w ww.e l s e v i e r . c o m / l o c a t e / b j p
Original
Article
The
effect
of
the
carotenoid
bixin
and
annatto
seeds
on
hematological
markers
and
nephrotoxicity
in
rats
subjected
to
chronic
treatment
with
cisplatin
Lucéia
F.
Souza
a,∗,
Carlos
Henrique
Pagno
a,
Niara
da
S.
Medeiros
b,
Sílvia
Barbosa
c,
Paula
C.P.
dos
Santos
b,
Alessandro
Rios
a,
Matilde
Achaval
c,
Erna
V.
de
Jong
aaInstitutodeCiênciaeTecnologiadeAlimentos,UniversidadeFederaldoRioGrandedoSul,PortoAlegre,RS,Brazil
bDepartamentodeBioquímica,FaculdadedeCiênciasdaSaúde,CentroUniversitárioMetodista,PortoAlegre,RS,Brazil
cDepartamentodeCiênciasMorfológicas,InstitutodeCiênciasBásicasdaSaúde,UniversidadeFederaldoRioGrandedoSul,PortoAlegre,RS,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received23September2015
Accepted7March2016
Availableonline7April2016
Keywords: Cisplatin Bixin Annatto
Celldifferentiation
Nephrotoxicity Antioxidant
a
b
s
t
r
a
c
t
Thisstudyassessedtheprotectiveeffectofthecarotenoidbixinandannattoseedsagainstpossible nephrotoxicityinducedwithasingleperitonealadministrationofpharmacologicalcisplatininmale Wistarrats.After48h,thebloodcelldifferentialcountshowedasignificantreductioninneutrophil countsinratsthatreceivedadietrichinbixinwhencomparedtothegroupthatreceivedonlycisplatin. Theuseofcisplatinledtoanincreaseinkidneyweight.Thecarotenoidbixinattenuatedrenalinjury, char-acterizedbyincreasedpolymorphonuclearinfiltration.Noprotectiveeffectwasobservedwithrespectto Annatto.Theseresultsdemonstratetheroleoftoxiccisplatinandsuggestthatbixinaffordsaprotective effectagainstcisplatin-inducednephrotoxicityinadultWistarrats.
©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Cisplatin [cis-diamminedichloroplatinum(II) – CDDP] is a chemotherapeuticagentusedaloneorincombinationwithother antineoplasticagentsinthetreatmentoflung,brain,throat, esoph-agus,stomach,colon,bladder,testicular,ovariananduterineand othercancers.However,cisplatinnephrotoxicityistheprinciple dose-limitingfactorfortheuseofcisplatin(Santosetal.,2012).It hasbeensuggestedthatthegenerationofreactiveoxygenspecies andlipidperoxidationisresponsibleforthecisplatin-inducedrenal tubularimpairment(ChirinoandPedraza-Chaverri,2009).
Thephysiologicalfunctionsofpatientsundergoing chemother-apyshouldbemonitoredusingperiodiclaboratoryteststoanalyze parametersofglobalrenalfunction,suchascreatinineandurea, andobtainthebloodcellcounttodeterminegeneralimmunestatus (Sodréetal.,2007).
Carotenoidsarecompoundsthathave antioxidantproperties capableofsequesteringfree radicals.Theyare foundin various foodsandmayhaveaprotectiveeffectagainstoxidativedamage
∗ Correspondingauthor.
E-mail:luceia.souza@ufrgs.br(L.F.Souza).
caused,forexample,bychemotherapy(AntunesandBianchi,2004; Riosetal.,2009;Bautistaetal.,2004).
Annattoisanaturalcolorextractedfromtheoutercoatingof theseedsoftheAnnattotree(BixaorellanaL.,Bixaceae),whichis nativetotheAmazonregioninSouthAmerica(Mercadante,2001; Gomes,2007).Itscolorvariesfromyellowtored,andthemost abundantcarotenoidistrans-bixin(1),whichaccountsforaround 80%ofthetotalpigmentcontentoftheseeds(Mercadante,2001; Giulianoetal.,2003;Bautistaetal.,2004).Inadditiontoitscoloring action,annattoalsohasfunctionalpropertiesthatformthebasis ofa varietyof rolesand actionsin livingorganisms duetothe presenceofantioxidantcompounds(Krinsky,1994).Studieshave shown thatbixin hasbeneficial effects,including thereduction ofoxidativestressanditsabilitytoactasachemo-preventative agentandtoreducethenephrotoxiceffectsofantitumoragents (BertramandBortkewicz,1995;Agneretal.,2005;Riosetal.,2009).
