Propriétés neurophysiologiques

Des modifications dans les propriétés électrophysiologiques conduisant à une diminution de l’exci-tabilité neuronale ont été mises en évidence dans l’hippocampe de rat exposés à des fragments (120, 300 et 600 mg) d’uranium appauvri en intramusculaire pendant 6 et 12 mois (Pellmar et al. 1999). Ce résultat suggère que l’uranium pourrait modifier l’efficacité de la transmission synaptique.

Des modifications dans l’hippocampe des oscillations de typeβ/γjouant un rôle dans les processus de mémorisation (Fries et al. 2001,Persaran et al. 2002) ont été observées chez des rats âgés de 2 mois exposés à l’uranium appauvri à partir de la naissance à la concentration de 2 mg/kg/jour (Dinocourt et al. 2015). Il a été démontré qu’une diminution des activitésβ/γserait associée à des altérations dans la reconnaissance d’objets (Steullet et al. 2010). Cette étude suggère que l’uranium pourrait altérer la reconnaissance d’objets des animaux, comme observé par l’équipe de Lestaevel (Lestaevel et al. 2013) par modification de activités électriques. Cependant, d’autres investigations sont nécessaires pour confirmer ces données.

The neurotoxicology of uranium (août 2015, Toxicology).

Review

The neurotoxicology of uranium

Céline Dinocourt¹,Marie Legrand,IsabelleDublineau²,PhilippeLestaevel*

InstitutdeRadioprotectionetdeSûretéNucléaire(IRSN),PôledelaRadioprotectiondel’Homme,ServicedeRadiobiologieetd’Epidémiologie,Laboratoirede RadiotoxicologieExpérimentale,BP17,F-92262Fontenay-aux-Roses,France

ARTICLE INFO

Articlehistory: Received6July2015

Receivedinrevisedform5August2015 Accepted11August2015

Availableonline12August2015 Keywords: Sleep Locomotion Cognition Oxidativestress Neurophysiology Uranyl ABSTRACT

Thebrainisatargetofenvironmentaltoxicpollutantsthatimpaircerebralfunctions.Uraniumispresent intheenvironmentasaresultofnaturaldepositsandreleasebyhumanapplications.Thefirstpartofthis reviewdescribesthepassageofuraniumintothebrain,anditseffectsonneurologicalfunctionsand cognitiveabilities.Veryfewhumanstudieshavelookedatitscognitiveeffects.Experimentalstudies showthatafter exposure,uranium canreach thebrainand leadtoneurobehavioralimpairments, includingincreasedlocomotoractivity,perturbationofthesleep-wakecycle,decreasedmemory,and increased anxiety.The mechanismsunderlying theseneurobehavioraldisturbances are not clearly understood.Itisevidentthattheremustbemorethanonetoxicmechanismandthatitmightinclude differenttargetsinthebrain.Inthesecondpart,wethereforereviewtheprincipalmechanismsthathave beeninvestigatedinexperimentalmodels:imbalanceoftheanti/pro-oxidantsystemandneurochemical andneurophysiologicalpathways.Uraniumeffectsareclearlyspecificaccordingtobrainarea,dose,and time.Nonetheless,thisreviewdemonstratesthepaucityofdataaboutitseffectsondevelopmental processesandtheneedformoreattentiontotheconsequencesofexposureduringdevelopment.

ã2015ElsevierIrelandLtd.Allrightsreserved.

