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AccumulationpatternofDechloranePlusandassociatedbiologicaleffectsonratsafter90dofexposureYanLia,c,LehuanYub,JiansheWanga,JiangpingWub,BixianMaib,,JiayinDaia,aKeyLaboratoryofAnimalEcologyandConservationBiology,InstituteofZoology,ChineseAcademyofSciences,Beijing100101,PRChinabStateKeyLaboratoryofOrganicGeochemistry,GuangzhouInstituteofGeochemistry,ChineseAcademyofSciences,Guangzhou510640,PRChinacGraduateUniversityofChineseAcademyofSciences,Beijing100049,PRChinahighlights"WeexposedDPonratsindifferentdoselevelstodetecttheaccumulationpatternandbiologicaleffects.
"DPpreferentiallyaccumulatedintheliverratherthaninmuscleatallexposurelevels.
"Syn-DPwaspronetoaccumulateinalltissuestestedathighexposureconcentration(10and100mgkgg1).
"Thevalueoffantiisdifferentinvariousexposureconcentration.
"Noobservable-effectinhistopathologyanddeathduringthewholeexperiment.
graphicalabstractarticleinfoArticlehistory:Received5May2012Receivedinrevisedform18October2012Accepted31October2012Availableonline11December2012Keywords:DechloranePlusTissuedistributionpatternBiotransformationBiologicaleffectsabstractRecentstudieshaveindicatedthatDechloranePlus(DP)iswidespreadintheenvironments.
However,differentisomer-specicenrichmentpatternofsyn-DPandanti-DPwasreportedinbiologicalsamplesfromtheeld.
Inthisstudy,Sprague–DawleyratswereconsecutivelyexposedtocommercialDP25bygavagefor90datdifferentdoses(0,1,10,and100mgkg1d1)toinvestigatetheaccumulationpatternofsyn-DPandanti-DPinliver,muscle,andserumofrats.
ThepossiblebiologicaleffectsofDPonratswerealsoexamined.
ResultsshowedthatDPpreferentiallyaccumulatedintheliverratherthaninmuscleatallexposurelevels.
Nosignicantstereoselectivityofanti-DPorsyn-DPintissueswasobservedinthelowDPexposuregroups(0and1mgkg1d1)withfantivalues(denedastheconcentrationoftheanti-DPdividedbythesumofconcentrationsofanti-andsyn-DP)rangingfrom0.
74to0.
78.
However,fantivaluesreduced(fantirangedfrom0.
26to0.
30)signicantlyinthehighDPexposuregroups(10and100mgkg1d1)andsyn-DPwaspredominantinalltissues.
Biochemicalparametersinserum,themRNAexpressionlevelsofcertainenzymesandtheiractivitiesinliverweredetected.
Therewasnoobservable-effectinhistopathologyanddeathduringtheexperiment,althoughthemRNAexpressionlev-elsofsomegenesinthelowdosagegroupdecreasedsignicantlyandenzymeactivityofCYP2B2increased.
2012ElsevierLtd.
Allrightsreserved.
1.
IntroductionDechloranePlus(DP)isanunregulatedchlorinatedameretar-dantusedasareplacementoftoxicDechloraneandMirex,whichwasrstintroducedintothemarketinthe1960s.
Commercial0045-6535/$-seefrontmatter2012ElsevierLtd.
Allrightsreserved.
http://dx.
doi.
org/10.
1016/j.
chemosphere.
2012.
10.
106Correspondingauthors.
Tel.
:+862085290146;fax:+862085290706(B.
Mai),tel.
:+861064807185;fax:+861064807099(J.
Dai).
E-mailaddresses:nancymai@gig.
ac.
cn(B.
Mai),daijy@ioz.
ac.
cn(J.
Dai).
Chemosphere90(2013)2149–2156ContentslistsavailableatSciVerseScienceDirectChemospherejournalhomepage:www.
elsevier.
com/locate/chemosphereDPhastwoisomers(syn-DPandanti-DP),withtheratioof1:3(Tomyetal.
,2007),whichhasbeenwidelyemployedinproductssuchaselectricalwires,cablecoatings,plasticroongmaterials,andhardconnectorsincomputersandtelevisions(WeilandLevchik,2004).
