Effects of 4-nonylphenol on blood cells of the African catfish Clarias gariepinus (Burchell, 1822

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Effects of 4-nonylphenol on blood cells of the African catfish Clarias gariepinus (Burchell, 1822
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  This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:http://www.elsevier.com/copyright  Author's personal copy TissueandCell   43 (2011) 223–229 ContentslistsavailableatScienceDirect Tissueand   Cell  journal   h   omepage:www.elsevier.com/locate/tice Effects   of4-nonylphenol   on   blood   cells   of    the   African   catfish   Clarias    gariepinus (Burchell,   1822) Imam   A.   Mekkawy a , b ,   Usama   M.   Mahmoud a ,   Alaa   El-DinH.   Sayed a , ∗ a  ZoologyDepartment,FacultyofScience,AssiutUniversity,71516Assiut,Egypt  b BiologyDepartment,FacultyofScience,TaifUniversity,Taif,SaudiArabia a   rtic   l   e   in   fo  Articlehistory: Received21February2011Receivedinrevisedform21March2011Accepted21March2011 Available online 17 April 2011 Keywords: 4-NonylphenolBloodCellMicronucleus Clariasgariepinus ab   s   t   r   a   c   t Inthepresentwork,   thedestructiveeffects   ofthe4-nonylphenolon   one   of    the   most   economicallyimportant   Nile   fishes,   namelyAfrican   catfish( Clariasgariepinus )   were   studied.   Apoptosis,   erythrocytesalterations,micronucleus   test   and   blood   parameters   countwereused   as   biological   indicators   todetectthoseeffects.   After   exposure   tosublethalconcentrationsof    4-nonylphenol(0,   0.05,0.08   and   0.1mg/l),apoptoticred   blood   cellswithmanymalformations   andmicronucleatederythrocytes   wererecorded.Decrease   in   the   blood   parameterssuch   as   redblood   cells   (RBCs),hemoglobin   (Hb),packagecell   vol-ume   (PCV),   meancorpuscular   hemoglobinconcentration(MCHC),platelets,   whiteblood   cells   (WBCs),lymphocytes,basophils,   monocytes   andincrease   in   meancorpuscular   volume(MCV),meancorpuscularhemoglobin   (MCH),   neutrophils,   eosinophils   indicated   the   negative   effects   of    4-nonylphenol.   Itwas   con-cludedthat,   the   4-nonylphenolcaused   genotoxicity   in   erythrocytes   with   manymalformations   in   shapeandnumber   indicatedwithotherblood   parameters. Published by Elsevier Ltd. 1.Introduction Pollutionisoneofthe   majorproblemsinour   countries.Chemicalpollutionappearsto   beduetoincreaseofindustrialization.Alargenumberofchemicalscancontaminateaquaticenvironmentsandtheiranimalsincludingfishandamphibiansduringtheiradultlifeandsensitivestagesofdevelopment(Radhaiahetal.,1987).Theirtoxicityappearsbecauseoftheirpersistenceintheenvironmentandtheiraccumulationinthebiotatissue.Nonylphenolethoxylate(NPE)hasbeenfoundinaquaticenvi-ronments,particularlyinriverwater(Clarketal.,1992;Tsudaetal.,2000;Riveroetal.,2008).Thiscompoundiswidelyusedintheman-ufactureofnon-ionicsurfactants,lubricants,stabilizerpolymers,antioxidants,alkylphenolchemicals,detergents,paints,anaerobictreatedsewagesludge,polystyrenetubes,insecticidesandherbi-cides(Gigeretal.,   1984;Marcominietal.,   1990;Sotoetal.,1991;Cox,1996).IntheaquaticenvironmentNPEbreaksdownto4-nonylphenol(NP),whichismorestableandpersistent(Sakai,2001;Kimetal.,2002;Uguzetal.,2003;Soneetal.,2004;Riveroetal.,2008).Nonylphenolanditsethoxylatesare   acutelytoxicto   awidevarietyofanimalsincludingbees,spiders,fish,molluscsandcrus-taceans(Cox,1996),andalso   estrogenic(Flouriotetal.,   