Contents

1. Introduction ....................................................................................................... 59 2. PartI:brainaccumulationandbehavioraleffects ......................................................................... 59 3. Themechanismsofuraniumpassageintothebrainanditslocalization ....................................................... 59 4. Behavioraleffects ................................................................................................... 62 4.1. Epidemiologicalstudies ........................................................................................ 62 4.2. Experimentalstudiesonanimalsexposedtouraniumduringadulthood ................................................. 62 4.2.1. Effectonlocomotion ................................................................................... 62 4.2.2. Effectsonthesleep-wakecycle ........................................................................... 63 4.2.3. Effectsoncognitivefunctions ............................................................................ 63 4.3. Effectsonanimalsexposedtouraniumduringdevelopment .......................................................... 63 5. Conclusion ........................................................................................................ 63 6. PartII:cellularandmolecularmechanismsunderlyingtheeffectsofuranium .................................................. 64 7. Mechanisticpathwaysinadultbrains .................................................................................. 64 7.1. Oxidativestress ............................................................................................... 64 7.2. Neurotransmitterpathways ..................................................................................... 66 7.2.1. Cholinergicsystemandneurobehavioralfunctions ........................................................... 66 7.2.2. Monoaminemetabolism ................................................................................ 66 7.3. Neurophysiologicalproperties ................................................................................... 67 8. Mechanisticpathwaysinbrains“underconstruction” ...................................................................... 68 8.1. Conclusion ................................................................................................... 68

*Correspondingauthor.Fax:+33158358467.

E-mailaddresses:celine.dinocourt@irsn.fr(C.Dinocourt),marie.legrand@irsn.fr(M.Legrand),isabelle.dublineau@irsn.fr(I.Dublineau),philippe.lestaevel@irsn.fr

(P.Lestaevel).

1Presentaddress:IRSN,DirectiondelaStratégieduDéveloppementetdesPartenariats,ServiceProgrammesetStratégiesScientifiques. 2

Presentaddress:IRSN,PôledelaRadioprotectiondel’Homme.

http://dx.doi.org/10.1016/j.tox.2015.08.004

0300-483X/ã2015ElsevierIrelandLtd.Allrightsreserved.

Toxicology337(2015)58–71

ContentslistsavailableatScienceDirect

Toxicology

9. Majorconclusionsandfutureinvestigations ............................................................................. 68 Acknowledgments .................................................................................................. 69 References ........................................................................................................ 69

1.Introduction

Anextensiveliteraturehasalreadydocumentedthedeleterious effectsof environmentaltoxic pollutantssuchasmetals onthe brainandnervoussystem(LiuandLewis,2014).Uraniumisthe heaviestnaturallyoccurringmetallicelementandisfoundinthe Earth’s crust. Due to its presence in soil, rocks, surface and underground water, air, plants, and animals, it occurs in trace amountsinmanyfoodsandindrinkingwater.Itsreleaseintothe environment raisesquestionsaboutitseffectsonhumanhealth (ATSDR(AgencyforToxicSubstancesandDiseaseRegistry),2013). Naturaluraniumcontainsthreeisotopes:uranium-238, uranium-235,anduranium-234.An‘enriched’formofuranium,inwhichthe uranium-235 concentrationisenhanced, is requiredtoproduce energy both in nuclear reactors and nuclear weapons. The remaining uranium mixture (after the enriched uranium is removed) has reduced concentrations of the uranium-235 and uranium-234 isotopes;this is knownasdepleted uranium.The relativelyhighavailabilityandlowcostofdepleteduraniumhave ledtothedevelopmentofvariouscivilianandmilitaryapplications (Bleise et al., 2003). Toxicity due to uranium is radiological, becausetheelementemitsradiations,andalsochemicalduetoits being a heavy element. Because depleted and natural uranium producesverylittleradioactivitypermassofuranium,thehealth effects from exposure of humans and animals to uranium are usuallyattributedtothechemicalpropertiesofuranium.

Afterexposuretouranium,sometargetorgansoftoxicityhave beenidentified,suchaskidneys,liver,lungsandbrain.Itiswell knownthattheuraniumtoxicitythresholdvariesalsoaccordingto thetimeandtherouteofitsexposure.

