Metabolome disruption of pregnant rats and their offspring resulting from repeated exposure to a

21 Pages·2017·4.39 MB·English

RESEARCHARTICLE
Metabolome disruption of pregnant rats and
their offspring resulting from repeated
exposure to a pesticide mixture
representative of environmental
contamination in Brittany
NathalieBonvallot1,2*,Ce´cileCanlet2,FlorenceBlas-Y-Estrada2,RoselyneGautier2,
a1111111111 MarieTremblay-Franco2,SylvieChevolleau2,SylvaineCordier1,Jean-PierreCravedi2
a1111111111
a1111111111 1 UnivRennes,EHESP,Inserm,Irset(Institutderechercheensante´,environnementettravail)—UMR_S
1085,Rennes,France,2 INRAUMR1331Toxalim,UniversityofToulouse,INP,ENVT,EIP,UPS,
a1111111111
UMR1331,Toulouse,France
a1111111111
*[email protected]
Abstract
OPENACCESS
Citation:BonvallotN,CanletC,Blas-Y-EstradaF, Theuseofpesticidesexposeshumanstonumerousharmfulmolecules.Exposureinearly-life
GautierR,Tremblay-FrancoM,ChevolleauS,etal. mayberesponsibleforadverseeffectsinlaterlife.Thisstudyaimedtoassessthemetabolic
(2018)Metabolomedisruptionofpregnantrats
modificationsinducedinpregnantratsandtheiroffspringbyapesticidemixturerepresentative
andtheiroffspringresultingfromrepeated
exposuretoapesticidemixturerepresentativeof ofhumanexposure.Tenpregnantratswereexposedtoamixtureofeightpesticides:aceto-
environmentalcontaminationinBrittany.PLoS chlor(246μg/kgbw/d)+bromoxynil(12μg/kgbw/d)+carbofuran(22.5μg/kgbw/d)+chlor-
ONE13(6):e0198448.https://doi.org/10.1371/
mequat(35μg/kgbw/d)+ethephon(22.5μg/kgbw/d)+fenpropimorph(15.5μg/kgbw/d)+
journal.pone.0198448
glyphosate(12μg/kgbw/d)+imidacloprid(12.5μg/kgbw/d)representingthemainenviron-
Editor:PieterSpanoghe,UniversiteitGent,
mentalpesticideexposureinBrittany(France)in2004.Anothergroupof10pregnantrats
BELGIUM
servedascontrols.Femaleswerefedadlibitumfromearlypregnancy,whichisfromgesta-
Received:September18,2017
tionalday(GD)4toGD21.UrinesampleswerecollectedatGD15.Attheendoftheexpo-
Accepted:May18,2018 sure,mothersandpupswereeuthanizedandblood,liver,andbrainsamplescollected.1H
Published:June20,2018 NMR-basedmetabolomicsandGC-FIDanalyseswereperformedandPCAandPLS-DA
usedtodiscriminatebetweencontrolandexposedgroups.Metabolitesforwhichthelevels
Copyright:©2018Bonvallotetal.Thisisanopen
accessarticledistributedunderthetermsofthe weresignificantlymodifiedwerethenidentifiedusingtheKruskal-Wallistest,andp-values
CreativeCommonsAttributionLicense,which wereadjustedformultipletestingcorrectionusingtheFalseDiscoveryRate.Themetabolo-
permitsunrestricteduse,distribution,and
micsanalysisrevealedmanydifferencesbetweendamsofthetwogroups,especiallyinthe
reproductioninanymedium,providedtheoriginal
plasma,liverandbrain.ThemodifiedmetabolitesareinvolvedinTCAcycle,energyproduc-
authorandsourcearecredited.
tionandstorage,lipidandcarbohydratemetabolism,andamino-acidmetabolism.These
DataAvailabilityStatement:Allrelevantdataare
modificationssuggestthatthepesticidemixturemayinduceoxidativestressassociatedwith
withinthepaperanditsSupportingInformation
files. mitochondrialdysfunctionandtheimpairmentofglucoseandlipidmetabolism.Theseobser-
vationsmayreflectliverdysfunctionwithincreasedrelativeliverweightandtotallipidcontent.
Funding:Thisworkwassupportedbygrantsfrom
theEHESP-SchoolofPublicHealth(Young Similarfindingswereobservedforglucoseandenergymetabolismintheliveroftheoffspring,
Researchers)andtheFrenchMinistryof andoxidativestresswasalsosuggestedinthebrainsofmaleoffspring.
EnvironmentinthenationalprogramGrenellede
l’EnvironnementProgrammeLOLF189Funding
(EXPiOmark).
