glyphosateresistantsuchassoycrops.ConsideringthewideuseofGBH/GMOagricu - PDF document

glyphosateresistantsuchassoycrops.ConsideringthewideuseofGBH/GMOagriculture,studiesofthepossibleimpacts glyphosateanditsprincipalmetabolite,AMPA,altercellcyclecheckpointsbyinterferingwiththephysiologicalDNArepairmachinery.SeveralGBHswereassayed,andtheyinducedcell-cycledysfunctionfromtheÞrstcelldivisioninseaurchinembryos(10,11).Thethresholdconcentrationforthiseffectis500-to4000-foldlowerthanthatsprayedoncropsintheÞeld.EightmillimolarglyphosateinducesadelayinthekineticsoftheÞrstcellcleavageofseaurchins,alteringtheentryintoS-phasebyinterferingwiththeactivationoftheCDK1/cyclinBcomplex(6,12).Thisfailureofcell-cyclecheckpointsisknowntoleadtogenomicinstabilityandthepossibledevelop-mentofcancer.InagreementwiththeseÞndings,genotoxicity domainsintheposteriorneuralplate:medial,intermediate,andlateral,whichcorrespondtomotorneurons(m),interneurons(i),andsensoryneurons(s),respectively(Figure1C)(39).Treatedembryosshowedadown-regulationinthethreestripesofprimaryneurons(Figure1D).TocorroborateiftheeffectisspeciÞcallyduetotheactiveprincipleoftheherbicideandnottoadjuvantspresentinformulations,glyphosatewasinjectedintoonecellatthe2-cellstageandslug,krox-20,andN-tubulinwererevealedatstages15,asbefore.Theseembryosshowedanimportantdown-regulationofslug(Figure1E,arrow),resemblingtheeffectsofGBHonthismarkeratthisstageofdevelopment.AlthoughKrox-20didnotcompletelydisappearfromr3asinGBH-treatedembryos,theexpressionclearlydecreasedinthisrhombomereaswellasinr5,indicatingthatglyphosatealsoaltersrhombo-mericpatterning(Figure1E;arrowheads).Normally,atstage18,theneuralcresthasformedthreepremigratoryblocksfromwhichthreedifferentsegmentssegregate:mandibularcrestsegment,hyoidcrestsegment,andbranchialcrestsegment(MCS,HCS,andBCS;Figure1F).TheÞrstsegmentcontributestotheMeckel,quadrate,andethmoid-trabecularcartilages;thehyoidcrestsegmenttotheceratohyalcartilage,andthebranchialsegmenttothecartilagesofthegills(40).Glyphosate-injectedembryosshowedthatthesegregationprocessclearlyaffectedtheinjectedside(Figure1F,arrow),suggestingthatthederivedcartilagesmaybeaffectedatlaterstagesduringdevelopment.WhenhybridizedwithN-tubulin,theseembryosshowedadecreaseinthenumberofprimaryneuronsinthethreestripescorrespondingtomotorneurons,interneurons,andsensoryneurons(Figure1G,arrows),resem-blingtheeffectsofGBHtreatments,althoughwithmilderconsequencesforthismarker.Inconclusion,theeffectsofGBH-treatedandglyphosate-injectedembryosrepresentequivalentphenotypesdespitethefactthattheyarenotidentical.Theadjuvantpresentinthecommercialformulationmayexplainthedifferences.Takentogether,theseresultsindicatethatbothGBHandglyphosateimpairneuronaldifferentiation,rhombomericformation,andthepatternoftheneuralcrestduringinductionandsegregation.GBHandGlyphosateProduceHeadDefectsandImpairtheExpressionofDorsalMidlineandCephalicMarkers.BecausecraniofacialdefectswereobservedinhumansresidinginareaschronicallyexposedtoGBH,wedecidedtoexplorewhethergenesinvolvedinheaddevelopmentarealteredasaconsequenceoftreatmentwithGBHorinjectionofglyphosate.Shhactsasamorphogencontrollingmultipledevelopmentalprocesses.Duringearlyvertebrateembryogenesis,shhexpressedinmidlinestructuressuchasthenotochord,prechordalmeso-derm,andßoorplatecontrolsleft-rightasymmetry,neuronidentity,neuralsurvival,anddorso-ventralpatterningoftheneuraltube(41,42).Moreover,ShhsecretedbytheprechordalmesodermisresponsibleforresolvingthebrainandtheretinaÞeldintotwoseparatehemispheresandeyes,preventingcyclopia(43).Shhexpressionwasdramaticallyreducedinthedorsalmidlineatneurulastages,especiallyintheprechordalmesoderminGBH-treatedembryos.Theanteriorlimitoftheshhexpressiondomainismovedcaudallyintreatedembryos,inrelationtothepax6domain(comparegreenarrowheads,Figure2A-C).Pax6isessentialforeyeformationinawiderangeofspecies.Itisexpressedintheeyeprimordiaofvertebratessuchasthemouse,chicken,Xenopus,zebraÞsh,andhumans,aswellasininvertebratessuchasDrosophila(44-47).EmbryosincubatedwithGBHshowedadistinctdown-regulationofthepax6territory(comparewhitearrowheads;Figure2A-C).Moreover,intreatedembryos,the C)andtailbud(D-I)stages.