Nonylphenol
effects
on
human
prostate
non
tumorigenic
cells
Maurizio
Forte
a,b,
Mariana
Di
Lorenzo
a,
Albino
Carrizzo
c,
Salvatore
Valiante
a,
Carmine
Vecchione
c,d,
Vincenza
Laforgia
a,b,
Maria
De
Falco
a,b,*
aDepartmentofBiology,UniversityofNaples,“FedericoII”,Naples,Italy
bNationalLaboratoryonEndocrineDisruptorsofInteruniversityConsortiumINBB,InstituteofGeneticandBiophysics(IGB)“ABT”,CNR,Naples,Italy c
IRCCSNeuromed,DepartmentofAngio-Cardio-Neurology,Pozzilli(Isernia)86077,Italy
d
DepartmentofMedicineandSurgery,UniversityofSalerno,Baronissi84081,Italy
ARTICLE INFO
Articlehistory: Received31March2016
Receivedinrevisedform23May2016 Accepted30May2016
Availableonline1June2016
Keywords:
Endocrinedisruptingchemicals(EDCs) Xenoestrogens
Estrogens Nonylphenol Prostate
ABSTRACT
Nonylphenol(NP)isanindustrialchemicalwithestrogenicactivitybothinvivoandinvitro;estrogens
playacriticalroleinthedevelopmentofprostateandmaybethecauseofsomepathologicalstates,
including cancer.Inthis studyweexaminedtheeffectsofNPonhumanprostatenontumorigenic
epithelialcells(PNT1A)investigatingoncellproliferation,interactionwithestrogenreceptors(ERs)and
geneexpressionofgenesinvolvedinprostatediseases.WefoundthatNPaffectscellproliferationat
106M,promotingacytoplasm-nucleustranslocationofERaandnotERb,likethenaturalestrogen17b
-estradiol(E2).Moreover,weshowedthatNPenhancesgeneexpressionofkeyregulatorsofcellcycle.
EstrogenselectiveantagonistICI182780inpartrevertedtheobservedeffectsofNP.Theseresultsconfirm
theestrogenicactivityofNPandsuggestthatothertransductionpathwaysmaybeinvolvedinNPaction
onprostate.
ã2016ElsevierIrelandLtd.Allrightsreserved.
1.Introduction
Nonylphenol (NP) is generated by the degradation of non-ylphenolethoxilates(NPEs).NPEsarechemicalswidelyusedas nonionicsurfactantsinthemanufactureofrubberandplasticfor domestic,industrialandagriculturalproducts(Fiegeetal.,2000; Langford, 2002; Vazquez-Duhalt et al., 2005). Due to its high hydrophobicity and low solubility NP accumulates in several environmentalmatrices,suchasseas,rivers,soils,groundwaters and sediments,in a range between1013to 106M(Berryman etal.,2004;Careghinietal.,2015;Vazquez-Duhaltetal.,2005).It was also found as a contaminant of food and drinking water (Gyllenhammaretal.,2012;Maggionietal.,2013;Soaresetal., 2008).HumanexposuretoNPmayoccurbyinhalation,cutaneous absorptionandingestionofcontaminatedfoodorwater(Guenther etal.,2002;Sotoetal., 1991).Inthisregard,NPwasfoundinhuman amnioticfluid,urineandplasmasamples,breastmilk,fetalcord serum,placentaandmaternalblood,withlevelsinthesetissues generallyvaryingfrom1010to109M(Calafatetal.,2005;Huang etal.,2014).Hovewer,insomecases,concentrationsofNPhave
beenreportedtobemuchhigherinhumansamples.Inthisregards inbreastmilkofhealthyItalianwomen,Ademolloetal.(Ademollo etal.,2008)detectedabout107MofNPaswellasinurineandin plasmaoftextileandhousekeepingworkerswerefoundthesame NPlevels(Chenetal.,2005).Instead,inmaternalcordblood,Chen et al. (Chen et al.,2008)founda concentrationof NPof about 106M.
