Characterization of Developmental Neurotoxicity of As, Cd, and Pb Mixture: Synergistic Action of ...

16 Pages·2010·1.14 MB·English

TOXICOLOGICALSCIENCES118(2),586–601(2010)
doi:10.1093/toxsci/kfq266
AdvanceAccesspublicationSeptember9,2010
Characterization of Developmental Neurotoxicity of As, Cd, and Pb
Mixture: Synergistic Action of Metal Mixture in Glial and Neuronal
Functions
Asit Rai,*,† Shailendra Kr Maurya,* Priyanka Khare,‡ Abhinav Srivastava,* and Sanghamitra Bandyopadhyay*,1
*DevelopmentalToxicologyDivision,IndianInstituteofToxicologyResearch,CouncilofScientificandIndustrialResearch,Lucknow226001,India; D
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†DepartmentofBiochemistry,FacultyofScience,JamiaHamdard(HamdardUniversity),NewDelhi110062,India;and‡EcotoxiologyDivision,IndianInstitute w
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ofToxicologyResearch,CouncilofScientificandIndustrialResearch,Lucknow226001,India lo
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1Towhomcorrespondenceshouldbeaddressed.Fax:[phone].E-mail:[email protected]. d fro
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ReceivedApril26,2010;acceptedAugust17,2010 h
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genesis, myelination, and apoptosis in the central nervous ://a
Neurotoxicityofindividualmetalsiswellinvestigatedbutthat system(CNS)(RiceandBarone,2000).Thelimitedcapacityof ca
d
of metal mixture (MM), an environmental reality, in the de- e
the developing CNS to compensate for the cell loss and the m
veloping brain is relatively obscure. We investigated the combi- ic
disruptions in neural networking results in compromised .o
natorialeffectofarsenic(As),cadmium(Cd),andlead(Pb)onrat u
neuronal functions (Bayer, 1989) and increased risk of p
brain development, spanning in utero to postnatal development. .c
MM was administered by gavage to pregnant and lactating rats, neurodegeneration (Grandjean and Landrigan, 2006). om
and to postweaning pups till 2 months. The pups exhibited Heavy metals including arsenic (As), cadmium (Cd), and /to
x
behavioral disturbances characterized by hyperlocomotion, in- lead (Pb) have received attention as both environmental con- sc
cthreeabsleododg-rbiprasitnrebnagrtrhie,ra(nBdBlBea)rwnainsga-smsoecmiaotreyddweifithcidt.oDsei-sdrueppetinodnenotf teatmali.n,a2n0ts06a;nFdowpoletrenettiaall.,n2e0u0ro4t;oJxaidchoalovgeictaall.,h2az0a0r7d)s.E(Bxpreonsduerer i/article
increase in deposition of the metals in developing brain. to the metals in utero and in infancy is associated with risk of -a
b
Astrocytes were affected by MM treatment as evident from their impaired cognitive development (Hu, 2000; Landrigan et al., stra
reduced density, area, perimeter, compactness, and number of c
processes, and increased apoptosis in cerebral cortex and 1an9d75b),ehsauvbicolrinalicaalnobmraainliedsy(sTfusnacitieotna(lL.,a2n0p0h3ea;rWetriaglh.,t 2e0t0a5l).,, t/118
cfiebrreilblealrlyuma.ciTdihce pmroetteailns i(nGdFuAcePd) seyxnperregsissitoicn rdeudruicntgionbrainingldiae-l 2006). Studies with single metal exposure have demonstrated /2/58
that As, Cd, or Pb infiltrate the immature blood-brain barrier 6
velopment; however, postweaning withdrawal of MM partially /1
(BBB) and accumulate in developing brain (Lidsky and 6
restored the levels of GFAP in adults. To characterize the toxic 5
1
mechanism, we treated rat primary astrocytes with MM at Schneider, 2003; Valkonen et al., 1983; Wang et al., 2007a; 67
concentrations ranging from lethal concentration (LC)10 to LC75 Xi et al., 2010). Pb uptake through the BBB disrupts Ca2þ 3 b
ofthemetals.Weobservedsynergisticdownregulationinviability transport mechanism (Marchetti, 2003) and promotes activa- y g
u
andincreaseinapoptosisoftheastrocytes,whichwereinducedby tion of mitogen-activated protein (MAP) kinases in apoptotic e
s
proximal activation of extra cellular signal-regulated kinase glial cells (Posser et al., 2007). The sequestration of Pb at the t o
n
(ERK) signaling and downstream activation of Jun N-terminal level of the choroid plexus undermines brain growth and 1
0
kinase(JNK)pathway.Furthermore,riseinintracellularcalcium affects learning and cognitive functions of CNS (Marchetti, A
iaopnop(t[oCsais21in]i)thaenadstrreoaccyttievse.Toaxkyegnentosgpeethcieers,tgheenseeroabtisoenrvaptrioomnsoaterde 2003). In vivo and in vitro studies have revealed that acute or pril 2
thefirsttoshowthatmixtureofAs,Cd,andPbhasthecapacityto chronic Cd exposure enhances oxidative stress in astrocytes 019
and accumulates reactive oxygen species (ROS) that induces
inducesynergistictoxicityinastrocytesthatmaycompromisethe
astrocytic death (Yang et al., 2008). Perinatal exposure to Cd
BBB andmay cause behavioraldysfunction indevelopingrats.
Key Words: environment; metal toxicants; GFAP; apoptosis; inducesanxiety(MinettiandReale,2006)andreduceslearning
brain damage. ability of offspring (Ishitobi et al., 2007). Chronic exposure to
As,evenatasubmicromolarconcentration,promotesoxidative
stress (Garcia-Chavez et al., 2006) and induces neuroglial
damageinhumanbrain(Jinetal.,2004).IntoxicationwithAs
The developing brain is vulnerable to injury from toxic presents deficits in spontaneous locomotor activity (SLA) and
metals that interfere with the critical developmental processes, alterations in learning-memory task during postnatal develop-
i.e., cellular proliferation, migration, differentiation, synapto- ment (Rodriguez et al., 2002).
(cid:1)TheAuthor2010.PublishedbyOxfordUniversityPressonbehalfoftheSocietyofToxicology.Allrightsreserved.
Forpermissions,pleaseemail:[email protected]
METALMIXTUREANDDEVELOPMENTALNEUROTOXICITY 587
Current knowledge of metal-induced neurocellular damage, The terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick end
however, is primarily confined to single metal exposure, and labeling (TUNEL) kit was purchased from Roche (Indianapolis, IN).
