Modifications dans les processus de prolifération et de mort cellulaire aux stades

L’objectif de cette première étude était de mettre en évidence les effets d’une exposition à l’uranium dès le stade in utero sur la morphologie structurale du cerveau et sur les processus de mort et de prolifération cellulaire aux stades prénatals E13, E18 et aux stades postnatals P0, P5 et P21. La mor-phologie structurale du cerveau et plus particulièrement du télencéphale aux stades prénatals et de l’hippocampe aux stades postnatals a été étudiée par coloration au crésyl violet. Le processus de mort cellulaire a été étudié par immunohistochimie de la caspase-3 activée et par la technique fluorojade. Enfin, le processus de prolifération cellulaire a été analysé par incorporation du BrdU.

Nos résultats ont mis en évidence une diminution du nombre de cellules marquées au fluorojade dans le neuroépithélium cortical des embryons E13 exposés à l’uranium à 40 et à 120 mg/L (NC : témoin=3,70±1,60x10−2mm2, UA40=2,40±0,50x10−2mm2, UA120=1,78±0,58x10−2mm2, p<0,01). Au stade E18, nos résultats montrent également une diminution du nombre de cellules marquées au fluorojade dans le neuroépithélium cortical des fœtus E18 exposés à l’uranium à 120 mg/L (NC : témoin=3,39±1,11x10−2mm², UA40=4,81±1,72x10−2mm², UA120=1,96±1,54x10−2mm², p<0,05). Aucune modification dans le nombre de cellules marquées à la caspase-3 n’a été observée aux stades prénatals. De plus, une augmentation du marquage BrdU dans le neuroépithélium denté des fœtus E18 exposés à 120 mg/L a été observée (témoin=0,51±0,41x10−2mm2, UA40=0,82±0,31x10−2mm2, UA120=1,77±0,63x10−2mm², p<0,01), mais aucune modification n’a été observée dans le neuroé-pithélium des embryons E13.

Aux stades postnatals, une augmentation du nombre de cellules marquées au fluorojade est obser-vée dans le gyrus denté des ratons P0 et P5 exposés à 120 mg/L (P0 : témoin=1,21±0,15x10−2mm2, UA40=1,31±0,15x10−2mm², DU120=2,01±0,28x10−2mm², p<0,01 et P5 : témoin=2,24±0,54x10−2

mm2, UA40=2,64±0,87x10−2mm2, UA120=3,99±1,04x10−2mm2, p<0,05). Cette augmentation est associée à une augmentation du nombre de cellules marquées à la caspase-3 uniquement au stade P5 (GD : témoin : 0,59±0,85x10⁻³mm2, UA40=1,13±0,90x10−3mm2, UA120=2,96±1,87x10−3mm2, p<0,05). Au stade P21, aucune modification n’a été observée sur le processus de mort cellulaire. Enfin, une diminution du nombre de cellules ayant incorporées le BrdU est observée dans le gy-rus denté des rats P21 exposés à 120 mg/L d’uranium (DG : témoin=3,62±1,47, UA40=2,54±0,71, UA120=1,85±0,45, p<0,05), mais aucune modification n’est mise en évidence au niveau du gyrus denté des ratons P0 et P5.

Nos résultats montrent pour la première fois que l’exposition à l’uranium pendant le développe-ment cérébral entraîne des modifications dans les processus de neurogenèse, principaledéveloppe-ment à la plus forte concentration d’uranium. Ces modifications semblent opposées entre les stades préna-tals et postnapréna-tals. En effet, l’exposition à l’uranium augmente la prolifération cellulaire dans le neu-roépithélium denté des fœtus et diminue le processus de mort cellulaire des embryons et des fœtus au cours du développement prénatal. Au stade postnatal, l’uranium entraîne une diminution de la prolifération cellulaire dans le gyrus denté des rats P21 et une augmentation de la mort cellulaire dans le gyrus denté des ratons P0 et P5. Bien que ces modifications n’entraînent pas de modifica-tion morphologique majeure dans le développement des structures cérébrales, elles peuvent avoir un impact sur les autres étapes de la neurogenèse, notamment sur la différenciation neuronale et la migration qui a fait l’objet d’une analyse particulière (cf. 3.2.2).

