2001).Tau

proteincanformaggregatesthatcontributetoanumberofneurodegenerativediseasesofgeneral

interest(Bueeetal.,2000;Muntaneetal.,2008;Wangetal.,2003).

Aims

As sucralose is approved to be used during pregnancy and in children’s food, it is important to

considertheeffectsthatsucralosecancauseduringtheBGS.Severalstudiesregardingthetoxicityof

sucralose have been made, but no data is available regarding effects of this compound after

exposureduringtheBGS.Thereforetheaimofthepresentstudywastoevaluateifsucralose,after

repeatedexposureduringtheperiodofbraingrowthspurt,affectstheneonatalexpressionofGAP‐

43, CAMKII, synaptophysin and tau, important proteins involved in neuronal growth, survival, and

synaptogenesis, which have earlier been used to indicate toxicity from xenobiotics after neonatal

exposure(Johanssonetal.,2009a,b;Viberg,2009a,b,c;Vibergetal.,2003a,b,2008a,b,c).

MaterialsandMethods

AnimalsPregnant Naval Medical Research Institute (NMRI) mice were purchased from B&K (Sollentuna,

Sweden) andwere individually housed in plastic cages in a roomwith an ambient temperatureof

22°Canda12/12hcycleoflightanddark.Theanimalsweresuppliedwithstandardizedpelletfood

(Lactamin, Stockholm,Sweden)and tapwaterad libitum. Cageswere inspected fornewbornpups

10

twotimesaday.Thedayofbirthwasdesignatedday0.Followingparturition,thesizeofthelitters

wasadjusted to8–14pupswithin72hafterbirth,by thekillingofexcess femalepups.The litters

were housed with their respective dams until the end of experiment. Animal experiments were

conducted in accordance with and after approval from the local ethical committee (Uppsala

UniversityandAgriculturalResearchCouncil)andbytheSwedishAnimalWelfareAgency.

ChemicalsSucralose (< 98%) was purchased from Sigma, Sweden. The substance was dissolved in a NaCl

solution containing 12.5mg/mL of sucralose;NaCl solutionwas used to guarantee good solubility

andabsorbanceofthecompoundregardingthehighwatersolubilityofsucralose.

Treatments

On postnatal day (PND) 8, 9, 10, 11 and 12 sucralosewas administered orally, in a volume of 10

mL/kgbodyweight,dailydose125mg sucralose/kgbodyweight, viaametal gastric tube.Control

miceweregiven10mL/kgbodyweightoftheNaClsolution.Onlymalemicewereusedintheprotein

assayinordertocomparewithearlierdevelopmentalneurotoxicologicalstudiesonPBDEs,ketamine

and other known neurotoxic substances (Eriksson et al., 2002, 2001; Johansson et al., 2009a,b;

Vibergetal.,2003a,b,2004a,2007,2008a,b,c).Femaleswerekeepandtreatedduringthestudyin

order tomaintain the conditionsof care from themother. In this study, the control and sucralose

groupscomprised10‐12mice,eachfrom5differentlitters.

Samplecollection

Animals were sacrificed by decapitation on postnatal day 13, 24 hours after the last dosing. The

brainsweredissectedout:thehippocampusandthecortexfromeachbrainwereisolatedondryice,

flash‐frozeninliquidnitrogenandthenstoredat–80°Cuntilfurtherprocessing.

Samplepreparation

CortexandhippocampuswerehomogenizedwithaPotter‐ElvehjemhomogenizerinaRIPAcelllysis

buffer (50mMTrisHCl,pH7.4,150mMNaCl,1mMEDTA,1mMEGTA,1%TritonX‐100,20mM

sodiumpyrophosphate, 2mM sodiumorthovanadate, 1% sodiumdeoxycholate; AssayDesign Inc,

cat#80‐1045)withtheadditionof0.5%proteaseinhibitorcocktail(ProteaseInhibitorCocktailSetIII,

Calbiochem,Germany).Thehomogenatewasthencentrifugedat14,000gfor15minat4°C,andthe

protein contentof the supernatantwasmeasuredusing theBCAmethod (Piercekit) andaWallac

1420spectrophotometer.Subsequently,thesupernatantwasstoredat‐80°Cuntiluse.

