www.bjorl.org
Brazilian
Journal
of
OTORHINOLARYNGOLOGY
ORIGINAL
ARTICLE
The
effectiveness
of
eugenol
against
cisplatin-induced
ototoxicity
夽
Muhammed
Sedat
Sakat
a,∗,
Korhan
Kilic
a,
Fazile
Nur
Ekinci
Akdemir
b,
Serkan
Yildirim
c,
Gizem
Eser
c,
Ahmet
Kiziltunc
daAtaturkUniversity,FacultyofMedicine,DepartmentofOtorhinolaryngology,Erzurum,Turkey
bAgriIbrahimCecenUniversity,HighSchoolofHealth,DepartmentofNutritionandDietetics,Agri,Turkey cAtaturkUniversity,FacultyofVeterinary,DepartmentofPathology,Erzurum,Turkey
dAtaturkUniversity,FacultyofMedicine,DepartmentofBiochemistry,Erzurum,Turkey
Received6May2018;accepted19July2018 Availableonline7August2018
KEYWORDS Cisplatin-induced ototoxicity; Eugenol; DPOAE; Oxidativestress; 8-Hydroxy-2 -deoxyguanosine Abstract
Introduction:Ototoxicityreferstocellulardamageorfunctionimpairmentdevelopinginthe
innerearinassociationwithanytherapeuticagentorchemicalsubstance,andstillrepresents theprincipalside-effectrestrictingtheuseofcisplatin.
Objective:Theaimofthisstudywastoperformabiochemical,functionalandhistopathological investigationofthepotentialprotectiveeffectofeugenolagainstcisplatin-inducedototoxicity.
Methods:Thestudy wasperformed with24femaleSpragueDawleyrats. Distortionproduct
otoacousticemissionstestswereperformedonallanimals,whichwererandomizedintofour equalgroups.Asingleintraperitonealdoseof15mg/kgcisplatinwasadministeredtocisplatin group,whiletheeugenolgroupreceived100mg/kgeugenolintraperitonealforfiveconsecutive days.100mg/kgeugenolwasadministeredtocisplatin+eugenolgroupfor5days.Onthethird day,theseratswerereceivedasingledoseof15mg/kgcisplatin.Thecontrolgroupwasgiven 8mL/kg/dayintraperitonealsalinesolutionforfivedays.Thedistortionproduct otoacoustic emissionstestwasrepeated24hafterthefinaldrugadministration.Allanimalsweresacrificed, andthecochleasweresubsequentlyusedforbiochemicalandhistopathologicalexaminations.
Results:Cisplatincausedoxidativestressinthecochlea,impairedthecochlearstructureand significantlyreducedsignalnoiseratiolevels.Administrationofeugenoltogetherwithcisplatin reversedtheseeffectsandprovidedfunctional,biochemicalandhistopathologicalprotection.
夽 Pleasecitethisarticleas:SakatMS,KilicK,AkdemirFN,YildirimS,EserG,KiziltuncA.Theeffectivenessofeugenolagainst
cisplatin-inducedototoxicity.BrazJOtorhinolaryngol.2019;85:766---73.
∗Correspondingauthor.
E-mail:mssakat@gmail.com(M.S.Sakat).
PeerReviewundertheresponsibilityofAssociac¸ãoBrasileiradeOtorrinolaringologiaeCirurgiaCérvico-Facial.
https://doi.org/10.1016/j.bjorl.2018.07.007
1808-8694/©2018Associac¸˜aoBrasileiradeOtorrinolaringologiaeCirurgiaC´ervico-Facial.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).
Conclusion: The study findings represent the first indication inthe literature that eugenol may protectagainst ototoxicitybyraisinglevelsofantioxidantenzymesandloweringthose ofoxidantparameters.
© 2018 Associac¸˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Published by Elsevier Editora Ltda. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/). PALAVRAS-CHAVE Ototoxicidade induzidapela cisplatina; Eugenol; EOAPD; Estresseoxidativo; 8-Hidroxi-2 -deoxiguanosina
Aeficáciadoeugenolcontraaototoxicidadeinduzidapelacisplatina Resumo
Introduc¸ão: Aototoxicidaderefere-seaodanocelularoucomprometimentodafunc¸ãodaorelha internaassociadoaqualqueragenteterapêuticoousubstânciaquímicaeaindarepresentao principalefeitocolateralquerestringeousodacisplatina.