H3CO CO2H
1
OInanattempttominimizethesideeffectscausedbycisplatin, thisstudyevaluatestheantioxidantactivityofthecarotenoidbixin
http://dx.doi.org/10.1016/j.bjp.2016.03.005
0102-695X/©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://
and annatto seedson cisplatin-inducednephrotoxicity inadult maleWistarratsbyexaminingbiochemicalandhistologicalaspects toassessrenalfunctionandmorphologyandperformingcomplete bloodcounts.
Materialsandmethods
Plantmaterial
Thisstudyexaminedannatto(BixaorellanaL.,Bixaceae)fromthe Eldoradoaccess,ICN(189644)10.VII.2009,collectedandidentified byJ.M.Wiest.
Assays
Animals
ThebiologicalassaywasperformedintheVivariumoftheFood ScienceDepartmentoftheInstituteofFoodScienceandTechnology oftheFederalUniversityofRioGrandedeSulinBrazil.The ani-malswerehousedinventilatedroomsatatemperatureof24±2◦C witha12–12hlight–darkcycleand60±5%humidityandgivenad libitumaccesstofoodandwater.
Theanimalsweredividedatrandomintofourgroups:CG (con-trolgroup), CPG (cisplatingroup), CPBG (cisplatin/bixingroup), and CPAG (cisplatin/annatto group). The biological trial lasted 28 days. The CG group received commercial rodent feed, the CPG group received commercial feed plus cisplatin (5mgkg−1 body weight), the CPBG group received commercial feed with bixin(0.065mgkg−1 bodyweight)andcisplatin(5mgkg−1 body weight),andtheCPAGgroupreceivedcommercialfeedwith cis-platin(5mgkg−1 bodyweight)andannatto(0.500mgkg−1 body weight).Thedosesofbixinandannattousedinpretreatmentwere basedondatafoundintheliteratureonnephroprotectiveeffects (FAO/WHO, 2007;Antunes andBianchi, 2004).Asingledoseof cisplatin(5mgkg−1)wasadministered48hbeforetheendofthe experimenttoinduceoxidativestress.
Obtainingannattoandbixincrystals
Bixincrystalswereobtainedusingamethodologydevelopedby RiosandMercadante(2004).Theseedswerefirstwashedinhexane andmethanoltoremovehydrophilicandhydrophobicimpurities andthebixinwasthenextractedusingethylacetate.Theextract obtainedthroughthis processwasdried ina rotary evaporator (T<30◦C)and redilutedindichloromethane. Forthe crystalliza-tionprocess,theextractwasheatedonahotplate(T<50◦C).After beingchilled,absoluteethanolwasaddedandtheextractwasthen cooledinanicebathandplacedinafreezerat14±2◦Cfor24h. Thebixincrystalswerefilteredandwashedwithchilledabsolute ethanol,driedinavacuumovenfor24h,andthenstoredat−18◦C untiluse.
Purificationofbixin
Thepurityofbixinwasdeterminedusingamethoddescribed byTocchiniandMercadante(2001),inwhichthebixinisextracted fromannattoseedspreviouslywashedwithpetroleumetherand amixtureofmethanolanddichloromethane(1:1).After concen-tration,theextractwassuccessivelypurifiedbysilicagelthinlayer chromatographyusingbothethylacetateandpetroleumether(3:2) as mobilephases. In the firstchromatography, silica gel layers (Merck)werepreparedinthelaboratoryandthetwoseparated bandsandeverythingremainingattheoriginwerescrapedoffand eluted withthemethanol/dichloromethanemixture(1:1). After concentration,thebandwasappliedagaintoreadysilicaplatesand splitintotwobands.ThemajoroneatRf0.61wasscrapedoffand
elutedwiththemethanol/dichloromethanemixture.Thepattern obtainedshows99%purity,checkedbyHighPerformanceLiquid Chromatography(HPLC)(Agilent1100series).
Cisplatin
Cisplatin [cis-diamminedichloroplatinum (II),CDDP; CAS No. 15663-27-1]waskindlydonatedinitscommercialformbyQuiral QuímicadoBrasilS.A.(Platinil®).
