Stress response to cadmium and manganese in Paracentrotus lividus developing embryos is mediated by nitric oxide

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Stress response to cadmium and manganese in

Paracentrotus lividus developing embryos is

mediated by nitric oxide

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AQUATIC TOXICOLOGY · NOVEMBER 2014

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Oriana Migliaccio

Stazione Zoologica Anton Dohrn di Napoli

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Immacolata Castellano

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Available from: Immacolata Castellano Retrieved on: 07 January 2016

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AquaticToxicology156(2014)125–134

ContentslistsavailableatScienceDirect

Aquatic

Toxicology

jo u r n al h om ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / a q u a t o x

Stress

response

to

cadmium

and

manganese

in

Paracentrotus

lividus

developing

embryos

is

mediated

by

nitric

oxide

Oriana

Migliaccio

a

_,

_Immacolata

_Castellano

a

_,

_Giovanna

_Romano

b

_,

_Anna

_Palumbo

a,∗

a_Laboratory_of_Cellular_and_{Developmental}_Biology,_Stazione_Zoologica_Anton_Dohrn,_Villa_Comunale,₈₀₁₂₁_Naples,_Italy b_Laboratory_of_Functional_and_Evolutionary_Ecology,_Stazione_Zoologica_Anton_Dohrn,_Villa_Comunale,₈₀₁₂₁_Naples,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received14July2014

Receivedinrevisedform8August2014 Accepted12August2014

Availableonline20August2014 Keywords: Cadmium Development Manganese Nitricoxide Paracentrotuslividus Stress

a

b

s

t

r

a

c

t

Increasingconcentrationsofcontaminants,oftenresultingfromanthropogenicactivities,havebeen reportedtooccurinthemarineenvironmentandaffectmarineorganisms.Amongthese,themetalions cadmiumandmanganesehavebeenshowntoinducedevelopmentaldelayandabnormalities,mainly reflectingskeletonelongationperturbation,intheseaurchinParacentrotuslividus,anestablishedmodel fortoxicologicalstudies.Here,weprovideevidencethatthephysiologicalmessengernitricoxide(NO), formedbyl-arginineoxidationbyNOsynthase(NOS),mediatesthestressresponseinducedby cad-miumandmanganeseinseaurchins.WhenNOlevelswereloweredbyinhibitingNOS,theproportion ofabnormalpluteiincreased.Quantitativeexpressionofapanelof19genesinvolvedinstressresponse, skeletogenesis,detoxificationandmultidrugeffluxprocesseswasfollowedatdifferentdevelopmental stagesandunderdifferentconditions:metalsalone,metalsinthepresenceofNOSinhibitor,NOdonor andNOSinhibitoralone.Thesedataallowedtheidentificationofdifferentclassesofgeneswhose metal-inducedtranscriptionalexpressionwasdirectlyorindirectlymediatedbyNO.Theseresultsopennew perspectivesontheroleofNOasasensorofdifferentstressagentsinseaurchindevelopingembryos.

1. Introduction

Seaurchinsarekeyspeciesinstructuringmarineecosystems throughtheirgrazingactivity.Amongthem,Paracentrotuslividus, thedominantseaurchin intheMediterraneanSea, locally con-trollingthedynamicsofseaweedandseagrasses(Boudouresque and Verlaque, 2013), was considered a good model systemto studytheresponseofmarineorganismstoenvironmentalstress. Indeed,thetransparencyofembryos/larvae,aswellastheir sen-sitivitytopollutants,madethisorganismparticularlysuitablefor embryotoxicitytestsandformonitoringorriskassessment pro-grams(Beirasetal.,2003).P.lividusactivatesdifferentprotection strategiesagainstavarietyofstress-inducingagents,suchasheat shock(Giudiceetal.,1999),metals(RoccheriandMatranga,2009; Pinsinoetal.,2010),UVBradiation(Bonaventuraetal.,2006), X-rays(Matrangaetal.,2010;Bonaventuraetal.,2011),naturaltoxins (Romanoetal.,2010;Varrellaetal.,2014)andendocrinedisrupter compounds(Sugnietal.,2010).Thesestrategiesincludeactivation ofMAPkinases(Bonaventuraetal.,2005),inductionof metallothi-oneins(Ragusaetal.,2013),caspase3(Agnelloetal.,2007)and

∗ Correspondingauthor.Tel.:+390815833293;fax:+390817641355. E-mailaddress:anna.palumbo@szn.it(A.Palumbo).

differentheatshockproteins(hsps)(Roccherietal.,2004;Romano etal.,2011;Marroneetal.,2012).

ContinuousexposureofP.lividusembryostosubacute/sublethal cadmium concentrations causes abnormal development with defectsinskeletonelongationandpatterning(Russoetal.,2003), coupledwithageneralreductionofproteinsynthesis,increased levelsofspeciﬁchsps(Roccherietal.,2004)anddifferential metal-lothioneinexpression(Ragusaetal.,2013).Parallelexperiments haverevealedthat theamountofincorporatedcadmium insea urchinembryoshighlyincreasedwithtime(Agnelloetal.,2007). Reduction and lack of arms and skeleton elongation were also observedinchroniclong-termexposuresatlowercadmium con-centrations,mimickingmoderatelyandhighlypollutedseawaters (Filostoet al.,2008).Cadmiumtreatmentalso induced apopto-sis (Agnello et al., 2007; Filosto et al., 2008) and authophagy (Chiarelli et al., 2011). A functional relationship between the two processeshasrecentlyalsobeensuggested(Chiarellietal., 2014).