Diaminobenzidine tetrahydrochloride (DAB) substrate kit and Vectashield
there has been increasing demand for cumulative hazard
mediumEliteABCkitwerepurchasedfromVectorLaboratories(Burlingame,
assessment of metals in mixture in the brain (Rodriguez et al.,
CA). Omniscript RT Kit and SYBR-green qPCR Kit were from Qiagen
1998; Wright and Baccarelli, 2007). The effect may be either (Valencia,CA).
dose additive, interactive (synergistic or antagonistic), or
AnimalsandTreatments
independent of each other. Of the very few reports on metal
Allanimal-handlingprocedureswerecarriedoutfollowingtheregulations
mixture (MM), studies with early-life low doses of Pb þ Cd–
of Institutional Animal Ethics Committee and with their prior approval for
associatedexposurehaverevealedgreateroxidativestressthan
usingtheanimals.ThepregnantfemaleWistarratswereprocuredfromIndian
withsingle Pb orCd (Zhangetal., 2009).The mixture altered InstituteofToxicologyResearchandwerehousedina12-hdayandlightcycle
cerebellarandstriatalfunctionsthatrelatedtochangesinmotor environmentwithadlibitumavailabilityofdietandwater.
D
activity (Antonio et al., 2002) and anxiety (Leret et al., 2003) Thepregnantfemaleratsweredividedintoninegroupsandtreatedwiththe o
w
in the long term. The synergistic toxic effect of Pb and Cd mcoenttailnsue(Tdaibnleth1e)lathctraotuingghrgaatsvaagnedfproosmtwgeeasntiantgionpudpasytil5l.2Tmheontrtheas.tmTehnetMwMas nloa
absorption in the brain cells of cerebellum, cortex, and d
wastreatedattwodifferentconcentrations(13and103)toobservethedose- e
hiniptphoecammixptuusreis(Gaucceotunatla.,b2le00f9o)r.tThheereenfhoaren,cewdeChNypSotdhaemsizaegde dfreepqeunednetlnyt oefcfceucrtrionfgthcoenMceMntraotnionratofbrtahienmdeevtaellsopinmegnrto,u1n3dwbateeirngsotuhrecemsoosft d from
that concurrent exposure to As along with Cd and Pb in India (Jadhav et al., 2007). The single metals were treated at two different h
concentrations.Onewasthesameasthatin103andtheotherwasthreetimes ttp
drinkingwatermayhavegreater-than-additive/synergistictoxic s
responses to brain development. ofit. ://a
The number of pregnant rats per treatment group was 30. After ca
Here,wedemonstratedthetoxiceffectofAs,Cd,andPbon standardization of litters (culling), equal numbers of male and female pups de
m
rat brain development. We investigated the behavioral impair- were taken for each experiment, and pups from different litters were ic
ments induced by the MM in developing rats. Because independentsubjects(Holsonetal.,2008). .o
u
To determine the toxic effect of metals on glial damage and behavioral p
abnormal blood-brain communication is a key mechanism .c
aberrationsduringratbraindevelopment,studieswerecarriedoutatpostnatal o
underlying neuronal dysfunction (Shalev et al., 2009), we m
examined the effect of the MM on BBB integrity and glial day(P)16andadultP60rats. /tox
BehavioralStudy s
fibrillary acidic protein (GFAP) levels. We further focused ci/a
oanntatghoenitsotxicic) mofechtahneismMMof aocntionthe(adGdiFtiAveP,-esxypnreersgsiisntgic, roart usinSgpotnhteanceoomuspulotecroizmeodtoOrpatcot-iVviatyri.meLxoc(oCmoolutimvebubsehIanvsitorurminernattss,wCaoslustmudbiuesd, rticle
OH)systemaspreviouslydescribed(Alietal.,1990).Thelocomotormarkers -a
astrocytes.Collectively,ourdatastronglysuggestedthatsingle b
monitored were the distance traveled, number of stereotypic movements and s
metal risk assessment underestimates the toxic impact of the rearings,andtimemoving(inminutes). tra
c
metals present in mixture and sheds new light on the harmful t/1
Grip strength. Vehicle and MM-treated rats were subjected to forelimb 1
role of environmental metal contaminants in pediatric and 8
grip strength test using a digital grip strength meter (ColumbusInstruments) /2
long-term CNS complications. followingthestandardprocedureasdescribedpreviously(Terryetal.,2003). /5
8
Eachratwastestedfivetimes,witha10-speriodbetweentwosuccessivetrials. 6/1
Thefiverecordingswereaveragedtoobtainafinalreadingforeachindividual. 6
5
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MATERIALS ANDMETHODS TABLE1 b
y
Metal Treatment Given toPregnant, Lactating, and gu
ReagentsandAntibodies e
Na-arsenite,Pb-acetate,Cd-chloride,Na-orthovanadate,NaF,PonceauSstain, Postweaning Rats st on
Evansblue(EB),PMSF,proteaseinhibitorcocktail,MTT[3-(4,5-dimethylthiazol- 1
Group1:Vehicle Water(vehicle) 0
a2N--,ytNol)c,-No2p#,,hNe5#ro--dtle,itpHrhaoaeecnceyhtlitscettr3a3azc2oi5ldi8umstteatirnba,krpoisom(layidc-eeLt]-o,lyxsy1imn,2ee,-tBhdyiiscl(h2l-oearsmotfleirnu)oopreh(sBecnAeoiPnxTyd)Aiea-tchAeaMtnaet)e-, Group2:MM(13) CNPbda(ACCls22OH:03:O.0029.)3828:p00p.p2mp2m0þppmþ April 20
(DCF-DA),Fluo3AM,andmammaliantissueproteinextractionreagentwere Group3:MM(103) Pb(CH2O ):2.220ppmþ 19
2 3 22
procuredfromSigmaChemicalCo.(StLouis,MO).Fluo3AMwasobtained
CdCl:0.98ppmþ
2
fromMolecularProbes(Carlsbad,CA).TheAlexaFluorsecondaryantibodies,
NaAsO:3.80ppm
2
PD98059,SB203580,LY294002,andSP600125,cellculturereagents,sample
Group4:Pbindividualtreatment Pb(CHO ):2.220ppm
2 3 22
loadingbufferforWesternblotting,andproteinmarkerswerepurchasedfrom
Group5:Cdindividualtreatment CdCl:0.98ppm
2
Invitrogen (Carlsbad, CA). The supersignal west femto maximum sensitivity
Group6:Asindividualtreatment NaAsO:3.80ppm
2
substrate for Western blotting was purchased from PIERCE Biotechnology
Group7:Pbindividualtreatment Pb(CHO ):6.660ppm
2 3 22
(Rockford, IL). Rabbit polyclonal antibodies to extracellular signal-regulated
(threetimesgroup4)
kinase (ERK1/2) and Jun N-terminal kinase (JNK1/2),and phospho-ERK1/2
Group8:Cdindividualtreatment CdCl:2.94ppm
2
andphospho-JNK1/2formswerepurchasedfromCellSignalingTechnology
(threetimesgroup5)
(Danvers, MA). Mouse monoclonal antibody to GFAP was obtained from
Group9:Asindividualtreatment NaAsO:11.4ppm
2
Millipore (Temecula, CA). Mouse monoclonal antibodies to b-actin,
(threetimesgroup6)
peroxidise-conjugated secondary antibodies were from Sigma Chemical Co..
