The BPA-substitute bisphenol S alters the transcription of genes related to endocrine, stress

17 Pages·2017·2.56 MB·English

RESEARCHARTICLE
The BPA-substitute bisphenol S alters the
transcription of genes related to endocrine,
stress response and biotransformation
pathways in the aquatic midge Chironomus
riparius (Diptera, Chironomidae)
O´scarHerrero1*,Mo´nicaAquilino1,PalomaSa´nchez-Argu¨ello2,RosarioPlanello´1
a1111111111 1 GrupodeBiolog´ıayToxicolog´ıaAmbiental,FacultaddeCiencias,UniversidadNacionaldeEducacio´na
Distancia,UNED,Madrid,Spain,2 LaboratoriodeEcotoxicolog´ıa,DepartamentodeMedioAmbiente,
a1111111111
InstitutoNacionaldeInvestigacio´nyTecnolog´ıaAgrariayAlimentaria,INIA,Madrid,Spain
a1111111111
a1111111111
*[email protected]
a1111111111
Abstract
BisphenolS(BPS)isanindustrialalternativetotheendocrinedisruptorbisphenolA(BPA),
OPENACCESS
andcanbefoundinmanyproductslabeled“BPA-free”.Itsusehasgrowninrecentyears,
Citation:HerreroO´,AquilinoM,Sa´nchez-Argu¨ello
andpresentlyitisconsideredaubiquitousemergingpollutant.Todatethereisalackof
P,Planello´R(2018)TheBPA-substitutebisphenol
Saltersthetranscriptionofgenesrelatedto informationontheeffectsofBPSoninvertebrates,althoughtheyrepresentmorethan95%
endocrine,stressresponseandbiotransformation ofknownspeciesintheanimalkingdomandarecrucialforthestructureandproperfunction
pathwaysintheaquaticmidgeChironomus
ofecosystems.Inthisstudy,real-timeRT-PCRwasusedtodeterminetheearlydetrimental
riparius(Diptera,Chironomidae).PLoSONE13(2):
effectsofBPSonthetranscriptionalrateofgenesinthemodelspeciesChironomusriparius,
e0193387.https://doi.org/10.1371/journal.
pone.0193387 specificallythoserelatedtotheecdysonepathway(EcR,ERR,E74,Vtg,cyp18a1)crucial
forinsectdevelopmentandmetamorphosis,stressandbiotransformationmechanisms
Editor:CherylS.Rosenfeld,UniversityofMissouri
Columbia,UNITEDSTATES (hsp70,hsp40,cyp4g,GPx,GSTd3)thatregulateadaptiveresponsesanddeterminesur-
vival,andribosomebiogenesis(its2,rpL4,rpL13)whichisessentialforproteinsynthesis
Received:October31,2017
andhomeostasis.While24-hourexposureto0.5,5,50,and500μg/LBPShadnoeffecton
Accepted:February11,2018
larvalsurvival,almostallthestudiedgeneswereupregulatedfollowinganon-monotonic
Published:February21,2018
dose-responsecurve.Geneswiththegreatestincreasesintranscriptionalactivity(fold
Copyright:©2018Herreroetal.Thisisanopen changerelativetocontrol)wereEcR(3.8),ERR(2),E74(2.4),cyp18a1(2.5),hsp70(1.7),
accessarticledistributedunderthetermsofthe
hsp40(2.5),cyp4g(6.4),GPx(1.8),andGST(2.1),whileothersincludingVtg,GAPDH,and
CreativeCommonsAttributionLicense,which
selectedribosomalgenesremainedstable.Wealsomeasuredthetranscriptionalactivityof
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal thesegenes24hoursafterBPSwithdrawalandageneraldownregulationcomparedtocon-
authorandsourcearecredited. trolswasobserved,thoughnotsignificantinmostcases.OurfindingsshowedthatBPS
DataAvailabilityStatement:Allrelevantdataare exposurealteredthetranscriptionalprofileofthesegenes,whichmayhaveconsequences
withinthepaper. forthehormonesystemandseveralmetabolicpathways.Althoughfurtherresearchis
Funding:Thisresearchwassupportedby neededtoelucidateitsmodeofaction,theseresultsraisenewconcernsaboutthesafetyof
MinisteriodeEconom´ıayCompetitividad(Spain) BPAalternatives.
contractgrantnumberCTM2015-64913-R(http://
www.idi.mineco.gob.es).Thefunderhadnorolein
studydesign,datacollectionandanalysis,decision
topublish,orpreparationofthemanuscript.