Manganesetreatmentresultedindevelopmentalabnormalities, especiallyattheskeletonlevel,directlycorrelatedtomanganese accumulationindevelopingembryos.Anincreaseofspeciﬁchsps withoutinductionofapoptosiswasreported(Pinsinoetal.,2010). Manganeseoverloadcaused interferencewithcalciumand per-turbationof phosphorylationof the MAPkinases ERKand P38, http://dx.doi.org/10.1016/j.aquatox.2014.08.007

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resultingintheformationofembryoswithoutskeletons(Pinsino etal.,2011,2013).

Recently,it hasbeenshown that inseaurchin embryosthe physiologicalmessengernitricoxide(NO),producedbyl-arginine oxidationbyNOsynthase(NOS),isinvolvedinthestressresponse inducedbythediatomaldehydedecadienal(Romanoetal.,2011). Atlowdecadienalconcentrations,NOprotectedembryosfromthe toxiceffectofthealdehyde,contributingtotheactivationofhsp70 geneexpression,whereasathighconcentrationsNOmediated ini-tialapoptoticeventsthroughthegenerationofperoxynitrite.The involvementofNOinthestressresponsehasalsobeenreportedin otherorganisms.HeatstressactivatedNOproductioninsponges (Giovineetal.,2001),andsalinityandlightstressgaverisetoNO burstsinculturemediaofmarinemicroalgae(Zhangetal.,2006). NOwasalsoacentralplayerinthesurveillancesysteminamarine diatominresponsetodecadienaltreatment(Vardietal.,2006)and incoralbleaching(BouchardandYamasaki,2008).

Our continuing interest in thebiological functions of NO in marineinvertebrates(Mattielloetal.,2010,2012;Ercolesietal., 2012;Castellanoetal.,2014;Migliaccioetal.,2014)hasprompted usto investigateif NOalso mediates theresponse of P.lividus embryos toother stress agents, such as metal ions. We chose cadmiumand manganese,two metals withdifferentproperties andknowntoaffectseaurchindevelopment(Russoetal.,2003; Filostoetal.,2008;Pinsinoetal.,2010).Cadmiumisanon-essential metal,without anybiological role and it is a potentpollutant, whereasmanganeseisanessentialelement,naturallyoccurring metalwhich becomes toxic athigh concentrations(Flicket al., 1971;CICAD,2004).

Here, we show that NO is produced in seaurchin embryos inresponse tocadmiumand manganeseandits levelsregulate directlyorindirectlythetranscriptionalexpressionofsome metal-inducedgenesinvolvedinstressresponse,skeletogenesis,aswell asindetoxiﬁcationandmultidrugefﬂuxprocesses.

2. Materialsandmethods 2.1. Ethicsstatement

P.lividus(Lamarck)seaurchinswerecollectedfromalocation that is not privately-owned norprotected in any way, accord-ing to the authorization of Marina Mercantile (DPR 1639/68, 09/19/1980conﬁrmedon01/10/2000).Theﬁeld studiesdidnot involveendangeredorprotected species.Allanimal procedures wereincompliance withtheguidelines oftheEuropeanUnion (directive609/86).

2.2. Gametecollection

SeaurchinswerecollectedduringthebreedingseasonbySCUBA diversintheGulfofNaples,transportedinaninsulatedboxtothe laboratorywithin1haftercollectionandmaintainedintankswith circulatingseawater.Theanimalswereacclimatedforaminimum of10daysuntiluseandkeptat18±2◦Cinacontrolledtemperature chamberata12:12light:darkcycle.Thedensityofthe individ-ualsinthetankswasmaximally1animal/5L.Every3daysanimals werefedusingfreshmacroalgae(Ulvasp.).Feedingwasinterrupted for2·daysbeforeexperimentalsampling.Veryrarespontaneous spawningor mortalitieswere observed during the acclimation period.Toinducegameteejection,seaurchinswereinjectedwith 0.5MKClsolutionthroughtheperibuccalmembrane.Eggsfrom individualfemaleswerewashedthreetimeswith0.22␮mﬁltered seawater.Concentratedspermwascollecteddry,mixingsamples fromatleastthreedifferentmalesandkeptundilutedat+4◦C.Ten ␮Lofspermmixwasdilutedin10mLSWjustbeforefertilization

andanaliquot(100␮L)ofthissolutionwasaddedto100mLof eggsuspension.Spermtoeggratiowas100:1forbothcontrolsand treatedembryos.Thefertilizationsuccesswasapproximately90%. 2.3. Embryoculture,treatmentsandmorphologicalanalysis

Eggs (150eggs/mL) were fertilized as described above and allowed to develop at 18±2◦C in a controlled temperature chamber ata 12:12light:dark cycle. After5minfrom fertiliza-tion, the metals at different concentration were added under carefulagitation.Nominalmetalconcentrationswere5×10−7M (0.09mg/L),10−6M(0.18mg/L),5.2×10−6M(0.96mg/L),10−5M (1.83mg/L), 2×10−5M (3.66mg/L), 3×10−5M (5.49mg/L) for cadmium (cadmium chloride, Sigma–Aldrich, Milan, Italy) and 1.8×10−5M (3.6mg/L), 3.6×10−5M (7.2mg/L), 7.8×10−5M (15.4mg/L),15.5×10−5M(30.8mg/L),31.2×10−5M(61.6mg/L), 62.4 ×10−5M(123.2mg/L)formanganese(manganesechloride tetrahydrate, Sigma–Aldrich, Milan, Italy). Stock solutions of 10−4M cadmium and 31.2×10−4M manganese in sea water werepreparedanddilutedtotheﬁnal experimental concentra-tion. The same protocol was followed for treatments withthe slow releasing NO donor (Z)-1- {N-(3-Aminopropyl)-N-(4-(3-aminopropylammonio)butyl)-amino}-diazen-1-ium-1,2-diolate)