588 RAIETAL.
TABLE 2
MM Enhanced SLA andGripStrength, andReduced Learning-Memory Performance in DevelopingRats
Stereotypic Learning-memory
Totaldistance Rearings Timemoving movements Gripstrength No.oferrors Learning-memory (7thday)
(cm/5min) in5min (min)in5min (in5min) (newton) intrainingphase (24h)(%saving) (%saving)
Vehicle 14±0.8 6±1 1.7±0.03 7±1 154±6.0 11.67±0.67 44 33.75
13 17±1.3* 24±2* 2.1±0.03* 9±1* 173±2.0* 13.33±0.33* 37.82* 4.97**
103 21±2.2* 50±4** 2.8±.0.03** 16±2** 200±8.0** 14.66±0.33* 34.32** 0.23***
Note.TheMM-treatedP16ratswereassessedforSLAandgripstrength.TheMM-treatedP60ratswereassessedforlearning-memoryperformance(refertothe
‘‘MaterialsandMethods’’section).Dataareexpressedasmeansof±SEofeightpostnatalratsfromdifferentlitters. D
o
*p<0.05,**p<0.01,and***p<0.001(comparedwithvehicle). w
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Y-maze. Learning-memory test for vehicle- and MM-treated rats was arms with the lights off (dark arms) were unsafe areas with electric de
d
c1a9r8r2ie)dwoiutht amsidneosrcrmiboeddifipcraetvioionuss.lyThuesintrgainYi-nmgazaepp(aWraetutzselwaansdaMYat-tmhiaezse, fdouortinshgoctekst(i1n–g5. TmhAe).trTaihneinsgafseesasnidonuncsoanfseisateredasofwe3r0etarribalistrapreilryasnhiimftaeld. from
with electrifiable grid-floored three alleys and 15-W light bulb at the end Running into the dark alley of the Y-maze was counted as an error (E). h
ofthealleys.Whentheratswerebeingtested,onlyonearmwithitslight Retention of the brightness discrimination was tested after 24 h and after ttp
s
on(bright arm)wasasafeareawithout footshock,whereastheothertwo 7daysofinitiallearningusinga30-trialrelearningsession;performancein ://a
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FIG.1. MMpromotedBBBpermeabilityindevelopingratbrain.EB(3%)wasinjectedthroughthefemoralveinofthevehicle(V)–andMM(13and103)–treated
(A)P16and(B)P60rats.Thewhole-brainsampleswereincubatedinformamide,andtheODofsupernatantwastakenat620nm.AverageofODratiosoftheMM-
treatedovervehicle-treatedratswasdetermined.Datarepresentmeans±SEoffourpupsfromfourdifferentlitters.**p<0.01and***p<0.001(comparedwithV).
EBwasinjectedinvehicle(V)–andMM-treated(103)-P60rats,and20-lmcryostatsectionsofcerebralcortex(coronal)andcerebellum(transverse)wereobserved
under fluorescence microscope. (C) Representative photomicrograph (310 magnification) showing EB fluorescence in the posterior lobe of rat cerebellum.
(D) Representative photomicrograph (310 magnification) showing EB fluorescence in white matter of rat cerebellum. (E) Representative photomicrograph (310
magnification)ofEBfluorescenceinthecorpuscallosum.ThesectionsarerepresentativesoffourP60ratsfromfourdifferentlitters.
METALMIXTUREANDDEVELOPMENTALNEUROTOXICITY 589
TABLE 3
Levels of As,Cs, and PbinMM-TreatedDeveloping RatBrain
P16 P60
As(ppb) Cd(ppb) Pb(ppb) As(ppb) Cd(ppb) Pb(ppb)
Group1:vehicle bdl bdl bdl bdl bdl bdl
Group2:MM(13) 14.25±3.10* 17.25±4.17* 60.72±6.01** 33.7±5.05* 52.68±6.88** 87±5.23**
Group3:MM(103) 63.72±7.35** 52.56±5.38** 135.11±11.20*** 87.23±9.28** 106.70±9.17*** 128.62±12.67***
Group4:Pbindividualtreatment bdl bdl 143.18±13.34*** bdl bdl 150.82±13.01***
Group5:Cdindividualtreatment bdl 53.67±4.82** bdl bdl 95±8.63** bdl
Group6:Asindividualtreatment 64.56±9.16** bdl bdl 90.17±7.52** bdl bdl D
o
Group7:Pbindividualtreatment bdl bdl 288.22±15.65*** bdl bdl 270.57±21.01*** w
n
(threetimesgroup4) lo
a
Group8:Cdindividualtreatment bdl 139±13.01*** bdl bdl 201±29.768*** bdl d
e
(threetimesgroup5) d
Group9:Asindividualtreatment 108.32±10.42*** bdl bdl 120.63±12.39*** bdl bdl fro
m
(threetimesgroup6) h
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speNcotrtoep.hWothoomlee-tberra.iDnastaamepxlpersefsrsoemdaPs1m6eaanndsP±60SEraotsfwfoeurrepsunpapsffrroomzenfoiunrldiqifufiedrennittrloitgteerns,.abcdidl,dbiegleoswteddeatnecdtitohnelmimeitta.lcontentdeterminedbyatomicabsorption s://ac
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*p<0.05,**p<0.01,and***p<0.001(comparedwithvehicle). d
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ic
relearningwasconsideredastestformemoryandexpressedaspercent(%) stages,anesthetized,andperfusedandthebrainwasfixedandcryoprotectedas .ou
p
saving(Serota,1971). described previously (Sinha et al., 2009; Zhu et al., 2001). Five-micron .c
transversesectionsweremadefromthecerebellumandcoronalsectionsofthe om
%saving ¼ ðEtraining(cid:1)EtestÞ3100=Etraining cerebralcortexusingcryomicrotome(MicromHM520;Labcon).Thesections /to
were then mounted on 3-aminopropyltriethoxysilane-coated slides. Immuno- xs
c
peroxidasestainingforGFAP(monoclonal,1:400)wascarriedoutwithDAB i/a
AnalysisofEBExtravasationinBrain chromogen and ABC kit as described previously (Otani et al., 1999) and rtic