PLOSONE|https://doi.org/10.1371/journal.pone.0193387 February21,2018 1/17
TheBPA-substitutebisphenolSinducestranscriptionaldisruptionintheaquaticmidgeChironomusriparius
Competinginterests:Theauthorshavedeclared Introduction
thatnocompetinginterestsexist.
EvidencedemonstratedinrecentyearsaboutthehealthhazardsofbisphenolA(BPA)—used
inplastics,receiptpaper,foodpackaging,andothermaterials—haspromptedindustriesto
removethisxenobioticfromtheirproducts.RisksarebasedprimarilyontheabilityofBPAto
actasanendocrine-disruptingchemical(EDC)[1],itsubiquitouspresenceintheenvironment
[2],andthepotentialhazardsarisingfromcontinuousexposureofanimalsandhumans[3].
Thus,manufacturersaresubstitutingBPAwithalternativesubstances,suchasotherbisphe-
nols,inanattempttocircumventinternationalrestrictions.
BisphenolS(BPS)isaBPAanalogusedinavarietyofindustrialapplicationssuchaswash
fasteningagentsincleaningproducts,electroplatingsolvent,andaconstituentofphenolic
resin[4].Itcanalsobefoundincannedsoftdrinks,cannedfoodstuffsandthermalreceipt
papers,includingproductsmarkedas“BPA-free”[5,6].Becauseofitslowerestrogenicactivity
anditsgreaterstabilityagainstheatingandsunlight,BPSisconsideredasaferalternativeto
BPA[7].AlthoughsomeBPA-likeeffectsarehypothesizedbecauseoftheirsimilarchemical
structures,BPSispresentlyunregulatedandcanbeusedwithoutrestriction,whichhasledtoa
sharpincreaseinitsproductionanduse[8].Consequently,BPShasbeendetectedindust,
water,sediment,sewagesludgeandeffluentsamples[9,10],andinhumanurineandblood
serum[11],atconcentrationsthataregenerallylowerthanBPA,butofthesameorderofmag-
nitude[12].
WhileourunderstandingofBPStoxicityisstilllimited,availabledatasuggeststhatBPS
maynotbeacompletelybenignsubstituteforBPA.IthasbeenreportedthatBPSalteredbasal
testosteronesecretionbymouseandhumanfetaltestes[13].Inrodents,BPSdecreasedbody
weight[14],inducedoxidativestressandmorphologicalalterationsinthemalereproductive
system[15],andaffectedtranscriptionofgenesrelatedtothedopamine-serotoninsystems
[16].Inzebrafish,BPSalteredtheembryonicnervousandendocrinesystems[17],and
induceddevelopmentalandreproductiveabnormalities[18].Invitrostudieshavefoundthat
BPSinducesmorphologicalandbiochemicalalterationsindifferenttypesofhumanblood
cells,altersthemeioticcycleofporcineoocytes,andhasweakestrogenicactivity[19–21].Dif-
ferentcelllineshavebeenusedtodemonstratethatBPShasestrogenicpotential,disrupts
estradiol-inducedcellsignaling,modifiesgeneexpression,induceslipidaccumulationanddif-
ferentiation,andalterssteroidogenesisandinhibitshormoneproduction[22–24].
Despitethefactthatinvertebratesrepresentmorethan95%ofknownspeciesintheanimal
kingdomandareextremelyimportantregardingecosystemstructureandfunction,todate
thereislittleinformationontheeffectsofBPSintheseorganisms.Studieswiththenematode
Caenorhabditiselegansdescribedseverereproductivedefects[25]andbehavioralchanges[26]
causedbyexposureto125–500μMor0.1–10μMBPS,respectively.Thelackofinformationis
amajorconcern,giventhatBPSisaubiquitouspollutantthatcanbefoundinalmostanyenvi-
ronmentalcompartment,isabletointeractwithnumerousinvertebratespecies,andthusmay
disrupttrophicchainsandalterthenaturalbalanceofecosystems.