Cell proliferation and cell death are disturbed during prenatal and

postnatal brain development after uranium exposure (2016,

Neurotoxicology).

Legrand M, Elie C, Stefani J, Florès N, Culeux C, Delissen O, Ibanez C,

Lestaevel P, Eriksson P, Dinocourt C.

Fulllengtharticle

Cell proliferation and cell death are disturbed during prenatal

and postnatal brain development after uranium exposure

M. Legrand^a,C. Elie^a,J. Stefani^a,N.Flore`s^a,C. Culeux^a,O. Delissen^a,C.Ibanez^a, P. Lestaevela,P. Erikssonb,C. Dinocourta,*

a

LaboratoiredeRadioToxicologieExpe´rimentale,ServicedeRadiobiologieetd’Epide´miologie,InstitutdeRadioprotectionetSuˆrete´ Nucle´aire,B.P.17, 92262FontenayauxRosesCedex,France

bDepartmentofEnvironmentalToxicology,UppsalaUniversity,Norbyva¨gen18A,75236Uppsala,Sweden

1. Introduction

During development, the brain is sensitive to injury from neurotoxicagents(RiceandBarone,2000).Developmentalexposure toheavymetalscaninterferewiththeprocessesofneurogenesis that lead to neuron generation during central nervous system formation:cellproliferation,migration,differentiation, synaptogen-esisandcelldeath(GotzandHuttner,2005).

Uranium (U) is a heavy metal naturally found in the environment. Civilian and military uses of U increase its concentrationintheenvironment(Briner,2010),andthuscivilian populations can be chronically exposed to U by ingestion of contaminatedfoodandwater(ATSDR,2013).Depleteduranium (DU)isaby-productofuraniumenrichmentfornuclearenergyor weapons.Itisanemergingenvironmentalpollutantandhasthe same chemical toxicity as natural uranium, but is 40% less radioactive.AlthoughDUaccumulatesmorein thekidneys and bones,thebrainisalsoatargetorgan(Lestaeveletal.,2005b).After chronic ingestion, U found in the brain of adult rats was heterogeneously distributed in the cortex, striatum and hippo-campus(Paquetetal.,2006).Umaybeabletocrossthe blood-brainbarrierbyvasculartransfer(Lemercieretal.,2003),butthe precisemechanismofthistransferisnotclearlydemonstrated.

Epidemiologic studies show that cognitive impairment increasesinpopulationsexposedtoU(Howland,1948;McDiarmid

NeuroToxicology52(2016)34–45

ARTICLE INFO

Articlehistory: Received1July2015

Receivedinrevisedform13October2015 Accepted13October2015

Availableonline23October2015 Keywords: Neurogenesis Heavymetal Braindevelopment Telencephalon Dentategyrus ABSTRACT

Thedevelopingbrainismoresusceptibletoneurotoxiccompoundsthanadultbrain.Itisalsowellknown thatdisturbancesduringbraindevelopmentcauseneurologicaldisordersinadulthood.Thebrainis knowntobeatargetorganofuranium(U)exposureandpreviousstudieshavenotedthatinternalU contamination of adult rats induces behavioral disorders as well as affects neurochemistry and neurophysiologicalproperties.Inthisstudy,weinvestigatedwhetherdepleteduranium(DU)exposure affectsneurogenesisduringprenatalandpostnatalbraindevelopment.Weexaminedthestructural morphologyofthebrain,celldeath andfinallycellproliferationinanimalsexposedtoDU during gestationandlactationcomparedtocontrolanimals.OurresultsshowedthatDUdecreasescelldeathin thecorticalneuroepitheliumofgestationalday(GD)13 embryosexposedat40mg/Land120mg/Land ofGD18 fetusesexposedat 120mg/L withoutmodificationof thenumber ofapoptoticcells.Cell proliferationanalysisshowedanincreaseofBrdUlabelinginthedentateneuroepitheliumoffetuses fromGD18at120mg/L.Postnatally,celldeathisincreasedinthedentategyrusofpostnatalday(PND)0 andPND5exposedpupsat120mg/Landisassociatedwithanincreaseofapoptoticcellnumberonlyat PND5. Finally, a decrease in dividing cells is observed in the dentate gyrus of PND21 rats developmentallyexposedto120mg/LDU,butnotatPND0andPND5.TheseresultsshowthatDU exposureduringbraindevelopmentcausesoppositeeffectsoncellproliferationandcelldeathprocesses betweenprenatalandpostnataldevelopmentmainlyatthehighestdose.Althoughthesemodifications donothaveamajorimpactinbrainmorphology,theycouldaffectthenextstepsofneurogenesisand thusmightdisruptthefineorganizationoftheneuronalnetwork.