1995;Cox,1996)andhavesublethaleffectsincludingreducedfertility,irreg-ularheartbeatandlossofmovement(Cox,1996).Nonylphenol ∗ Correspondingauthor.Tel.:+20882412381;fax:+20882342708. E-mailaddress: alaa h254@yahoo.com(A.H.Sayed). isnota   singlechemicalcompoundbutreferstoafamilyofcom-poundsallofwhichhaveacentralaromaticorbenzeneringandaninecarbonchain(Cox,1996).Nonylphenolissomewhatsolubleinwater(VincentandSneddon,2009)butismorepersistentintheenvironmentthantheparentnonylphenolexthyolate(Aheletal.,1993;Cox,1996).Nonylphenolsbioaccumulatesinaquaticorganismsandthebreakdownproductsofthesecompoundsarepersistentintheenvironment(Aheletal.,1993;Cox,1996).Theadverseeffectsof nonylphenolsanditsethoxylatesincreaseduetotheirbioaccumu-lationinfish(Aheletal.,1993;Cox,1996).Thebioconcentrationfactor(BCF)ofnonylphenolinfishvariesfrom3to1300(Ekelundetal.,1990;Aheletal.,   1993).Relativelylowconcentrationsof nonylphenolandnonylphenolethoxylatescancausedeathtofish(Salanitroetal.,   1988;Cox,1996;Servos,1999;Staplesetal.,2004;Vazquez-Duhaltetal.,   2005).AsstatedbytheEnvironmentalPro-tectionAgency(EPA),forfreshwater,theaquaticlifeshouldnotbe   affectedif    the1haverageconcentrationofnonylphenoldoesnotexceed28  g/Lonaveragemorethanonceeverythreeyears(VincentandSneddon,2009).Lethalconcentrationof0.032ml/lnonylphenolwas   previouslydeterminedfor Oreochromisniloticus (Riveroetal.,   2008).Bloodisagoodindicatortodeterminethehealthofan   organ-ismandhematologicalparametersare   importantindiagnosingthefunctionalstatusofexposedanimalstotoxicants( Joshietal.,2002a).Asa   resultofassociationbetweenthecirculatorysys-temandtheexternalenvironment,thehematologicalvariableswereusedtodeterminetheeffectsofexternalstressorsandtoxic 0040-8166/$–seefrontmatter. Published by Elsevier Ltd. doi:10.1016/j.tice.2011.03.006  Author's personal copy 224  I.A.Mekkawyetal.    /TissueandCell 43 (2011) 223–229 substances(WendelaarBonga,1997).Ithasbeensuggestedthathaematology,biochemicalchanges,growthrateandoxygencon-sumptionoffishbeusedinpollutantstoxicitydetection(Wepener,1997).Damagetobloodandheamapoieticorgansinfishmaybeassociatedduetoeitherchangeinenvironmentalconditions(DeWildeandHouston,1967;GardnerandYevich,1969a)orwaterbornpollutants(McMay,1929;Dawson,1935;Reichenbach-Klink,1966;GardnerandYevich,1969b).Bloodcellindices(RBCs,WBC,andDLCcounts)aregoodindicatorsofsystemicresponsetoexter-nalstressandanychangesthereforereflectedintheirmorphologyanddistributionintheblood(SrivastavaandChoudhary,2010).Davisetal.(2008)reportedthatnaturalstressorsandexogenousstressorsintheenvironmentelicitastressresponseinfishleuko-cytesprofile.Thedetectionofmicronucleus(MN)infishhelpsus   toknowthestatusofwaterquality,thehealthofspeciesandpotentialrisk(Al-SabtiandMetcalfe,1995).Thestudyofmicronucleustestandabnormalerythrocytesmorphologyinfishesbyvariouschem-icalshavebeenreported(GrisoliaandStarling,2001;Ferraroetal.,2004;TalapatraandBanerjee,2007).Fisherythrocytesareespe-ciallyfavouredformicronucleustest(Bushraetal.,   2002)anditsfeasibilityhasalreadybeenestablishedin Clariasgariepinus (Baharietal.,1994).Itwasstatedbymanyauthorsthatabnormalnuclearmorphologyisanindicatorofgenotoxicdamageinfish(Bombailetal.,2001;TalapatraandBanerjee,2007).Alternatively,variousabnormalmorphologicalformsoferythrocytesareeffectiveindica-torsofcytotoxicity(Bushraetal.,2002).Apoptoticcellsareformedby   activelydyingcellsthatincludecellshrinkage,membranebleb-bingandchromatincondensation(Murakawaet   