Sincewaterusuallyconstitutesthemainvehiclefordispersionofanthropogenicpollutants,
aquaticecosystemsareespeciallysensitivetotheirpresence.Aquaticinsectsareamongthe
mostimportantcomponentsofafreshwaterecosystem’sbiota,andthefamilyChironomidae
(Diptera)ispredominantinabundanceanddiversity.Inthisregard,Chironomusmidgeshave
routinelybeenusedtoassesswaterqualitybymeansofclassicalecotoxicologicalendpoints,
suchassurvival,growth,immobilization,development,andreproduction,amongothers[27].
Complementarytothosetraditionalmethods,molecularbiomarkersinChironomusripar-
iusincludinggenetranscriptionandenzymeactivityhavebeendemonstratedinrecentyears
tobeeffectivefortheearlydetectionofchemicaltoxicity,constitutinganimportanttime-and
PLOSONE|https://doi.org/10.1371/journal.pone.0193387 February21,2018 2/17
TheBPA-substitutebisphenolSinducestranscriptionaldisruptionintheaquaticmidgeChironomusriparius
cost-effectivealternativeforlarger-scaleevaluations.Thus,C.ripariuslarvaehavebeenusedto
assesstranscriptionalalterationsinducedbyavarietyofpollutantsunderdifferentstresscon-
ditions,andtotesttheireffectsontheactivityofenzymesinvolvedinphaseIandphaseII
detoxpathways.Therehavebeenmanyeffectsrelatedtoendocrinedisruption,specificallythe
ecdysonehormone,causedbyBPA,phthalates,UVfilters,biocides,metals,nonylphenol(NP),
andnanoparticles(e.g.[28–31]).Furthermore,stage-andsex-dependenttranscriptionalmod-
ulationofseveralecdysone-relatedgeneshasbeenreportedthroughoutC.ripariusdevelop-
ment[32],andalsosomeendocrineresponsesinnaturalpopulationsexposedtocomplex
mixturesofchemicals[33].Thesealterationsontheecdysonepathwaycouldultimatelyaffect
otherecdysteroidssuchas20-hydroxyecdysone(20E),theactiveformoftheecdysonehor-
mone,whichcontrolstheecdysis(molting)andmetamorphosisofarthropods.
Theaimofthepresentstudywastodetermineforthefirsttimethetranscriptionalalter-
ationsinducedinC.ripariuslarvaebyexposuretoenvironmentally-relevantconcentrationsof
BPS.Thepathwaysstudiedincludetheecdysonehormonalpathway,whichplaysacentralrole
duringinsectdevelopmentandmetamorphosis,severaldetoxicationandstressresponsegenes
thatareinvolvedinresistancemechanismsandsurvival,anddifferentribosomalgeneswhich
areimportantformaintenanceofproteinsynthesisandhomeostasis.
Materialsandmethods
Testanimals
C.ripariuspopulationsweremaintainedatthefacilitiesoftheLaboratoryforEcotoxicologyof
INIAfollowinglaboratoryconditionsdescribedbySa´nchezetal.[34].Briefly,larvaewere
rearedinglassaquariawithsandsedimentandspringwaterunderstaticflowandgentle
aeration,aphotoperiodof16/8hoflight/darknessandatemperatureof20±1˚C.Commercial
fishfood,TetraDiscus1(530mg)wasaddedthreetimesaweekandtherearingcontainercov-
eredtopreventescapeofmidges.Emergingadultswerelefttopermitmatingandoviposition.
Afterremovalofadultsbyaspiration,eggropeswerecollectedandusedtoinitiatethenext
generation.