(spermine NONOate, sperNO) (Alexis, San Diego, California) and spermine (Sigma–Aldrich, Milan, Italy). Incubations with 1-(2-trifluoromethylphenyl)imidazole(TRIM) (Alexis,SanDiego, California)wereperformed20minbeforefertilization.Stock solu-tionsof2×10−4MTRIM(in10−6MDMSO),4×10−4MsperNO(in 10−2MNaOH),4×10−4Mspermine(inseawater)wereprepared anddilutedtothefinalexperimentalconcentrationsof2×10−5M, 5×10−5Mand10−4MTRIM,4×10−5MsperNOand 4×10−5M spermine. Experiments were performed in triplicate using the eggscollectedfromthreedifferentfemales.Thedevelopmentwas followed usingan inverted microscope(Zeiss Axiovert135TV) andpicturesweretakenusingaZeissAxiocamconnecteddirectly tothemicroscope.Morphological observationswereperformed approximately48hpostfertilization(hpf)onpluteicollectedand fixed in 4% formalin. The percentage of normal and abnormal pluteiwas determinedby counting at least300 embryosfrom each well. Abnormalities were recorded following the criteria reportedinPaganoetal.(1986).Embryoswereconsiderednormal iftheysatisfiedallthemorphologicalcriteriadefinedelsewhere (Radenac et al., 2001;Kobayashi and Okamura,2005), namely: (1) reachedthe pluteus stage of development,(2) exhibited a good body symmetry,(3) showedfully developed skeletalrods and(4)displayedawelldifferentiatedgut.Allthemorphologies thatdidnotsatisfytheabove-mentionedcriteriaweregrouped and referred to as abnormal. In particular, the abnormalities wereclassifiedin twodifferentlevelsaccording totheseverity ofthealterations,takingintoaccounttheclassificationreported inCarballeiraetal.(2012)withsomemodifications.ThelevelA wasrepresentedbypluteicharacterizedbyincorrectarrangement of skeletal rods, such as larvae withcrossed tip,separated tip andfusedarms.ThelevelBwasrepresentedbylarvaeshowing incomplete or absent skeletal rods and development blocked, such as plutei with folded tip, fractured ectoderm, fertilized eggsandblastula,gastrula,exogastrula, prismaandpre-pluteus stages.

2.4. Nitricoxide(NO)determination

The endogenous NO levels were measured by monitoring nitriteformationbytheGriessreaction(Greenetal.,1982). Fer-tilized eggs were treated as described above. Samples (about 15,000embryos/larvae) were collected at 2-cell stage, 8-cell stage,earlyblastula, swimmingblastula,prismaandpluteusby

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O.Migliaccioetal./AquaticToxicology156(2014)125–134 127

centrifugationat1800rcffor10mininaswingoutrotorat4◦C. Thepelletwaswashedwithphosphatebufferedsalineandthen frozeninliquidnitrogenandkeptat−80◦_C._Samples_were

homog-enizedinphosphatebuffer(1:2,w/v)andcentrifuged(12,000rcffor 30minat4◦C),andthesupernatantswereanalyzedfornitrite con-tent.Aliquots(300␮L)wereincubatedfor2h,atroomtemperature (25◦C)withnitratereductase(1U/mL)andenzymeco-factorsFAD (100␮M)andNADPH(0.6mM).Sampleswereincubatedfor10min inthedarkwith300_␮Lof1%(w/v)sulphanilamidein5%H3PO4

and then for 10minwith 300␮L of 0.1% (w/v) N-(1-naphthy)-ethylenediaminedihydrochloride.Theabsorbanceat540nmwas determinedandthemolarconcentrationofnitriteinthesample wascalculatedfromastandardcurvegeneratedusingknown con-centrationsofsodiumnitrite(0–100␮M).NOineachsamplewas determinedintriplicate.Theefﬁcacyofnitratereductionbynitrate reductasewasdeterminedonknownconcentrationsofnitrateand nitrite recoverywas 90–100% over the entire range of sodium nitrite.Thecoefﬁcientofvariationbetweenthedifferent experi-mentswaslessthan5%.

2.5. RNAextractionandcDNAsynthesis

About1500fertilizedeggsweretreatedasdescribedaboveand collectedbycentrifugation asreportedin theabove paragraph. TotalRNA wasextracted from each developmental stageusing RNAqueous-Microkit (Ambion) according to themanufacturer’s instructions. Theamountof total RNAextracted wasestimated by the absorbance at 260nm and the purity by 260/280 and 260/230nm ratios by Nanodrop (ND-1000 UV–Vis Spectropho-tometer; NanoDrop Technologies). The integrity of RNA was evaluatedbyagarosegelelectrophoresis.IntactrRNAsubunits(28S and18S)wereobservedonthegelindicatingminimaldegradation oftheRNA.Foreach sample,600ngoftotal RNAextractedwas retrotranscribedwithiScriptTM_cDNA _Synthesis_kit_(Biorad),

fol-lowingthemanufacturer’sinstructions.cDNAwasdiluted1:5with H2OpriortouseinRealTimeqPCRexperiments.