PLOSONE|https://doi.org/10.1371/journal.pone.0198448 June20,2018 1/21
Metabolomicsandpesticideexposure
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
Introduction
Pesticidesarecommonlyusedinagriculture.Theirapplicationcanleadtoenvironmentalcon-
tamination.Althoughhumanexposureisnotfullyelucidated,numerousstudieshaveshown
thatproximitytoareasofagriculturalpesticideusemaybeasourceofpesticideexposure,in
additiontodomesticordietarysources[1–5].Exposuretopesticidesishazardoustohuman
health,particularlyinoccupationalsituations,inwhichincreasedrisksofcancer,developmen-
taldefects,orneurologicproblemshavebeenobserved[6,7].Suchconclusionsaremorediffi-
culttodrawforlow-doseenvironmentalexposuretopesticides.However,fetusesandinfants
areparticularlysusceptibletotoxicants.Ananalysisofpublishedstudiesshowsthatprenatal
exposuretosomepesticidesmayinducemalformations(herbicides)[8–10],affectfetalgrowth
(herbicides,organophosphorous)[11–13],orareassociatedtobehavioraldisorders(organo-
phosorous,pyrethroids)[14–23].
Metabolomicsisapromisingapproachtostudytheassociationsbetweenenvironmental
exposuresandhealtheffects.Overthelastfewyears,manytoxicologicalstudieshavedemon-
stratedthatmetabolomicsisapowerfulmethodfordetectingchangesinthemetabolomeof
individualsexposedtopesticides.Arecentcomprehensivereviewshowedthatexposuretopes-
ticidesmixturesmayinducemetabolicmodificationspotentiallylinkedwithphysiopathological
disturbances[24].Endosulfan,atrazine,andchlorpyrifosincombinationcanpotentiallyinduce
changesinamino-acidmetabolism,thecitratecycle,theureacycle,andglucosemetabolism
throughanoxidativedisturbanceinmice[25,26].Exposuretoorganophosphorusmixtures
(chlorpyrifosandcarbaryl,ordichlorvos,dimethoate,acephate,phorate)inducesdisturbances
inenergyandlipidmetabolisminrats[27–29].Alteredlipidmetabolismhasalsobeenrecently
confirmedforexposuretoorganophosphorus.:hepaticdysfunction,suchasnon-alcoholicfatty
liverdisease,hasbeenobservedinratsexposedtodichlorvos+acephate+phorate+dimethoate
[30].Energymetabolismhasalsobeenshowntobealteredbyexposuretopyrethroids(deltame-
thrin)incombinationwithdichlorvos[31].Toxicologicalstudiesareusuallycarriedoutincon-
ditionswhicharenotrepresentativeofhumanexposures,inparticularbecausetheyinvestigate
simplemixturesathighdoses.Onlyonestudyhasbeendesignedtoinvestigatethemetabolic
effectsofcomplexmixturesofpesticidesatlowerdoses:Mehrietal.investigatedtheeffectin
miceofamixtureoflow-doses(calculatedfromtheirrespectiveacceptabledailyintakesdefined
bythejointFAO/WHOmeetingonpesticideresiduesandextrapolatedtothemiceonthebasis
ofmeanbodyweightvalues)ofsixpesticidesfrequentlyfoundinfruitsandvegetablesgrownin
Franceandshowedametabolicsignaturelinkedtooxidativestressandglucoseregulation[32].
Morethan60%ofthesurfaceareainBrittany(France)isdevotedtoagriculturalactivities,
with50%devotedtocerealsandcorn.Inthe2000’s,almostalllandinthisregionreceivedat
leastfourdifferenttreatmentstocontroltheproliferationofannualgrasses,fungi,andinsects.
In2004,acetochlor(chloroacetanilideherbicide),carbofuran(carbamateinsecticide),chlor-
mequat(quaternaryammoniumplantgrowthregulator),ethephon(organophosphorusplant
growthregulator),fenpropimorph(morpholinefungicide),imidacloprid(neonicotinoid
insecticide),glyphosate(glycinederivativeherbicide),andbromoxynil(nitrileherbicide)con-
stitutedalmost90%ofthepesticidesusedoncerealsandcorn.Apreviousstudybasedon83
pregnantwomenfromthePELAGIEcohortshowedchangesintheurinarymetabolomeof
womenlivingintownswithhighagriculturalcerealactivities[33].Thesechangesarepoten-
tiallyrelatedtoenergymetabolism.Theobjectiveofthepresentstudywastoexaminethe
effectsinpregnantratsofarealisticpesticidemixturebasedonsubstancesappliedtocropsin
Brittanytosupportthesepreviousobservations.
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Metabolomicsandpesticideexposure
Materialandmethods
Chemicalsandexposure
Methanol(purity(cid:21)99.8)waspurchasedfromMerck(Darmstadt,Germany).Ultrapurewater
wasproducedwithaMilli-Qsystem(Millipore,SaintQuentinenYvelines,France)andwas
usedforpesticidedissolution.Theeighthigh-purity((cid:21)98%)pesticides(pestanal1analytical
standards)acetochlor(CAS34256-82-1,BatchSZB9314XV),bromoxynil(CAS1689-84-5,
BatchSZB8021XV),carbofuran(CAS1563-66-2,BatchSZB9064XV),chlormequat(CAS999-
81-5,BatchSZB8248XV),ethephon(CAS16672-87-0,BatchSZBB021XV),fenpropimorph
(CAS67564-91-4,BatchSZB9243XV),glyphosate(CAS1071-83-6,BatchSZB9320XV),and
imidacloprid(CAS138261-41-3,BatchSZB9112XV),werepurchasedfromFluka,Sigma
Aldrich(LesUlis,France).Theywereincorporatedintotheratdiet,atanominaldosecorre-
spondingtothesameproportionastheirrespectiveenvironmentalexposurebasedonFrench
usein2004(https://www.airbreizh.asso.fr/),toreachatotaldoseof447μg/kgbw/d,whichcor-
respondstothesumoftheirrespectiveacceptabledailyintake.Theoreticalandingesteddoses
calculatedfromfoodconsumptionduringtheexperimentareshowninTable1.