visualizedunderopticalmicroscope(NikonInstechCo.Ltd).Tenfieldsineach le
femRoaratslwveeirne.aTnheesthraettsizwederaentdhe3n%trEaBnsicnarsdailainlleywpearsfiunsjeedctaenddsltohwelbyrathinroiusgohlatthede sectionwerecapturedusing340objectiveforevaluation.Image-ProPlus5.1 -abs
and incubated in formamide as previously described (Lin et al., 2010). The software (Media Cybernetics Inc., Silver Spring, MD) was used for image tra
capturing. c
smuepaesrunraetdantuswinagsacoSllPeEctCedT,RaAndmtahxePoLpUticSa3l84desnpseicttyrop(OhoDto)maette6r20(Mnomlecwulaasr TheimageswerethenimportedintoImage-J1.42q(http://rsb.info.nih.gov/ t/11
devices, Sunnyvale, CA) to determine the relative amount of EB in each ij/; developed by Wayne Rasband, National Institutes of Health, Bethesda, 8/2
MD).ThenumberofGFAP-immunoreactive(ir)cellsandthearea,perimeter, /5
sample.TheODratiowasderivedfromtheODoftheMM-treatedanimalsover 8
compactness, and number of processes in the GFAP-ir cells were quantified 6
thatofthevehicle-treatedanimals. /1
withtheShapedescriptorspluginofthesoftware. 6
Fluorescence study of EB extravasation was carried out as described 5
1
previously (Duran-Vilaregut et al., 2009). Briefly, EB-injected rats were DetectionofApoptosis(invivo)inGFAP-irAstrocytes 67
perfused; cerebral cortex and cerebellum dissected, postfixed in 4% para- 3
formaldehyde (PFA), and cryoprotected with 30% sucrose; and 20-lm-thick InsitudetectionofapoptosiswascarriedoutbyTUNELassayinP16and by
cryostaticsectionsobtainedusingcryomicrotome(MicromHM520;Labcon, P60 rats. Briefly, four pups from four different litters were taken at the gu
Germany). The cryostat sections were visualized under fluorescence micro- developmentalstages,anesthetized,andperfusedandthebrainwasfixedand es
scope (Nikon Instech Co. Ltd, Kawasaki, Kanagawa, Japan) after being cryoprotected as described previously (Sinha et al., 2009; Zhu et al., 2001). t on
coverslippedonVectashieldmedium(VectorLaboratories). Five-microntransversesectionsfromthecerebellumandcoronalsectionsfrom 1
0
the cerebral cortex were made using cryomicrotome (Microm HM 520; A
EvaFlouratdioenteromfiMnientgaltsheinleBvrealisnofAs,Cd,andPbinbrain,whole-brainsamples Lfluabocreosnc)e.inF-olarbethleedTdUUTNPELintahsesapyr,esaenlcaebeolfinTgdTreaatc3ti7o(cid:2)nCwfoars1cha.rTrioedinovuesttiwgaitthe pril 2
0
weresnapfrozeninliquidnitrogenandkeptina(cid:1)80(cid:2)Cfreezertillanalysis. whethertheapoptoticcellswereastrocytes,thesectionswereimmunostained 19
Samples for Pb, Cd, and As estimation were prepared by acid digestion as withanti-mouseGFAPantibody(1:400dilutioninTBST[10mMTris,pH8.0,
previouslydescribed(Ongetal.,2006;SinghandRana,2007),andanalysis 150mMNaCl,0.01% Tween20]) accordingtomanufacturer’s protocol. The
was carried out using atomic absorption spectrophotometer equipped with sections were then incubated with Alexa Fluor 546–conjugated (fluorescent
avaporgenerationassembly(VarianAAS250þcoupledwithVGA77;Varian color: red; Abs/Em: 555/565) goat anti-mouse antibody (1:200 dilution) as
AustraliaPvtLtd[manufacturingsite],Mulgave,Australia).Detectionlimitof previously described (Bandyopadhyay et al., 2007); counterstained with
theinstrumentwas1ppb(BehariandPrakash,2006). Hoechst 33258 (0.2mM) for 10 min; and visualized under a fluorescence
microscope (Nikon Instech Co. Ltd) after being coverslipped on Vectashield
ImmunohistochemistryandQuantitativeEstimationof medium(VectorLaboratories).
GFAP-ImmunoreactiveAstrocytes
ProteinExtractionandWesternBlotting
Immunoperoxidase staining with 5-lm cryostat sections of cerebellum
(transverse)andcerebralcortex(coronal)wascarriedoutforGFAPantibody. Cerebralcortexandcerebellartissuesfromfivetosevenpostnatalratswere
Briefly,fourpupsfromfourdifferentlittersweretakenatthedevelopmental harvested, snap frozen in liquid nitrogen, and stored at (cid:1)80(cid:2) C until further
590 RAIETAL.
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FIG.2. MMreducedsizeandnumberofGFAP-irastrocytesindevelopingratcortexandcerebellum.Five-micron-thickcryostatsectionsofcerebralcortex
(coronal section) and cerebellum (transverse section) from vehicle (V)– and MM (103)–treated P16 and P60 rats were stained for GFAP using peroxidase
conjugateandDABchromogen.(A)Representativephotomicrograph(340magnification)ofGFAP-irastrocytesinP16ratcerebralcortex.(B)Representative
photomicrograph (340 magnification) of GFAP-ir astrocytes in P16 rat cerebellum. (C) Representative photomicrograph (340 magnification) of GFAP-ir
astrocytesinP60rat cerebralcortex.(D)Representativephotomicrograph(340magnification)of ratGFAP-irastrocytesinP60rat cerebellum.Thesections
arerepresentativesoffourP16orP60ratsfromfourdifferentlitters.