ß2015ElsevierInc.Allrightsreserved.

* Corresponding author.Present address:IRSN,Direction de laStrate´giedu De´veloppementetdesPartenariats,ServiceProgrammesetStrate´giesscientifiques. Tel.:+33158359183;fax:+33158358467.

E-mailaddresses:marie.legrand@irsn.fr(M.Legrand),christelle.elie@irsn.fr

(C.Elie),johanna.stefani@irsn.fr(J.Stefani),nicole.flores@irsn.fr(N.Flore`s),

cecile.culeux@gmail.com(C.Culeux),olivia.delissen@irsn.fr(O.Delissen),

chrystelle.ibanez@irsn.fr(C.Ibanez),philippe.lestaevel@irsn.fr(P.Lestaevel),

per.eriksson@ebc.uu.se(P.Eriksson),celine.dinocourt@irsn.fr(C.Dinocourt).

ContentslistsavailableatScienceDirect

NeuroToxicology

http://dx.doi.org/10.1016/j.neuro.2015.10.007

etal.,2002).Experimentalstudiesonadultratshavedemonstrated that DU exposure by ingestion impairs cognitive functions. Subchronic and chronicexposureto DUinducedan increaseof paradoxicalsleep(Lestaeveletal.,2005a),areductioninspatial memory capacities, an increase in anxiety and a decrease of locomotor activity(Briner and Davis,2002; Briner and Murray, 2005;Houpertetal.,2005).

Several mechanisms by which DU would be able to cause neurotoxicityandalterbehaviorhavebeenexplored.Alterationsof thecholinergicsystemaswellasofdopaminergicand serotonin-ergicmetabolism(Bensoussanetal.,2009;Bussyetal.,2006)and disturbanceofpro-/antioxidantbalancehavebeendescribedafter chronicingestionofDU(BrinerandMurray,2005;Lestaeveletal., 2009).

LittleisknownaboutthetoxicityofUonthebrainduringits development.Mostdataontheperinatalandpostnataleffectsof DUinrodentshavefocusedondevelopmentaltoxicityandfetal development effects (Domingo, 2001; Paternain et al., 1989).

BrinerandAbboud(2002)showedthatmiceexposedtoDUduring gestationandlactationexposedmothersviadrinkingwaterhada decreaseoflocomotoractivityattheageof3weeksthancontrols. Parentalexposuretoenricheduraniumaswellasduringgestation and lactation showed changes in postnatal development and decreasedspatialworkingmemoryoftheoffspring(Houpertetal., 2007). In contrast, Sanchez et al. (2006) did not observe any disorders in spatiallearning afterexposuretonatural uranium. RecentstudiesshowedthatDUexposurefrombirthto10weeks causesimpairmentofobjectrecognitionmemoryassociatedwitha decreaseofacetylcholineconcentrationandacetylcholinesterase activity (Lestaevel et al., 2013). In these exposed animals, a decreaseoflocomotoractivityandadisturbanceofanxietywithan increaseofoxidativestresswerealsodescribed(Lestaeveletal., 2015).Thus,possibleeffects ofUonneurobehaviorand mecha-nismsunderlyingtheseeffectsduringthesensitiveearlylifestages ofratsareinsufficientlyexplored.

Byitschemicalproperties,DUtoxicitycanbelikenedtoheavy metaltoxicity,ofleadorcadmium,forexample.Itiswellknown thatchronicexposuretoheavymetalpresentintheenvironment during pregnancy and in infancy is associated with a risk of impaired cognitivedevelopment and behavioral alterations(Liu and Lewis,2014).Themechanismsbywhichheavymetalsalter behavior remain to be determined, but neurogenesis may be involved(Chowetal.,2008;DouandZhang,2011;LiuandLewis, 2014).