Objetivo: Oobjetivodesteestudofoirealizarumainvestigac¸ãobioquímica,funcionale histopa-tológicadopotencialefeitoprotetordoeugenolcontraaototoxicidadeinduzidapelacisplatina.
Método: O estudo foi realizado com 24 ratos fêmeas Sprague Dawley. Testes de emissões
otoacústicasporprodutodedistorc¸ãoforamrealizadosemtodososanimais,osquaisforam ran-domizadosemquatrogruposiguais.Umaúnicadoseintraperitonealde15mg/kgdecisplatina foiadministradaaogrupocisplatina,enquantoogrupoeugenolrecebeu100mg/kgdeeugenol intraperitoneal por cinco diasconsecutivos. Foramadministrados 100mg/kg deeugenol ao grupocisplatina+eugenoldurante5dias.Noterceirodia,estesratosreceberamumadoseúnica de15mg/kgdecisplatina.Ogrupocontrolerecebeu8mL/kg/diadesoluc¸ãosalina intraperi-tonealporcincodias.Otestedeemissõesotoacústicasporprodutodedistorc¸ãofoirepetido 24horasapósaadministrac¸ãofinaldomedicamento.Todososanimaisforamsacrificadoseas cócleasforamposteriormenteutilizadasparaexamesbioquímicosehistopatológicos.
Resultados: Acisplatinacausouestresseoxidativonacóclea,prejudicou aestruturacoclear
ereduziusignificativamenteosníveisdarelac¸ãosinal/ruído.Aadministrac¸ãodeeugenol jun-tamentecomacisplatinareverteuessesefeitoseforneceuprotec¸ãofuncional,bioquímicae histopatológica.
Conclusão:Os achadosdo estudorepresentam a primeira indicac¸ão naliteraturade que o
eugenol pode proteger contra aototoxicidade, eleva os níveis de enzimas antioxidantese diminuiosníveisdosparâmetrosoxidantes.
© 2018 Associac¸˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Publicado por Elsevier Editora Ltda. Este ´e um artigo Open Access sob uma licenc¸a CC BY (http://
creativecommons.org/licenses/by/4.0/).
Introduction
Cancer is one of the leading causes of death worldwide and a source of severe social disruption. The preva-lence of cancer is rising all the time, thus resulting in increaseduseofchemotherapeuticagents.1Oneimportant
chemotherapeutic agent is platinum-containing cisplatin (cis-diamminedichloroplatinumII).Thismoleculeconsistsof adivalentPtcentralatomtogetherwithfourligandsof cis-positionedpairsofchlorineatomsor aminegroups.1Since
itsdiscoveryithasbeenemployedtotreatvarioustypesof cancer.However,side-effectssuchasnephrotoxicity, neuro-toxicityandototoxicityseverelylimititsuse.Nephrotoxicity canbepreventedbymeansofsalinehydrationand manni-toltherapy,butneurotoxicityandototoxicitystillrepresent restrictingfactors.
Ototoxicity refers to inner ear damage characterized by transient or permanenthearing loss. Cisplatin-induced ototoxicitygenerallymanifestsintheformofirreversible, progressive, and bilateral sensorineural hearing loss,
particularly at high frequencies. This loss may rarely be unilateral or asymmetrical. The degree of hearing loss is dose-dependentand is alsorelated to the frequency and method of application.2 Since this clinical manifestation
resultsin severe disturbancesof qualityof life andsocial communication,variousagents havebeenusedtoprevent cisplatin-induced ototoxicity. The most desirable charac-teristic of such agents is that they should be capable of reducingtheototoxiceffectofcisplatinwhilepreservingits antitumoreffectiveness.VitaminE,N-acetylcysteine, dex-amethasone, resveratrol and whortleberry have all been employedfor thispurpose.3---7 However,none hasemerged
asindisputablyprotectiveagainstcisplatin-induced ototox-icity. There is also no agent currently recommended for routineuse.