investigation. SDS polyacrylamide gel electrophoresis and Western blotting and ERK1/2, previously described protocol was followed (Bandyopadhyay
weredonewiththetissuesfollowinganoptimizedprotocol(Sinhaetal.,2009) etal.,2006).Theworkingdilutionsfortheprimaryantibodieswereasfollows:
withGFAPandb-actinantibodies.TodeterminethephosphorylationofJNK1/2 GFAP(monoclonal,1:1000),b-actin(monoclonal,1:1000),JNK1/2(polyclonal,
METALMIXTUREANDDEVELOPMENTALNEUROTOXICITY 591
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FIG.2. Continued. E-H.MMpromotedapoptosisinGFAP-irastrocytesindevelopingratcortexandcerebellum.Five-micron-thickcryostatsectionsof s
cerebral cortex (coronal section) and cerebellum (transverse section) from vehicle (V) - and MM (10X) -treated P16 and P60 rats were stained for TUNEL, trac
followedbyGFAPandnuclearHoechstcostain.(E)Representativephotomicrograph(X40magnification)ofapoptoticTUNEL-positivecells(greenfluorescence, t/1
1
marked with white arrowhead), GFAP (red fluorescence), nucleus (blue fluorescence), and the three merged in the same field in P16 rat cerebral cortex. 8
/2
(F) Representative photomicrograph (X40 magnification) of apoptotic TUNEL-positive cells (green fluorescence, marked with white arrowhead), GFAP (red /5
fluorescence),nucleus(bluefluorescence),andthethreemergedinthesamefieldinP16ratcerebellum.(G)Representativephotomicrograph(X40magnification) 86
ofapoptoticTUNEL-positivecells(greenfluorescence,markedwithwhitearrowhead),GFAP(redfluorescence),nucleus(bluefluorescence),andthethreemerged /1
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inthesamefieldinP60ratcerebralcortex.(H)Representativephotomicrograph(X40magnification)ofapoptoticTUNEL-positivecells(greenfluorescence, 51
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markedwithwhitearrowhead),GFAP(redfluorescence),nucleus(bluefluorescence),andthethreemergedinthesamefieldinP60ratcerebellum.Thesections 7
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arerepresentativesoffourP16orP60ratsfromfourdifferentlitters. b
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1:1000),ERK1/2(polyclonal,1:1000),phospho-JNK1/2(polyclonal,1:1000), As,Cd,Pb,orMMatconcentrationsrangingfrom0.01to200lMfor18hin st o
aanndti-prahbobspithoIg-EGRK(fo1r/2th(peolpyocllyocnlaol,na1l:1p0r0i0m).arTyheanwtiobrokdiniegs)dialuntdionasntfio-mrsoeucsoendIagrGy ahTumheidiinfiveodlvteismsueentcoufltsuirgenianlcinugbaptaotrhawta3y7s(cid:2),Csuwchitahs5P%I3CkOin2a-s9e5,%MEaiKr.1/2,P38- n 10
(forthemonoclonalprimaryantibodies)conjugatedtohorseradishperoxidase MAPK, and JNK1/2, in MM-induced astrocyte toxicity was determined by A
p
were1:1000inPBSplus0.2%TritonX-100.Thesamplesweredetectedby incubating the astrocytes with the MM and cotreating with LY294002 ril 2
chemiluminescence with the supersignal west femto maximum sensitivity (10lM), PD98059 (10lM), SB203580 (10lM), or SP600125 (10lM), which 0
1
substrate.Relativeexpressionofeachproteinwasdeterminedbydensitometric are inhibitors to PI3 kinase, MEK1/2, P38-MAPK, and JNK1/2 pathways, 9
quantification of blots using the VersaDoc Gel Imaging System (BioRad, respectively.
Hercules,CA). The time course of phosphorylation of MEK1/2 (assessed by ERK1/2
phosphorylation) and JNK1/2 in MM-treated astrocytes was determined by
CellCulture
incubatingthecellswithMMfor0,5,10,15,30,and60min.
Astrocytes. Rat pups(P1) weredecapitated, thebrainwas removed,and CytotoxicityAssay
astrocytes were cultured from the rat brain as described previously (Tanaka
et al., 1998). The purity of astrocytes was 94–97% as determined by the MTT cell viability assay, a colorimetric assay, was used to determine
immunofluorescencestainingofGFAP. astrocyteviability.Theastrocytesweregrownto80%confluence,pre-incubated
inreducedserum(0.5%FBS)mediumfor2h,andthentreatedwithAs,Cd,Pb,
TreatmentofCellswithMetalsandCellSignalingInhibitors
orMMatconcentrationsrangingfrom0.01to200lMfor18hinreducedserum
The astrocytes were grown to 80% confluence, pre-incubated in reduced mediuminahumidifiedtissuecultureincubatorat37(cid:2)Cwith5%CO-95%air.
2
serum(0.5%fetalbovineserum[FBS])mediumfor2h,andthentreatedwith Thecellswerethenincubatedwith10llMTT(10.4mg/ml)andanoptimized
592 RAIETAL.
protocol was followed to determine cell viability (Sanders et al., 2000). indicating intracellular ROS production was measured using a fluorescent
Absorbancewasmeasuredat595nm,withbackgroundsubtractionat655nm. microplatereaderwithexcitationat485nmandemissionat530nm.
TheLCof themetalsonastrocyteswasanalyzedbytheGraphPadPrism3.0
EvaluationofMetal(s)InteractioninCombination
software.
Tocharacterizetheinteractionbetweentheheavymetalsfortheireffectson
TUNELAssay astrocyteviability,apoptoticindexandGFAPlevel,acombinationindex(CI)
TheinsitudetectionofapoptosisinastrocyteswascarriedoutwiththeIn was calculated using the software Calcusyn (Biosoft, Manchester, United
SituCellDeathDetectionfluoresceinkit(RocheAppliedScience)forTUNEL Kingdom). CI values less than 1.0 indicated synergism (Zhao et al., 2004),
whichsuggestsgreaterthanexpectedbasedoneffectaddition.