Several human populations may be exposed to uranium, notably workersinvolved in one of the different stages of the nuclear fuel cycleleading tothe production of electricity from uraniuminnuclearpowerreactors:exploration,uraniummining, milling, uraniumconversion,enrichment,fabrication of nuclear fuelandreprocessing.Inadditiontotheseworkers,Gulfwarand Balkan Veterans were exposed to depleted uranium used in munitions and armors that were extensively used from the 1991GulfWar(Bleiseetal.,2003)aswellaspopulationslivingin environmentaffectedbydepleteduranium-munitionsuse.Finally, generalpublicmaybeexposedtouraniuminsomecountriesand countieswithhighnaturaluraniumlevelsindrinkingwater(Canu etal.,2011).

Forallthesereasons,uraniumhasbeenthesubjectofseveral studies,particularlyonitshealtheffects.So,ourreviewdiscusses theavailabledatapublishedinthelast10yearsonuraniumeffects on cerebral functions. We therefore wrote two parts: the first describesthepassageofuraniumintothebrainanditsimpacton behavior,whilethesecondfocusesonthecellularandmolecular mechanismsofactionunderlyingitscentraleffects.

2.PartI:brainaccumulationandbehavioraleffects

Uranium is primarily an alpha emitter and represents a radiation hazard only after internal exposure. Uranium enters thehumanbodybyingestionandinhalationofairborne uranium-containingdustparticlesoraerosols.Uraniumisabsorbedfromthe intestine or the lungs, enters the bloodstream, and is rapidly deposited in the tissues, predominantly kidneys and bone, or excreted in the urine. Uranium’s majoradverse effect is renal

toxicityafteracuteexposure(Fukudaetal.,2006),whichismore difficultto demonstrate after chronic exposure (Poisson et al., 2014).Concerningthebrain,uranium contentwasmeasuredin wholebrainorinspecificcerebralstructuresfollowingdifferent types of exposure: intraperitoneal injection (Lestaevel et al., 2005b),ingestionviadrinkingwater(Bellésetal.,2005;Gilman et al.,1998;Ortega et al.,1989; Paquetet al., 2006),inhalation (Houpert et al., 2007c; Monleau et al., 2005) and following implanteddepleteduraniumpellets(Pellmaretal.,1999b).Areal accumulation ofuranium incerebral structureswas notalways demonstratedafteringestion,sincestudiesreportedcomparable uraniumquantityinuranium-contaminatedanimalsand uncon-taminatedanimals(Houpertetal.,2005;Lestaeveletal.,2005a, 2009).Nevertheless somecerebral structures,suchasstriatum, cortex orhippocampus, accumulate more uranium than others (Barberetal.,2005;Paquetetal.,2006).Severalstudiesindicated theabsenceofacleardose-dependentaccumulationofuranium (Bellésetal., 2005;Dublineauet al.,2014; Gilmanetal.,1998; Ortega et al.,1989). Finally, uranium accumulation was clearly observedatlongterminratswithimplanteddepleted uranium-pellets(Fitsanakisetal.,2006;Pellmaretal.,1999a,b).Thus,the issueontheuraniumpresenceincentralnervoussystemisstillon the table and deserves further investigations to resolve it. Differentialmechanismsofpassagehavebeenproposedtoexplain thiscerebralpresenceofuranium.

3.Themechanismsofuraniumpassageintothebrainandits localization

Apossiblevasculartransferofuraniumhasbeensuggestedby onlyastudycarriedoutbyLemercieretal.(2003)showingthat uranium didnotimpairtheintegrityof theblood–brainbarrier (BBB).In vitro, uraniumdoesnot inducetoxicity onRBE4 cells (Dobsonetal.,2006).Themechanismoftheblood–braintransfer, however,remainsunknown.Uraniummaybindwithothermetals transporters,i.e.transferrin,ferritinortransporterdivalentmetal type 1 (DMT1), known to be not evenly spread on the brain (Hémadietal.,2010;Konietzkaetal.,2014).Inthebone,Basset et al.(2013) suggest thatfetuin A isa majorproteintarget for uranium (Basset et al., 2013).Thus uranium coulduse similar transportersorbloodproteinstopassthroughthebrainbarrieras demonstratedinotherorgans.Theseassumptionshavenotbeen confirmed yet and need more investigations within vitro BBB models.