Eugenol is a yellowish oily fluid obtained from the clove plant (Eugenia caryophyllata), which is endemic in various parts of the world. The oil has been shown to exhibitantimicrobial,analgesic,anti-inflammatory, antiox-idant,anti-mutagenic,andanti-carcinogeniceffects.8 The
antioxidant activity of eugenol compounds derives from theirmethoxy-phenolicstructure.9Studieshaveshownthat
theantioxidantactivity ofeugenolprotectsagainst oxida-tive tissue damage in a range of experimental models. Eugenolisnon-toxic,non-mutagenic,andnon-carcinogenic, and is considered safe by the U.S. Food and Drug Administration.10
Ourscanoftheliterature revealednoprevious studies of the effect of eugenol on ototoxicity induced by cis-platin. The aim of this study was therefore to perform a biochemical, functional and histopathological investiga-tionofwhethereugenolexhibitsaprotectiveeffectagainst cisplatin-inducedototoxicity.
Methods
Thestudywasapprovedbythelocalethicalcommitteeof AtaturkUniversity(n◦E.1700283394-11:142).Theprinciples oftheCareandUseofLaboratoryAnimalsGuidelinewere appliedthroughout thestudy.Twenty-four femaleSprague Dawleyratsweighing250---300gwerehousedinspecialcages withadlibitumaccesstochow andwaterthroughout the experiment.Thecageswerekeptinaroomequippedwitha fullyautomaticlightingsystemsettoa12hlight/12hdark cycle.Temperature wasfixed at 23±2◦C andhumidity at 45%---50%.
Eugenol was supplied by the Sigma---Aldrich Chemical Co.,andcisplatinfromKocakFarmaCo.(Kocak, Istanbul, Turkey).Atthebeginningofthestudy,ananesthesiamixture was prepared with ketamine hydrochloride (Ketasol 10%, RichterPharma Ag, Wels, Austria) and xylazine (Alfazyne 2%,AlfasanInternationalBV,Voerden,Netherlands)atdoses of 40mg/kg and 5mg/kg, respectively and administered intraperitonealytoallratsfor otoscopicexamination.The DistortionProductOtoacousticEmissions(DPOAE)test was thencarriedoutusingappropriateprobes.The24ratswere thenrandomized intofourgroups of six animalseach. An experimentalototoxicitymodelwasestablishedwitha sin-gle Intraperitoneal (ip) dose of 15mg/kg cisplatin in the cisplatin group (Group Cis). The eugenol group (Group E) received 100mg/kg eugenol ip on five consecutive days. Thecisplatin+eugenolgroup(GroupCE)received100mg/kg eugenol ip for five consecutive days. On the third day of thestudy,theseratsalsoreceivedasingleip infectionof 15mg/kgcisplatin.The controlgroup(GroupC)wasgiven 8mL/kg/day ip saline solution for five days. The second DPOAEtestwasapplied24hafterthefinaldrug administra-tion,againunderketamine+xylazineanesthesia.Allanimals weresacrificedafterthetest.Thecochleasofallratswere removedafterthedissectionofthetemporalbones.Theleft cochleasweresetasideforhistopathologicalstudyandthe rightcochleasforbiochemicalanalysis.
DPOAEmeasurement
ThetestwasperformedusinganOtometricsMadsenCapella device after the otoscopic examination of the rats under generalanesthesia. Priortoeach test,the probewas cal-ibratedwithanautomated system andplacedagainstthe external ear canal. DPOAE measurement of the bilateral ears was then carried out in a silent environment using
appropriate probes. Measurements consisted of DPgrams. PrimarystimuliintensitiesofL1=65dBandL2=55dBwere specified.Twofrequencies,f1/f2=1.22,wereemployedin orderto elicitthe most powerfulresponse. DPgram mea-surementswerecarriedoutat8frequenciesbetween2002 and10000Hz. Signal Noise Ratio(SNR)values equaltoor greaterthan3dBwereassumedtorepresentpositivity.