assay,asperthemanufacturer’sinstructionandvisualizedunderafluorescence
TheU.S.EnvironmentalProtectionAgencyhasselecteddoseadditionasthe
microscope(NikonInstechCo.Ltd).Briefly,theastrocytesweregrownto80%
confluence,pre-incubatedinreducedserummediumfor2h,andthentreated no-interactiondefinitionformixtureriskassessment,sothatsynergismwould
represent toxic effects that exceed those predicted from dose addition
withAs,Cd,Pb,orMMinreducedserummediumataconcentrationrangeof
(Hertzberg and MacDonell, 2002). Synergistic interactions have implications
LC10toLC100ofthemetalsfor18hinahumidifiedtissuecultureincubatorat D
of gain (Berenbaum, 1985) and the combined effects are greater than the o
37(cid:2)C with 5% CO2-95% air. The cells were counterstained with Hoechst w
33258. The TUNEL-positive cells were counted in five randomly selected additiveeffectofthecomponents(Wessinger,1986). nlo
fields.Around10,000cellsineachcoverslipwerescored.Theapoptoticindex StatisticalAnalysis ad
e
wasexpressedasthenumberofTUNEL-positivecellsper100nuclei(Hoechst d
stained).Image-ProPlus5.1software(MediaCyberneticsInc.)wasusedfor Dataarepresentedasmean±SEoftheindicatednumberofexperiments. fro
cellcounting. StatisticalanalysiswascarriedoutinSPSS9.0software(SPSSInc.,Chicago, m
IL).Datawereanalyzedbyone-wayANOVA,followedbyStudent-Newman, h
Assayfor[Ca2þ]i KeulsposthoctestorStudent’st-testwhenappropriate. ttps
Theastrocytesweregrownto80%confluenceat4000cells/wellin96-well ://a
c
poly D-lysine-coated black view plates (VWR no. 62406–036; Falcon), pre- RESULTS ade
incubated in reduced serum medium for 2 h, and then treated with MM in m
reducedserummediumfor2min,5min,10min,30min,1h,2h,and24hin ic
Effect of MM on SLA, Grip Strength, and Learning-Memory .o
ianchrueamsiediinfie[dCat2isþs]uiewacsulmtueraesuirnecdubuasitnogr Fatluo337(cid:2)ACMwaisthan5%indiCcOat2o-r9d5y%eaafitre.rAthne Performance in Developing Rats up.co
addition of metals (single or in mixture) to the culture wells following an IthasbeenpreviouslyreportedthatexposuretoinorganicAs m
optimized protocol (Arey et al., 2005). The fluorescentsignalswereread by /to
(Rodriguez et al., 2002), Cd (Ishitobi et al., 2007), or Pb x
fluorescenceimagingplatereaderSynergyHT(BioTek,Winooski,VT). s
(Marchetti, 2003) caused behavioral alterations in developing ci/a
AssayforROSGeneration rats.We,therefore,investigatedwhetherMMtreatmentaltered rtic
The production of ROS, mainly H2O2, was assessed using DCF-DA, SLA, grip strength, and learning-memory performance in le-a
followed by semiquantitative fluorometric measurements as described pre- developing rats. The rats exhibited dose-dependent increase bs
viously(Izawaetal.,2009).Theastrocytesweregrownto80%confluenceat in the distance traveled, number of stereotypic movements, tra
c
4000 cells/well in 96-well poly D-lysine-coated black view plates (VWR no. numberofrearings,movementtime,andgripstrength(Table2). t/1
62406–036;Falcon);pre-incubatedinreducedserummediumfor2h;treated 1
withMMfor0min,30min,60min,2h,orovernight;andthenincubatedwith MM treatment also demonstrated dose-dependent reduction in 8/2
DCF-DA to a working concentration of 20 lg/ml. The fluorescent level learning-memory performance (Table 2). /58
6
/1
6
5
1
6
7
3
TABLE 4 b
y
SizeandDensity ofAstrocytes inVehicle-Treated andMM-Treated P16andP60Rats g
u
e
s
Astrocyte t o
n
1
0
Count/0.081mm2 Surfacearea(lm2) Perimeter(lm) Compactness No.ofprocesses A
p
Cerebralcortex V-P16 35±1.20 1762±107.75 149±17.50 0.88±0.02 8±1.00 ril 2
0
103-P16 7.71±1.25*** 722.42±60.25*** 87.91±12.20*** 0.722±0.05** 6.16±0.23** 19
V-P60 34±0.75 2750.92±15.7631 184.91±6.231 0.90±0.03 7.04±0.80
103-P60 14.25±0.75*** 1283.48±91.561*** 126.83±13.25** 0.86±0.05* 5.10±0.50**
Cerebellum V-P16 30±0.80 1450.29±52.75 137.76±12.50 0.86±0.042 6.97±0.20
103-P16 8.44±0.23*** 768.5±20.02** 108.23±8.92** 0.84±0.05* 5.4366±0.02**
V-P60 29.75±0.75 2058.2±120.35 160.18±17.50 0.90±0.035 5.12±0.30
103-P60 16.25±0.25*** 1091.74±52.6*** 115.33±9.711** 0.79±0.025** 4.20±0.2**
Note.Five-microncryostatsectionsofcerebralcortexandcerebellumweremadefromvehicle(V)–andMM(103)–treatedP16andP60rats,immunostained
withGFAP,andimagesobtainedwithopticalmicroscopy.Theimageswerethenanalyzed(refertothe‘‘MaterialsandMethods’’section).Thenumber,surface
area,perimeter,andcompactnessofastrocytes,andnumberofastrocyteprocessesinV-andMM-treatedratsweredetermined.Dataexpressedasmeans±SEof
fivepupsfromfivedifferentlitters.
*p<0.05,**p<0.01,and***p<0.001(comparedwithV).