NPbelongstothesubclassofendocrinedisruptingchemicals (EDCs)thatmimictheendogenousestrogens,called xenoestro-gens (Falconer et al., 2006; Wozniak et al., 2005), that also includesdioxins,polychlorinatedbiphenyls, hexachlorocyclohex-ane,octylphenolandbisphenolA(Kuoetal., 2012;Forteetal., 2016).EstrogenicactivityofNPhasbeenreportedbothinvitro(de Weertetal.,2008;Sotoetal.,1991;Whiteetal.,1994)andinvivo, in reproductive and in non reproductive tissues,such as brain (Blometal.,1998;Lawsetal.,2000;Nageletal.,1999;terVeld etal.,2008;Xiaetal.,2008;DeFalcoetal.,2014,2015)andithas been shown that NP interacts with estrogen receptors (ERs), competing withthe natural estrogen 17
b
-estradiol (E2) (Bechi etal.,2006;Kwacketal.,2002;Whiteetal.,1994),althoughwith lessspecificity(Bechietal.,2010;Blometal.,1998;Nageletal., 1999).Estrogens predominantly bind two nuclear receptors: the estrogen receptor alpha (ER
a
) and the estrogen receptor beta* Corresponding author at: Department of Biology, University of Naples, “FedericoII”ViaMezzocannone8,Naples,80134,Italy.
E-mailaddress:madefalco@unina.it(M.DeFalco).
http://dx.doi.org/10.1016/j.tox.2016.05.024
0300-483X/ã2016ElsevierIrelandLtd.Allrightsreserved.
ContentslistsavailableatScienceDirect
Toxicology
2000);thenon-genomicpathwayisnotmediatedbyER
a
orERb
butthroughtheG-proteincoupledreceptor,GPR30,thatlocalizes in the plasma membrane activating rapid responses such as increasedlevelsof c-AMP(Filardoet al.,2007;Lin etal., 2009; Wangetal.,2010).Severalstudieshavesuggestedtheroleofestrogensinnormal and aberrant growth of prostate, alone or in synergy with androgens(Hoetal.,2011)andepidemiologicalandexperimental studiesunderlinearelationshipbetweenestrogens/xenoestrogens andpathogenesisofprostatecancer(PCa)(Bostwicketal.,2004; Hoetal.,2006).NeonataltreatmentwithBisphenolA(BPA),a well-knownxenoestrogen,wasreportedtoinducehigh-gradeprostatic intraepithelialneoplasiainSprague-Dawleyrats(Hoetal.,2006) andtoincreasecell proliferationofurogenitalsinusepithelium (UGE)intheprimaryprostaticductsofCD1mice(Timmsetal., 2005). BPA was also found to increase the number of basal epithelialcellsintheadultprostateofBALB/cmice(Oguraetal., 2007).Recently,Tarapore etal. (2014)foundinprostate cancer patientshighBPAurinarylevelscomparedtononprostatecancer patients.
Despitetherelationshipbetweenestrogenandprostate,the precisefunctions of thetwo ER subtypesin thisgland remain unclear; several authors have reported differential expression patternsofthetworeceptorsbetweentheepithelialandstromal compartmentoftheprostate,withER
a
localizedpredominantly inthestromaandERb
intheepithelium(Fixemeretal.,2003; Leavet al., 2001; Tsurusaki et al., 2003; Weihua andWarner, 2002).Consideredthisbackgroundandgiventhehumanexposureto EDCs,theestrogen-likeactionofNPisconceivabletoinfluencethe normalgrowthofprostateandtobethecauseofsomepathological states of this gland, affecting the male reproductive functions. Thus, in this study we evaluated the effects of NP on the proliferationofhumannon tumorigenicprostate cells (PNT1A), whichisresponsivetosexhormones(Stephen etal.,2004),the cellularlocalizationofER
a
andERb
afterexposuretoNPandgene expressionofgenesinvolvedinpathologicalstatesoftheprostate suchas cyclinD, Ki67,p53 and IL1-b
. We performedthe same experimentstreatingcellswiththenaturalestrogenE2andwith theselectiveantagonistofestrogenreceptorsICI182,780(Osborne etal.,2004).Thisstudyaimstofacilitatetheunderstandingofthe mechanismsby which xenoestrogens and estrogens may exert theiractivityonprostate.2.Materialandmethods
2.1.Cellculture
PNT1A cells (a human prostate cell line established by immortalizationofadultprostateepithelialcells)wereobtained fromtheEuropeanCollectionofCellCulture(ECACCSalisbury,UK). PNT1AcellsweregrowninredphenolfreeRPMI-1640medium (LONZA, Basel, Switzerland), supplemented with 10% dextran-coatedcharcoal fetal bovineserum (FBS)(GIBCO,Grand Island, NY), 2mM L-glutamine and antibiotics (100U/mL penicillin/
streptomycin,10
m
g/mLgentamicin)inahumidifiedincubatorat 37C and 5% CO2. When confluent, the cells were detached(Sigma Aldrich, St. Louis, MO) and were dissolved in DMSO (InvitrogenCarlsbad,CA).NP,E2andICIweredilutedwithculture mediumatfinalconcentrationsfrom1012to106MforNPandE2 and105MforICI.Inalltheexperimentswiththeinhibitor,ICIwas added1hpriortostarttreatments.FinalconcentrationofDMSOin themediumdidnotexceed0.01%.