Preparationofthecontaminateddiet
Commercialpowdereddietformulatedforgrowth,pregnancy,andlactation(A03,SAFE)was
usedforthisstudy.Wefirstincorporatedthepesticidesintoapremixbatch,becauseofthe
extremelylowquantitiesaddedtothediet,bythoroughlymixingtherequiredquantityofeach
pesticidewith50gpowdereddiet.Briefly,allpesticidesweredissolvedinmethanol(10mL
totalvolume),exceptglyphosate,whichwasdissolvedinwater(1mL),andbothalcoholicand
aqueoussolutionswereaddedtothediet.Themixturewasthenhomogenizedfor15min
usingaVirtis45Blade-typehomogenizer(Gardiner,NewYork).Theresultingmixwaskept
for24hatroomtemperaturetoallowthemethanolandwatertoevaporate.Themixwasthen
Table1. Ingesteddosesofpesticidesbydams(μg/kgbw/day),calculatedfromatotaldosebasedonthesumoftheacceptabledailyintake(ADI)ofeachpesticide
andtheproportiontotheirlevelofenvironmentalexposureinBrittany(France).
Pesticide: Theoreticaloral Dietaryintakebasedonfood ADIa(PODbusedforits Proportionofenvironmentalpesticide
Name(chemicalclass) dose consumption derivation)inμg/kgbw/day exposureinBrittany(%)c
Acetochlor 290 246 3.6(LOAEL:1080) 64.83
(chloroacetanilide)
Bromoxynil(nitrile) 14 12.0 10(NOAEL:1000) 3.16
Carbofuran(carbamate) 27 22.5 0.15(LOAEL:30) 6.03
Chlormequat(quaternary 42 35.0 40(NOAEL:4000) 9.33
ammonium)
Ethephon(organo- 27 22.5 30(NOAEL:27000) 6.03
phosphorus)
Fenpropimorph(morpholine) 18 15.5 3(NOAEL:300) 4.02
Glyphosate(glycine 14 12.0 300(NOAEL:31000) 3.17
derivative)
Imidacloprid(neonicotinoid) 15 12.5 60(NOAEL:3700) 3.43
aacceptabledailyintakefromEFSAandAgritox,datafromtheDecisionoftheEuropeanUnion,availableatwww.efsa.europa.euandwww.agritox.anses.fr,respectively;
bPOD:pointofdeparture(NOAEL:noobservedadverseeffectlevel,orLOAEL:lowestobservedadverseeffectlevel);
ctheproportionsofpesticideusedinthisstudyweredefinedfromaregistryofpesticide’semissionsachievedin2003inBrittany(France)byaregionalassociationfor
airqualityhttps://www.airbreizh.asso.fr/).
Thisregistrywasbasedondifferentpractice’ssurveysimplementedonthewatercatchmentscale,thephysicalchemicalcharacteristicsofthepesticides,andthe
spreadingquantitiesatthesquare-kilometerscale.
https://doi.org/10.1371/journal.pone.0198448.t001
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Metabolomicsandpesticideexposure
combinedwith50gdietandputinaDangoumauball-mill(Prolabo,Fontenay-sous-Bois,
France)tobereducedtoapowder.Thispremixwasstoredat4˚Cinglassflasksuntilmixing
andpelletingofthediet.Thesameprocedurewasusedforthecontroldiet,exceptthatnopes-
ticidewasaddedtothealcoholicandaqueoussolutions.Beforepelleting,alldietingredients
andthepremixweremixedforapproximately20mininamixingbowl(Santos,AsmoSud,
Toulouse,France).
Animals
TwentyfemaleWistarCrl:WI(Han)ratswerepurchasedfromCharlesRiverLaboratories,
L’Arbresle,France.Ratswerematedthedaybeforereceivingthediet,consideredtobegesta-
tionalday(GD)1.Animalswereacclimatedfortwodaysbeforeexposure.Animalsweremain-
tainedundercontrolledtemperatureandlight(21˚C±2˚C,12-hourslight/darkcycle).The
meanbodyweightwas212gatthebeginningoftheexperiment.Females(10perlot)werefed
controlorcontaminateddietsfromGD4toGD21.Foodconsumptionandbodyweightwere
measuredeverytwodays.AtGD13,eightfemalespergroupwereplacedinmetaboliccages
andacclimatedfortwodaysbeforea24-hurinecollectiononGD15(theendoftheorgano-
genesis).AtGD21,approximately12hbeforetheexpectedtimeofparturition,animalswere
sacrificedbycervicaldislocationfollowedbyexsanguination.Bloodsamplesweretakenfrom
thefacialarteryofthedams,addedtoaglassvialcontainingheparin,andplacedonicebefore
centrifugationandfreezingat-20˚C.Atthesametime,thefetuseswerewithdrawnfromthe
uterusofthedamsforbloodrecovery.Foreachindividual,theliver(themedianlobefor
dams,andapoolofthreeentireliversfromeachlitterformaleandfemalefetuses)andthe
brain(entirefordams,andapoolofthreeentirebrainsfromeachlitterformaleandfemale
fetuses)wereexcised,weighed,andrapidlyfrozeninliquidnitrogen.