11

Slot­blotanalysis

Theamountofprotein (between3and4µg)wasdiluted toa final volumeof200µLwith sample

buffer(120mMKCl,20mMNaCl,2mMNaHCO3,2mMMgCl2,5mMHEPES,pH7.4,0.05%Tween‐

20,0.2%NaN3)andapplied induplicate toanitrocellulosemembrane (0.45mm,Bio‐Rad)usinga

Bio‐Dot SFmicrofiltration apparatus (Bio‐Rad). The proteinswere fixed on themembranes in 25%

isopropanoland10%aceticacidsolution,washed,andblockedfor1houratroomtemperaturein5%

non‐fat dry milk solution containing 0.03% Tween 20. The membranes were then incubated

overnightat4°Cwithamousemonoclonalanti‐CaMKIIantibody(ChemiconMAB8699,1:10,000),a

rabbit polyclonal anti‐GAP‐43 antibody (Chemicon AB5220, 1:5000), a mouse monoclonal anti‐

synaptophysin antibody (Calbiochem 573822, 1:10,000) or amouse anti‐tau antibody (Santa Cruz

32274, 1:1000). Immunoreactivity was detected using a horseradish peroxidase‐conjugated

secondary antibody against mouse (KPL 074‐1806, 1:20,000) or against rabbit (KPL 074‐1506,

1:20,000) and an enhanced chemiluminescent substrate (Pierce, Super Signal West Dura). The

intensityofbandswasquantifiedusingimagingonaIR‐LAS1000Pro(FujiFilm,Tokyo,Japan).

StatisticalanalysisTo compare the levels of one protein within the two groups of exposure (control and sucralose‐

treatedanimals),thedatafromtheslot‐blotanalysisweresubjectedtoastudent’st‐test.Differences

wereconsideredsignificantwhenp<0.05.

Results

There were no visual sings of toxicity in the sucralose‐treatedmice at any given time during the

experimentalperiod,norwerethereanysignificantdifferencesinthebodyweightsinthesucralose‐

treatedmice,comparedwiththecontrolgroup(datanotshown).

EffectsofsucraloseonGAP­43,CaMKII,synaptophysinandtauproteinlevelsinneonatalhippocampusandcortex

Protein levels ofGAP‐43, CaMKII, synaptophysin and tau in cortex andhippocampusofmice, 24

hoursaftertreatmentwith125mgsucralose/kgbwonpostnatalday8to12,arepresentedinfigure

3.Student’st‐test indicatednosignificantchanges inanyoftheproteins incortexorhippocampus

comparedtothecontrolgroup.

12

Figure3.Protein levelof(a)GAP‐43,(b)CaMKII, (c)synaptophysinand(d)tauincortexandhippocampusof

miceexposedtoeither0.9%NaCl(Control)or125mgsucralose/kgbodyweightonpostnataldays8to12and

sacrificed24hafterlastexposure.ThedataweresubjectedtoStudent´st‐test.Theheightofthebarsrepresent

themeanvalue±SD.Thenumberofobservationforeachgroupis10‐12.

13

Discussionandconclusion

Ithasbeenshown inseveralstudiesthatprotein levelsofGAP‐43,CaMKII,synaptophysinandtau,

present inhippocampusandcortex inneonatalmicearegood indicatorsof futuremalfunctions in

thebrain(Garciaetal.,2003;Mundyetal.,2008;O’CallaghanandMiller,1988a,b;Radioetal.,2008;

RadioandMurdy,2008).Thisisduetothefactthattheaboveproteinsarecriticallyinvolvedinthe

processesunderlyingbraindevelopment.GAP‐43,CaMKII,synaptophysinandtauareproteinswhich

expression peaks during the brain growth spurt (Viberg, 2009c; Viberg at al. 2008b), are highly

enrichedinthenervoussystemandaresignallingproteinsthatregulateneuronalprocesses(survival,

growth, and synapatogenesis)(Oestricher et al., 1997; Frankland et al., 2001; Rongo and Kaplan,

1999;Navoneetal.,1986;WiedenmannandFranke,1985;WangandLiu,2008;Weingartenetal.,

1975;VilaOrtiz,2001).

Earlierstudieshaveshownthatduringtheperiodofrapiddevelopmentofthebrain(BGS)laboratory

animalsarevulnerableto insultsofxenobiotics likePFOS,PFOA,ketamineandPBDE209,andthat

thereisonlyaslightpotentialforsubsequentrepair,thereforetheconsequencescanbepermanent

(Davinsonanddobbing,1968;RiceandBarone,2000).Thesecompoundshavechangedthelevelsin

hippocampus and/or cortex of all or some of the proteins used in this study (Johansson et al.

2009a,b;Viberg,2009a,b,c;Vibergetal.2008a,b,c).

For example, ketamine significally increased CaMKII in hippocampus 24h after exposure, also

increasedlevelsofGAP‐43inhippocampusanddecreasedlevelsofGAP‐43incortexforthehighest

ketamine dose were seen (Viberg et al. 2008c). In a study with PBDE 209, the protein analysis

showed that CaMKII and GAP‐43 increased significantly in hippocampus and GAP‐43 decreased in

cortex 7 days after exposure to PBDE 209 (Viberg et al. 2008b). Another study showed that

synapthophysin levels increasedsignificantly inbrain tissueand taudidnotpresentany significant

change(Viberg,2009c).