2.6. GeneexpressionbyrealtimeqPCR

Inthisarticlethetermgeneexpressionreferstotranscriptional expression,althoughitisacknowledgedthatgeneexpressioncan alsobe regulated, e.g., at translation or mRNA/protein stability level.ForrealtimeqPCRexperimentsthedatafromeachcDNA sam-plewerenormalizedusingPl-Z12-1asendogenouscontrol.Itslevel remainedconstantduringdevelopment(Costaetal.,2012;Ragusa etal.,2013).TheprogramsgeNormVBAappletforMicrosoftExcel andNormFinderversion19(2009)wereusedtoconfirmPl-Z12-1 asthereferencegene.Theanalyzedgenesandthespecificprimers, usedforamplification,arereportedinsupplementaryTable1.For Pl-Z12-1,hsp70,hsp60,hsp56,sm30,sm50,p16,p19,msp130, bmp5-7,fg9/16/20,mt4,mt5,mt6,mt7andmt8weusedprimersreported intheliterature.InthecaseofNOS,abc1a,abc4a,abc1b,abc8b, spe-cificprimersweredesignedonthebasisofnucleotidesequence withthehelpofPrimer3.TheamplifiedfragmentsusingTaqHigh FidelityPCRSystem(Roche)werepurifiedfromagarosegelusing QIAquickGelextractionkit(Qiagen)andspecificityofPCR prod-uctswascheckedbyDNAsequencing.Specificityofamplification reactionswasverifiedbymeltingcurveanalysis.Theefficiencyof eachprimer pairwascalculatedaccordingtostandardmethods curvesusingtheequationE=10−1/slope.Fiveserialdilutionswere setuptodetermineCtvaluesandreactionefficienciesforallprimer pairs.Standardcurvesweregeneratedforeacholigonucleotidepair usingtheCtvaluesversusthelogarithmofeachdilutionfactor.PCR efficiencieswerecalculatedforcontrolandtargetgenesandwere foundtobeabout2.DilutedcDNAwasusedasatemplateina reac-tioncontainingafinalconcentrationof0.3␮Mforeachprimerand

1×FastStartSYBRGreenmastermix(totalvolumeof10␮L).PCR ampliﬁcationswereperformedinaViiATM₇_Real_Time_PCR_System

(AppliedBiosystems)thermalcyclerusingthefollowingthermal profile:95◦Cfor10min,onecycleforcDNAdenaturation;95◦Cfor 15sand60◦Cfor1min,40cyclesforamplification;72◦Cfor5min, onecycleforfinalelongation;onecycleformeltingcurve analy-sis(from60◦Cto95◦C)toverifythepresenceofasingleproduct. Eachassayincludedano-templatecontrolforeachprimerpair.To captureintra-assayvariabilityallRealTimeqPCRreactionswere carriedoutintriplicate.FluorescencewasmeasuredusingViiATM

7Software(AppliedBiosystems).Theexpressionofeachgenewas analyzedandinternallynormalizedagainstPl-Z12-1usingRelative ExpressionSoftwareToolsoftware(REST)basedonthemethodby

Pfafﬂetal.(2002).Relativeexpressionratiosabovetwocycleswere consideredsigniﬁcant.

2.7. Statisticalanalysis

Data arepresentedasmeans±SD andanalyzedbyOne-way ANOVA(P<0.05)withTukey’sMultipleComparisonTestand Two-wayANOVA(P<0.05),withBonferroniposthoctestasreportedin ﬁgurelegends.StatisticswasperformedwithGraphPadPrism4.0 forWindows(GraphpadSoftware,SanDiego,CA,USA).

3. Results

3.1. InvolvementofNOintheabnormaldevelopmentinducedby cadmiumandmanganese

PreviousstudieshavereportedthattreatmentofP.lividus fer-tilizedeggswithcadmiumandmanganeseresultedinabnormal development.An increase in thenumber of pluteiwith abnor-malskeletalpatterninganddevelopmentaldelaywasdependent onmetalconcentration(Russo etal.,2003; Filostoet al.,2008; Pinsinoetal.,2010).Wetreatedfertilizedeggsofseaurchinwith cadmiumandmanganeseattheconcentrationsreportedin Sec-tion2.After48h,morphologicalobservationswereperformedin ordertoassesstheproportionofnormalandabnormalplutei.In detail,cone-shapedlarvaewithfourfullydevelopedarmsand com-pleteskeletalrodswereconsiderednormal,whereaslarvaewith defectsinarmandskeletonelongation,larvaewithareductionof rudimentgrowthanddevelopmentallydelayedlarvaewere con-sideredabnormal,followingthecriteriareportedinSection2.The exposuretoincreasingconcentrationsofcadmiumresultedinan increaseofpercentageofabnormalplutei(Fig.1)whichreached values of 20±1.87%, 28±0.86% and 38±2.98% at 5×10−7M, 10−6Mand5.2×10−6Mcadmium,respectively,comparedto con-trol(8±1.33%)intheabsenceofcadmium.Atthehighestcadmium concentrationsof10−5M,2×10−5Mand3×10−5Mthe percent-ageofabnormallarvaewas62±4.27%,78±3.12%and100±1.15%, respectively(datanotshown).Defectswerefoundmainlyinthe arms,whichappearedmalformedorabsent.Oftenlarvaeshowed somearmsshorterthantheothersorinmanycasesthepost-oral armswereabsent.Moreover,adelayindevelopmentcomparedto controlpluteiwasobserved:treatedlarvaeweremuchsmallerin sizeandwereoftenatgastrulaorprismastage.

As that with cadmium, manganese treatment resulted in a signiﬁcant increase in the percentage of abnormal plutei withincreasingmanganese concentration(Fig. 1).Inparticular, the effect was signiﬁcant starting from 1.8×10−5M at which 17±1.29%ofthelarvaeappearedabnormal.Atthisconcentration, defectsmainlyconcernedthesizeofthelarvaeandthearm elon-gationandnolethaleffectswereobserved,asshownbythenormal swimmingbehaviorofthelarvae.Athighermanganese concentra-tionsof3.6×10−5Mand7.8×10−5M,thepercentageofabnormal

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Fig.1.Effectsofcadmiumandmanganeseonseaurchindevelopment.Fertilized eggsweretreatedwiththeindicatedcadmiumandmanganeseconcentrationsin theabsenceandpresenceofTRIM,asdescribedinSection2.Developmentwas monitoredafter48hpf.Signiﬁcantdifferencescomparedtothecontrol:*P<0.05, **P<0.01,***P<0.001.Signiﬁcantdifferencescomparedtothecorresponding cad-miumandmanganeseconcentrations:#P<0.05,##P<0.01,###P<0.001.One-way ANOVA(P<0.05),withTukey’sMultipleComparisonTest.Darkgreen:normalplutei. Lightgreen:abnormalplutei.N=3.

larvaewas29±3.36and 43±2.64%, respectively.At concentra-tionvaluesof15.5×10−5M,31.2×10−5Mand62.4×10−5M,an increaseinthemalformationsregardingarmelongation,skeletal abnormalities,developmentaldelayandmortalitywasobserved andthepercentageofabnormallarvaewas58±3.17%,69±4.22% and98±5.11%,respectively(datanotshown).