Thestudywasconductedinaccreditedanimalcarefacilities(#C3155513)byanapprovedstaff,
andanimalcarewasinaccordancewiththeguidelinesoftheEuropeanCouncilonAnimalsused
inExperimentalStudies.Allexperimentalprotocolsandprocedureswereapprovedbythelocal
InstitutionalAnimalCareandUseCommitteesfromINRA:"Comite´d’EthiquedePharmacologie-
ToxicologiedeToulouseMidi-Pyre´ne´es"(CEEA#86/MinistryofHigherEducation,Researchand
Innovation)(TOXCOM/0124/JPC,nationalnumberAPAFiS#4303_2016022912425905_V3),
accordingtotheDirective2010/63/EUrecommendations.
Metabolomicsanalyses
Samplepreparation. Urinesamples:Afterthawingatroomtemperatureandvortexing,
500μLurinewasmixedwith200μLphosphatebuffer(pH7.39)preparedinD Otowhich
2
wasaddedsodium3-trimethylsilyl-1-[2,2,3,3,-2H ]-propionate(TSP,1mM).Thephosphate
4
bufferisusedtominimizevariationsinchemicalshiftvaluesintheacquiredNMRspectradue
topHdifferences.TSPservedasachemicalshiftreference(δ0ppm)andD Oasafield-fre-
2
quencylockfortheNMRspectrometer.Eachsamplewasvortexedandcentrifugedfor10min
at6,080gtoremoveanyprecipitate.Then,600μLaliquotsweretransferredtostandard5mm
—NMRtubesforanalysis.
Plasmasamples:Afterthawingatroomtemperatureandvortexing,200μLserumwas
mixedwith500μLD O.Eachsamplewasvortexedandcentrifugedfor10minat6,080gand
2
600μLaliquotsweretransferredtostandard5mm—NMRtubesforanalysis.Liverandbrain
samples:ExtractionprocedureswerederivedfromthemethoddescribedbyWatersetal.[34].
Tissuesamples:Liver(100mg)andwholebrainwerehomogenizedusingaPolytron
PT2100inacetonitrile/H O(50/50,v/v)containing0.1%BHTinanice-waterbath.Homoge-
2
nateswerecentrifugedat5,000gfor10minat4˚Candthesupernatantsremovedand
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Metabolomicsandpesticideexposure
lyophilizedbeforebeingreconstitutedin600μLD Ocontaining0.25mMTSP(asachemical
2
shiftreferenceat0ppm).
1HNMRspectraacquisition. All1HNMRspectrawereacquiredat300KonaBruker
AvanceDRX-600,operatingat600.13MHz(BrukerBiospin,Wissembourg,Germany),
equippedwithanautosamplerandaninverse1H-13C-15Ncryoprobe.Forurinesamples,1H
NMRspectrawereacquiredusingastandardpulsesequenceNOESYtosuppresswaterreso-
nance.Arelaxationdelayof2sandmixingtimeof150mswereusedand256freeinduction
decays(FIDs)werecollectedinto32kdatapointsusingaspectralwidthof20ppmwithan
acquisitiontimeof1.36s.1HNMRspectrawereacquiredusingtheCarr-Purcell-Meiboom-
Gill(CPMG)spin-echopulsesequencewithpre-saturation,withatotalspin-echodelay(2nt)
of240mstoattenuatebroadsignalsfromproteinsandlipoproteins.Atotalof128transients
werecollectedinto32kdatapointsusingaspectralwidthof20ppm,arelaxationdelayof2s
andanacquisitiontimeof1.36s.
1HNMRdatapreprocessing. Allfreeinductiondecayswerethenmultipliedbyanexpo-
nentialfunctionwithalinebroadeningfactorof0.3HzpriortoFouriertransformation.All
spectrawerereferencedtothechemicalshiftofTSP(δ0.00).AllNMRspectraweremanually
phase-andbaseline-correctedusingTopspin(V2.1,BrukerBiospin,Germany)andACD/NMR
Processor(AcademicEdition,ACDLabs,Canada).Thespectralregionscontainingresidual
water,solvents,andurearesonanceswereremovedandthespectradigitizedto469to766buck-
ets(accordingtothebiologicalmedia)correspondingto0.01ppmintervalsusingtheAMIX
softwarepackage(V3.9.11,BrukerBiospin,Germany).Eachintegratedregionwasdividedby
thetotalspectralintensitytonormalizevalues.Thispartiallyremovesconcentrationdifferences
betweensamples.