2.3.MTTassay
The effects of NP or E2 on PNT1A cells proliferation was evaluated using the 3-[4,5-dimethylthiazol-2-yl]-3,5 diphenyl tetrazoliumbromide(MTT) test(SigmaAldrich,St. Louis,MO). Cells were seeded in 200
m
L of growth medium(5104 cells/well)in96-wellplatesandhormonedeprived(1%FBS)for24h. Then, NP or E2 was added after dilution to an appropriate concentration(from1012Mto106M),withorwithout105M ICI.Controlcellsweretreatedwithvehicle(DMSO0.01%).Thetest wasperformedfor24hofincubation.Aftertheincubationperiod, 10
m
LofaMTTsolutionwasaddedtoeachwell.After4hof37C incubation, the culture medium was gently aspirated and replaced by 100m
L of DMSO/isopropanol (1:1) in order to dissolvetheformazancrystals.Theabsorbanceofthesolubilized dye, which correlates with the number of living cells, was measured with a microplate reader at 570nm. The test was performedintriplicate.2.4.Fluorescencemicroscopy
PNT1Acellswere seededin 4-well chamberslide (Sarstedt, Nürnbrecht,Germany)overnightatadensityof5104/well.After
24hserumstarvation(1%FBS),cellswereincubatedwith106M NP or106ME2, withorwithout 105MICI for fourdifferent times:15min,1h, 2hand 6h.Controlgroupwastreatedonly withvehicle(DMSO0.01%).Controlandtreatedcellswerefixed withmethanolfor10minatRT,permeabilizedwith0.25%Triton X-100for10min,washedinPBS,andblockedin5%normalgoat serum (NGS) for 1h at RT. Then cells were subjected to immunofluorescence protocol using a mouse monoclonal anti-humanER
a
(SantaCruzBiotechnology,SantaCruz,CA,Cat. sc-8005) and a mouse monoclonal anti- human ERb
antibodies (SantaCruz,Cat.sc-373853),diluted1:100in1%NGSfor24hat 4C. FordetectionofERa
andERb
,secondarygoatanti-mouseAlex Fluor488 (Cat.A11001, Invitrogen, Carlsbad,CA), dilution 1:200in1%NGSwasused.Cellnucleiwerecounterstainedwith 0.1
m
g/mL Höechst (Invitrogen, Carlsbad, CA, Cat. H3570). Negativecontrol for ERa
and ERb
wasperformed byavoiding incubation with the primary antibodies (Supplementary data Fig. S1). Fluorescent images were taken on an Axioskop (Carl Zeiss, Milano, Italy) epifluorescence microscope using a 40 objective.AxiocamMRc5andtheacquisitionsoftwareAxiovision 4.7 (Carl Zeiss) were used to capture the images in different channels (AlexaFluor 488,Höechst33258).Threeindependent immunofluorescence experiments were performed for each experimental conditions and different fields were randomly chosenfordataanalysis.Then,imageswereprocessedwiththe Image J software (developed by Wayne Rasband, National InstitutesofHealth,USA).2.5.Proteinextractionandseparation
Nuclear and cytoplasmatic proteins were extracted from PNT1Acellsafter two andsix hoursof treatment with106M NP,106ME2, with or without 105MICI. Control cells were treatedwith0.01%ofDMSOandwesternblotwasperformedfor detectionof ER
a
and ERb
.Different buffers were prepared to isolate cytoplasmic/membrane and nuclear proteins: harvest buffer(10mMHEPESpH7.9,50mMNaCl,0.5Msucurose,0.1mM EDTA, 0.5% Triton X-100 and freshlyadded 1mM DTT,10mM tetrasodiumpyrophosphate,100mMNaF,1mMPMSF,4mg/mL Aprotininand2mg/mLPepstatinA),bufferA(10mMHEPESpH 7–9,10mMKCl,0.1mMEDTA, 0.1mM EGTA andfreshlyadded 1mMDTT,1mMPMSF,4mg/mLAprotininand2mg/mLPepstatin A)andbufferB(10mMHEPESpH7.9,500mMNaCl,0.1mMEDTA, 0.1mM EGTA 0.1% NP-40 and freshly added 1mM DTT, 1mM PMSF,4mg/mLAprotininand2mg/mLPepstatinA).10cm cell disheswithconfluentPNT1Acellswereplacedonicefor10min andwashed twicewithice coldPBS.Then100m
L ofPBS-EDTA 1mM was added and cells were scraped and transferredto a microcetrifugetube.Thecollectedcellswerethencentrifugedat 3000rpmfor5minat4Candthenresuspendedincoldharvestbuffer,incubatedonicefor5minandsubsequentlycentrifugedat 1000rpmfor10mintopelletnuclei.Thenthesupernatantwas transferredinto a newtube and centrifugedat 14000rpm for 15min,inordertoclearthesupernatant.Thislattercontainsthe cytoplasmic and membrane proteins. Nuclear pellet was then resuspendedin bufferA,centrifugedat1000rpm,the superna-tantwasdiscarded. Then4volofbufferBwereaddedandthe tubeswerevortexfor 15minat4Ctoloosenpellet. Finally,a centrifugationfor10minat14,000rpmat4Cwasperformedand thesupernatantthatcontainnuclearextractwastransferredinto anewtube.ProteinconcentrationwasdeterminedwithBradford assay(Biorad).