Basedontheseresults, furtherexperimentswereperformed toinvestigatethepossibleinvolvementofNOintheresponseof developingseaurchin embryostocadmiumandmanganese.To this aim, metaltreatments were performed in the presence of theNOSinhibitor,1-(2-triﬂuoromethylphenyl)imidazole (TRIM), which interferes with the binding of both _l-arginine and the cofactorBH4totheenzyme.Todetectanymorphologicalvariation

due toa reduction inthe endogenous NOlevels,we chosethe

Fig.2. Morphologicalanalysisoftheabnormalpluteifollowingdifferenttreatments. Abnormalpluteiobtainedfromcadmiumormanganeseexposureintheabsence andpresenceofTRIM,wereexaminedatthemorphologicallevel.Theseverityof themalformationswereindicatedasAandB,accordingtothecriteriareportedin Section2.Signiﬁcantdifferencescomparedtothecontrol:**P<0.01,***P<0.001. Signiﬁcantdifferencescomparedtothecorrespondingcadmiumandmanganese concentration:#P<0.05,##P<0.01,###P<0.001.Two-wayANOVA,Bonferroni’s posttest(P<0.05).White:levelA.Black:levelB.N=3.

metalconcentrationswhichcausedtheformationoflessthan50% ofabnormallarvae.Thesewere5×10−7M,10−6M,5.2×10−6M cadmiumand1.8×10−5M,3.6×10−5M,7.8×10−5Mmanganese. ThecombinedtreatmentsofTRIMwithcadmiumormanganese (Fig.1)resultedinanincreaseinthepercentageofabnormallarvae comparedtothetreatmentwithmetalalone,atallconcentrations tested.TRIMaloneat2_×10−5M,5_×10−5Mand10−4Mdidnot affectpluteimorphology.ThissuggestedtheinvolvementofNOin thestressresponseinducedbythesemetals.

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Fig.3.TotalNOconcentrationinseaurchindevelopingembryos.Embryostreatedwithcadmium10−6_M_and_manganese_3.6_×₁₀−5_M_in_the_absence_and_presence_of_TRIM,

wereexaminedatdifferentdevelopmentalstagesfornitritecontent,asreportedinSection2.Significantdifferencecomparedtothecontrol:*P<0.05,**P<0.01,***P<0.001. Significantdifferencecomparedtothecadmiumtreatment:###P<0.001.Significantdifferencecomparedtothemanganesetreatment:§§§P<0.001.Two-wayANOVA, Bonferroni’sposttest(P<0.05).N=3.

A further morphological analysis of the abnormal plutei, obtained under these experimental conditions, was performed (Fig.2).Theabnormalitieswereclassifiedintwodifferentlevels, AandB,asdescribedinSection2.Theydifferedonthebasisofthe severityofthealterations.AandBwereabnormalpluteiwithmild andseveremalformations,respectively.Theexposuretoincreasing cadmiumandmanganeseconcentrationsledtoanincreaseofboth levelsofmalformations,althoughwithdifferentratiosbetweenthe twolevels(Fig.2).ThecombinedtreatmentsofmetalswithTRIM resultedinasignificantincreaseofabnormalitiesatbothlevelsat lowermetalconcentrationandatthelevelBathighermetal con-centrations.Nosignificantdifferencesbetweenthetwolevelswere foundinlarvaetreatedwithTRIMalone(datanotshown).

Tofurtherinvestigatetherole ofNOintheresponse to cad-miumandmanganese,theNOcontentoftreatedP.lividusembryos wasexaminedatdifferentdevelopmentalstages.Indetail,fertilized eggswereincubatedwithmetalconcentrationsresultingina per-centageofabnormallarvaeofabout30%,i.e.10−6Mcadmiumand 3.6×10−5MmanganeseintheabsenceandpresenceofTRIM.Then, theembryosatthefollowingstageswerecollected:2cell,4cell, earlyblastula,swimmingblastula,prismaandpluteus.NOlevels weremeasuredastotalnitriteusingtheGriessassay.Asigniﬁcant increaseinnitritewasobservedafter48htreatmentofembryos withcadmiumormanganese(Fig.3).Asexpected,NOlevelswere reducedinthepresenceofTRIM.TheNOSinhibitoralsodecreased NOlevelsofcontrolsamplesatalldevelopmentalstages.

3.2. NO-mediatedabnormaldevelopment:Geneexpression

Thequantitativeexpressionlevelsofaseriesofgenesimplicated indifferentfunctionalresponsesinseaurchins,includingstress, skeletogenesis,detoxificationandmultidrugefflux,wasfollowed. Thegenesencodingheatshockproteinshsp70,hsp60andhsp56 wereexaminedasstressgenes.Thegenesinvolvedin skeletoge-nesisincludedgenesencodingthespiculematrixproteinssm30, sm50, msp130,the twoproteinsinvolved in skeleton formation p16,p19,thefibroblastgrowthfactorfg9/16/20,codingfora pro-teininvolvedinprimarymesenchymecellmigrationandskeletal morphologyandthegrowthfactorbmp5–7.Thegenesencoding metallothioneinsmt4,mt5,mt6,mt7andmt8wereinvestigatedas genesinvolvedinmetaldetoxification,whereasthegenes encod-ingabctransportersabc1a,abc1b,abc4aandabc8bwereexamined formultidrugeffluxintheprotectionsystem.Moreover,the expres-sionofNOSwasfollowedinrelationtoNOproduction.Pl-Z12-1was usedasacontrolgeneforRealTimeqPCRexperimentsbecauseits expressionremainedapproximatelyconstantinalldevelopmental

stagesof seaurchin(Ragusaetal.,2013)and indifferentmetal exposures(datanotshown).Fig.4showedtherelativeexpression ratiosofthegenesinvolvedinthestressresponse(a), skeletogen-esis(b),detoxiﬁcation(c)andmultidrugefﬂux(d)aftercadmium (A)andmanganese(B)treatmentwithrespecttocontrolembryos whichdevelopedinseawaterwithoutmetals.