(A)Controlembryohybridizedwithshh(arrow)andpax6(whitear-rowheads).(B-C)Embryosexposedto1/5000dilutionofGBH.Noticethedramaticreductionofshhexpressionintheembryonicdorsalmidline(arrows)andthecaudaldisplacementoftheanteriorlimit(greenarrowheads)(85%,n)33).Theexpressionofpax6isreduced,andthedomainisnotproperlyresolvedintheeyeÞeld(lightbluearrowheads)(85%,n)33).(D,E)Controlembryos.(D)Normalexpressionofshhinthenotochord(n),ßoorplate(fp),andprechordalmesoderm(pm)andofotx2intheeye(e),forebrain(fb),andmidbrain(mb).Thespacebetweenbarsindicatesthesizeofthebrain.(E)Normalexpressionofsox9inthepharyngealarches(pa),oticplacode(op),eye(e),genitalridge(gr),andnotochord(n).(F,G)1/5000GBH-treatedembryos.(F)Reducedexpressionofshhandotx2(92%,n)24)in ).Rowaspreviouslyusedasatooltoblockretinoid-mediatedsignaling,producingavarietyofmorphologicalchangesinthefrogembryo.Themostseverephenotypesshowedanteriorandposteriortruncations,areduc-tionorlossofeyesandoticvesicles,andageneraldisorganiza-tionofbranchialarches(22).Moreover,maternalinsufÞciencyofvitaminA(theprecursorofRA)orRAinexcessinvertebratescauseawiderangeofteratologiceffects(18,57,58).Allthisevidencedemonstratesthatvertebratesrequireapreciselyregulatedsupplyofretinoidsduringembryogenesis.ConsideringthatthephenotypesobtainedinouranalysispredominantlyresemblethoseofRAexcess,wewonderedifGBHtreatmentsareabletoincreaseendogenousRAactivity.Toanswerthisquestion,wemeasuredthelevelsofRAsignalingbytakingadvantageofthereporterplasmidRAREZ(32),as 21,59,60).Aspreviouslyreported(22),embryosincubatedwith0.5or1MRoalonealsodisplayedaconcentration-dependentshorten-ingoftheA-Paxisandreductionofheadstructures,whichwasconÞrmedbyareductionoftheotx2domain(Figure4D,E;comparethespacebetweenbarswithB).Wealsoobservedamorediffusestainingofshh,mainlyintheprechordalmeso-derm,incomparisonwiththatofsiblingcontrols(Figure4D,E; exposedtoGBH.MisregulationofRA,shh,andAreInvolvedinCephalicMalformationsandNeuralCrest-DerivedPheno-typesReminiscentoftheEffectsofGBHandGlyphosate. Whole-mountinmunoßuorescenceanalysisofPax6at8somites.(A,D)ControlembryoshowingPax6expressionintheopticvesicles signalingcouldbemediatingtheeffectsofGBHtreatments.GBHIncreasestheActivityoftheMorphogenRA,LeadingtoTeratogenicEffects.InXenopusembryos,theendogenousactivityofretinoidsgraduallyincreasesduringearlyembryogenesisandisÞnelyregulatedinspace.Atlategastrula,arostral-caudalgradientfrom0.01to0.16µMRAisestablished,withthehighestlevelsattheposteriorendoftheembryo.Thegradientpersistsattheearlyneurulastage(stage13-14).SynthesisanddegradationofRAseemtobethemechanismsthatleadtothisunevendistribution(74).ThisgradientexplainswhylowdosesofappliedRAprimarilyaffectthecephalicregionandincreasingthedosesbeginstoaffectthetrunk(20,21).Moreover,maintaininganormalendogenousdistributionofRAisimportantforaxespatterningandorga-nogenesisnotonlyinXenopus(74,22,38)butalsoinotherVertebratessuchaszebraÞsh(75-77),chicken(78-80),and