Biochemicalanalysis
Followingremovaloftherightcochleas,thesewerewashed withphysiologicalsalinesolutionandstoredat−80◦Cuntil analysis. On the day of study, each cochlear tissue was groundinliquidnitrogenusingacommercialTissueLyserII grindingjarkit(Qiagen,Hilden,Germany).Alltissue spec-imens were homogenized in KCl buffer 900L 0.1M KCl per0.1gtissue.Supernatantwasobtainedbycentrifuging thehomogenate at4◦Cfor30minat 13000rpm. Malondi-aldehyde(MDA)levelsinthehomogenateweredetermined spectrophotometricallywiththepresenceofthiobarbituric acid,usingthemethoddescribedbyOhkawaetal.11Values
wereexpressed asnmoL/mL. Superoxide Dismutase(SOD) activitywasdeterminedusingthemethoddescribedbySun etal.12andwasexpressedasU/mL.Glutathioneperoxidase
(GPx)activitywasalsodeterminedspectrophotometrically followingthetechniquedescribedelsewherebyPagliaand Valentine,13 andconcentrationswereexpressedasIU/L.
Histopathologicalanalysis
Forhistopathological evaluation, the leftcochlear tissues werefixedin10%formalinsolutionfor48h.Aftersoftening for96---120hinOsteosoft(Merc,HC313331,Germany) solu-tionfordecalcification,theywerewashedinrunningwater for24h.Beforebeingembeddedinparaffinblocks,tissues were passed througha seriesof alcohol andliquid paraf-fin. Sections 4m in thickness were collected fromeach block and transferred onto glass slides. Preparates were nextstainedwithMasson’strichromeandphotographswere taken. The presence of lesionswas evaluatedunder light microscopyonasemi-quantitativescaleofnone(−),slight (+), moderate (++)and severe (+++).In semi-quantitative histopathologicalexamination; hyperemiain thestria vas-culariswasclassifiedasavesseldiameter≤0.4---1m (−), 1---2m(+),3---5m(++),and≥5m(+++).Degenerationin thespiral gangliawasdefined asa degeneratedcell num-berof0(−),3---5(+),6---10(++),and≥10(+++).Structural impairmentanddecreasedcellnumbersinouterhaircells weredefinedasanimpairedcellnumberof0(−),3---5(+), 6---10(++),and≥10(+++).
Immunohistochemicalexamination
For immunoperoxidase analysis, adhesive slides (poly-l-lysine) were used.After the transferof sections tothese slides, theywerepassed throughxylolandalcohol series, prior to deparaffinization and drying. They were then washedindistilledwaterfor5min.Sectionswereexposed toheatinamicrowave,fourtimesfor5mineach,in anti-gen retrieval (citrate buffer, pH 6.1) in orderto prevent
core antigen masking. They were then removed from the microwaveandlefttocoolfor30minatroomtemperature. Followingtheseprocedurestheywerewashedwithdistilled wateranddried.Aglasspenwasthenusedtodelineatethe areassurroundingthesectionofinterest.Theywerewashed for 5min inPhosphate Buffer Solution(PBS) which have a pHvalueof7.2andkeptin3%H2O2for10minfor inactiva-tionofendogenousperoxidase.FollowingwashinginPBSfor 5---10min,specimenswereincubatedfor5minwithprotein blockcompatiblewithallprimaryandsecondaryantibodies forthepreventionofnon-specificbackgroundstaining. Fol-lowingincubation, the excessblocksolutionwasremoved fromtheslidesurfaces.PrimaryantibodiesPBSinthe con-trolgroupand8-hydroxy-2-deoxyguanosine(8-OHdG)were placedontothesideswithouttheapplicationofawashing procedure.Dependingontheprimaryantibodyconcerned, they were then kept either for 1h at room temperature or overnight at +4◦C. They were washed twice for 5min withPBSandincubatedfor10---30minatroomtemperature withbiotinylatedsecondaryantibodies.Sectionswereagain washed withPBS and kept in streptavidin---peroxidase for 10---30min.TheyweresubsequentlywashedagainwithPBS inthesamemanner.Inthenextstage,3-3diaminobenzidine (DAB) chromogen wasdropped onto the sectionsand left insitufor5---10minforchromogenabsorption.Background staining was provided by keeping the specimens 1---2min inMayer’s hematoxylin.The specimenswerethen washed undertap waterandsubsequently passed throughalcohol andxylolseries,beforebeingcoveredandsubjectedto anal-ysisbymeansofalightmicroscope(LeicaDM1000).Immune positivitywasgradedasnone(---),slight(+),moderate(++) orsevere(+++)whichwasdefinedasapositivecellnumber of0(−),3---5(+),6---10(++),and≥10(+++).