Amongstressgenes,hsp70andhsp60wereup-regulatedatthe pluteusstageof2.5-and2.4-fold,respectively,bycadmiumand manganesetreatments(Fig.4Aa,Ba).Theexpressionofthe skele-togenicgenessm30,p16andmsp130increasedattheearlyblastula stage2.8-,2.2-and3.8-fold,respectively,aftercadmiumtreatment (Fig.4Ab).Incontrast,theexpressionlevelsofthegenesinvolved in skeletogenesiswerenot affectedatthis stageby manganese exposure(Fig.4Bb).Attheswimmingblastulastage,inthe pres-enceofcadmium,theexpressionofsm30wasdown-regulated(2.4 fold),whereasmsp130andfg9/16/20expressionwasup-regulated, showinga6-foldincreaseforbothgenes(Fig.4Ab).Atthesame stage,manganesetreatmentinducedtheexpressionofp16,msp130 and fg9/16/20 of 2.2-, 5.8- and 5.7-fold, respectively (Fig. 4Bb). At theprisma stage,no variation in the expressionof skeleto-genicgeneswasobservedwitheithercadmiumormanganese.At thepluteusstage,followingcadmiumtreatment,wefounda 3.2-and 3.9-foldup-regulationofsm30andp19,respectively,and a down-regulationofmsp130of3.9-fold(Fig.4Ab).Inthepresence ofmanganese,theexpressionofsm30andp19wasup-regulated by3.8-and3.9-fold,respectively(Fig.4Bb).Theexpressionofthe detoxifyinggenesstartedtochangeattheprismastageaftermetal treatment.Duringcadmiumexposure,theexpressionofmt4,mt5, mt6andmt8wasup-regulatedby2.9-,4.3-,4.1-,2.3-fold, respec-tively(Fig.4Ac).Manganeseexposurecauseda2.8-and2.7-fold up-regulationofmt5andmt8,respectively(Fig.4Bc).Atthepluteus stage,cadmiumtreatmentinducedanup-regulationofmt4,mt5 andmt8by2.3-,4.9-and2.4-fold,respectively,whereasmanganese causeda2.6-folddown-regulationofmt6expression(Fig.4Ac,Bc). Regardinggenesencodingabctransportersinvolvedinmultidrug efﬂux,theexpressionofabc4awasup-regulated(2.6-fold)by cad-miumattheswimmingblastulastage(Fig.4Ad).Cadmiumalso caused down-regulationof abc8b(2.9fold)and abc1a(3.3-fold) attheprismaandpluteusstages,respectively(Fig.4Ad).In con-trast,manganesetreatmentresultedonlyinthedown-regulation (3.3-fold)ofabc1aatthepluteusstage(Fig.4Bd).Nosigniﬁcant variationswerefoundfortheexpressionofNOS,atany develop-mentalstageoraftercadmiumandmanganesetreatments(data notshown).

Toinvestigatetheinvolvement ofNOin thetranscriptionof the genesaffected by cadmium or manganese treatment, their

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Fig.4. Geneexpressionanalysisindevelopingembryosaftermetaltreatment.Fertilizedeggsweretreatedwithcadmium10−6_M_(A)_and_manganese_3.6_×₁₀−5_M_(B)_and

differentdevelopmentalstages(2-cell,8-cell,earlyblastula,swimmingblastula,prismaandpluteus)wereexaminedforthetranscriptionalexpressionofgenesinvolvedin stress(a),skeletogenesis(b),detoxification(c)andmultidrugefflux(d)byRealTimeqPCR.Dataarereportedasafolddifferenceintheexpressionlevelsoftheanalyzed genes,comparedtocontrol(mean±SD),embryosdevelopedinseawaterwithoutmetals.Folddifferencesgreaterthan±2(seedottedhorizontalguidelinesatvaluesof2 and−2)wereconsideredsignificant.

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Fig.5. Geneexpressionanalysisindevelopingembryostreatedwithmetalsunder lowlevelsofNO.Fertilizedeggsweretreatedwith10−6Mcadmium+TRIM10−4M (A)and3.6×10−5Mmanganese+TRIM 10−4M(B).Samplescollectedatearly blastula,swimmingblastula,prismandpluteusstagewereexaminedforgene expressionbyRealTimeqPCR.Dataarereportedasafolddifferenceinthe expres-sionlevelsoftheanalyzedgenes,comparedtocontrol(mean±SD),embryosinsea waterwithoutmetals.Folddifferencesgreaterthan±2(seehorizontalguidelines atvaluesof2and−2)wereconsideredsigniﬁcant.

expressionwasanalyzed underreducedlevelsofNO,i.e.inthe presenceoftheNOSinhibitorTRIM.Theexpressionofthesegenes wasreportedwithrespecttothatofthemetalalone(Fig.5).During cadmium+TRIMexposure,sm30wasup-regulated(2.6-fold)atthe swimmingblastulastage.Atthepluteusstage,mt4(2.5-fold)and mt6(4.3-fold)wereup-regulated,whereashsp70(3.6-fold),sm30 (2.5-fold),p19(2.9-fold)andabc1a(2.9-fold)weredown-regulated (Fig.5A).Inthecaseofmanganese,additionofTRIMcaused down-regulation(3.8-fold)offg9/16/20attheswimmingblastulastage (Fig.5B).