ThreetypesofDPproductsarecurrentlyavailable(DP-25,DP-35,andDP-515),whichareidenticalinchemicalcom-positionbutdifferinparticlesize(Zhuetal.
,2007).
Annualproduc-tionofDPisestimatedat$5000tonsintheEuropeanUnionandismarketedworldwide(Renetal.
,2008),whilecurrentproductioninChinaisestimatedat$300tons(http://info.
china.
alibaba.
com/news/detail/v4-d1001424159.
html,inChina).
Recently,DPwaslistedbytheEuropeanCommissionasapossiblereplacementforthenewlyrestricteddecabromodiphenylether(Deca-BDE)forsev-eralapplicationssuchelectricalproducts(Sverkoetal.
,2011),whichmightleadtoincreasingusageinnewlyindustrializingcountriesinAsiawherethemajorityoftheseproductsaremanufactured.
PreviousstudieshaveinvestigatedtheoccurrenceandbehaviorofDPintheatmosphere,sediment,soil,water,andbiotaincludingwildlifeandhumans,demonstratingthatDPisapersistent,bioac-cumulativeenvironmentalcontaminant,andsusceptibletolong-rangeatmospherictransport(Sverkoetal.
,2011;Xianetal.
,2011).
Oneofthemostdiscussedissuesamongresearchersregard-ingtheenvironmentalbehaviorofDPisthestereoisomericfrac-tionationofDPinvariousenvironmentalmatrices.
Whileselectivedepletionsofanti-DPduringlong-rangetransportwereobservedintheatmospherefromtheGreatLakesregionandalongthenorthernAtlanticOcean(Molleretal.
,2010;Sverkoetal.
,2011),nosignicantstereoisomericfractionationsofDPwerefoundinairsamplesacrossChina(Renetal.
,2008).
TheDPisomerprolesmeasuredinbiotasamplesaremorecomplicated.
Wuetal.
(2010)describednetsyn-isomerenrichmentintheaquaticfoodwebfromareservoirinane-wasterecyclingsiteinChina.
Tomyetal.
(2007)reportedanti-isomerbiomagnicationsinthefoodwebatLakeWinnipegbutanabsenceofastereoisomerbiomagni-cationsinthefoodwebatLakeOntario.
InbaldeagleplasmafromtheGreatLakesregion,thecontentofanti-DPwasabouttwotimesthansyn-DP(Venieretal.
,2010).
NosignicantstereoselectiveenrichmentsweredetectedwithinherringgulleggsfromtheLau-rentianGreatLakesofNorthAmericaorwhitestorkeggsfromSpain(GauthierandLetcher,2009;Munoz-Arnanzetal.
,2011),butpreferentialaccumulationofsyn-DPwasobservedinwaterbirdsfromtheabove-mentionede-wasterecyclingsite(Zhangetal.
,2011).
FromafewstudiesofDPinmammals,apreferentialdepletionofanti-DPwasmeasuredinserumsamplesofresidentsfromane-wastedismantlingregioninChina,withlittlestereose-lectivityofDPfoundinserumsamplesofpeoplelivinginanearbyregion(Renetal.
,2009).
Onlyslightdepletionofanti-DPwasre-portedinasmallnumberofpetdogserumsamplesfromtheUni-tedStates(VenierandHites,2011).
TheseavailabledataonDPstereoselectivitygiveapicturethatbiotasamplesfrome-wastesitesgenerallyhavemorestereoselectivitythanothersamplesintheenvironment,leadingtothespeculationthatstereoselectivityofDPinorganismsmaydependonexposureconcentration.
VerylittledataexistsonthetoxicityofDP(Brocketal.
,2010;Crumpetal.
,2011).
Arecentstudyexaminedthereproductivetox-icityofDPinratsandfoundthatDPdosesof5000mgkg1hadnoeffectonin-lifeparametersorclinicalandanatomicpathologydur-ingthe28-drepeatdosetoxicityphase,andnoeffectswereob-servedonreproductiveorfertilityindicesoronfetalgrowthinthedevelopmentalandreproductivetoxicityphase(Brocketal.
,2010).
Additionally,Crumpetal.