METALMIXTUREANDDEVELOPMENTALNEUROTOXICITY 593
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FIG.3. MMsuppressedGFAPexpressionindevelopingratbrain.Thecerebralcortexandcerebellumofvehicle(V)–andMM-treated(13or103)P16 xs
c
andP60ratswereimmunoblottedforGFAPandb-actin.(A)RepresentativeWesternblotanddensitometricanalysesofrelativeGFAPexpressionnormalized i/a
withb-actininratcerebralcortexatindicatedpostnataldays.Datarepresentmeans±SEoffourpupsfromfourdifferentlitters.*p<0.05,**p<0.01,and rtic
***p < 0.001 (compared with V). (B) Representative Western blot and densitometric analyses of relative GFAP expression normalized with b-actin in rat le
cerebellumatindicatedpostnataldays.Datarepresentmeans±SEoffourpupsfromfourdifferentlitters.*p<0.05,**p<0.01,and***p<0.001(comparedwith -ab
s
V).(C)RepresentativeWesternblotanddensitometricanalysesofrelativeGFAPexpressionnormalizedwithb-actinincerebralcortexofP60ratsafterMM tra
treatmentorpostweaningMMwithdrawal(WD).Datarepresentmeans±SEoffourpupsfromfourdifferentlitters.*p<0.05and***p<0.001(comparedwith c
V).(D)RepresentativeWesternblotanddensitometricanalysesofrelativeGFAPexpressionnormalizedwithb-actinincerebellumofP60ratsafterMMtreatment t/11
8
orpostweaningMMwithdrawal(WD).Datarepresentmeans±SEoffivepupsfromfivedifferentlitters.*p<0.05and***p<0.001(comparedwithV). /2
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8
6
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Effect of MM on BBB Permeability in Developing Rat Brain to deposition of the metals in rat brain. Because the metals 6
5
Increase in BBB permeability is a potentially important couldreachthefetusthroughplacenta(Benitezetal.,2009;Xi 16
7
cause of brain dysfunction including behavioral disorders et al., 2010), and lactating off-springs through mother’s milk 3 b
(Shalev et al., 2009). Moreover, it has been reported that As (Counter et al., 2007; Kippler et al., 2009; Samanta et al., y g
u
and Cd disrupted the integrity of BBB (Zheng, 2001) and 2007),weinvestigatedwhethermetal(s)treatment(groups1–9) es
exposure to Pb disrupted the BBB (Lidsky and Schneider, caused theirdeposition in developing brain. Adose-dependent t o
n
2003). We, therefore, determined the effect of MM on BBB increase in the levels of As, Cd, and Pb was observed in the 10
permeability by measuring EB extravasation. We observed postnatal rat brain (Table 3). Ap
dose-dependent increase in EB extravasation in MM-treated ril 2
postnatal rat brain (Figs. 1A and 1B). Transverse section of EffectofMMontheNumber,Area,Perimeter,Compactness, 01
9
cerebellum (Figs. 1C and 1D) and coronal section of cerebral Processes, and Apoptosis of Astrocytes in Developing Rat
cortex (Fig. 1E) exhibited marked BBB damage, as evident Brain
from the increased red fluorescence (indicative of EB leakage) Astrocytes are involved in the maintenance of BBB (Choi
in adult rats. and Kim, 2008; Pekny et al., 1998), and loss of BBB
integrity is associated with astrocyte damage under neuro-
Levels of As, Cd, or Pb in Metal-Treated Developing Rat pathological conditions (Prior et al., 2004; Willis et al.,
Brain 2004).BecauseweobservedthatMMpromotedpermeability
Previous studies have demonstrated that exposure to of the developing rat BBB, we studied the response of the
inorganic As (Garcia-Chavez et al., 2006), Cd (Lafuente astrocytes to MM. We observed reduction in the GFAP-ir
etal.,2001),orPb(Benitezetal.,2009;Raderetal.,1981)led astrocytecount,area,perimeter,compactness,andnumberof
594 RAIETAL.
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FIG.3. Continued. E-H.MMsuppressedGFAPexpressionsynergisticallyindevelopingratbrain.(E)RepresentativeWesternblotshowingGFAP-and t/1
1
beta-actin-irbandsinwholebrainofP16ratstreatedwithvehicle,singlemetal(group4,5,or6ofTable1),andMM(10X).Fordensitometricanalysis,brainfrom 8/2
single metaltreated P16 rats was isolated and GFAP level suppression for each metal determined through Western blotting. The fold-suppression in GFAP /5
8
expressionbythethreesinglemetalswasadded(Ad)andcomparedwiththatbyMM.Densitometricdatarepresentmeans±SEoffivepupsfromfivedifferent 6
/1
litters.***p<0.001(comparedwithAd).(F)RepresentativeWesternblotshowingGFAP-andbeta-actin-irbandsinwholebrainofP16ratstreatedwithvehicle, 6
5
singlemetal(group7,8,or9ofTable1),andMM(10X).Fordensitometricanalysis,brainfromsinglemetal-treatedP16ratswasisolatedand1/3rdofGFAPlevel 1
6
suppressionforeachmetaldetermined.Thefold-suppressioninGFAPexpression(1/3rdvalues)bythethreesinglemetalswasadded(Ad)andcomparedwiththat 7
3
byMM.Densitometricdatarepresentmeans±SEoffivepupsfromfivedifferentlitters.***p<0.001(comparedwithAd).(G)RepresentativeWesternblot b
y
showingGFAP-andbeta-actin-irbandsinwholebrainofP60ratstreatedwithvehicle,singlemetal(group4,5,or6ofTable1),andMM(10X).Fordensitometric g
u
analysis, brain from single metal-treated P60 rats was isolated and GFAP level suppression for each metal determined through Western blotting. The fold- e
s
suppressioninGFAPexpressionbythethreesinglemetalswasadded(Ad)andcomparedwiththatbyMM.Densitometricdatarepresentmeans±SEoffivepups t o
fromfourdifferentlitters.***p<0.001(comparedwithAd).(H)RepresentativeWesternblotshowingGFAP-andbeta-actin-irbandsinwholebrainofP60rats n 1
treatedwithvehicle,singlemetal(group7,8,or9ofTable1),andMM(10X).Theblotrepresentsdataoffiveratsfromfivedifferentlitters.Fordensitometric 0 A
analysis,brainfromsinglemetal-treatedP60ratswasisolatedand1/3rdofGFAPlevelsuppressionforeachmetaldeterminedthroughWesternblotting.Thefold- p
suppressioninGFAPexpression(1/3rdvalues)bythethreesinglemetalswasadded(Ad)andcomparedwiththatbyMM.Densitometricdatarepresentmeans± ril 2
SEoffourpupsfromfourdifferentlitters.***p<0.001(comparedwithAd). 01
9
processesinthecerebralcortexandcerebellum(Figs.2A–D, adulthood (Figs. 3A and 3B). Upon withdrawal of MM
Table 4). We further observed that the MM promoted treatment from weaning, the GFAP level was significantly
apoptosisinthedevelopingcerebralandcerebellarastrocytes restored in adults (Figs. 3C and 3D). This suggests that the
(Figs. 2E–H). decrease in level of GFAP was primarily caused by MM
treatment.