After inhalation or instillation exposure of rats, uranium is transferreddirectlyfromthenasalcavitytotheolfactorybulbs,as wellasthroughthebloodstream(Monleauetal.,2005;Tournier etal.,2009).Thispassagegoesviathecerebrospinalfluidalongthe olfactorynerve(Ibanezetal.,2014).

Thenextquestionstoconsiderarethecellularandsubcellular localizations of uranium in the brain. Use of a laser micro-dissectiontechniquecombinedwithinductivelycoupledplasma massspectrometry(LMDICP–MS)hasrecentlymadeitpossibleto show uranium in the pyramidal cell layer of the mouse hippocampus, in braintissue stainedwith uranium solutionat 100mg/L(Sussulini and Becker, 2015).Rouas et al. (2010) also demonstratedinvitrothatafterexposureatlowconcentrations, uraniumislocalizedmainlyinthenucleusinneuronal(IMR-32) celllines.Atleveloftheproximaltubulecellsofthekidneys,the uptakeofuraniumismediatedbyasodium-dependentphosphate

Table 1

Behavioral impairments observed after uranium exposure in experimental studies. Abbreviations: DU: depleted uranium; EU: enriched uranium.

60 C. Dinocourt et al. / Toxicology 33 7 (20 1 5 ) 58 – 71

co-transportersystem(Mulleretal.,2006).Theseco-transporters areperhapsalsoimplicatedatcerebrallevel.TypeIIIof sodium-phosphate co-transporters are present in the brain and are localized in neurons, astrocytes and endothelial cells (Inden etal.,2013).Nevertheless,themaintypesofsodium-phosphate co-transportersinthekidneysaretypeII(subtypeIIaandIIb)andare notexpressedinbrain(Hilfikeretal.,1998).Theseresultsraisethe questionofhowuraniumpenetratesintobraincellsandthusopen newperspectivesforstudyingthemechanismsofitstoxicity.

Inconclusion,thestudiestounderliethemechanismsofhow uranium could enter into the brain are still in progress. After inhalation,uraniumisabletobypasstheBBBtopassintothebrain. It may also cross the BBB without alterations, but the exact mechanismsofthistransferarestillunknown.

Thenexthalfdescribestheneurobehavioraldisturbancesthat thispassageintothebraincauses.

4.Behavioraleffects

Behavioral effects in humans and in various other animal specieshavebeenreportedafteruraniumexposure. Epidemiologi-calsurveysofhumansdonotalwaysmakeitpossibletoconnect behavioraldisorderstospecificcausalfactor,becauseitisdifficult tocontrolallenvironmental variables.However,certain param-eters can becontrolledin experimental animal models. Conse-quently,rodents,inparticular,havebeenusedinseveraltypesof experimentstostudybehavioralresponses.

4.1.Epidemiologicalstudies

Behavioral disturbances have been observed in workers exposedtohighconcentrationsofinhaleduranium.Theseworkers showedsigns ofdepression, apprehension,motor and language disorders(Howland,1948).Inlonger-termexposures, epidemio-logicstudiesreportcontradictoryresultsaboutdeathsfrombrain tumors and centralnervous system cancersin nuclearworkers (includingminers).Mortalityfromcancerofthebrainandcentral nervoussystemwasfoundinexcessinthepost-55Frenchcohortof uraniumminers(Rageetal.,2015;Vacquieretal.,2011),butthere is no evidence of an association with cumulated exposure to radionuclidesarisingfromuraniumoredust.Similarly,nodose– responsetrendswerefoundinnuclearfuelworkersbetweenbrain andcentralnervoussystemcancermortalityandcumulatedlung dose equivalent used for the evaluation of internal radiation exposure (Boice etal., 2006;Carpenteret al.,1988; Checkoway et al.,1988).No increase ofcentral nervoussystem cancerwas observedinGulfwarveterans(Macfarlaneetal.,2003;Stormetal., 2006).