al.,2001;TalapatraandBanerjee,2007)andthatindicatorofabnormalcelldivisions(Cavasetal.,2005;TalapatraandBanerjee,2007).Africancatfish( C.gariepinus )isoneofthemostimportanttrop-icalculturedfishduetohighgrowthrate,highstocking-densitycapacities,highconsumeracceptabilityandhighresistancetopoorwaterqualityandoxygendepletion(AppelbaumandKamler,2000;AkinwoleandFaturoti,2007;Adewoluetal.,   2008;Karamietal.,2010).Africancatfish( C.gariepinus )isdistributedthrough-outAfrica(NguyenandJanssen,2002).Moreover,ithasbeenusedinfundamentalresearchandconsideredasanexcellentmodelfortoxicologicalstudies(Degroot,1987;Volckaertetal.,1994;NguyenandJanssen,2002;Osmanetal.,2007a;Mekkawyetal.,2010),sinceithasawelldocumentedbiology(Volckaertetal.,1994;Osmanetal.,2007b).Thepresentworkwastherefore,undertakentoexaminetheabilityof4-nonylphenoltoinduceapoptosis,micronucleus,morphologicalalterationsinblooderythrocytesandotherhema-tologicalparametersofthecatfish, C.gariepinus . 2.Materialsandmethods  2.1.Specimencollection SpecimensofadultCatfish C.gariepinus were   collectedfromtheRiverNileatAssiutandthenweretransportedtoFishBiologyLaboratoryatZoologyDepartment,FacultyofScience,AssiutUni-versity.Thefish(500–1200g)werefedonacommercialpelletdiet(3%ofbodyweightperday)andkepttogetherin100l   rectangulartankscontainingtapwater(conductivity2000l/cm;pH7.5;oxy-gen88–95%saturation;temperature27–28 ◦ C;photoperiod12:12light:dark).After2weeksacclimatization,fisheswereusedfortheexperimentalsetup.  2.2.4-Nonylphenol NonylphenolwasobtainedfromSigma–Aldrich(Schnelldrof,Germany).  2.3.Experimentalsetup Theadaptedadultfishweresubdividedintofourgroups(10fishper   each):control,4-nonylphenol-treatedgroup(0.05mg/ldailyfor15days),4-nonylphenol-treatedgroup(0.08mg/ldailyfor15days),and4-nonylphenol-treatedgroup(0.1mg/ldailyfor15days).Inthepresentstudy,therangeofNPexposureswas   0.05–0.1mg/landtheseconcentrationswerechoseninaccordancewithenvi-ronmentallyobservedvalues.Theconditionsoftheexperimentwereas   that   ofacclimatizationwithchangingallthetapwaterandconcentrationsof4-nonylphenoleveryday.  2.4.Hematologicalparameters Bloodsamplesweretakenfromthecaudalveinintoheparinizedtubes.TheconcentrationofHbandbloodcellscountwereimme-diatelyestimated.Othersamplesofbloodwerecentrifugedat5000rpmfor10min   andserumsampleswerestoredinpolyethy-leneEppendorftesttubesat   − 20 ◦ Cuntilserumanalysis.TheRBCs,WBCs,bloodplatelets,haematocrite(HCT),hemoglobin(Hb)weredeterminedbyusingautomatedtechnicalanalyser(Min-drayBc-2800).Meancorpuscularvolume(MCV),meancorpuscularhemoglobin(MCH),andmeancorpuscularhemoglobinconcentra-tion(MCHC)were   calculatedusingtheformulaementionedbyDacieandLewis(1991).MCHC(g/dl) = HbHct  ×   100MCH   (pg) = HbRBCs  × 10MCV   (mm 3 ) = HctRBCs  ×   10DifferentialWBCswerecountedusingbloodsmearsstainedwithGiemsastainaccordingtoTavares-DiasandMoraes(2003).  2.5.Apoptosisdetection ApoptoticerythrocytesweredetectedusingAnnexinV-EGFPApoptosisDetectionKit(Cat.No.K104-25,100,400),Biovisionresearchproducts,980LindaVistaAvenue,Mountainview,CA94043,USA).TheprocedureusedtodetecttheapoptosisinRBCswereas,aftercollectionofbloodinheperinizedtubescentrifuga-tionoccurredat50,000rpmobtaining1–5 × 10 5 cells,resuspensioncellsin100  lof1 × bindingbuffer,thenadditionof5  lAnnexin-EGFPand5  lofpropidiumiodide.Incubationatroomtemperaturefor5mininthedarkandthenplacingthecellsuspensiononglassslideandcoveredwithaglasscoverslip.FinallyobservationcellsunderaZeissAxioplan2fluorescencemicroscope(200 × )   providedwithadigital3CCDcolorvideocamera(Sony,AVT-Horn).  