Likewise, DUmight impair cognitive functionsby acting on neurogenesis.Therefore,theaimofourstudywastodetermineif DUexposuredisturbsneurogenesisduringbraindevelopmentin rats.Morphogenesis,cellproliferationandcelldeathwerestudied attheprenatal,neonatalandpostnatalstages.Wefocusedonthe telencephalon during embryonic and fetal brain development sinceitwillgiverisetothefuturecortexandhippocampus,which are involvedincognitivefunctions.Inpupsand youngrats,we focused on thedentate gyrus(DG), which develops principally afterbirthandisoneofthemainneurogenicareasattheadult stageinvolvedinsynapticplasticityandthusincognitivefunctions (MingandSong,2005).

2. Materialandmethods

2.1. Animalsandcontaminationprotocol

Pregnant female Sprague–Dawley rats were received from Charles River(L’Arbresle,France)and usedfortheexperiments. Pregnant femaleswerehoused aloneunderstandardconditions with food and water provided ad libitum (light on: 8.00am/ 8.00pm, temperature:228C18C).Bodyweight,foodandwater

consumptionweremeasuredweekly.Pregnantfemaleswereexposed toDU(Areva-Cogema,France)viadrinkingwaterfromthefirstday aftermating(gestationalday1:GD1).ThespecificactivityofDUis 14.103Bq/g andits isotopic composition is 238U:99.73%, 235U: 0.255%,234U:0.01%.Pregnantratsweredividedintothreegroups: control group, 40mg/L DU group and120mg/L DUgroup. These concentrationswerechosen frompreviousstudies(Houpertetal., 2005; Brinerand Murray,2005).Based on waterconsumption of 100mLdaily,ratswereexposedto2or6mg/kg/day,respectively. Thesedosesareapproximatelyequalto1/100and1/33ofacuteoral LD50ofUinadultrats(Domingoetal.,1987).DUwasdissolvedin standardizedmineralwater.Theanimalsofthecontrolgroupdrank mineralwaterofthesamecomposition.Thenumberoffemaleswas calculated withrespectto thenumberofoffspringneeded forall experiments.

Pregnant females were divided in five time points during development: gestational day (GD) 13 (21 females), GD18 (18females),postnatalday(PND)0(15females),PND5(15females) andPND21(18females).Embryos,fetuses,neonatalandpostnatal ratsweretakenforanalysisasfollows:42embryosfrom21littersat GD13,33fetusesfrom17littersatGD18,30maleandfemalepups from15littersatPND0,30maleandfemalepupsfrom15littersat PND5and33malepupsfrom18littersatPND21.

All animal procedures were approved by the Animal Care CommitteeofIRSNandwereconductedinaccordancewithFrench legislationandEuropeanlegalrequirements(Decree86/609/EEC) concerning the protection of animals used for experimental purposes.ScientistscertifiedbytheFrenchMinistryofAgriculture performedallproceduresinanimals.

2.2. Tissuecollectionandpreparation

Pregnantratsweredeeplyanesthetizedwithisoflurane5%/air 95% inhalation and sacrificed by exsanguination. Kidneys of pregnant females were removed in order to measure the concentration of U at each time point (GD13: n=12; GD18: n=18,PND0andPND5:n=15,PND21:n=18).Uconcentrations were also measured in whole bodies of embryos from GD13 (n=12),fetusesfromGD18(n=18),inthebottomofthestomach, wherethekidneysarelocalized,ofpupsfromPND0(n=24)and PND5(n=24)andinkidneysfromPND21rats(n=24).Allsamples wereweighedandstoredat208CuntilassaybyICP–MS.