SincethefirstGulfwarin1991,symptomsofseveralveterans remained unexplained (Blanck et al., 1995). The correlation between these symptoms and the depleted uranium exposure wasnotclearlydemonstrated.MonitoringveteransofGulfWarI hasshownareductionincognitiveabilities7yearsafterexposure viawoundsandpenetrationofuraniumfragments,correlatedwith uraniumconcentrationsinexcretedurine(McDiarmidetal.,2002). However,furtherstudiesperformedbetween12and20yearsafter exposurehavenotconfirmedtheseobservations(McDiarmidetal., 2006, 2013). These cognitive disorders may be correlated to depleted uranium but might also be due to the psychological reactionsinducedbywar.Stress-relatedhealthcomplaintsarea common sequel and may add to the confusion about possible healthconsequences.Moreover,notalloftheembeddedfragments contained depleted uranium. Indeed, some fragments were primarily composed of copper, lead, iron, and zinc, which are knowntocauseneurologicaleffects(Squibbetal.,2012).

Finally,Goasguenetal.(1982)reportedthecaseofamanaged 50yearswitha6-yearhistoryofseveralneurologicaldisorders, manifestedbylossofbalance,motorweakness,andlimbatrophy. Extensiveclinicalexaminationsledtothedetectionofsignificant amounts of uranium in his feces. In the absence of any other apparent cause, these neurological deficits were attributed to uraniumexposure,fromaclipboardthatthepatienthandleddaily forthreeyears.Noreportsaboutthispatientandhissubsequent medicalmonitoringappeartoexist.

Inconclusion,fewscientificstudieshavedirectlyaddressedthe health effects of uranium on humans. Larger studies with epidemiologically appropriate sampling and assessment tools needtobeconductedonpopulationslivingaroundnuclearplants, forexample.Thispaucityofhumanstudieshasledtosomeanimal experimental studies to improve our knowledge of uranium’s behavioraleffects.

4.2.Experimentalstudiesonanimalsexposedtouraniumduring adulthood

Afterexposuretouranium,locomotion,sleep-wakecycleand cognitive functions can be modified (Table 1). Summary of literatureexamining behavioral responsestouranium exposure inrodentsispresentedinTable1.Thisoverviewofavailabledata indicatesthedose,thetimeofexposureandthespecies. 4.2.1.Effectonlocomotion

After inhalation of uranium between 0.5 and 18mgU/m3 duringseveralweeks(8h/day,5days/week,duringuntil5weeks), neurological signs including gait instability and lassitude have beenobservedindogsandcats(BerkeandRothstein,1949).Inrats exposedto190mgU/m3asdepleteduraniumdioxide30min/day, 4 days/week for 3 weeks, locomotor and rearing behaviors increased significantly compared with controls on day 1 after theendoftheinhalationperiod,butnotonday5(Monleauetal., 2005).

Another neurobehavioral study examined the effects of depleted uranium alloy pellets (between 4 and 20 pellets) implantedinthegastrocnemiusmuscleofmaleandfemaleadult rats(Arfstenetal.,2007).After150days,theauthorsconducted threetestsfromthebatteryofbehavioralassessmentsoftoxicity. The first test measured spontaneous locomotor activity and stereotypicmovements, thesecondtheintegrity ofthehearing reflexcentersinthebrainstem,andthethirdsocial interactions betweenrats.Noneurobehavioralperturbationsassociatedwith depleted uranium implantation were observed in any of these tests.Theauthorsdetermined,however,thatfurtherstudiesare needed, with more implants and longer exposure, before any definitive conclusion can be reached about the impact on rat behavior of intramuscularly implanted depleted uranium frag-ments.