2.6.Micronucleustestanderythrocytesalterations Bloodsmearswereobtainedbythecaudalincisionon   cleangreasefreemicroscopicslidesaftercompletionofadesiredexpo-sure.Thesmearswerefixedinabsolutemethanolfor10min   afterdryingatroomtemperature.Slideswerestainedwithhaema-toxylinandeosin.It   wasfollowedbydehydrationinascendinggradesofalcohol(30,50,70,and90%,absolute),Finallytheslideswereclearedinxyleneandpermanentlymountedby   DPX(PascoeandGatehouse,1986).Manyslideswereselectedonthebasisof stainingquality,thencoded,randomizedandscoredblindly.In   eachgroup10,000cells(aminimumof1000perslide)wereexamined(Al-SabtiandMetcalfe,1995)at   40 × objectiveand10 ×   eyepiecefor  Author's personal copy I.A.Mekkawyetal.    /TissueandCell 43 (2011) 223–229 225  Table1 Apoptosis,micronucleustestandalterederythrocytes(mean ± SD)%afterexposuretodifferentdosesof4-nonylphenolofadultsAfricancatfish Clariasgariepinus (averagebetween   parentheses).Test TreatmentsControl0.05mg/l4-nonylphenol0.08mg/l4-nonylphenol0.1mg/l4-nonylphenolApoptosis(%)0.8 ±   1.03(0.0–3)a3 ± 1.41(1–5)b   6.1 ± 1.72(3–8)c   23.9 ± 4.22(18–32)dMicronucleustest(%) 0.2  ±   0.42(0.0–1)a 1  ± 0.816(0.0–2)b 1.9  ± 0.99(1–4)c4.2 ± 2.44(1–9)dAlterederythrocytes(%)0.7 ±   0.48(0.0–1)a2.1 ± 1.19(0.0–4)b12 ± 3.26(5–18)c   42 ± 15.98(12–64)dDifferentlettersindicatesthereisasignificantdifferenceat(  p ≤ 0.05). micronucleatedandmorphologicallyalterederythrocytesinsepa-ratestudies.TheestablishedcriteriaforidentifyingMN   (Schmidt,1975)werestrictlyfollowedtoensureauthenticscoring.In   mor-phologicallyalterederythrocytes,theimportantvariationssuchasacanthocytes,teardroplikecells,sicklecells,andswelledcellsechinocytes,alterationofnuclearmorphology,enucleatederythro-cytesandvacuoleswereobserved.  2.7.Statisticalanalysis Thebasicstatistics,means,standarddivisionsandrangeswereestimated.Thepatternofvariationwasone-wayanalysisofvari-anceusingtheSPSSpackage(SPSS1998)atthe0.05significancelevel.TheTukey-HSDtestwasconsideredformultiplecomparisonsanddesignedtoverifythefrequencyofapoptoticcells,MN   inci-denceanderythrocytealterations.Dunnett t  -teststreatonegroupasacontrol,andcompareallothergroupsagainstit.  2.8.Ethicalstatement  Allexperimentswerecarriedout   inaccordancewiththeEgyp-tianlawsandUniversityguidelinesforthe   careofexperimentalanimals.AllproceduresofthecurrentexperimenthavebeenapprovedbytheCommitteeoftheFacultyofScienceofAssiutUniversity,Egypt. 3.Results  3.1.Apoptosisdetectionanderythrocytesalterations AsshowninTable1,theapoptoticcellspercentageappearsinthegroupstreatedwith4-nonylphenolmorethanthecontrolone.Also,thisincreasedwith4-nonylphenoldosesincrease.Fig.1showsthebloodsmearofnormalfishandrepresentsthenormalstructureofbloodofthecatfish, C.gariepinus .   Thebloodiscomposedofnucleatederythrocytes(Er);roundedwithacen-trallylocatedroundednucleus,thrombocytes(T);were   foundovalwiththenucleusnearlyfillingthecellwithdarkpurplestain.Therearetwo   typesoflymphocytesaccordingtotheirsize,theircytoplasmisweaklybasophilic,nongranularwithlargeroundedanddarklystainednucleus,largelymphocytes(LL)arecircularin   shape,nongranularleucocyteswithlargeroundedanddarklystainednucleus,smalllymphocytes(SL);   as   thatoflargelympho-cytesbutsmallerinsize,monocytes(M);nongranularleucocyteswithkidney-shapednucleusandweaklybasophiliccytoplasm,neutrophils(N);granulatedleucocytes,withafaintpurplecolor,roundedinshapewitheccentricallynucleuswhichexhibitsa   vari-etyofshapes,eosinophils(E);granulatedandroundedleucocytes,theystainbrightpurple,theirnucleushavenodefiniteshape,beingsometimesrounded,kidney-shapedorlobulated,basophils(B);basophilicgranulatedleucocytes,few   innumber,roundedorovalwitheccentricallyandvariablyshapednucleus.Exposureofcatfishtosublethalconcentrationsof4-nonylphenol(0.05mg/l,0.08mg/land0.1mg/l)resultedinmorphologicalchangesintheredbloodcellsandappearance Fig.1. Bloodfilmofcontrolcatfish Clariasgariepinus showingthe   roundedshapeofthenucleatederythrocytes(Er),thrymbocytes(T),largelymphocytes(LL),smalllymphocytes(SL),monocyte(M),neutrophils(N),eosinophil(E),andbasophil(B).