Forhistologyandimmunochemistry,embryosfromGD13and fetusesfromGD18werecollected,fixedin4%paraformaldehyde (PFA)for1hatroomtemperature(RT)andovernightat48C.Brains ofPND0andPND5pupswerecollectedandfixedinthesameway as embryosand fetuses. PND21ratswereanesthetizedwithan intraperitoneal injection of 60mg/kg sodiumpentobarbital and wereperfusedtranscardiallywith4%PFA.Brainswereremoved andpost-fixedfor1hin4%PFAatRTandovernightat48C. All headsofembryosandfetusesandbrainsofratpupswerewashed inPBS1Xsolutionandstoredina30%sucrosesolutionat48C.They werethen embeddedin Tissue-TekOCT compound,frozen in a liquidnitrogen/isopentanemixtureandkeptat808Cuntiluse. Embryosandheadsoffetusesweresagittallysectionedandbrains ofpupswerecoronallysectionedwithacryostatatathicknessof 10mmor20mm.Brainsectionswerecollectedonsuperfrostslides andstoredat808Cuntiluse.

2.3. Uraniumconcentration

Sampleswerepreparedby adding8mLofultrapure67–69% nitricacidand2mLofhydrogenperoxideandthenmineralizing themina1000Wmicrowave(EthosTouch;MilestoneMicrowave LaboratorySystems;Italy)witha20-minrampto1808Candthen 10min at 1808C. U content from mineralized samples was

determinedwithaninductivelycoupledplasmamass spectrome-ter (ICPMS-QX7-serie 2, Thermoelectron, France) withbismuth (1mg/L)astheinternalstandard.TheICP-MSlimitofdetectionfor Uis104mg/L.ValueswereexpressedasngU/gtissue.

2.4. Nisslstaining

Brain sections were hydrated in alcohol of descending concentration(95–80–70%)andstainedfor45sinacresylviolet solution.After a stepoftissuedifferentiationinacetic formalin solution, sections were dehydrated in alcohol of ascending concentration (80–95–100%), fixed in xylene and then cover-slippedwithPermount(FishierScientific,NewJersey).

Nisslstainingwas usedtodeterminethe generalhistological characteristicsofthebrain,especiallyinthetelencephalicregionfor prenatalstagesandinthehippocampusforneonatalandpostnatal stages.Nisslstainingallowedustochoosesimilarlevelsofthebrain tocomparethecontrolandexposedanimalgroups.Twolevelsof embryonicandfetalbrains(lateralandmedian)wereselectedand wefocusedonthetelencephalonarea(cortical,hippocampaland dentate neuroepithelia). Atthe differentneonatal and postnatal stages, 4 levels of the brain were selected and the DG of the hippocampus was analyzed. From each prenatal, neonatal and postnatal brain, adjacent sectionswere processed for: (1) BrdU immunohistochemistry;(2)neuronalcelldeathmethod(fluorojade C)and(3)apoptosisdetection(activatedcaspase-3).Brainsections fromcontrolandexposedanimalswerealwaysprocessedinparallel. 2.5. BrdUinjectionandimmunohistochemistry

In order to investigate the effect of DU on proliferation of progenitor cells, 5-bromo-20deoxyuridine (BrdU, Sigma Aldrich, L’IsleD’AbeauChesnes,France;50mg/kgpreparedinsterileNaCl) wasinjectedintraperitoneallyintopregnantfemalesonGD13and GD18orintoratpupsatPND0,PND5andPND21.Cellproliferation wasexamined4hafterBrdUinjectionatalltimepoints,exceptfor PND21ratsthat receivedtwoinjectionsofBrdUat48and24h before sacrifice. For immunochemistry, brain sections were rehydrated in phosphate-buffer saline 1X (PBS) and were incubated in 0.1% trypsin-0.1% CaCl2 diluted in distilled water for20minat378C.SlicesweretreatedtodenaturetheDNAin2M HCLfor30minandwererinsedtwicefor5mininaboratebuffer (55mLof0.2Mboricacid/45mL0.05Msodiumtetraborate,pH 8.4,SigmaAldrich,L’IsleD’AbeauChesnes,France).Sectionswere rinsed three times in PBS and incubated in a primary mouse monoclonal anti-BrdUantibody(1:50,Dako,Trappes, France)in PBS containing0.3% TritonX-100(PBS-T) for30minat RTand overnight at 48C. Slices were washed three times in PBS and incubatedwithcyanine3-conjugateddonkeyanti-mousediluted inPBS-T(1:200,JacksonImmunoresearch,France)for2hatRT. Slides were then rinsed and mounted using Vectashield Dapi-containingmedium(VectorLaboratories,Burlingame).