Statisticalanalysis
StatisticalanalysiswasperformedbyusingSPSS17.0 soft-ware. Analysis of DPOAE measurements and biochemical valueswasperformedusingtheShapiro---Wilktestand his-tograms. One-way ANOVAwas usedfor comparisons when normaldistributionwasestablished,andtheKruskal---Wallis test in case of non-normal distribution.Analysis of varia-tions among data elicited by means of semi-quantitative histopathologicalexaminationwasalsoperformedusingthe Kruskal---Wallistest.TheTukeytest wasemployedforpost hocanalysiswhenANOVAwasappliedandtheMannWhitney
Utestfortwo-waycomparisonswhensignificantdifferences weredeterminedwiththeKruskal---Wallistest.Foralltests,
p<0.05wasregardedasstatisticallysignificant.
Results
Biochemicalanalysisoftheratcochleasrevealeda signifi-cantincreaseinMDAlevelsinthecisplatingroupcompared to the control group, while significant decreases were observedinSODandGPxactivities.Thisshowsthatcisplatin causes oxidative stress in the cochlea and that oxidative stress maybeoneof themechanismsinvolved in ototoxi-city.ThelevelsofMDAaswellastheactivitiesofSODand GPx were similarin theeugenol and control groups. MDA levelsweresignificantlylowerinthecisplatinpluseugenol
Biochemical results GPx (IU/L) SOD (IU/L) MDA (nmol/mL) 0 0.1 aa a b b b a a a a a a 0.2
Group CE Group E Group C Group Cis
0.3 0.4 0.5 0.6 0.7
Figure1 Biochemicalresults.Thelettersneareachbar indi-cate the statistical comparison. Different letters indicate a significantdifferenceatthep<0.05level,whilethesame let-tersindicatenosignificantdifference.
comparedtothecisplatinonlygroup.Wealsodeterminedno significantdifferenceinMDAlevelsofcisplatinpluseugenol group compared tothe control group. Similarly, SOD and GPxactivitiesincisplatinpluseugenolgroupwerefoundto besignificantlyhigherthancisplatinonlygroup,butno sig-nificantdifferencewasdeterminedbetween cisplatinplus eugenoland controlgroups. Thesefindings show that cis-platincauses oxidative stressin the ratcochlea,but that administrationofeugenolpreventsoxidativestress.The bio-chemicalanalysisresultsareshowninFig.1.
NosignificantvariationwasdeterminedamongSNR val-uesinallDPgramsonthefirstdayofthestudy.Therewas alsonosignificantdifferencebetween SNRvaluesof right and left ears in all groups on days 1 and 6. Comparison ofthetest resultsonthesixthday withthoseofthefirst dayrevealedasignificantdecreaseinSNRvaluesinthe cis-platingroup. Cisplatin group SNRvalues onthe sixth day were significantly lower than those of the control group. Thisshowsthatcisplatinleadstofunctionaldamageinthe ratcochlea.Therewasnosignificantdifferencebetweenthe SNRvaluesoftheratstreatedwitheugenolandeitherthe controlgrouporthefirstdayvalues.Nosignificant differ-encewasalsoobservedbetweentheSNRvaluesoftherats administeredcisplatintogetherwitheugenolandeitherthe controlgroupor thefirstday values.Inaddition,theSNR valuesof thecisplatinpluseugenol groupwere significan-tly higher than those of the rats receiving cisplatin only. These findings indicate that with its ototoxiceffect, cis-platinreduced SNRvalues at all frequencies butthat the administrationofeugenolincombinationwithcisplatin pre-ventedthisdecreaseinSNRvalues.TheDPOAEresultsare summarizedinFig.2.