(2011)determinedtheeffectsofDPexposureoncytotoxicity,embryotoxicityandmRNAexpres-sionlevelsinthedomesticchickenusinginvitroandinovoap-proaches;nooverttoxiceffects(hepatocytesandpippingsuccess)wereobserveduptothehighestnominaldosegroupof500ngg1egg,andnoneoftheelevengenetargetsidentiedasresponsivetothebrominatedameretardant,hexabromocyclodo-decane,changedafterDPtreatment.
AlthoughthefewavailabletoxicitydataindicatethatDPhasnosignicantlyoverttoxiceffectsontestorganisms,additionalmolecularmarkersshouldbeana-lyzedtoidentifythepotentialtargetsthatmightberesponsivetoDP,andsubchronic/chronictoxicitystudiesonDPareneededtoevaluatetheriskofDPontheenvironmentandhumansbeforeitisusedasanalternativeforDeca-BDE.
Inthisstudy,maleSpragueDawley(SD)ratswereorallyadmin-istratedcommercialDP-25mixedwithcornoil(existingserviceisused)for90datdifferentdoses.
AnothertwogroupswereexposedtoaknownamountofDP-25for45dfollowedby45ddepuration.
TheDPlevelsinliver,muscle,andserumweremeasuredafterexposuretoinvestigatethetissuedistributionofDPanditsstereo-isomericfractionationsfollowingdifferentdoses(intherangeof0–100mgkg1d1).
ThedechlorinatedDPproductsandotherpos-sibleDPmetabolitesinvarioustissueswerealsoscreenedusingGC/ECNI-MSandGC/EI-MS.
WealsoassessedpossibletoxicityofDPbymonitoringasuiteofbiochemicalparameters,includingthy-roidhormonelevels,thirteenclinicalchemistryparameters,there-sponsetoenvironmentalpollutantsofmRNAexpressionlevels,andtheactivityofcertainenzymes.
2.
Materialsandmethods2.
1.
AnimalsForty-twoMaleSDrats(35-d-old)withaverageweightof110gwereobtainedfromtheWeitongLihuaExperimentalAnimalCentral(Beijing,China)andwerehousedindividuallyinamassair-displacementroomwitha12-hlight–darkcycleat20–26°Candarelativehumidityof50–70%.
After1weekacclimation,theywereseparatedintosixgroups:theratsinfourgroupswereexposedtoconsecutivedosagesof0,1,10,and100mgkg1d1for90d,whiletheothertwogroupswereexposedto0and100mgkg1d1for45dfollowedby45dofdepurationduringwhichtheratswerefedunfortiedfood.
Allratswereeuthanizedaftertheirexposureperiod.
Liver,muscle,andserumwereimme-diatelycollectedfromeachratandwerefrozeninliquidnitrogenandstoredat20or80°Cuntilthechemicalanalysisandtoxicexperiments,respectively.
2.
2.
ChemicalsandchemicalanalysisCommercialDP25(P99%purity)wasobtainedfromJiangsuAnponCo.
,LtdandcornoilwaspurchasedfromSigma–Aldrich(Oakville,ON,Canada).
Theanti-DP,syn-DP,anti-Cl11-DP,andanti-Cl10-DPstandardswerepurchasedfromWellingtonLaborato-riesInc.
(Guelph,ON,Canada).
Allsolventsandreagentsusedintheseexperimentswereofanalyticalgrade,andorganicsolventswereredistilledusingtheglasssystem.
TheBDE-77,BDE-181,BDE-128,andBDE-118werepurchasedfromAccuStandard(NewHaven,US).
TheDPwasscannedbyanAgilent6890gaschromatographcoupledwithanAgilent5975Cmassspectrometer(GC/MS)usingelectroncapturenegativeionization(ECNI)intheselectiveion-monitoring(SIM)modeandseparatedbyaDB-XLB(30m0.
25mm0.
25lm,J&WScientic)capillarycolumn.
Theinitialoventemperaturewassetas110°C(heldfor1min),rampedat8°Cmin1to180°C(heldfor1min),andthen2°Cmin1to240°C(heldfor5min),2°Cmin1to280°C(heldfor15min),andnally10°Cmin1to310°C(heldfor10min).
Wemanuallyinjected1lLofsampleinthepulsedsplitlessmode.