2.6.Westernblot
50
m
gofproteinextractsforeachsamplewasseparatedby10% SDS-PAGE and transferred onto a nitrocellulose membrane. Membraneswereblockedwith5%milkinTBS-Tweenfor 2hat roomtemperatureandthenincubatedwithprimaryantibodiesin TBS-Tweenand5%milkovernight.Blockedmembraneswerethen incubatedwithanti-ERa
(1:500) oranti-ERb
(1:500) and with mouseanti-humanb
-tubulin(1:2000)(SantaCruz,Cat.sc-5274) ormouseanti-humanHDAC2(1:2000)(SantaCruz,Cat.sc-55542) overnight and then detected using appropriate horseradish peroxidase-coupled secondary antibody (Santa Cru, Cat. sc-2005)andvisualizedwithenhancedchemiluminescence (Amer-sham,ThermoFisherScientific,Milano,Italy).Thepurityofnuclear andcytoplasmicfractionswasconfirmedusinganti-HDAC-2and anti-b
-tubulin, respectively. All antibodies have been used to probe the same experimental membrane. In detail, before incubationwithanotherprimaryantibody,themembraneshave beenstrippedwiththestrippingsolution:100mM 2-Mercaptoe-thanol,1%SDS,62.5mMTris-HClpH6.7andincubatedat50Cfor 30minwithagitationand,subsequently,themembranehavebeen re-equilibratedinTBSandthenblockedwith5%milkinTBS-Tween for2hatroomtemperature.Therenderingofstrippinghasbeen tested by evaluating the ECL- signal after treatment with the strippingsolution.Onlywhenthesignalofthepreviousantibody wasabsent,themembranewasincubatedwithanewantibody. Immunoblotting data were analyzed using ImageJ software to determineopticaldensity(OD)ofthebands.TheODreadingwas normalized on anti-b
-tubulin and anti-HDAC2 to account for variationsinloading.Foreachtimeoftreatment(i.e.2h,6h,2hwith ICI) were analyzed data of three independent western blotting.
2.7.RNAextractionandRT-qPCR
mRNAexpressionlevelsofestrogentargetgeneswereanalyzed using real-time PCR. Total RNA from PNT1A control cells and treated for24hwith106ME2 or 106MNP,withor without 105MICIwasextractedusingTrizol(LifeTechnologies,Carlsbad, CA).AfterpurificationfromgenomicDNAwithTURBODNA-freeTM
Kit (Ambion, Life Technologies), the total amount of RNAwas quantified with a NanoDrop spectrophotometer. cDNAs were synthesizedfrom1
m
gRNAusingtheHighCapacitycDNAReverse Transcriptase(LifeTechnologies)andquantitativePCR(RT-qPCR) was performed by using the 7500 Real-Time PCR System and SYBR1SelectMasterMix2Xassay(AppliedBiosystem,FosterCity, CA,USA).Allprimersused(Table1)weredesignedaccordingtothe sequencespublishedonGenBankusingPrimerExpresssoftware version3.0andprimerefficienciesweretestedpriortoperform qPCR.TheamountoftargetcDNAwascalculatedbycomparative threshold (Ct) method and expressed by means of the 2DDCtmethod(LivakandSchmittgen,2001)usingtheglyceraldehyde 3-phosphatedehydrogenase(GAPDH)ashousekeepinggene,which expression was not affected by the treatment. Three separate experiments(n=3)wereperformedforRT-qPCRandeachsample wastestedintriplicate.