TofurtherdemonstratethatNOaffectedhsp70,mt4,mt6,sm30, p19,fg9/16/20andabc1aexpression,theirmRNAlevelswere mea-suredaftermodifyingendogenousNOlevelswiththeNOSinhibitor TRIMand theNO donorsperNO(Fig.6).Ascontrol forsperNO treatment,weusedspermine,theproductderivedfromsperNO afterNO release.Under low levelsof NO withTRIM,we found a down-regulationof sm30(2.2-fold)atthe swimmingblastula stageand anup-regulationofsm30(3.2-fold)andp19(3.6-fold) anddown-regulationofabc1a (3.1-fold)atthepluteusstage.In thepresenceofsperNO,attheswimmingblastulastage,sm30was up-regulated(4.4-fold)and fg9/16/20 wasdown-regulated (3.6-fold).Atthepluteusstage,hsp70,mt4andabc1awereup-regulated 4.2-,3.7-,2-fold,respectively,whereas mt6,sm30andp19were down-regulated2.3-,4.3-and3.6-foldrespectively.

Additional experiments were performed to detect if the expression of some genes was affected only by the combined treatment of metal+TRIM and not by metal or TRIM alone.

Fig.6.GeneexpressionanalysisindevelopingembryosunderdifferentNOlevels. Fertilizedeggsweretreatedwith10−4MTRIMor4×10−5MsperNO.Samples, col-lectedatswimmingblastulaandpluteusstage,wereexaminedforgeneexpression byRealTimeqPCR.Dataarereportedasafolddifferenceintheexpressionlevels oftheanalyzedgenes,comparedtocontrol(mean+SD),embryosinseawateror inthepresenceofspermineinthecaseofsperNO.Folddifferencesgreaterthan±2 (seehorizontalguidelinesatvaluesof2and−2)wereconsideredsigniﬁcant.

Duringcadmium+TRIMexposure,hsp60andfg9/16/20were down-regulated(2.6-and2.3-fold)attheswimmingblastulaandpluteus stage,respectively. Inthecase ofmanganese,theexpressionof hsp60(3.8-fold),sm30(5.7-fold),sm50(3.5-fold)andp16(2.49-fold) increasedasaresponsetothemetal+TRIMexposureattheearly blastulastage,whereasmt8(2.48-fold)andabc1b(3.7-fold)were down-regulated.Attheprismastage,anup-regulationoffg9/16/20 (2.5-fold)wasobserved.

4. Discussion

Theresultsofthisstudyexpandedpreviousinvestigationson theeffectsofcadmiumandmanganeseonseaurchindevelopment (Filostoetal.,2008;Pinsinoetal.,2010),providingnewinsights atphenotypicandgenelevelsandhighlightingtheinvolvementof NOinthestressresponsecausedbythesemetals.

In this paper, a vis-a-vis comparison of the effects of cad-miumandmanganeseonthedevelopingembryosofP.lividuswas performedforthefirsttime, thusallowingustoidentify analo-giesanddifferencesbetweenthetwometals.In bothcases,the percentage of abnormal plutei increasedwith increasing metal concentrations. However, some differences were evident. Cad-miumexposureledtoimportantskeletalmalformationswithhigh levelsoflarvaewithdelayeddevelopment,whereas manganese treatmentcausedmorespecifictypesofmalformations, particu-larlyinthearms,withalowerpercentageoflarvaewithgeneral delayindevelopment,thusrevealingalowertoxicitycompared tocadmium,inagreementwithpreviousexperimentsinArbacia crassispina(KobayashiandOkamura,2005).Moreover,aseriesof genesencoding proteinsinvolved in thestressresponse, skele-togenesis,detoxificationandmultidrugeffluxweredifferentially modulatedbythesemetalsatenvironmentallyrelevant concentra-tions.Indetail,theconcentrationofcadmium(10−6M)usedinthis studycorrespondstohighlypollutedseawaterandthatof man-ganese(3.6×10−5M=7124␮gL−1)isclosetothemaximumvalue (10,000␮gL−1)reportedin naturalwaterfor thismetal(CICAD, 2004).Thedifferentresponseofseaurchinembryostocadmium andmanganeseisprobablycorrelatedtothedifferentroleofthese metals. Cadmiumis apotentpollutant(Flicket al.,1971),toxic evenatverylowconcentrations(Foulkes,2000);itdoesnothave anybiologicalroleanditspresenceintheenvironmenthasgrown

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inthelast yearsbecauseofitslargeuseinsomeindustrial and agriculturalactivities(Ruleetal.,2006).Manganese,ontheother hand,isanaturallyoccurringmetalrequiredintraceamountsby theorganismswhereitplaysanumberofessentialrolesinmany metabolicfunctions,incellularprotection,replicationmechanisms andinbonemineralizationprocesses(ATSDR,2008;Daly,2009; Santamaria,2008).However,theexposuretohighmanganese lev-elscancausetoxicity(CICAD,2004;Gerberetal.,2002;Limaetal., 2008)and,duetothemassiveproductionofmanganese-containing compounds,this metalisbeingconsideredasanemergent pol-lutant,especiallyintheaquaticenvironment(Satyanarayanaand Saraf,2007).