Repeatedinjections of 0.1 or1mg/kg of uraniumfor 7 days impairedthemotorcoordinationofrats(Abou-Doniaetal.,2002). Itispossiblethaturanium-inducedlocomotordeficitsarelinkedto anoverstimulationofthereceptorsmodulatedbyexcitatoryamino acids.Administrationofdepleteduraniumat4,8or10mgkg/day to rats via their drinking water for 2 weeks, 3 or 6 months significantlyincreasedlocomotoractivityin males(Bellésetal., 2005; Briner and Murray, 2005). In the same experimental conditions, female locomotor activity was modified but not significantly(BrinerandMurray,2005).Theresistanceoffemales touraniummaybedue toa difference inhormonalregulation. Theseresultssuggestedthattheroleofthehypothalamo–pituitary axismustbeconsideredinuraniumneurotoxicity.

In summary, locomotor activities are impaired in animals exposedtouranium(Table1).However,theexactmechanismby which uranium induces these locomotor effectsremains to be elucidated.Nitric oxide,uraniumreplacementofcalciumin the electrophysiologicalsystem,andtheroleofmotoneuronsmightall playaroleintheseimpairments.

4.2.2.Effectsonthesleep-wakecycle

Threedaysafterinjectionof144mgofdepleteduranium/kg,a centraleffectmanifestedbyshorterrapideyemovement(REM) sleep ( 18% compared with controls) was observed in rats (Lestaevel etal., 2005b).Thiseffect onREM sleepis explained byadecreaseofthenumberandthemeandurationofREMsleep episodes.Anotherstudyexaminingtheeffectsofuraniumonthe sleepcyclefoundthatmoretimewasspentinREMsleepbyrats exposedtoenricheduraniumat2mg/kg/dayinmineralwaterfor both30and60days(Lestaeveletal.,2005a).ThisincreaseinREM sleepwasduetoanincreaseinthenumberofREMsleepepisodes ratherthaninepisodeduration.Thefindingthattheseincreasesin theamountofREMsleepoccurredprimarilyduringthedaylight period (rats’ normal sleeping period) suggests that circadian rhythms were unaffected. The neurophysiological mechanisms underlyingREMsleepincreasesareonlyverypartiallyunderstood. Aroleforthehypothalamo–pituitaryaxisinsleepregulationhas beensuggested,notablyaroleforglucocorticoidsinthe modula-tionofREMsleep(Bornetal.,1991).

These early neurophysiological perturbations of REM sleep mightsubsequentlyinduceotherneurologicaleffects.Thissleep playsafundamentalroleinmemoryprocesses,anditsmodi fica-tioncouldinducetheirimpairment(Jouvet,1994).

4.2.3.Effectsoncognitivefunctions

Barber et al. (2007) showed that intramuscular injectionof 1mg/kg of depleted uranium caused rapid, but only transient impairmentofworkingmemoryinrats.Asignificantdecreasein spatialworkingmemorywasalsoobservedonday6aftertheend of prolonged (3-week) inhalation exposure to190mg depleted uranium/m³(Monleauetal.,2005).

Rats exposedto4%enriched uraniumat 2mg/kg/day for1.5 monthsviadrinkingwaterhaveshowedsignificantlydecreased spatial workingmemoryand increased anxiety(Houpert etal., 2005).Inanotherstudy,atestforspatialworkingmemoryina Y-maze was conducted after 3, 6, and 9 months of exposure to enriched uranium (Houpertet al., 2007a). The spatial working memory was decreased after 3 and 9 months of exposure. According to the investigators,these results may indicate that enricheduraniumdisruptsmemoryinspatialtasksinvolvingthe hippocampus but not in differenttasks in which hippocampal functioningis lesscrucial.Inthesameexperimentalconditions, depleteduraniumhadnosignificanteffectonmemoryoranxiety (Houpertetal.,2005).Animbalanceofreactive oxygenspecies (ROS)andlipidperoxidationintheentorhinalcortexmightexplain thedifferentbehavioraleffectsinducedbyenrichedanddepleted uranium(Lestaeveletal.,2009).

Finally,thespatial workingmemoryofApolipoprotein E / miceexposedto2mgofdepleteduranium/kg/dayfor3months