Histopathologicalexaminationrevealednormal architec-tureintheorganofCorti,striavascularisandspiralganglia inboth thecontrol andeugenol groups (Fig.3A---C). How-ever,thecisplatingroupexhibitedsignificanterosioninthe striavascularistogetherwithdegenerationandan edema-tousappearanceintheconnectivetissuelayerofendothelial cells.Spiralganglionnervecellswerealsoseverely degen-erated.Impairedmorphologywasobservedintheouterhair cellsoftheorganofCorti,andthesecellshadalso significan-tlydecreasedinnumbersduetodegenerationandnecrosis (p<0.05)(Fig.3B).Significantlyfewerofthesedegenerative andnecroticfindingswereobservedtheCis+eugenolgroup
DPOAE results 40 35 30 25 20 15 10 * * * * * * * * 5 0 2002 Hz 2500 Hz 3154 Hz 4004 Hz *: Statistically significant Frequencies
All groups (Day1) Control (Day 6) Cisplatin (Day 6) Eugenol (Day 6) Cis+Eugenol (Day 6) 5000 Hz 6299 Hz 7998 Hz 10000 Hz
DP
O
AE amplitude (dB SPL)
Figure2 DPOAEresults.
(p<0.05),andeugenolappearedtoprovidesignificant pro-tectionagainstthem(Fig.3D).Histopathologicalfindingsin cochleartissuesfromthedifferentgroupsaresummarized inTable1.
8-Hydroxy-2-deoxyguanosine(8-OHdG)stainingwas per-formedinordertoidentifyoxidativeDNAdamagefromthe immunohistochemicalperspective.No8-OHdG immunopos-itivitywasobservedineitherthecontroloreugenolgroups (Fig.4A---C).Severeimmunopositivitywasdeterminedin spi-ralganglioncellsandouterhaircellsinthecisplatingroup (Fig.4B).IntheCis+eugenolgroup,slight8-OHdG expres-sionwasobservedinspiralganglioncellsandouterhaircells (Fig.4D).Therewasasignificantdecreasein8-OHdG expres-sionsbetweenCis+Eugenolandcisplatingroups(p<0.05).
Figure3 Histopathologicappearanceofthecochlea,H&E,bar:50m.(A)Controlgroup.Normalhistopathologicalstructureofthe cochlea(arrow,striavaskularis;arrowhead,outerhaircells).(B)Cisplatingroup.Hyperemia,degenerationanderosioninthestria vascularis(arrows),severedecreaseinthenumberofouterhaircells(arrowheads).(C)Eugenolgroup.Normalhistopathological structureofthecochlea(arrow,stria vaskularis;arrowhead,outerhair cells).(D)Cisplatin+eugenolgroup:mildhyperemiain thestriavaskularis(arrows),normalhistopathologicalstructureofouterhaircellswithmilddecreaseinthenumberofthesecells (arrowhead).
Table1 Histopathologicfindingsofthecochlea.
Controlgroup Eugenolgroup Cisplatingroup Cis+eugenolgroup Hyperemia,degenerationand
necrosisinstriavaskularis
--- --- +++ ++
Degenerationinspiralganglia --- --- +++ +
Decreaseinthenumberofouterhair cellswithstructuraldeformation
--- --- +++ +
Figure4 Immunohistochemicalexaminationofthecochlea,IP,bar:50m.(A)ControlGroup.Negative8-OHdGexpressionin spiralganglia.(B)Cisplatingroup.Severe8-OHdGimmunopositivityinspiralganglia(arrow).(C)Eugenolgroup:negative8-OHdG expressioninspiralganglia.(D)Cisplatin+eugenolgroup:mild8-OHdGimmunopositivityinspiralganglia(arrow).
Discussion
Ototoxicity describes cellulardamage or function impair-ment occurring in the inner ear in association with any therapeuticagentor chemicalsubstance.Oneofthe ther-apeuticagentsimplicatedinototoxicityiscisplatin,widely employedasachemotherapeuticagent.Recentstudieshave reportedthatcisplatinexhibitsitscytotoxiceffectthrough bothnuclearandcytoplasmicsignalingpathways.14
Molecu-larmechanismscapableoffullyaccountingforthecytotoxic effectofcisplatinhaveyettobeidentified.However, stud-ieshaveimplicatedincreasedReactiveOxygenSpecies(ROS) production, a decrease in the antioxidant system, DNA damage,oxidative changesinproteins andincreasedlipid peroxidationinthe developmentof ototoxiceffects.15 We
employed8OHdGstaininginordertorevealoxidativeDNA damagefromtheimmunohistochemicalpespective.Severe immunopositivitywasidentifiedinspiralganglioncellsand outerhair cellsinthecisplatingroup,indicatingthe pres-enceofoxidativestress.