(400 × ). ofsomepathologictypesofcells.ThemajoralterationsofRBCsinthefishtreatedwith0.05mg/l4-nonylphenol(Fig.2)areshownby   echinocytesorcrenatedcells(Cr);wheretheredbloodcellsdevelopirregularcellsurfacewithnumerousprojections,acanthocytes(Ac);crenatedcellswithlessprojectionsfromthesurface,teardroplikecells(Tr),theirshapelooksliketearwithpointedapices.Fig.3showsothermorphologicalchangesinRBCsinfishtreatedwith0.08mg/l4-nonylphenol,thatare   sicklecells(Sk)whichvaryinshapebetweenelliposoidal,boat-shapedandgenuinesickles,swelledcells(Sc);theircytoplasmfilledwithwaterandmicronucleus(Mn);one   ormoreMnpercellinthemostobservations.Alsointhistreatedgroup,crenatedcells,acantho- Fig.2. Bloodfilmoftreatedcatfish Clariasgariepinus with0.05mg/l4-nonylphenolshowing   crenatedcells;echinocytes(Cr),teardroplikecells   (Tr)andacanthocyte(Ac).(400 × ).  Author's personal copy 226  I.A.Mekkawyetal.    /TissueandCell 43 (2011) 223–229 Fig.3. Bloodfilmoftreatedcatfish Clariasgariepinus with0.08mg/l4-nonylphenolshowingsicklecells(Sk),swelledcells(Sc)   anddistinctmicronucleus(Mn).(400 × ). cytesandteardroplikecellswerefrequent(Fig.3).Besides,lysisof    someredcells,wheresomecellsappearstobestackedtogether.Thefishexposedto0.1mg/l4-nonylphenolshowsmanyalter-ationsintheirblood(Figs.4–8)suchassomeofredbloodcellslosetheirnormalshape,acanthocytesandcrenatedcellswereapparentbesidelysisofsomecells,stickingoftheredbloodcellsandfrag-mentationsofsomecellswereobserved,teardroplikecellsandsicklecellsbecomecommonandobviousdisintegrationofcellwallofsomecellswithvariationintheirnucleishape.Fig.4showssicklecells,swelledcells,distinctmicronucleusandstickingoftheredbloodcells.Bloodfilmoftreatedfishwith0.1mg/l4-nonylphenolasinFig.5aandbshowsstickingalteredcellswithdisintegrationof theredbloodcells(arrows).Fig.6recordedcommonmalformationsin   RBCswhichbecomesticking,altered,acquireddifferentshapesandpalecytoplasm(Pc).Themostcommonobservedchangeintheredbloodcellmorphologyaftertreatmentwith0.1mg/l4-nonylphenolwasthecellsstickingtogetherinachainlikeshape Fig.4. Bloodfilmoftreatedcatfish Clariasgariepinus with0.1mg/l4-nonylphenolshowingcrenatedcells(Cr),swelledcells(Sc),cellshaveprominentvacuoles(Va)and   distinctmicronucleus(Mn).(400 × ). Fig.5. Bloodfilmoftreatedcatfish Clariasgariepinus with0.1mg/l4-nonylphenolshowingstickingalteredcells;(aandb)noticedisintegrationofthe   cell   wallofRBCs(arrow).   (400 × ). Fig.6. Bloodfilmoftreatedcatfish Clariasgariepinus with0.1mg/l4-nonylphenolshowingstickingalteredcells.NoticeRBCsacquireddifferentshapeswithpalecyto-plasm(Pc).(400 × ). whichcalledRouleauxappearanceofRBCsasshowninFig.7aandb.Asignofkaryolysis;wherecellsnucleilosetheircentraldye,redbloodcellswithprominentvacuolesandpresenceofsicklecellswereobservedinfishtreatedwith0.1mg/l4-nonylphenolasshowninFig.8. Fig.7. Bloodfilmoftreatedcatfish Clariasgariepinus with0.1mg/l4-nonylphenolshowingRouleauxappearanceofRBCs;(aandb)(400 × ).
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