2.8.Statisticalanalysis
DatareportedingraphsareexpressedasmeanvaluesSEMfor the indicated number of independent determinations. The statistical significance was calculated by the one way ANOVA withBonferroni’smultiplecomparisontest,anddifferenceswere consideredstatisticallysignificantwhenthePvaluewasatleast <0.05.
3.Results
3.1.Cellproliferationassay
To determineifNPaffects cellularproliferation, PNT1Acells weretreatedwithincreasingconcentrationofNP(from1012Mto 106M)for24hofexposure;toassessanysimilarities,treatment wasperformedalsoexposingcellswithE2;thesameexperiments were alsocarried out inpresence of 105M ICI. NPstimulated PNT1Acells proliferation at thehighest concentrationwe used (106M)(Fig.1a).Atlowerconcentrations,wedidnotobserveany significant effects when compared to controlgroup.105M ICI inhibited the proliferation induced by 106M NP. Similarly, treatment with E2 stimulated PNT1A cells proliferation from 109M to 106M, with the greatest effect showed at 106M (Fig.1b).E2inducedproliferationisstronglyinhibitedbyadding ICI.Fig.1cshowsasE2hasagreatereffectcomparedtoNPon PNT1Acellsproliferation.
Table1
PrimersusedinqPCR.
Gene 50-Forward-30 50-Reverse-30
CyclinD CGTGGCCTCTAAGATGAAGGA CGGTGTAGATGCACAGCTTCTC CyclinE GATGACCGGGTTTACCCAAA CCTCTGGATGGTGCAATAATCC Ki67 CCCGTGGGAGACGTGGTA TTCCCGTGACGCTTCCA p53 TCTGTCCCTTCCCAGAAAACC CAAGAAGCCCAGACGGAAAC IL1-b ACGATGCACCTGTACGATCACT CACCAAGCTTTTTTGCTGTGAGT GAPDH CAAGGCTGTGGGCAAGGT GGAAGGCCATGCCAGTGA
3.2.Fluorescencemicroscopy
3.2.1.LocalizationofER
a
We investigatedonestrogen receptorsER
a
localizationafter 106M NP and 106M E2 treatment, in order to evaluate the interaction between NP and ERs. After 15min and 1h ERa
remained localized in the cytoplasm after both NP and E2 treatments(datanotshown).After2hoftreatment, NPdidnot affectERa
cellularlocalization,thatwaslocalizedinthecytoplasm, as in control. On the contrary,in E2 treated cells for 2h, ERa
localizedpredominantlyinthenucleus(Fig.2).After6hofexposure,bothinPNT1AcellstreatedwithNPand E2,ER
a
shiftedfromthecytoplasmtothenucleusthatappearedto be strongly positive, with a weak cytoplasmic fluorescence if comparedtocontrol(Fig.2).In PNT1A cells pre-treated with ICI ER
a
was found in the cytoplasmbothafter2and6hoftreatmentwithnucleicompletely negatives(Fig.3).3.2.2.LocalizationofER
b
ER
b
localizationaftertreatmentwith106MNPand106ME2 wasalsoinvestigated;dataafter15minand1h(datanotshown)as wellasafter2hand6hofexposuredidnotrevealanydifferences between control and exposed cells. ERb
was localized in the cytoplasmofPNT1Acells andcellnucleiappearedwithaweak signal(Fig.4).3.3.Westernblotanalysis
After separation of cytoplasmic and nuclear proteins we performedatranslocationstudyofER
a
andERb
withawestern blotanalysis,inordertoconfirmmicroscopyresultsafter106M NPandE2exposure.Densitometricanalyseswerenormalizedfor cytoplasmicand nuclear extracts withb
-tubulin(55Kda) and HDAC2(55KDa),respectively. After2hof exposure(Fig. 5)we foundERa
protein(molecularweight66KDa)inthecytoplasmof NPtreatedandnontreatedPNT1Acellswhereasopticaldensity values were significantly lower in E2 treated cells (Fig. 5a,b). Moreover,after2hnuclearproteinsrevealedasignalonlyinE2 treatedcells (Fig.5a).ERb
(56KDa) after2h of treatmentwas foundonlyincytoplasmicfractions(Fig.5a,c).NosignalforHDAC2 andb
-tubulininthecytoplasmandnucleusproteins,respectively, suggestthatproteinseparationwasperformedcorrectly.After6hofexposure(Fig.6)weobservedanuclear transloca-tionofER
a
bothinNPandE2treatedcells(Fig.6a,b).However, densitometry did not reveal significant differences in nuclear extractsbetweenNPand E2 treated cells (Fig. 6b).In contrast, valuesweresignificantlylowerinthecytoplasmintreatedcells comparedtocontrol(Fig.6b).ERb
wasfoundonlyincytoplasmaticfractions(Fig.6a,c)andtherewerenotsignificantdifferencesin opticdensitybetweentreatedandnontreatedcells(Fig.6c).