Animportantoutcomeofthisstudyisthedemonstrationthat NOwasproducedinresponsetocadmiumandmanganese treat-ments.Moreover,theexaminationofgeneexpressionaftermetal treatments,aswellasunderdifferentNOlevels,hasallowedusto getaninsightintotheinvolvementofNOinmediatingtheeffects ofcadmiumandmanganeseontranscriptionalgeneexpression.An overallpictureofourresultsisschematicallysummarizedin sup-plementaryTable2.Ourfindingthattheexpressionofthegenes sm30,hsp70andp19followingcadmiumtreatmentandfg9/16/20 aftermanganeseincubationwasreversedbyNOSinhibitionwith TRIMatsomedevelopmentalstages,revealedthatNOmediated theeffect of the metals onthe expression of thesegenes. The oppositeeffect ofsperNOand TRIMontheexpression ofthese genesindicates that NO levelscould play a role in their regu-lation.Afurtherinsightwasprovidedbythecomparisonofthe effectsofsperNOandmetalalone.Inparticular,cadmium treat-ment for 48h induced theexpression of hsp70,similar to that observedwiththeNOdonorsperNO.Thisresult,togetherwith dataonNOdetermination,suggeststhatNOformedaftercadmium treatmentcandirectlyup-regulatehsp70expression(Fig.7A).The involvementofNOininducingtheexpressionofhsp70hasbeen previouslyreportedinseaurchinembryosinthecaseofanother stressagent,thediatomtoxindecadienal(Romanoetal.,2011). Contrarytohsp70,theexpressionofsm30andp19regulatedby cadmium,aswellasfg9/16/10bymanganese,showedanopposite trendcomparedtosperNOtreatmentalone.Thisfindingsuggests thatthemetalsaffecttheexpressionofthesegenesnotdirectly butthroughax-factor,whichisregulatedbychangesinNO lev-elsaftermetaltreatment(Fig.7B).ThelackofeffectofTRIMon theexpression of mt4 and abc1a after cadmium treatment for 48hsuggestedthatNOisnotinvolvedintheregulationofthese genesbycadmium(supplementaryTable2).Thepicture emerg-ingfromthisstudywasfurthercomplicatedconsideringthatthe expression of some genes(hsp60, mt8, sm30, sm50, p16,abc1b andfg9/16/20)atcertaindevelopmentalstageswasaffectedonly bythecombinedtreatmentofthemetalswithTRIMandnotby sperNOor TRIM alone (Fig. 7C). This result suggests that dur-ingmetaltreatmentanalreadyunknownx-factorwasexpressed, which,inresponsetochangesinNOlevels,regulatedthe expres-sionofthestudiedgenes.Thelackofeffectof sperNOorTRIM indicatedthatthisfactorispresentonlywhenseaurchinembryos areexposed tometals. Overall thesefindings demonstrate that NOdifferentially regulatesgene expressionin response to cad-miumandmanganesetreatments.Inconclusion,withcadmium, NOdirectlyactivatedhsp70andindirectlyregulatedsomestress (hsp60) and skeletogenic (sm30, p19, fg9/16/20) genes. On the otherhand,withmanganese,NOindirectlyaffectedthe expres-sionofstress(hsp60),skeletogenic(sm30,sm50,p16,fg9/16/20)as wellasmultidrugeffluxgenes(abc1b)anddetoxification genes (mt8).

Theability ofNO toregulate gene expressionhasalsobeen demonstratedindifferentcellsandorganisms(Heemskerketal., 2007;Nakayaetal.,2000;Rossigetal.,2000).Thekineticsofgene activationbyNOwasinvestigatedinculturedﬁbroblastsexposedto

Fig.7. SchematicrepresentationoftheNOinvolvementinthemodulationofgene expressionindevelopingembryosofseaurchinexposedtocadmiumand man-ganese.(A)DirectinvolvementofNO:metalinducesanincreaseofNOlevelswhich up-regulateshsp70expression.(B)IndirectinvolvementofNO:metaltreatment causesavariationinNOlevelswhichaffectsanunknownfactor,regulatinggene expression.(C)IndirectinvolvementofNO:metaltreatmentaffectsanunknown factorwhichregulatesgeneexpressionthroughNO.

NOdonorandthreedistinctwavesofgeneactivitywereidentified (Hemishetal.,2003).Thefirstonewasgeneratedwithin30min oftreatmentandrepresentedtheprimarygenetargets,whereas thesubsequentwaveswereduetofurthercascadesofNO-induced geneexpression.SpecificsignalingpathwaysusedbyNOtoactivate geneexpressionhavebeenidentified,includingPI3-kinase,PKC, NF-kappaBandp53.IthasbeenreportedthatNOaffectedgene expressiondirectlyinfluencingtheactivityoftranscriptionfactors ormodulatingupstreamsignalingcascades,ormRNAstabilityand translationortheprocessingoftheprimarygeneproducts(Bogdan etal.,2001).

Inconclusion,ourﬁndingthatNOmediatedthestressresponse inducedby differentstress agents, suchascadmium and man-ganese(thiswork)anddecadienal(Romanoetal.,2011),pointed toNOasamediatorofdifferentenvironmentalstressagentsinsea urchins.

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Acknowledgements

ThisworkhasbeenpartiallyfundedbytheFlagshipRITMARE – TheItalian Research for theSea –coordinated bythe Italian NationalResearch Counciland fundedbytheItalianMinistryof Education,UniversityandResearchwithintheNationalResearch Program2011–2013.OrianaMigliacciohasbeensupportedbya SZNPhDfellowship.ImmacolataCastellanohasbeensupportedby aSZNpostdocfellowship.

We thank the Molecular Biology Service for gene sequenc-ing and Davide Caramiello and the service Marine Resources forResearch forassistance withliving organisms.We acknowl-edge Valeria Matranga for her helpful advice and discussion as external supervisor of Oriana Migliaccio PhD program. We acknowledge Thierry La Page for the use of the web site

http://octopus.obs-vlfr.fr/blast/blastoursin.html. AppendixA. Supplementarydata

Supplementary data associated with this article can be found,intheonlineversion,athttp://dx.doi.org/10.1016/j.aquatox. 2014.08.007.

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