Bloodcollectionanddosesofureaandcreatinine
Theanimalsweresedatedforbloodcollectionusing benzoadi-azepine(0.25mg100g−1bodyweight)andsodiumpentobarbital (4.6mg100g−1 body weight). A median incision was made in theventralpart oftheabdomen and bloodwas collectedfrom the ascending aorta. The blood was centrifuged for 10min at 5081.31×gforcetoseparateserumforureaandcreatinine analy-ses.
Ureaandcreatininelevelsweredeterminedusingcommercial kits (BioLiquid). Urea wasquantifiedusing a spectrophotome-ter (Micronal – B342II digital model) adopting the UV kinetic method,while creatininewasquantified usingthecolorimetric kineticmethod.Theresultswereexpressedinmg/dl.
Bloodcelldifferentialcount
Thebloodcollectedfromtheaortawasplacedintoflasks con-tainingEDTAsolution.Theanimalsweresubsequentlysacrificed. The blood smearswere stained(InstantProv – New Prov) and manuallyanalyzedtodeterminethebloodcelldifferentialcount usinganopticalmicroscope.Thebloodcelldifferentialcountwas expressedasapercentage(%).
Determinationofkidneyweightandpreparationforhistological analysis
The kidneys werewashed free of blood (byperfusion) with asalinesolution.Afixingsolutionwasthenaddedandthe kid-neyswereweighedusingaMicronalB600balanceandfixedusing Bouin’ssolutionfor24hatroomtemperature.
Thematerialwaspreparedforhistologicalanalysisusingroutine techniques(Prophetetal.,1994).Afterfixingfor24h,thesample tissueswerecryoprotectedusingsucrosesolutionsatincreasing concentrations(from15%to30%)andthenfrozeninnitrogen.Next, usingacryostat(Leitz,Digital1702,Germany)at−20◦C,eachpiece wascutat15mthicknessintervalsandtheslicewereplacedon glassslidesandstainedusinghematoxylinandeosin(HE)toassess tissuemorphology.Histologicalparameterswereobtainedusing anOlympus®Bx50opticalmicroscope(Olympus,Tokyo,Japan).
Ethicalaspects
ThisstudywasapprovedbytheAnimalResearchEthics Com-mitteeoftheFederalUniversityofRioGrandedoSul(reference number17809),beingconsideredethicallyandmethodologically adequateaccordingtoResolution196/1996and complementary itemsoftheBrazilianNationalHealthCouncil.
Statisticalanalysis
Table1
MeansandstandarddeviationsofbloodcelldifferentialcountsinWistarratssubjectedtodifferenttreatments.
CG(n=6) CPG(n=6) CPBG(n=6) CPAG(n=6)
Neutrophils(%) 16.5±4.9 24.6±9.2a 13.5±3.5a 18.33±3.7
Lymphocytes(%) 73.3± 3.9 67.4± 9.0 75.5± 5.7 72.50± 3.2
Monocytes(%) 8.0± 2.7 7.8± 1.9 10± 3.6 9.0± 4.1
Basophils(%) 0.2± 0.4 0.2± 0.4 0.3± 0.8 0.16± 0.4
Eosinophils(%) 1.3±1.0 0.4±0.9 0.3±0.8 0±0
CG,controlgroup;CPG,cisplatingroup;CPBG,cisplatin/bixingroup;CPAG,cisplatin/annattogroup.
aCPGstatisticallydifferentfromCPBG(p<0.05).
50.00
45.00
40.00
35.00
30.00
25.00
20.00
15.00
CG CPG CPBG CPAG
10.00
Concentr
ation (mg/dl)
5.00
0.00
Groups
Fig.1. Meanandstandarddeviationofbloodureadosing(mg/dl)inWistarrats
48hafteradministeringcisplatin.Controlgroup(CG),cisplatingroup(CPG),
cis-platin/bixingroup(CPBG)andcisplatin/annattogroup(CPAG),p>0.05.
Resultsanddiscussion
With respect to hematological analysis, the blood cell dif-ferential count showed a significant reduction in neutrophil counts(p<0.05)in thegroup thatreceiveda diet rich in bixin (13.5±3.5%)whencomparedtothegroupthatreceivedonly cis-platin(24.6±9.2%).Nosignificantdifferencewasfoundbetween thegroups(p>0.05)withrespecttootherbloodcells:lymphocytes, monocytes,basophilsandeosinophils(Table1).The immunolog-icalresponsecanbedividedintoadaptive(innateimmune)and non-adaptivecomponents,enhancingthephagocyticcapacityof cells,suchasmonocytes,macrophagesandneutrophils,whichact inthedestructionofantigensandconstitutethefirstlineofhuman defensemechanisms(PathakandPalan,2005).