Treatments
BPS(CASRN80-09-1;purity(cid:21)98%;Sigma-Aldrich,USA)wasdissolvedindimethylsulfoxide
(DMSO;CASRN67-68-5;purity(cid:21)99.9%;Sigma-Aldrich)toobtainastocksolutionof1g/L,
andstoredat−20˚C.WorkingsolutionsofBPS(0.5μg/L,5μg/L,50μg/L,500μg/L)werepre-
paredfreshforeachexperimentfromthestocksolutiondilutedinculturemedium,andcon-
tained0.05%DMSO.Experimentswerecarriedoutexclusivelyusingearlyphasesoffourth
instarlarvae,whichweredeterminedbasedonheadcapsulewidth.Groupsof20larvaein
glassvesselswereexposedto200mlofeachBPSsolutionfor24h(24hcondition).Atthesame
time,fortherecoverystudies,thesameconditionsweremaintainedandafterexposure,BPS
solutionswereremovedtoallowlarvaetorecover,maintainingtheminfreshculturemedium
foranadditional24h(24+24hcondition).Aftereachtimepointoftheexperiment,survival
valueswererecordedandfivelarvaefromeachvesselwerecollectedin1.5mlmicrotubesand
frozenimmediatelyondryiceforgeneexpressionstudies.Sampleswerestoredat-80˚Cuntil
RNAisolation;fourindependentexperimentswereperformed.
RNAisolationandcDNAsynthesis
TotalRNAwasextractedusingTRIzolReagent(Invitrogen,Germany),followingthemanu-
facturer’sinstructions.Subsequently,RNAwastreatedwithRNase-freeDNase(Roche,
PLOSONE|https://doi.org/10.1371/journal.pone.0193387 February21,2018 3/17
TheBPA-substitutebisphenolSinducestranscriptionaldisruptionintheaquaticmidgeChironomusriparius
Germany)andextractedwithphenol:chloroform:isoamylalcohol(Fluka,Germany)using
5PRIMEPhaseLockGelLighttubes(Quantabio,USA).PurifiedRNAwasresuspendedin
diethylpyrocarbonate(DEPC)water,quantifiedbyspectrophotometryat260nmusingaBio-
Photometer(Eppendorf,Germany),andstoredat-80˚C.
Aliquotscontaining5μgofisolatedRNAwerereverse-transcribedinaC1000Thermal
Cycler(Bio-Rad,USA)usingiScriptReverseTranscriptionSupermixforRT-qPCR(Bio-Rad),
accordingtothemanufacturer’sprotocol.TheobtainedcDNAwasconservedat-20˚Cand
usedasthetemplateforsubsequentqPCRanalyses.
Real-timeRT-PCR
Quantitativereal-timeRT-PCR(qRT-PCR)wasperformedinaCFX96Real-TimeDetection
System(Bio-Rad)usingtheQuantimixEasyKit(Biotools,Spain).Genesencodingactinand
the26Sribosomalsubunitwereusedasendogenousreferencecontrolstoanalyzetherelative
transcriptionalactivityofselectedtargetgenes:EcR,ERR,E74,Vtg,hsp70,hsp40,cyp18a1,
GAPDH,cyp4g,GPx,GSTd3,its2,rpL4,andrpL13.Primersequencesandefficienciesare
showninTable1.TheqRT-PCRwasrunusingthefollowingconditions:30sinitialdenatur-
ationat95˚C,followedby35cyclesof5sdenaturationat95˚C,15sannealingat58˚Cand10
selongationat65˚C.Toverifytheaccuracyofeachamplicon,ameltingcurveanalysiswasper-
formedafteramplification.CFXManager3.1software(Bio-Rad)wasusedtodeterminetotal
mRNAlevelsbynormalizingtheexpression(2−ΔCq)ofthetargetgenesagainstthetwoendoge-
nousreferencegenes.Eachsamplewasruninduplicatewells,andthreeindependentreplicates
wereperformedforeachexperimentalcondition.