Thepresent studyof sucralose isof interestbecause thechemical structureof sucralose,beingan

organochlorine, is similar to the structure of known toxic compounds, for example chlorinated

hydrocarbons used in various industrial processes and pesticides, such as PCBs (polychlorinated

biphenyls) and DDT, respectively. PCBs and DDT are known persistent contaminants of the

environment(Safeetal.,1987)PCBsproduceneurotoxiceffectsinhumans(TilsonandHarry,1994),

andDDThavebeenprovedtobeapotentneurotoxicantinbothvertebrateandinvertebratespecies

(Woolley,1982).Anotherreasonfortheinterestofthepresentstudyisthatsucraloseisapprovedto

14

be used during pregnancy and in children’s food, being present during the BGS in humans.

Furthermoretheconsumptionofsucraloseisincreasingandnodataisatpresentavailableregarding

effectsofthiscompoundafterexposureduringtheBGS.

This study indicates that repeatedexposureofneonatalmice to theartificial sweetener sucralose,

commonly used as non‐caloric sweetener and an option for solving dietary problems, has no

influenceonGAP‐43,CaMKII,synaptophysinandtaulevelsintheneonatalhippocampusandcortex.

Themethodused toexposeneonatalmice to sucralose, and themethod tomeasure the levelsof

GAP‐43,CamKII,synaptophysinandtau,havebeenusedinseveralstudiesandhavebeenoptimized,

establishing themost sensitive period for the neonatal brain inmice during theBGS, assuring the

antibodyspecificityforeachprotein,determiningthecorrectamountofproteinneededfortheslot

blot. All the studies have followed the same or similar procedure, which make the comparison

between them possible. (Johansson et al., 2009a,b; Viberg, 2009a,b,c; Viberg et al., 2003a,b,

2008a,b,c).

Theseresults,orlackofeffects,intheneonatalbrain,maybeduetothechemicalcharacteristicsof

sucralose.Firstofall,sucralosepresentsahydrophilicnature,andlowoctanolwaterpartition(28.3

g/100ml at 20°C; 0.3 octanol/water partition coefficient respectively) (Jenner and Smithson, 1989;

Convertunits.com,2000).Duetothesecharacteristicssucraloseispoorlysolubleinlipidsandpoorly

absorbed by the organisms, explaining why the largest portion of an oral dose of sucralose is

unabsorbedandexcreted10%intheurineandunchanged90%infeces(GriceandGolsmith,2000).

Secondofall,eventhoughsucralosehaschlorineinthestructure,extensivebiologicaldatasupports

thatsucralosehasstability,andthatdechlorinationdoesnotoccur(GriceandGolsmith,2000).Only

twominorurinarymetaboliteshavebeenidentified,onlyaccountingfor10%oftheurineasstudied

infivespecies(rat,rabbit,mouse,dogandman)aftersucraloseadministration(FinnandLord,2000).

Thesemetabolitesaremorereadlyabsorbedthansucralose,excretedessentiallyunchangedinurine.

Thereforeitisnotlikelythatmetabolitesofsucralosewouldaffecttheorganism(GriceandGolsmith,

2000)..

Third, sucralose is excreted rapidly from the body. For example, pharmacokinetic studies indicate

that the effective half life of sucralose is 13 hours inman (Grice andGolsmith, 2000). This rapid

elimination rate may not allow the compound to reach sensitive areas, or would not allow the

compound to cause any damage to the parts reached. So even if sucralose would present some

15

conjugativemetabolism,whichseemsnottobethecase,thetimeitwouldbepresent inthebody

andcausedamagewillbeveryshort.

Thecharacteristics referredtoearliercouldexplainwhysucralosedidnotaffect theprotein levels,

evenafterrepeatedexposureduringasensitiveperiodofbraindevelopment.Anotherexplanation

maysimplybethatsucraloseisnotadangerouscompoundfortheneonatalmice.

ItisstillunclearhowtoxiccompoundslikePFOS,PFOA,ketamineandPBDE209,changethelevelsof

the brain proteins. Almost all of them have bioacumulative potential and/or are highly lipophilic

ratherthanhydrophilic.Sucralosepresentsdifferentcharacteristics,forexample,itishydrophilicand

has a lack of bioaccumulation potential as discussed previously. Therefore these differences may

explain the lack of effects demonstrated in the current study, but it is not possible tomake any

conclusionregardingthisaspectuntilitisknownhowthesetoxiccompoundsoperateintheneonatal

brainandaffecttheproteinlevels.

InconclusionslotblotanalysisshowednosignificantdifferenceinGAP‐43,CamMKII,synaptophysin

andtaulevelsinhippocampusorcortexof13‐day‐oldmiceexposedto125mgsucralose/kgbwfrom

PND8to12,comparedtothecontrolgroup.Toconfirmthenon‐toxicityofsucraloseitwouldbeof

great interest to perform a behavioural study after neonatal exposure to sucralose and see if the

mice are affected. This is because it has been shown that behavioural tests are another sensitive

way,toevaluatetoxiceffectsofcompoundsafterneonatalexposure(Alhom,1994;Ericksson,1997;

Fredrikssonetal.,2000;Johansson,2009;Johanssonetal.,2008).

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