Ingivensituations,particularlyinthecaseofacute inflamma-tion,highlevelsofneutrophils occur.Thiswasobserved inthe presentstudy;however,whenthegroupthatreceivedcisplatin wascomparedwiththegroupthatreceivedcisplatin/bixin,it is evidentthatbixinapparentlyminimizedtheinflammatoryeffects ofcisplatin,giventhatitsignificantly(p<0.05)reducedthelevels ofneutrophils,indicatingareductioninantitumoragent-induced inflammation.
Nostatisticallysignificantdifferencewasfoundbetweenthe groups, withrespect todose in relationto plasma urealevels, althoughatendency(p=0.07)towarddecreasedurealevelswas observedinthegroupreceivingdietarybixin/cisplatinin compar-isontotheCGandgroupreceivingannatto/cisplatin,asshownin Fig.1.
Itisimportanttoevaluateplasmacreatinineand urealevels together,despitethefactthatthesetwoparametersdonotalways showconcomitanthighlevels.Incasesofprerenalattack, situa-tionssuchasprotein-richdiet,highproteinbreakdownrates,and dehydration,highlevelsofureamaybefoundtogetherwith nor-malcreatininelevels.However,thesetwodosageschangetogether
1.10
1.00
0.90
0.80
0.70 0.60
0.50
0.40
Concentr
ation (mg/dl)
0.30
0.20 0.10
0.00
Groups
CG CPG CPBG CPAG
Fig.2.Meanandstandarddeviationofbloodcreatininedosing(mg/dl)inWistar
rats48hafteradministeringcisplatin.Controlgroup(CG),cisplatingroup(CPG),
cisplatin/bixingroup(CPBG),andcisplatin/annattogroup(CPAG),p>0.05.
insituationsofpostrenalattack,whererenalobstructions, malig-nancy,renalexcretiondisordersandadecreaseintheglomerular filtrationrateoccur(Burtisetal.,2006).
Urearesultsfromthecatabolismoftheaminoacids,andis elim-inatedfromthebody,predominantlybythekidneys(Burtisetal., 2006).Ureaissynthesizedexclusivelybyhepaticenzymesanditis averyunstablemarker,sinceitisinfluencedbytheproteincontent ofdiets,andthusitisnotasaccurateascreatinineforevaluating renalfunction.However,itservesasacomplementaryindicatorin thediagnosisofrenalinjury(Sodréetal.,2007;Burtisetal.,2006). No significant difference between the groups was found (p>0.05)withregardtoserumcreatininedosage(Fig.2).Serum creatininedosageisthemostcommonlyusedindicatorfor mon-itoringtheevolutionofrenalfailure,sinceitistheleastvariable nitrogenouscompoundintheblood.Sinceitisaresidueof creat-inine,freecreatinineisnotreusedbythebody’smetabolism,but ratherexcretedthroughglomerularfiltration(Limaetal.,2003).
Themacroscopicexaminationofthekidneysof ratsfromall groupsshowedthattheyhadanormalappearance,withapink-red colorandfirmconsistency.Theresultsofkidneyweightin rela-tiontobodyweightrevealedpossibleswellingofthisorgan,which couldberelatedtolocalinflammatoryprocesses(Table2).
Table2
Meanandstandarddeviationofthe“finalbodyweight/kidneyweight”ratioinWistarratssubjectedtodifferenttreatments.
n Initialweight(g) Finalweight(g) Renalweight(g)
CG 6 250.31±13.80 375.71±24.81 1.36±0.10
CPG 6 244.81±16.90 369.42±21.48 1.41±0.21
CPBG 6 249.11±16.70 353.61±10.30 1.38±0.10
CPAG 6 253.01±28.80 347.11±24.89 1.23±0.10a
CG,controlgroup;CPG,cisplatingroup;CPBG,cisplatin/bixingroup;CPAG,cisplatin/annattogroup.
aCPGstatisticallydifferentfromCPBG(p<0.05).
CG
CPBG
CPG
CPAG
Fig.3.Photomicrographsofhematoxylinandeosinstainedkidneysectionsofratsinthecontrolgroup(CG),cisplatingroup(CPG),cisplatin/bixingroup(CPBG),and
cisplatin/annattogroup(CPAG),showingpolymorphonuclearinfiltrates(arrow).Magnification400×.
numberofinflammatorycells,werenotedinanimalsthatreceived onlycisplatin,suggestingasimilarassociationinthisgroup.