Effect of MM on GFAP Level in Developing Rat Brain We further investigated whether MM had synergistic or
We studied the effect of MM on the GFAP levels in the additive toxic effect on the level of GFAP in the developing
developingcerebralcortexandcerebellum.Weobserveddose- ratbrain.Wedeterminedtheeffectofthesinglemetals(groups
dependent decrease in the level of GFAP that persisted till 4–9 in the ‘‘Materials and Methods’’ section) or of MM (103
METALMIXTUREANDDEVELOPMENTALNEUROTOXICITY 595
TABLE5 astrocyteswiththeMMandcotreatedthecellswithLY294002
LCValuesof As, Cd,andPbin RatPrimaryAstrocytes (10lM), PD98059 (10lM), SB203580 (10lM), or SP600125
(10lM), which are inhibitors to PI3 kinase, MEK1/2, P38-
LC10(lM) LC25(lM) LC50(lM) LC75 MAPK, and JNK1/2 pathways, respectively. Inhibitors them-
selves were nontoxic to astrocytes. Treatment with PD98059
As 9.99 24.99 49.95 74.93
or SP600125 promoted viability of MM-treated astrocytes
Cd 2.01 4.80 10.05 15.08
Pb 15 37.5 75.0 112.5 (Fig. 4A) but LY294002 or SB203580 did not, suggesting the
involvementofMEK1/2andJNK1/2pathwaysinMM-induced
Note.The80%confluentratprimaryastrocyteswerepretreatedinreduced toxicity.
serummediumfor2handthentreatedwithAs,Cd,andPbatconcentrations We then studied the time course of phosphorylation of
ranging from 0.01 to 200lM in reduced serum medium for 18 h, and cell
MEK1/2 (assessed by ERK1/2 phosphorylation) and JNK1/2.
viabilitywasdetermined(refertothe‘‘MaterialsandMethods’’section). D
We observed that phosphorylation of ERK1/2 started before o
w
concentration)ontheGFAPlevel.Weobservedthatthesumof 5 min, reached its peak at 10 min, and declined to less than nlo
a
the basal level in 30 min (Fig. 4B). The phosphorylation of d
thefold-reductioninGFAPlevelbysinglemetalswaslessthan e
tshyenefrogldistriecdGucFtAioPnsinupGprFeAssPiobnybMyMMM(F.igs. 3E–H), suggesting JdNecKli1n/e2dstthaertreedaftaetr1t0o mthien,braesaaclheledveiltsinpea3k0 amtin15(Fmigin.,4aCn)d. d from
Because MM-induced phosphorylation of ERK1/2 preceded h
ttp
EffAecsttroofcyMteMCounltuVrieability and Apoptosis of Rat Primary pohfosJpNhKor1y/l2atiownassofEJRNKK11//22,wdeepdeentdeermnti.neWdwehestihmeurlatacntievoautisolyn s://ac
a
incubated the primary astrocytes with PD98059 and MM, d
WeinvestigatedthetoxicmechanismofactionofMMinthe e
m
and observed that PD98059 blocked MM-stimulated phos-
rat primary astrocytes. ic
phorylation of JNK1/2 (Fig. 4D). But upon incubation of the .o
We first determined the effect of As, Cd, or Pb on the u
p
astrocyte viability. The individual metals induced cell death, cells with SP600125 (10lM) and MM, the former failed to .co
block MM-stimulated phosphorylation of ERK1/2 (Fig. 4E). m
athned itnhdeilveitdhuaallcomnecteanlstrawtieornes d(LeCte1rm0,iLneCd25,(TLaCb5le0, a5n).dWLCe75t)heonf ThesedatasuggestedthatMMtoxicityinratprimaryastrocytes /toxs
treated the cells with MM and observed that MM induced involvesanupstreamMEK1/2activationfollowedbyJNK1/2. ci/a
greater cell death than the sum of cell death induced by rtic
individual metals (Table 6), suggesting that As, Cd, and Pb Effect of MM on [Ca2þ]i and ROS Generation in the Rat le-a
induced synergistic reduction in astrocyte viability. Primary Astrocytes bs
tra
We determined the effect of As, Cd, Pb, or of MM on the It has been previously reported that induction of apoptosis c
astrocyte apoptotic index. We observed that the MM by metals correlates with [Ca2þ]i release and ROS generation t/118
synergistically promoted apoptosis in the astrocytes (Table 6). (Yangetal.,2008)inastrocytes,andtherefore,weinvestigated /2
/5
the effect of MM on them. 8
6
EffectofMMonActivationoftheMAPKSignalingPathways We treated the astrocytes with MM and observed that the /1
6
in the Rat Primary Astrocytes MM triggered [Ca2þ] release. The [Ca2þ] release reached its 51
i i 6
Possibleinvolvementofthesignalingpathways,suchasPI3 peak after 30 min of MM treatment (Fig. 5A). Similarly, MM 73
b
kinase, MEK1/2,P38-MAPK,and JNK1/2, was determined in triggered ROS generation, and the ROS generation reached its y
g
MM-induced toxicity in the astrocytes. We incubated the peak after 1 h of MM treatment (Fig. 5B). u
e
s
t o
TABLE 6 n
1
MM-Induced SynergisticReduction in ViabilityandIncrease inApoptosis inRatPrimaryAstrocytes 0
A
p
Viability% Apoptoticindex% ril 2
0
As,Cd,andPbconcentrationinMM 1
9
Ad MM Ad MM
As(3.33lM)þCd(0.67lM)þPb(5lM) 90.59±0.46 72.25±5.39*** 10.31±0.62 22.865±0.67***
As(8.33lM)þCd(1.67lM)þPb(12.5lM) 75.25±2.97 53.09±3.21*** 25.25±0.73 43.23±1.74***
As(16.67lM)þCd(3.33lM)þPb(25lM) 50.32±1.76 27.96±2.73*** 49.87±2.03 67.56±3.82***
As(25lM)þCd(5lM)þPb(37.5lM) 25.16±0.97 7.43±1.52*** 73.98±2.56 98.42±5.05***
Note.The80%confluentratprimaryastrocyteswerepretreatedinreducedserummediumfor2handthentreatedwithAs,Cd,andPborMMat1/3ofLC ,
10
LC ,LC ,andLC ofthemetalsinreducedserummediumfor18h,andcellviabilityandapoptosisweredetermined(refertothe‘‘MaterialsandMethods’’
25 50 75
section).Theviabilityandapoptoticindexoftheastrocytesforeverysinglemetalwereadded(Ad).Thiswascomparedwithviabilityandapoptoticindexof
MM-treatedcells.Datarepresentmeans±SEoffourindependentexperimentsintriplicates.
***p<0.001(comparedwithAd).