Western blot for ER
a
localization performedin presence of 105MICI aftertwo (Fig. 7a,b) and 6hof treatment(Fig.7c,d) revealedERa
exclusivelyincytoplasmproteins,withaweaksignal in the nuclear extracts after 6h of exposure (Fig. 7c). No statisticallysignificant differenceswereshowed comparingnon treatedandtreatedcells(Fig.7b,d).3.4.RT-qPCRanalysis
InordertoinvestigateifNPisabletoaffectgeneexpression, RT-qPCR analysis ofgenes involved in cellcycle regulationand in pathological states of prostate were investigated after 24h of exposure.NPenhancedmRNAlevelsofCyclinD(Fig.8a),CyclinE (Fig.8b),Ki67(Fig.8c)andIL1-
b
(Fig.8e)whileitdidnotaffectp53 expression(Fig.8d).Interestingly,ICIreducedgeneexpressionof CyclinD(Fig.8a)andKi67(Fig.8c)inPNT1AcellstreatedwithNP, whileitdidnotinhibitgeneexpressionofCyclinE(Fig.8b)orIL1-b
(Fig.8e).E2significantlyaffectedgeneexpressionup-regulating CyclinD(Fig.8a)andKi67(Fig.8c). Thisinductionwasstrongly inhibitedbyICI(Fig.8a,c).4.Discussion
EDCs are receiving more and more attention by scientific community,duetotheirabilitytomimicendogenoushormones andalteringthemetabolismoforganisms(DeFalcoetal.,2014).NP belongstothefamilyofxenoestrogensanditsestrogenicactivityis welldocumentedbothwithinvitro(deWeertetal.,2008;Soto etal., 1991;Whiteetal., 1994)andinvivostudies(Lawsetal.,2000; terVeldetal.,2008).Inthisworkweseektoinvestigatetheeffects of NP on humanprostate cells PNT1A, precisely evaluating its estrogenicactionintermsofproliferation,interactionwithERsand gene analysis of genes involved in cell cycle regulation and aberrant physiology of prostate. Few studies investigated the effectsofNPonprostate,bothoncellularandanimalmodels.These findings did not characterize any molecular mechanisms and resultsoftenappeartobeinconflict.Forexample,Leeetal.showed thatNPisabletoreducetheweightoftheprostateinrats,inadose dependent manner (Lee, 1998). In the same study, authors demonstratedthat ICI revertedthis effect.Similarly, Whoet al. obtainedthesameresultbutwithhigherconcentrationofNP(Woo etal.,2007).Moreover,gestationalexposuretoNPwasreportedto affectprostatemorphologyinF1rats(Jieetal.,2010).Incontrast, other authorsfailed to demonstrateany adverse effects on rat prostatecaused byNP(Inagumaet al.,2004;Odum andAshby, 2000).Thesecontrastingdatamaybeexplainedbythetimeand thewayofNPdosage,aswellasbythedurationoftreatment.We conductedourexperimentsalsotestingtheeffectsofE2andICI.
Fig.1.MTTassayafter24hofexposuretononylphenol(NP)and17b-estradiol(E2)aloneorincombinationwithICI182,780(+I).NPstimulatesPNT1Acellsproliferationat 106M(a)whileE2at109M,108M,107Mand106M(b).105MICI(I)revertsthiseffect.E2showsthegreatesteffectonproliferationifcomparedtononylphenol(c).In
graphisreportedtheabsorbancemeasuredat570nmwhichcorrelateswiththenumberoflivingcells.a,responsesignificantlydifferentthanthevehiclecontrol(P<0.05);a’, responsesignificantlydifferentthanthevehiclecontrol(P<0.01);b,responsesignificantlydifferentthancellswithoutICI(P<0.05).