GC-FIDanalysisoffattyacidmethylestersintheliver. Tissuesampleswerehomoge-
nizedusingtheFastprepsystem(40s)inmethanol/water(83/17v/v).Dichloromethane(2
mL)wasaddedtothehomogenatesand,aftervortexing,adichloromethane/water(50/50v/v)
mixture(4mL)wasadded.After15minat4˚C,theextractswerecentrifuged(2,870gfor15
minat4˚C),andthelowerlipophilicphaserecoveredandevaporatedtodrynessunderanitro-
genstream.Theliversamplesfromadultfemalesandalloffspringwereretakenby5mLand1
mLofdichloromethane,respectively.Analiquotcorrespondingto5mgoftissuewassampled
and5μgTG17(glyceryltriheptadecanoate,SigmaAldrich,lesUlis,France)addedasinternal
standardtoverifythecompletenessofhydrolysis.AfterhydrolysisinKOH-methanol(0.5M)
for30minat56˚C,FAsweretransmethylatedwith1mLBF -methanol,10%wt(60minat
3
80˚C).Oncecooleddown,1mLMilli-Qwaterand2mLheptanewasaddedtothemethylated
FAsandthemixturemanuallyshaken.Aftercentrifugation(500g,5min),theupperlayer
containingFAmethylesters(FAMEs)wastransferredtoaglasstubeandevaporatedtodry-
ness.Heptane(200μL)wasthenaddedandthesampletransferredtoavial.FAMEswereana-
lyzedonaTRACE1310gaschromatograph(ThermoScientific,LesUlis,France)equipped
withasplit-splitlessinjectoroperatedinthesplitlessmodeandaflame-ionizationdetector.
FAMEswereseparatedonaFAMEWAXTMcolumn(30m,0.32mminternaldiameter,
0.25μmfilmthickness)fromRestek(Lisses,France)usingheliumascarriergasataconstant
flowof1.0mL.min-1.Theinjectortemperaturewassetat225˚Candtheoventemperaturewas
programmedasfollows:1minisothermalstepat130˚C,from130˚Cto245˚Cat2˚C.min-1
andthen8minat245˚C.FAMEswereidentifiedbycomparingsampleretentiontimesto
thoseofcommercialstandardmixtures(MenhadenoilandFoodIndustryFAMEMix,Restek)
usingXcalibur2.2software(ThermoScientific).
Discriminantanalysisaccordingtoexposuregroups. TheNMRspectralandGC-FID
dataintegrationwereimportedintotheSIMCA-P+softwarepackage(version12.0,Umetrics)
formultivariatestatisticalanalysis.Principalcomponentanalyses(PCA)wasperformedto
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Metabolomicsandpesticideexposure
separatetheexposedandcontrolgroupsandremoveoutliers,ifnecessary.ThePLS-DA
methodwasthenappliedtoidentifypotentialmetabolitescorrespondingtothebucketswith
variableimportanceintheprojection(VIP)above1.Cross-validationwasusedtodetermine
thenumberoflinearcombinationstobeincludedinthePLS-DAmodel.Thequalityofthe
modelwasgivenbythetwoparameters,R2Y(proportionofexplainedvariance)andQ2Y
(predictiveability).TheQ2valuewasevaluatedusingaseven-foldcross-validation.Apermu-
tationtest(200iterations)wasconductedforeachPLS-DAmodeltotesttheirvalidity.Ifno
separationwasobtainedwithpreliminaryPCAorPLS-DA,weusedorthogonalsignalcorrec-
tion(OSC)filteringtodecreasevariabilityintheX-matrix(spectraldata)notcorrelatedwith
theexposuregroups[35,36],whichincludeconfoundingfactorssuchasphysiological,experi-
mental,andinstrumentalfactors.
Anon-parametricKruskal-Wallistestwithacriticalp-valueof0.05wasfurtherusedto
determinewhethertherewasasignificantdifferencebetweenthetwogroupsforeachmetabo-
liteobtainedfromthePLS-DAmodels.P-valueswereadjustedformultipletestingcorrection
usingtheFalseDiscoveryRate[37].ThistestwasconductedusingRsoftware(version2.12.1).
Statisticallysignificantchangesbetweenthelipidcontentofliverandbrainfromtheexposed
andcontrolgroupswerealsoassessedusinganon-parametricKruskal-Wallistest(n=6to10
pergroup,p-value<0.05).
Metaboliteidentification. Spectralassignmentswerebasedonmatching1DNMRdata
toreferencespectrainahome-madereferencedatabase,aswellaswithotherdatabases(http://
www.bmrb.wisc.edu/metabolomics/;http://www.hmdb.ca/),andreportsintheliterature.
Results
CharacteristicsoftheanimalsarepresentedinTable2.Ourresultsdonotshowanysignificant
differencesinbodyweight,bodyweightgain,orabsoluteliverweightinexposeddamsrelative
tocontrols.Foodconsumptionwasslightlybutsignificantlyincreasedinexposeddamsin
comparisontocontroldams,butthedifferencedisappearedafteradjustingforbodyweight.