Thetemperatureoftheinjectionportwassetat280°C.
The2150Y.
Lietal.
/Chemosphere90(2013)2149–2156monitoredandquantitativeionswereasfollows:m/z653.
8and651.
8forDPisomers(syn-DPandanti-DP),m/z618and620foranti-Cl11-DPandsyn-Cl11-DP,andm/z584and586foranti-Cl10-DP.
Thesyn-Cl11-DPwaspreviouslyidentiedasadechlorinatedproductofsyn-DPbyWangetal.
(2011).
Semi-quantitationwasachievedtosyn-Cl11-DPbyreferencetotheresponseofanti-Cl11-DPonGC/MS.
Wealsoattemptedtoscreensomepossiblemeth-oxy-and/ormethylsulfone-DPinratserumandliver.
SuchDPmetabolites,ifpresent,wouldionizesimilartoOH-CBand/orMeSO2-CBbyscavenginganelectrontoshowadominantM-inENCIionsource.
Correspondingm/zvaluesofpotentialoxidativemetabolitesweredemonstratedbyTomyetal.
(2008).
2.
3.
SamplepreparationDetailsoftheanalyticalproceduresforliverandmusclearede-scribedinthesupplementarydata.
PretreatmentforserumwasperformedasMalmberq(Malmbergetal.
,2005)withminormod-ication.
Twomilliliterofserumwasspikedwithsurrogate(BDE-181).
Theproteinwasdenaturedwith1mLofhydrochloricacid(6M)and6mLof2–propanol,andthemixtureswereshakenvig-orously.
TheDPanditsmetabolitesweretwiceextractedwith6mLof1:1hexane/methyltert-butylether(MTBE)(V/V=1:1)mixture.
Thecombinedorganicextractswerewashedwithapotas-siumchloridesolution(1%,3mL),andthesolventwasconcen-tratedtodrynessunderN2forgravimetriclipidweightdetermination.
Theextractwasredissolvedwith6mLofhexane,andtheaqueousphasecompoundswereseparatedfromneutralsbypartitioningwithpotassiumhydroxide(0.
5Min50%ethanol).
Theaqueousphasewasre-extractedagainwithhexane(6mL)forcompleteextraction.
Neutralphasewastreatedwith2mLofconcentratedsulfuricacidtoremovelipids,andsubsequentpro-cessingwaselutedwith40mLhexane/dichloromethane(V/V=1:1)inmulti-layersilicacolumn(i.
d.
=1cm)packedwith8cmneutralsilicaand8cmacidiedsilica.
Eluteswereconcen-tratedtoneardrynessunderN2andredissolvedin270lLofisooc-taneandspikedwithaknownamountofinternalstandard(30lLBDE-128)beforeinstrumentalanalysis.
2.
4.
QA/QCanddataanalysisProceduralblankscoveringthewholeprocedurewereper-formedinparallelwiththesamplesoneachbatchofextraction.
Theaveragerelativestandarddeviations(RSDs)amongtriplicatessamplesrangedfrom3%to9.
6%foralltargets.
Therecoveriesforsurrogatesrangedfrom71.
7%to112.
9%inalldetectedsamples.
Themethoddetectionlimit(MDL)wasdenedasthemeanvalueplus3-foldstandarddeviationforanalytes,whichweredetectedintheproceduralblanks(n=6)andforotherswhichwerenotde-tectedinblanks,asignal-to-noiseratiooftenwassetasMDL.
TheMDLsforsyn-DP,anti-DP,andanti-Cl11-DPwere108.
31,70.
44,and0.
98ngg1lwinmuscleandliver,andwere0.
20,0.
054,and0.
042ngml1forsyn-DP,anti-DPandanti-Cl11-DPinserum,respectively.
StatisticalanalysiswasperformedusingSPSS16.
0forWindows(SPSSInc.
,Chicago,IL)forcomparisonsofmeansbetweenandwithintreatmentgroups.
Thefantiwasdelegatedtoratioofanti-DP/(anti-DP+syn-DP),whichwasusedtoevaluatetheprolesofDPinthetissueofexperimentanimals.
One-wayanalysisofvariance(ANOVA)wasusedtodeterminedifferencesinconcentrationsandotherexperimentalstatistics.
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