Statisticalanalysis
StatisticalanalyseswereperformedusingSPSSStatistics22software(IBM,USA).Normality
andhomoscedasticityofdatawereassessedwithShapiro-Wilk’stestandLevene’stest,respec-
tively.NormalizedlevelsoftranscriptswereanalyzedwithANOVA,followedbyGamesHow-
ell’sorBonferroni’sposthoctests,whenappropriate.TheKruskal-Wallis’testwasusedwhen
datawerenothomogeneousornormallydistributed,andthedifferencesbetweenpairswere
establishedusingMann-Whitney’stests.Aprobability(P)value(cid:20)0.05wasusedasacutofffor
statisticalsignificance((cid:3))ofdifferencesbetweentreatmentsandcontrolsamples.
Results
AfterexposureoffourthinstarC.ripariuslarvaeto0.5,5,50and500μg/LBPS,therewasno
significanteffectonsurvivalrate,neitherafter24-hourcontinuousexposuretoBPSnorin
48-hourexperiments(24-hourexposureand24hofrecoveryinfreshculturemedium).Sur-
vivalpercentagesarepresentedinTable2.
TheresultsobtainedfromanalysisoftranscriptionalactivityofEcR,ERR,E74,andVtg(Fig
1),cyp18a1,hsp70,andhsp40(Fig2),GAPDH,cyp4g,GPx,andGSTd3(Fig3),its2,rpL4,and
rpL13(Fig4)areshown.Ingeneral,24-hourexposuretoincreasingdosesofBPSledtoan
invertedU-shapedoverexpressioncurveinwhichthemaximumtranscriptionalactivity
appearedattheconcentrationof50μg/L,whilewithdrawalofthecompoundledtoageneral-
izeddownregulation24hlater.Thispeaktranscriptionat50μg/Lwasstatisticallysignificant
comparedto5μg/Land500μg/LBPS,fortheendocrineanddetoxicationgenesanalyzed.
Genesencodingnuclearreceptorsweresignificantlyoverexpressedafter24hexposureto
5μg/LBPSorgreater,withmaximumlevelsreachedat50μg/LBPS,withameanincreasein
transcriptionalactivityforEcRandERRof3.8-foldand2-fold,respectively(Fig1Aand1C).
BothgenesweresignificantlyrepressedinalmostallconditionsafterBPSwithdrawal,falling
PLOSONE|https://doi.org/10.1371/journal.pone.0193387 February21,2018 4/17
TheBPA-substitutebisphenolSinducestranscriptionaldisruptionintheaquaticmidgeChironomusriparius
Table1. Primersusedforquantitativereal-timeRT-PCR.
Gene Description Primer(5’!3’) Efficiency(%)
26S 26Sribosomalribonucleicacid (F) TTCGCGACCTCAACTCATGT 96.6
(R) CCGCATTCAAGCTGGACTTA
actin Beta-actin (F) GATGAAGATCCTCACCGAACG 96.1
(R) CGGAAACGTTCATTACCG
cyp18a1 CytochromeP45018a1 (F) GTTTCACTCGAGACGATCCA 104.5
(R) TTTAGCGGCTTGAAATGTTG
cyp4g CytochromeP450family4subfamilyG (F) GACATTGATGAGAATGATGTTGGTG 101.7
(R) TAAGTGGAACTGGTGGGTACAT
E74 Earlyecdysone-induciblegene (F) TCTTACTGAAACTTCTTCAAG 105.4
(R) GCTTTGAGACAGCTTTGGAAT
EcR Ecdysonereceptor (F) TCTTCTCACGGCCATCGTCA 103.2
(R) GCTGCATCTTGTTTCGCCAC
ERR Estrogen-relatedreceptor (F) CTCAGCAAGTAAGGAGGAG 99.4
(R) CGTCTAATAATGTGATCGG
GAPDH Glyceraldehyde3-phosphatedehydrogenase (F) GGTATTTCATTGAATGATCACTTTG 103.9
(R) TAATCCTTGGATTGCATGTACTTG
GPx Glutathioneperoxidase (F) AAGTGTGGTTACACAGCTAAGCATT 100.4
(R) GATATCCAAATTGATTACACGGAAA
GSTd3 GlutathioneS-transferaseD3 (F) TGGTTGAAACGAGAGCACCA 109.3
(R) TCGGATATAGAGTGCCAGCATCG