Therelativeliverweightsofexposeddamswereslightlybutsignificantlyreducedthanthoseof
thecontroldams,andthiswasassociatedwithasignificantincreaseinlipidmasspergramof
liver.Thebrainsofexposeddamsweresignificantlyheavierthanthoseoftheexposedgroup
(p<0.05),butthedifferencedisappearedafteradjustingforbodyweight.Damsexposedto
pesticideshadfewerfemalefetusesthannon-exposeddams,butthenumberofoffspringper
litterwasthesame.Furthermore,themeanliverweightofmalefetusesintheexposedgroup
wassignificantlyhigherthanthatofthoseofthenon-exposedgroup(p<0.005),whereasthe
meanbrainweightoffetusesofbothgenderswashigher(p<0.01formales,p<0.05for
females).Thissuggestspossiblegender-specificsensitivitytothispesticidemixture,withmales
possiblybeingmoresensitivethanfemales.
Comparisonofmetabolicprofilesofexposeddamsversuscontroldams
Wecomparedthe1HNMRspectraofurine(atGD15),plasma(atGD21),andliverandbrain
extractsofexposedandcontroldams(rawdataareavailableinS1–S4Tables):thefingerprints
resultingfromdietaryexposuretothepesticidemixturewerecomprisedof35metabolites.
Urinarymetabolicprofiles. ThefirstPCAofurinemetabolitesrevealedachemicalshift
forcitrate,possiblyduetoadifferenturinarypHbetweenindividuals,despitetheadditionof
phosphatebuffer.Afterexcludingthespectralregionscorrespondingtothesignalsofthecit-
rateprotons(δ2.51–2.58ppmandδ2.65–2.73ppm),neitherpreliminaryPCAnorPLS-DA
separatedtheexposedgroupfromthecontrols.AnOSCfilteredPLS-DAmodelallowedusto
identifytwometabolites(hippurate,citrulline)forwhichsignificantdifferencesinthesignal
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Metabolomicsandpesticideexposure
Table2. Foodconsumptionandbodyandorganweightsfordamsandoffspring.
Parameters Controlgroup Exposedgroup
Dams (n=10) (n=10)
Foodconsumption(g) 294.3±18.3a 317.2±31.8
Relativefoodconsumption/bodyweight(%) 1.43±0.10 1.47±0.13
Bodyweightgain(g) 97.5±6.3 95.5±13.3
BodyweightatGD21(g) 302.7±12.6 312.2±22.5
Absoluteliverweight(g) 9.49±0.82 8.98±0.98
Relativeliver/bodyweight(%) 3.13±0.19 2.87±0.19(cid:3)b
Lipidmasspergramofliver(mg/g) 26.20±4.52 33.77±5.50(cid:3)(cid:3)
Brainweight(g) 1.44±0.18 1.57±0.07(cid:3)
Relativebrain/bodyweight(%) 0.48±0.06 0.50±0.03
Offspring
Numberofoffspringperlitter 11.39±1.6 10.28±1.6
Numberofmales 5.71±1.03 5.47±2.13
Numberoffemales 5.53±1.34 4.20±1.51(cid:3)
Liverweightc(g)
Males 0.45±0.08 0.58±0.10(cid:3)(cid:3)(cid:3)
Females 0.56±0.11 0.60±0.08
Lipidmasspergramofliver(mg/g)
Males 20.09±3.92 16.1±3.56(cid:3)
Females 15.73±3.42 15.18±2.17
Brainweightc(g)
Males 0.36±0.05 0.42±0.04(cid:3)(cid:3)
Females 0.39±0.02 0.41±0.03(cid:3)
aDatashownasthemean±SD.
bComparisonwiththeKruskalWallistest:
(cid:3)p<0.05;
(cid:3)(cid:3)p<0.01;
(cid:3)(cid:3)(cid:3)p<0.005.
cpoolofthreeindividualsperlitter.
https://doi.org/10.1371/journal.pone.0198448.t002
wereconfirmedusingtheKruskal-Wallistest.Differenceswerealsoshownforthreeother
metabolites(creatine,phenylacetylglycineandtaurine)withoutstatisticalsignificance
(0.05<p<0.08).CharacteristicsofthemodellingarepresentedintheTable3andscoreplots
intheFig1A.Additionally,aspecificanalysisofthespectralregionscorrespondingtothecit-
rateusingtheKruskal-Wallistestshowsastatisticalsignificantincreaseintheexposedgroup
comparedtothecontrolgroup(p=0,001).
Plasmametabolicprofiles. PreliminaryPCArevealedacceptableseparation,especially
onthesecondcomponent(Fig1B).Ratn˚23wasmoredistantthantheotherindividuals,
becauseofincreasedlipidandlactatecontent.PLS-DAmodellingallowedustoidentify11sig-
nificantlyalteredmetabolitesignalsfortheexposedgrouprelativetocontrols(higherforlip-
ids,aceto-acetate,glutamateanddimethylamineandlowerforcreatine,acetate,alanine,
glutamine,isoleucine,lysineandvaline),suggestingadisturbanceofenergy,amino-acid,and
lipidmetabolism(Tables3and4).
Livermetabolicprofiles. Aqueousextract:Afterexcludingtwoindividualswithahigh
contentof3-hydroxybutyrateandhighlydilutedspectra,thePCAshowedverygoodsepara-
tiononthefirstcomponentthatexplained44%ofthetotalvariabilitybetweengroups(Fig
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Table3. Resultsofthepartialleastsquaremodellingofmetabolicprofilesofdams(urine,plasma,aqueousliverandbraintissueextracts,andlipidiclivertissue
extracts).