Thehistologicalanalysis ofthekidney sectionsshowedthat characteristicstructureswereforminginthisorgan.Thepresence ofglomeruliwasnotedinthecorticalregion,surroundedbytwisted proximalanddistaltubules(Fig.3).
Therenaltissueoftheanimalsthatreceivedcisplatinshowed polymorphonuclear (PMN) infiltrates in the glomerular region, whichisacharacteristicofinflammatorychanges.However,PMN infiltrateswere not present in the kidneys of theanimals that receivedpretreatmentwithbixin(Fig.3).
PMN,attractedduringthereperfusionprocess,areconsidered important mediators during the beginning phase parenchymal injuryincasesofacuteischemicrenalfailure.Ontheotherhand,the rateofinfiltrationofmononuclearleukocytescanvarydepending onrenaldamage(Ysebaertetal.,2004).
Accordingto Faraco (1996), thenumber of PMN withinthe glomerularcapillariesisanindicatorofthelevelofinflammatory activityinglomerulopathies,includingacuteglomerulonephritis (Oliveira,2005).
Acute glomerulonephritis, in addition to the presence of macrophagesandPMN,ischaracterizedbyincreasedglomerular volumeandaproliferationofthemesangialcells(Faraco,1996; Oliveira,2005).Glomerularchangeswereobservedinthekidneys ofthe CPG,characterizedby PMNinfiltrates,which could indi-cateacuteglomerulonephritis.However,PMNinfiltrateswerenot
presentinthekidneysofanimalswhoreceivedpretreatmentwith bixin,which suggeststhatthis substancemayafford protection againstinflammationtypicallycausedbythisantitumoragent.
Theseresultscorroboratethefindingsofotherstudies concern-ingdamagecausedbycisplatin.AstudyconductedbyBehlingetal. (2006)evaluatingtheeffectofmultipledosesofquercetintoinhibit renaldamagecausedbycisplatinshowedthattheadministrationof thisflavonoidpreventsandsignificantlyreducesrenaldamage,and thattreatmentwithcisplatinwithoutquercetinincreasesplasma creatininelevels,lipidperoxidationandrenaldamage,asshownby thehistologicalanalysisofthisorgan.
Withrespecttotheproximalanddistaltubules,nodifferences wereobservedbetweenthegroups,showingthatneithercisplatin norpretreatmentwithbixin and annattohad anyeffect. These resultsarecontrarytothefindingsofBehlingetal.(2006)who, using thesame dose of cisplatinadopted in the present study (5mg/kg), noted morphological changes suchas tumor-tubular necrosis.However,thelengthofexposuretothedruginthestudy carriedoutbyBehlingwaslongerthanthatofthepresentstudy(5 and20days,ascomparedtojust2days,respectively).
Conclusions
Thefindingsofthisstudysuggestthatexposuretimewas suf-ficienttodemonstratethatcisplatincausedacuteinjuryandthat bixinprobablyhelpedtominimizeinflammation,asshownbythe lowlevelsofneutrophils.
However,pretreatmentwithbixinattenuateskidneydamage, preventinganincreaseinPMNs,andisapossibleprotectoragainst cisplatin-inducedacuteinflammatoryprocesses.
Ethicaldisclosures
Protectionofhumanandanimalsubjects. Theauthorsdeclare
thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).
Confidentialityofdata. Theauthorsdeclarethattheyhave
fol-lowed theprotocolsof theirworkcenter onthepublication of patientdata.
Right to privacy and informed consent. The authors have
obtainedthewritteninformedconsentofthepatientsorsubjects mentionedinthearticle.Thecorrespondingauthorisinpossession ofthisdocument.
Authors’contribution
LFS,CHPandNSM(PhDstudent)contributedtoorganizingthe laboratorywork,dataanalysisanddraftedthispaper.SB,PCPSand MAcontributedtothebiologicalstudies.EVJandAORwere respon-sibleforstudydesign,supervisedthelaboratoryworkandmadea criticalreadingofthemanuscript.Weconfirmthatthemanuscript hasbeenreadandapprovedbyallnamedauthors.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
WearegratefultoCAPESandtheCNPqfortheirfinancial sup-port.
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