hsp40 40kDaheat-shockprotein (F) TACGTGACGCTAGAGGAAA 108.1
(R) TTCCAGCCCGGCTT
hsp70 70kDaheat-shockprotein (F) ACTTGAACCAGTTGAGCGT 102.8
(R) TTGCCACAGAAGAAATCTTG
its2 Internaltranscribedspacer2 (F) TCATCAAAGCCGTTGTCT 97.2
(R) AATCGAATTGCAAACACC
rpL4 RibosomalproteinL4 (F) AACGCTTCAGAGCTGGACGTGG 110.7
(R) ATTCATCTTGTGTACGCTCATTG
rpL13 RibosomalproteinL13 (F) AAGCTGCTTTCCCAAGAC 103.3
(R) TTGGCATAATTGGTCCAG
Vtg Vitellogenin (F) GATTGTTCCATGTGCAG 96.8
(R) TTTGAGTATGGTGGAGAATC
https://doi.org/10.1371/journal.pone.0193387.t001
tovalues70%lowerthancontrols(Fig1Band1D).Similarly,theexpressionprofileofE74had
apeakafterexposureto50μg/LBPS(2.4-fold)(Fig1E)andageneraldownregulationafter24
hrecovery(upto80%lessthancontrol)(Fig1F).However,inthiscasedownregulationwas
notsignificantexceptatthelowestBPSconcentrationtested.Moresubtlechangeswere
observedforVtg,whichdidnotexhibitstatisticalsignificanceinanyoftheexperimentalcon-
ditions(Fig1Gand1H).
Table2. EffectofBPSonthesurvivalrate(%)ofC.ripariuslarvae(n=80)(mean±SD).
BPSconcentration[μg/L]
Control 0.5 5 50 500
24h 100±0.00 100±0.00 96.67±2.89 100±0.00 95±5.00
24+24h 91.67±8.33 100±0.00 88.64±12.65 94.44±4.81 88.89±19.25
https://doi.org/10.1371/journal.pone.0193387.t002
PLOSONE|https://doi.org/10.1371/journal.pone.0193387 February21,2018 5/17
TheBPA-substitutebisphenolSinducestranscriptionaldisruptionintheaquaticmidgeChironomusriparius
Fig1.TranscriptionalactivityofEcR,ERR,E74,andVtg.Box-and-whiskerplotsrepresenttheexpressionpatternsofEcR(A-B),ERR
(C-D),E74(E-F),andVtg(G-H),measuredbyreal-timeRT-PCR.C.ripariusfourthinstarlarvaewereexposedto0.5–500μg/LBPSfor
24h(24h;leftcolumn;blueseries),andmaintainedforanadditional24hinfreshculturemediumafterBPSwithdrawal(24+24h;right
column;greenseries).FourindependentexperimentswereperformedandRNAwasextractedfrom5larvaeforeachexperimental
condition(n=20).Resultswerenormalizedtocontrolvalues.Boxandwhiskersrepresentthe25–75percentileandtheminimum/
PLOSONE|https://doi.org/10.1371/journal.pone.0193387 February21,2018 6/17
TheBPA-substitutebisphenolSinducestranscriptionaldisruptionintheaquaticmidgeChironomusriparius
maximummeasuredvalues;themeanisrepresentedbyadot;thehorizontallineseparatingthelower(dark)andtheupper(light)area
representsthemedian.Asterisks((cid:3))indicatesignificantdifferences(p(cid:20)0.05)withrespecttocontrolvalues.
https://doi.org/10.1371/journal.pone.0193387.g001
Fig2.Transcriptionalactivityofcyp18a1,hsp70,andhsp40.Box-and-whiskerplotsrepresenttheexpressionpatternsofcyp18a1(A-B),hsp70(C-D),
andhsp40(E-F),measuredbyreal-timeRT-PCR.C.ripariusfourthinstarlarvaewereexposedto0.5–500μg/LBPSfor24h(24h;leftcolumn;blue
series),andmaintainedforanadditional24hinfreshculturemediumafterBPSwithdrawal(24+24h;rightcolumn;greenseries).Fourindependent
experimentswereperformedandRNAwasextractedfrom5larvaeforeachexperimentalcondition(n=20).Resultswerenormalizedtocontrolvalues.