Fig.2. LocalizationofERaafter2and6hofexposuretoNPandE2.E2promotestranslocationofERatothenucleusatbothtimeoftreatment,whileNPat6h.PNT1Acells wereplatedinchamberslideunderhormonedeprivedconditions.24hlater,cellsweretreatedwith106MNPor106ME2.ERa(AlexaFluor488)andnuclearstaining
WeshowedthatNPstimulatedPNT1Acellsproliferationafter24h of exposure at 106Mas well asE2 did. However,E2 affected PNT1Acells proliferationalsoat lowerconcentrations. Interest-ingly,ICI revertedNPandE2 proliferativestimuli. Theseresults suggestthatNPmayinterferewithnormalcellcycleofPNT1Acells asreportedbyotherauthorsindifferentcelllines(Choietal.,2011; Manenteet al.,2011).Recently, Ganet al.(Ganet al., 2015)in humanprostateepithelialcelllineRPWE-1showedareductionin cellviabilityafter24hexposuretoNP.Theincongruitybetween ourresultsandthoseofGanetal.canbeexplainedbythedifferent concentrationsused.Inthisstudy,noeffectswerereportedat106
NPandthedecreaseincellproliferationappearedtobeevident onlyathighconcentration(105–104M).
Withtwodifferentapproaches,westudiedthelocalizationof ER
a
andERb
inatimecourseanalysistestingtheconcentrations (106M)thatshowedthegreatesteffectoncellproliferation.We demonstratedthatE2inducedcytoplasm-nucleustranslocationof ERa
atbothtimetested,whileNPonlyaftersixhourofexposure. Surprisingly,bothE2andNPdidnotaffectERb
localization.ICI inhibitedtheERa
translocationobservedwithNPandE2alone.Bothproliferationandlocalizationdataconfirmtheestrogenic activityofNP.However,thegreatestbiologicalresponsesshowed
Fig.3. LocalizationofERaafter2and6hofexposuretoNPandE2combinedwithICI(+I).ICIinhibitsERanucleustranslocationatbothtimeconsidered.Cellswere pre-treatedfor1hwith105MICIandthentreatedwith106MNPor106ME2.ERa(AlexaFluor488)andnuclearstaining(Höechst)wereanalyzedbyimmunofluorescence. Scalebar10mm.
Fig.4. LocalizationofERbafter2and6hofexposuretoNPandE2.InalltheimagesERbislocalizedincellularcytoplasm.PNT1Acellswereplatedinchamberslideunder hormonedeprivedconditions.24hlater,cellsweretreatedwith106MNPor106ME2.ERb(AlexaFluor488)andnuclearstaining(Höechst)wereanalyzedby immunofluorescence.Scalebar10mm.
by E2 can be explained by its best binding affinity with ERs compared to NP (Laws, 2000). Notwithstanding the lowest responsestoNP,we canspeculatethat itsproliferativeeffects, asforE2,ismediatedbytheinteractionwithER
a
.Onthisissue,it iswellknowntheroleofERa
incellularproliferationprocessand carcinogenesisof prostate, whilesome authors suggested that ERb
seemstobeinvolvedinapoptosisofprostatecells(Hartman etal.,2012).Forexample,ithasbeenreportedinknockoutERb
mice prostatic hyperplasia and cancer (Weihua et al., 2001). Moreover,inmiceandratsprostate,likeinhuman,it hasbeen shownthatligandsthatinteractwithERb
mayreduce prolifera-tion(EllemandRisbridger,2009;Omotoetal.,2005;Prinsand Korach,2008).To assess if the presence of ER
a
in the nucleus led tothe activationoftranscription,weanalyzedgeneexpressionofE2gene targetsalsoknowntobederegulatedinpathologicalstateofthe prostate.WedemonstratedthatNPwasabletoupregulateCyclinD, Cyclin E, Ki67 and IL1-b
gene expression whereas E2 induced upregulationonlyofCyclinDandKi-67.Moreover,weshowedthat up-regulation of Cyclin D and Ki67 is mediated by estrogen signaling pathways, while the induction of Cyclin E and IL1-b
involved an estrogen independent pathways, since ICI did not revert this induction. These results of gene expression are in agreementwiththeshowedinducedproliferationcausedbyNP andE2.Inthisregard,itiswellknownthatCyclinDandCyclinE promotingG1/Sphasetransitionofcellcycle(KastanandBartek, 2004)andareoftenusedtoscreeningthecarcinogenicpotentialof EDCs(Diamanti-Kandarakis etal., 2009).Moreover,it hasbeen reportedthatoverexpressionofCyclinD,Ki67,CyclinEandIL1b
are aprognosticfactorsprostatecancer(Aaltomaaet al.,2006;Deyet al., 2013; Sfanos and De Marzo, 2012).In particular, down-regulationofCyclinDandCyclinEhasbeenshowntoinhibittumor progressionindifferentprostatecelllines(Alagbalaetal.,2006; Chinnietal., 2001;Lin etal., 2015).In addition,thenull effect showedforp53,thehallmarkofapoptosis,reinforcetheideathat NPhasaroleinpromotingprostatecellssurvival(Ganetal.,2011; Gumulecetal.,2014).