Samples Numberof Modelused Numberof R2 Q2 Interceptsb NumberofVIPC>
rats LVa 1
Urine 15 OSC-paretoscaled+PLS-DA(mean 2 94.8% 0.613 R2=(0.0,0.665)Q2=(0.0, 163
centered)d -0.249)
Plasma 20 PLS-DA(paretoscaled) 3 80.6% 0.413 R2=(0.0,-0.472)Q2=(0.0, 155
-0.232)
Liver–aqueous 14 PLS-DA(paretoscaled)e 1 81.2% 0.704 R2=(0.0,-0.277)Q2=(0.0, 87
-0.222)
Liver–Fatty 20 OSC-unitscaled+PLS-DA(unitscaled) 1 93.3% 0.835 R2=(0.0,0.291)Q2=(0.0, 7
acids -0.166)
Brain–aqueous 19 PLS-DA(paretoscaled)f 2 86.5% 0.552 R2=(0.0,0.514)Q2=(0.0, 165
-0.231)
aLV:latentvariables.
b200permutations.
cVIP:variableimportanceinprojection.
dexclusionofthecitrateregion(shift)andratn˚G2-18(outlierinapreliminaryPCA).
eexclusionofratsn˚G2-17andG0Mbis.
fexclusionofratn˚G2-Mter.
https://doi.org/10.1371/journal.pone.0198448.t003
1C).ThefirstcomponentofPLS-DAmodelexplained81.2%ofthedifference(Table3).There
wasasignificantlyhighersignalforglucoseandglycerophosphocholinefortheexposedgroup
andtoalesserextent(tendencywithoutsignificance),forcreatine,acetateand3-hydroxybuty-
rate.Aconcomitantdecreaseinthesignalforglutamate,glutamine,glycogen,glycerol,serine,
dimethylglycine,glycine,andtaurinesuggestsanimpactonenergy,glucose,andamino-acid
metabolismandpotentialoxidativestress(Table4).
Lipidfraction:Weundertookspecificanalysesofhepaticfattyacidsbecauseofthesig-
nificantdecreaseinrelativeliverweightandincreaseinlipidcontentintheliversof
exposeddams(seeTable2)andbecauseoftherelevanceoftheliverasatargetorganfor
pesticides[38].Therewasnostatisticallysignificantdifferenceinthesaturated/unsatu-
ratedfattyacidratiobetweentheexposedgroupandcontrols.NeitherPCAnorPLS-DA
separatedtheexposedandcontrolgroups.AnOSCfilteredPLS-DAmodelallowedusto
identifysevenfattyacidsforwhichsignificantdifferencesinthesignalwereconfirmed
usingaKruskal-Wallistest.ThefirstcomponentofthePLS-DAmodelexplained93.3%of
thevariability(Table3).Thesignalsforunsaturatedfattyacids(PUFAsC20:2ω6,C20:3ω6,
C20:4ω6;C22:4ω6,C22:5ω3,andMUFAC18:1ω7)andonesaturatedfattyacid(C18:0)
werehigherfortheexposedgroup(Table5),whereasthoseforC14:0,C16:0,C16:1ω7,
C18:1ω9,C20:1ω9,C18:2ω6,C18:3ω6,C18:3ω3,C20:3ω9,C20:5ω3,C22:5ω6,C22:6ω3
wereunaffected.
Brainmetabolicprofiles(aqueousextracts). Afterexcludingoneoutlier(withavery
concentratedspectrumwhichcontainsmethanol,probablyduetopartialevaporation),the
PCAproperlyseparatedtheexposedgroupfromthecontrols,especiallyonthefirstcompo-
nent(Fig1D).PLS-DAmodellingfollowedbyaKruskal-Wallistestallowedustoidentifysig-
nificantlyhighersignalsforN-acetylaspartate,lysine,inosine,oxidizedglutathioneand
ethanolamineinbrain,andlowersignalsforATP,ADP/AMP,lipids,glutamine,aspartate,
succinate,lactate,serine,glycerophosphocholine,phosphocholine,anduridine.Non-signifi-
cantincreasesincreatineandtaurinewerealsoobserved(Table4).Thesedifferencescouldbe
linkedtooxidativestress.
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Metabolomicsandpesticideexposure
Fig1.Two-dimensionalPLS-DAandPCAscoresplotofpregnantratsamplesintegrated1HNMRspectra.1a:GD15urinesamples(PLS-DA,A=2,R2=94.8%,
Q2=0.613);1b:GD21plasmasamples(PCA,A=4,R2=72.5%);1c:GD21liveraqueousextractsamples(PCA,A=3,R2=61.6%);1d:GD21brainaqueousextract
samples(PCA,A=4,R2=72.8%).Eachdotrepresentsanobservation(animal),projectedontofirst(horizontalaxis)andsecond(verticalaxis)PLS-DAorPCAvariables.