Boxandwhiskersrepresentthe25–75percentileandtheminimum/maximummeasuredvalues;themeanisrepresentedbyadot;thehorizontalline
separatingthelower(dark)andtheupper(light)arearepresentsthemedian.Asterisks((cid:3))indicatesignificantdifferences(p(cid:20)0.05)withrespectto
controlvalues.
https://doi.org/10.1371/journal.pone.0193387.g002
PLOSONE|https://doi.org/10.1371/journal.pone.0193387 February21,2018 7/17
TheBPA-substitutebisphenolSinducestranscriptionaldisruptionintheaquaticmidgeChironomusriparius
Fig3.TranscriptionalactivityofGAPDH,cyp4g,GPx,andGSTd3.Box-and-whiskerplotsrepresenttheexpressionpatternsof
GAPDH(A-B),cyp4g(C-D),GPx(E-F),andGSTd3(G-H)measuredbyreal-timeRT-PCR.C.ripariusfourthinstarlarvaewereexposed
to0.5–500μg/LBPSfor24h(24h;leftcolumn;blueseries),andmaintainedforanadditional24hinfreshculturemediumafterBPS
withdrawal(24+24h;rightcolumn;greenseries).FourindependentexperimentswereperformedandRNAwasextractedfrom5larvae
foreachexperimentalcondition(n=20).Resultswerenormalizedtocontrolvalues.Boxandwhiskersrepresentthe25–75percentile
PLOSONE|https://doi.org/10.1371/journal.pone.0193387 February21,2018 8/17
TheBPA-substitutebisphenolSinducestranscriptionaldisruptionintheaquaticmidgeChironomusriparius
andtheminimum/maximummeasuredvalues;themeanisrepresentedbyadot;thehorizontallineseparatingthelower(dark)andthe
upper(light)arearepresentsthemedian.Asterisks((cid:3))indicatesignificantdifferences(p(cid:20)0.05)withrespecttocontrolvalues.
https://doi.org/10.1371/journal.pone.0193387.g003
Fig4.Transcriptionalactivityofits2,rpL4,andrpL13.Box-and-whiskerplotsrepresenttheexpressionpatternsofits2(A-B),rpL4(C-D),andrpL13
(E-F),measuredbyreal-timeRT-PCR.C.ripariusfourthinstarlarvaewereexposedto0.5–500μg/LBPSfor24h(24h;leftcolumn;blueseries),and
maintainedforanadditional24hinfreshculturemediumafterBPSwithdrawal(24+24h;rightcolumn;greenseries).Fourindependentexperiments
wereperformedandRNAwasextractedfrom5larvaeforeachexperimentalcondition(n=20).Resultswerenormalizedtocontrolvalues.Boxand
whiskersrepresentthe25–75percentileandtheminimum/maximummeasuredvalues;themeanisrepresentedbyadot;thehorizontallineseparating
thelower(dark)andtheupper(light)arearepresentsthemedian.Asterisks((cid:3))indicatesignificantdifferences(p(cid:20)0.05)withrespecttocontrolvalues.
https://doi.org/10.1371/journal.pone.0193387.g004
PLOSONE|https://doi.org/10.1371/journal.pone.0193387 February21,2018 9/17
TheBPA-substitutebisphenolSinducestranscriptionaldisruptionintheaquaticmidgeChironomusriparius
Transcriptionalactivityofcyp18a1wassignificantlyupregulatedfrom0.5μg/LBPSand
reacheditsmaximum(2.5-foldincrease)at50μg/LBPS(Fig2A).WithdrawalofBPSledtoa
generalreductionofactivity(50%lessthancontrol),whichwasstatisticallysignificantatthe
highestdose(500μg/L)(Fig2B).Thetwogenesencodingheatshockproteinshadincreased
expressionlevelsinthepresenceofBPS,althoughtheyshowedadifferentialresponse.While
hsp40showedsignificantdifferencesabove5μg/LBPS(Fig2E),similartoothergenesinthis
study,hsp70showedanegative,lineardose-response,withsignificantdifferencesonlyatthe
twolowestdoses(Fig2C).BothgenesweredownregulatedafterBPSwithdrawal(upto60%
less)(Fig2Dand2E),withhsp40significantlylowerthanthecontrolatallconcentrations(Fig
2E).