Thisdata,togetherwiththeless estrogenicactivity ofNPin termsofproliferationandinteractionwithER
a
stronglysuggest thatNPmayactivatealsoothertransductionpathways,suchasthe G-proteincoupledestrogenreceptorGPR30(Filardoetal.,2007)or theandrogenreceptor(ARs)(Wangetal.,2010).Inthisregard,ina recent study, Kim et al. showed that NP induced human tumorigenic prostate cells (LNCaP) proliferation in a pathway mediatedby ARs(Kimet al.,2016).InadditiontoARs mediate pathways,in epithelialnontumorigenic cellsDU145,Ganet al. (Ganetal.,2014)provided evidencesabouttheinvolvementof GPR30inNPinduced proliferation,whenusedatconcentration from108to106M.Interestingly,accordingwithourdataand despite thedifferent prostate model used, in both studies, the concentrationthatshowedthebesteffectswas106Minboththe studies.ConsideringtheexposurelevelofNP,106Mrepresentsanhigh doseofNP,foundrarelyinhumansamples,suchasinbreastmilk andinumbilicalcordblood(Chenetal.,2005).Furthermore,itis farfromthetolerabledailyintakelimitvaluesforNP(Wooetal., 2007). Despite these considerations, it should not be under-estimatedtheadverseeffectsthatNPmayexertwhencombined with circulating estrogens or with other EDCs which we are simultaneouslyexposed.Thisphenomena,commonly knownas
Fig.5.WesternblotanalysisandnuclearandcytoplasmicquantificationofERaandERbafter2hofexposureto106MNPandE2.E2inducesnucleustranslocationofERa whileERbwasfoundonlyinthecytoplasmaticproteins(a).ThegraphsrepresentedtheOpticaldensity(OD)ratioofERaandERbnormalizedtotheODofTubulinbfor cytoplasmaticproteinsandtotheODofHDAC2fornuclearproteins(b).(N=3separateexperiments)a,responsesignificantlydifferentthanthecytoplasmaticcontrol (P<0.05);b’,responsesignificantlydifferentthanthenuclearcontrol(P<0.01)
Fig.6.WesternblotanalysisandnuclearandcytoplasmicquantificationofERaandERbafter6hofexposureto106MNPandE2.ERawasfoundinnuclearproteinsafterE2 andNPtreatmentwhileERbwasfoundonlyinthecytoplasmaticproteins(a).ThegraphsrepresentedtheOpticaldensity(OD)ratioofERaandERbnormalizedtotheODof TubulinbforcytoplasmicproteinsandtotheODofHDAC2fornuclearproteins(b).(N=3separateexperiments)a,responsesignificantlydifferentthanthecytoplasmatic control(P<0.05);b’,responsesignificantlydifferentthanthenuclearcontrol(P<0.01).
Fig.7.WesternblotanalysisandnuclearandcytoplasmicquantificationofERaafterpre-treatmentwith105MICI182,780(+I).ICIinhibitscytoplasm-nucleustranslocation ofERaafter2hofexposureto106ME2(a–b)andafter6hofexposureto106ME2andNP(c–d).ThegraphsrepresentedtheOpticaldensity(OD)ratioofERanormalizedto theODofTubulinbforcytoplasmicproteinsandtotheODofHDAC2fornuclearproteins(b,d).(N=3separateexperiments)nosignificantlydifferences.
“cocktail effect” is a feature widely accepted for EDCs risk management(Bergmanetal.,2012).
Inconclusion,wedemonstratedthatNPactsonPNT1Acells withsimilareffectsifcomparedtoE2,probablymediatedbyER
a
anditmaybeinvolvedinaderegulationofcellcycle,leadingto aberrantproliferationofprostateepithelialcells,whichinturn maycontributetopathological states,includingcancer.Weare alsoprovidingdataonthedualroleofERsinprostatecells.Notwithstandingthefindingsofthisstudy, furtherevidences remaintobeinvestigatedinordertobestcharacterizetheriskof NPexposureforprostatediseases.Inaddition,morecellularandin vivo models will be needed. However, our data may help epidemiologists to consider and monitoring the association betweenNPandprostatepathologies.
Acknowledgment
ThisworkwassupportedbyPORCAMPANIAFESR2007/2013, withtheproject“Modelliinvivodipatologieumane(MOVIE)”. AppendixA.Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.tox.2016.05.024.
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