2.6. Fluorojadestaining

BasedontheprotocoldescribedbySchmued(Schmuedetal., 2005),brainsectionsweredriedovernightat508Candimmersed

inasolutioncontaining1%sodiumhydroxidein80%alcoholfor 5min.Quicklyrehydrated,theywereincubatedin0.06% potassi-umpermanganatefor5min.Sliceswerethenrinsedin distilled waterandtransferredto0.0004%fluorojadeC(Millipore,France) solutionfor10min.Finally,sliceswerewashedindistilledwater, dried at 508C for several minutes, immersed in xylene and coverslippedwithPermount(FisherScientific).

2.7. Cleavedcaspase-3immunohistochemistry

BrainsectionswererehydratedinPBSandincubatedfor1hina blockingsolutioncontaining5%donkeyseruminPBS-TatRT.Then theywereincubatedinananti-cleavedcaspase-3antibody(1:200, Millipore,France)overnightat48C.Brainsectionswerewashedin PBS 1Xand incubatedfor 2hat RTwithalexa 488-conjugated donkey anti-rabbit diluted in PBS-T (1:200, Jackson Immunor-esearch,France). Sliceswerefinallywashedand mountedusing VectashieldDapi-containingmedium.

2.8. Microscopyandquantitativeanalyses

Nissl-stainedsliceswereobservedwithavisiblelightoptical microscope(LeicaDM4000B,Microvision,Evry,France)andslices stainedforBrdU,fluorojadeCorcleavedcaspase-3detectionwere analyzed with a fluorescence microscope (Zeiss, Axiophot, Germany). Histolab software (Microvision Instruments, Evry, France)wasusedtoanalyzeandquantifythestaining.

Quantitativeanalyseswereperformedincortical,hippocampal anddentateneuroepitheliaofbrainsfromembryosfromGD13and fetusesfromGD18andintheDGofhippocampusfromPND0and PND5.AtPND21,analyseswerefocusedonthegranularcelllayer oftheDG.

From each developmental stage, DU-exposed animals were comparedwithcontrols.WemeasuredtheBrdU-stainedarea/mm2

oneachstructureinembryosGD13,fetusesGD18andpupsfrom PND0andPND5.Thedensity(numberofBrdU-positivecells/mm2) inthegranularcelllayerofDGofPND21ratswasanalyzedand comparedbetweencontrolandDU-exposedgroups.Thenumberof fluorojade-positive cellsand the number of cleaved caspase 3-positivecells/mm2werequantifiedatallstagesofDUexposure. 2.9. Statisticalanalyses

Uconcentrationsinsampleswerecomparedbetweencontrol and DU-exposed groups with one-way analysis of variance (ANOVA)followedbyTukey’stest.

Bodyweightsofneonatalandpostnatalratsarepresentedas meanstandard deviation (SD) in Table 1. One-way ANOVA followedbyDunn’s methodortheHolm–Sidakmethodwasused tocomparethebodyweightsofthecontrolandexposedgroups.

Analyseswereperformedon5animalsfromdifferentlittersper groupforhistologicalstainingandimmunochemistry.Allresults aregraphicallypresentedasmeansSD.One-wayANOVAfollowed byDunn’smethodortheHolm–Sidakmethodwasusedtocompare thecontrolandexposedgroups.

Differences were considered statistically significant for p<0.05.AllstatisticalanalyseswereperformedusingSigmaplot

Table1

Uraniumconcentrationinkidneysofanimals.

GD13 GD18 PND0 PND5 PND21

Control 1.801.06 2.450.53 3.680.99 4.322.17 5.251.08

DU40 34.4714.81*** 76.3938.99*** 68.7732.39*** 124.4744.39*** 142.2957.50*** DU120 226.11148.08*** 152.9051.72*** 378.55147.49*** 555.07157.32*** 1722.16577.74*** Uraniumconcentration(ng/goftissue)inkidneysofpregnantandnursingfemaleratsforcontrolgroupandgroupsexposedto40and120mg/LDU.Resultsareexpressedas