ControlgroupisshownwithblacksquaresandthegroupexposedtoeightpesticideswidelyusedinBrittany(France)in2004(acetochlor,bromoxynil,carbofuran,
chlormequat,ethephon,fenpropimorph,glyphosate,imidacloprid)isshownwithredcircles.Theblackellipsedeterminesthe95%confidenceinterval,whichisdrawn
usingHotelling’sT2statistic.
https://doi.org/10.1371/journal.pone.0198448.g001
ComparisonofthemetabolicprofilesatGD21betweenexposedand
controlgroupsofoffspring
Plasmametabolicprofiles. Onlymalebloodprofileswereanalyzedduetoaninsufficient
quantityofbloodcollectedfromthefemalefetuses.EachNMRspectrumwasacquiredfrom
pooledbloodfromthreemalesperlitter.RawdataarepresentedasbuckettableinS5Table.
ThepreliminaryPCA(mean-centereddata)revealedgoodseparation,especiallyonthesecond
component(Fig2A).PLS-DAmodelling(characteristicsgiveninTable6)allowedtoidentify
significantlowerplasmalevelofglutamateandcholine,andahigherplasmaleveloflipidsin
exposedmalefetusesthancontrols(Table7).
Livermetabolicprofiles. Aqueousextract:malesandfemaleswereanalyzedseparately.
RawdataarepresentedasbuckettableinS6andS7Tables.Formales,thepreliminaryPCA
showedthreeoutliers.Afterexcludingthem,thePCAcorrectlyseparatetheexposedandcon-
trolgroups(Fig2B).Wefound13metaboliteswithalteredsignalsbetweenthegroupsafter
PLS-DAmodeling(Tables6and7),includingsignificantlowersignalsforcreatine/creatinine,
glutamate,aspartate,glycogen,glycerol,leucine,valine,andglutathione,andhighersignalsfor
glucose,lysine(withacetate),taurine,andglycerophosphocholineforthemalefetusesof
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Metabolomicsandpesticideexposure
Table4. Plasma,liverandbrainmetabolitelevelsthatweresignificantlydifferentbetweendamsfromtheexposedandcontrolgroups.
Plasma Liveraqueous Brainaqueous Biologicalrole
Hippurate Gutmicrobialmetabolism?
Citrulline Ureacycle
Creatine & %c %a Amino-acidsynthesis,storageenergy(phosphocreatine)
Phenylacetylglycine Minormetabolitesoffattyacids
Citrate TCAcycle
Succinate &b TCAcycle
ATP & Energysource
ADP/AMP & Energysource
Glycerol & Lipidcomponent,convertedtoglucoseforenergyproduction
Glycogen & Energystorage
Glucose % Energysource
Lactate & Energymetabolism
Alanine & Energysource,glucosemetabolismregulator
Glutamate % &b Neoglucogenesis,excitatoryneurotransmitter
Glutamine & & & Non-essentialamino-acid,roleinTCAcycle
Valine & Essentialamino-acidinvolvedinstress,energyandmusclemetabolism,roleincarbohydrate
synthesis
Acetate(withlysine) & %c Lipidandcarbohydratemetabolism
Lipids % & Lipidmetabolism
Aceto-acetate % Lipidmetabolism,cholesterolsynthesis
3-hydroxybutyrate %c Lipidmetabolism.Energysource
Isoleucine & ““
Lysine(withacetate) & % Essentialamino-acidinvolvedinstress,precursorofacetyl-coA
Serine & & Non-essentialamino-acidderivedfromglycine
Dimethylamine % Gutmicrobialmetabolismofcholine,hostco-metabolism
Dimethylgylcine & Byproductofhomocysteineandglycinemetabolism
Inosine % Purinemetabolism
Aspartate & Non-essentialamino-acidproducedfromglutamate,neurotransmitter.
N-acetylaspartate % Neuronalosmolyte,lipidsynthesis,derivedfromaspartateinbrain
Oxidizedgluthatione % Anti-oxidant
Glycine & Osmoprotector,defensemechanisms
Taurine & %c Membranesstabilizerinbrain,antioxidant,osmolyte
Glycero- % & Membranestabilizer,osmolyte
phosphocholine
Phosphocholine & Membranesstabilizer,osmolyte
Ethanolamine % Membranephospholipidsynthesis
Uridine & Nucleoside,synthesisofRNAmembrane,regulationofphysiologicalprocesses
awithcreatinine.
bclosetothesignalofglutamine.
ctendencybutwithoutstatisticalsignificance(0.05<p<0.08).
https://doi.org/10.1371/journal.pone.0198448.t004
exposeddams.Alaninewasalsodecreasedbutinanon-significantway.Thissuggestsamodi-
ficationinglucosemetabolism,potentiallyassociatedwithmitochondrialdysfunction.Oxida-
tivestressmayalsobepossibleforthesechanges.Forfemales,theresultswerelessinformative,
buttherewasaslightseparationbyPCA(Fig2C)andthePLS-DAmodelexplainedmorethan
85%ofthevariabilitybetweengroups(withalowerQ2thanformales,Table7).Thesignals
foronlyeightmetabolitesweredifferentbetweengroups,includingdecreasesinglycogen,
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