GAPDHremainedunalteredunderalltheexperimentalconditions(Fig3Aand3B).How-
ever,genesinvolvedinbiotransformationactivities(cyp4g,GPx,andGST)showedsimilar
responsestothoseobservedforEcR,ERR,cyp18a1,andhsp40,withsignificantoverexpression
after24hexposureto5μg/LBPSandamaximumvalueat50μg/L(6.4-fold,1.8-fold,and
2.1-fold,respectively)(Fig3C,3Eand3G).AllthesegeneswerealsorepressedafterBPSwith-
drawal,althoughourresultsdidnothavesignificantdifferences(Fig3D,3Fand3H).
Ribosomalgenesremainedfairlystable,althoughsomeeffectswereobserved.Thelowest
BPSdose(0.5μg/L)causedadropintranscription,significantforits2andrpL4(about25%
belowcontrols)(Fig4Aand4C).Onthecontrary,andsimilartothatobservedforothergenes
(althoughwithoutstatisticalsignificance),maximumtranscriptionwasobservedafterexpo-
sureto50μg/LBPS(Fig4A,4Cand4E).AfterrecoveryfollowingBPSremoval,bothits2and
rpL4wererepressed(Fig4Band4D),whilerpL13showedariseinitstranscriptionrate(upto
2-fold)thatwasnotsignificantduetodatavariability(Fig4F).
Discussion
ManyworksinrecentyearshaverevealedthedangerousnatureofBPA,notonlyregarding
hormonalalterationanditsimplicationinreproductionanddevelopment,butalsoother
healthdisorderssuchasbreastcancer,obesity,type-2diabetes,oxidativestress,neurodevelop-
ment,asthma,andcardiovasculardiseases[1].Withthisevidence,andthankstodifferent
socialandlegalpressures,industrieshavegraduallyreplacedBPAwithalternativesincluding
structuralanalogssuchasbisphenolsAF,AP,B,E,F,P,S,orZ.Thisreplacementhasbeencar-
riedoutwithoutstrictadherencetothe“precautionaryprinciple”,whichshouldbeusedwhen
preliminaryscientificevidenceindicatesthattherearereasonableconcernsaboutpotentially
dangerouseffectsontheenvironmentorhuman,animalorplanthealth[35].Thus,asmore
scientificstudiesshowadverseeffectsofthesechemicalalternatives[8,13,36],itmaybejustas
riskytobuyaproductlabeled"BPA-free"asoptingforthosecontainingBPA.
Theobjectiveofthepresentworkwastoshedlightonthepossibleadverseenvironmental
effectsofBPS,aBPAanaloguecommonlyusedin"BPA-free"products.Ourexperimentswere
carriedoutwithaquaticlarvaeofC.ripariusmidges,averyrelevantspeciesinaquaticecosys-
temsusedinstudiesonecotoxicityofwaterandsediments,andespeciallysuitableforrisk
assessmentsofpotentialendocrine-disruptingpropertiesofchemicals[37,38].Forthispur-
pose,weselectedBPSconcentrationsrangingfrom0.5to500μg/L,wherethelowestdosesare
ofthesameorderofmagnitudeasthosedetectedinnaturalenvironments,wastewater,and
theurineofexposedpopulations.Somemeanmaximumconcentrationsreportedinaquatic
environmentsare0.16and1.6μg/LforTaihuLake(China)[39,40],and7.2μg/LforAdyar
River(India)[41].Sunetal.[10]reportedvaluesrangingfrom0.183–0.746μg/L(influent)ina
wastewatertreatmentplantinChina.UrinesamplesfromJapan,USA,andChinacontained
0.933,0.304,and0.223μg/mlBPS,respectively[42].BPSispersistentinsedimentsandmore
PLOSONE|https://doi.org/10.1371/journal.pone.0193387 February21,2018 10/17