rescuedbyRotreatments.Therefore,theRAREZreporterdoesnotseemtobesensitiveenoughtodetectminimalvariationsinthelevelsofRAactivity.Thisreinforcestheimportanceofusingvertebrateembryosasbiosensorsfortestingpossibleteratogens.Moreover,ithasbeenrecentlyreportedthatTriadimefon,asystemicfungicidewithteratogeniceffectsinrodentmodels,producescraniofacialmalformationsinXenopuslaeVisbyalteringendogenousRAsignaling(93).Arsenic,anotherendocrinedisruptor,alsoincreasesRAsignalingatlow,noncytotoxicdoses,inhumanembryonicNT2cells(94).RAsignalingisoneoftheÞnestpathwaystotuneupgeneregulationduringdevelopment,andallthisevidenceraisesthepossibilitythatdisturbancesinRAdistributionmaybeamoregeneralmechanismunderlyingtheteratogeniceffectsofxenobioticsinvertebrates.Sincemechanismsofdevelopmentarehighlyconservedinevolutionamongvertebrates(95),wewouldliketostressthattheycouldbeusefulasverysensitivebiosensorstodetecttheundesirableeffectsofnewmolecules.ClinicalApproaches.InArgentina,theextensionofsoildevotedtotransgenicsoyreached19millionhectares.Twohundredmillionlitersofglyphosate-basedherbicideisusedforaproductionof50milliontonsofsoybeansperyear(96,97TheintensiveandextensiveagriculturalmodelsbasedontheGMOtechnologicalpackagearecurrentlyappliedwithoutcriticalevaluation,rigorousregulations,andadequateinforma-tionabouttheimpactofsublethaldosesonhumanhealthandtheenvironment,leadingtoaconßictingsituation.Inthiswork,wefocusedonsublethaldosesofGBHtoarriveatthethresholdsforteratogenicphenotypesinsteadoflethality.Inthelast10years,severalcountriesinLatinAmericahaveinitiatedstudiesabouttheenvironmentalconsequencesoftheuseofherbicidesandpesticides.InParaguay,anepidemiologicalstudyintheoffspringofwomenexposedduringpregnancytoherbicidesshowed52casesofmalformations(3),whichstrikinglyresemblethewidespectrumphenotypesresultingfromadysfunctionalRAorShhsignalingpathway.InArgentina,anincreaseintheincidenceofcongenitalmalformationsbeganto wereconcentratedinfamilieslivingafewmetersfromwheretheherbicidesareregularlysprayed.Allofthisinformationisextremelyworryingbecausetheriskofenvironmentally-induceddisruptionsinhumandevelopmentishighestduringthecriticalperiodofgestation(2to8weeks)(98).Moreover,thematurehumanplacentahasbeenshowntobepermeabletoglyphosate.After2.5hofperfusion,15%ofadministeredglyphosateistransferredtothefetalcompartment(99).AlloftheevidencereportedinthescientiÞcliteratureandtheclinicalobservationsintheÞeldwerenotsufÞcient,however, ,IraBlitzforotx2,Jean-PierreSaintJeannetforsox9,ThomasHollemannforpax6,andCliffTabinforc-shh.WearealsogratefultoAbrahamFainsodfortheRAREZplasmid,Dr.M.KlausforprovidingRo41-5253,andBruceBlumbergforusefuldiscussions.WethankAnaAdamoformaterialsupport,HugoRõ«os,EzequielVarela,andErnesto«lezforhelpinguswithchickenexperiments,andmembersofourlab(CeciliaAguirre,SabrinaMurgan,andDiegoRevinski)forhelpingwithembryosandreagentpreparations.WealsothankCarlosDavioandSandraVerstraetenforassistanceinluminiscencedetermination.A.E.C.isparticularlyindebtedtoBardeCao.A.R.P.andA.E.C.arefromConsejoNacionaldeInvestigacionesCientõ«ÞcasyTe«cnicas(CONICET)andUniversidaddeBuenosAires.V.G.wassupportedbyafellowshipfromANPCyT,andH.AwassupportedbyafellowshipfromUniversidaddeBuenosAires.S.L.L.isfromCONICET.Thisworkandtheauthorsarecompletelyindepen-dentfromindustry.TheauthorsdeclarenocompetingÞnancialandcommercialinterests.References(1)vomSaal,F.,andHughes,C.(2005)Anextensivenewliteratureconcerninglow-doseeffectsofbisphenolAshowstheneedforanewriskassessment.EnViron.HealthPerspect.113,926Ð933.(2)Myers,J.,Zoeller,R.,andvomSaal,F.(2009)AclashofoldandnewscientiÞcconceptsintoxicity,withimportantimplicationsforpublichealth.EnViron.HealthPerspect.117,1652Ð1655.(3)Benõ«tezLeite,S.,Macchi,M.A.,andAcosta,M.(2009)Malforma-cionesConge«nitasasociadasaagroto«xicos.Arch.Pediatr.Drug80,237Ð247.(4)Richard,S.,Moslemi,S.,Sipahutar,H.,Benachour,N.,andSeralini,G.E.(2005)Differentialeffectsofglyphosateandrounduponhumanplacentalcellsandaromatase.EnVironHealthPerspect113,716Ð720.(5)Haefs,R.,Schmitz-Eiberger,M.,Mainx,H.G.,Mittelstaedt,W.,andNoga,G.(2002)Studiesonanewgroupofbiodegradablesurfactants 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