The cochlea has a complex structure containing sev-eralcisplatin-sensitivecelltypes.Cisplatin-inducedototoxic effectsmanifestwithdamageinouterhaircells,thespiral ligament,supportcells,andstriavascularisandspiral gan-glioncells.Thestructuremostsensitive tocisplatininthe innerearisthehaircell.Theouterhaircellsinthebasalturn ofthecochleaarethemostaffectedcells.Thisexplainswhy thehearinglossthatoccursincisplatin-inducedototoxicity particularlyinvolveshighfrequencies.Thedamagemayalso cometoinvolvelowerfrequenciesastreatmentcontinues.1
Inourstudy,histopathologicalexamination ofthecochlea
inthe cisplatingroup revealed erosionin the stria vascu-laris,degenerationandedemaintheconnectivetissuelayer inendothelial cells,severedegenerationinspiralganglion nervecells,impairedmorphologyintheouterhaircellsof theorganofCorti,togetherwithadecreaseinthenumbers ofthesecellsduetodegenerationandnecrosis.
The cochlea possesses various defense mechanisms againstototoxiceffects,themost importantof which are antioxidant enzyme systems such as SOD, catalase, and GPx.Protectivesystemssuchasadenosinereceptors;heat shockproteinsandhemooxygenase-1arealsopresent. How-ever,theprotectiveeffectofthesemechanismsislimited, andcelldeathoccurswhentheototoxiceffectovercomes theendogenousdefensesystem.16Biochemicalexamination
in our study showed elevated MDA levels and decreased GPxand SODactivities in cochlear tissuesof ratreceived cisplatin which also showed the impairment of oxidative balance.
Cisplatin-inducedototoxicity exhibitsclinical effectsin theearlyphase.Inclinicalterms,ototoxicitymanifestswith symmetrical,bilateral, progressive,irreversibleand dose-dependent sensorineural hearing loss findings. DPOAE, an objective,rapidandnon-invasivetechnique,isatestwidely usedtoshowthishearinglossinexperimentalstudies.Since DPOAEreflectstheactivityofouterhaircells,itiseffective eveninshowing damageoccurringintheearly stages.We usedtheDPOAEtestinthisstudytoshowfunctionaldamage arisinginthecochlea.Thetestwasappliedtwicetoallrats, ondays1 and6. At all frequencies, theSNR valueswere significantlylowerinratsreceivedcisplatinthanbothday1 SNRvaluesandthoseofthecontrolgroup.Thisshowsthat
withitsototoxiceffect,cisplatincausedhearinglossatall frequenciesstudied.
Sinceototoxicityisthemost importantside-effect lim-itingthe useof cisplatinin clinicalpractice,studies have investigatedthe potentialotoprotectiveeffectsof various drugs.An ideal otoprotective agentmust provide reliable protectionwhile notcompromising theantitumoral effect ofcisplatin.Itmust alsohaveminimal side-effectsandbe easytoapply.The agentsthat havereceivedthegreatest attentioninthatcontextareantioxidantsthateliminatethe effectsofROS.Thepresent studyinvestigatedthe otopro-tectiveeffectofeugenol,withknownpowerfulantioxidant properties.
Eugenol is a yellowish oily fluid obtained from the clove plant which grows in various parts of the world. Theoilhasbeenshowntoexhibitantimicrobial,analgesic, anti-inflammatory,antioxidant,antimutagenic,and anticar-cinogenic effects.8 It was shown that, usage of eugenol
togetherwithcisplatin donothaveanegativedrug inter-action. Thus, in a study performed by Islam et al., it was suggested that, in the combination of eugenol and cisplatin,eugenolpotentiatestheanticarsinogenic proper-tiesof cisplatin by inhibitingNF-B pathway.17 Rao etal.
investigatedtheeffectofeugenolagainstcisplatin-induced nephrotoxicityanddeterminedthatitprotectsagainst cyto-toxicity through free radical scavenging activities.18 Binu
etal.investigatedthetherapeuticeffectofeugenolagainst arsenic-inducedcardiotoxicity. They reportedthatarsenic increased levels of MDA, a marker of lipid peroxidation, through its cytotoxic effect, and reduced levels of GSH, themostimportantnon-enzymaticendogenousantioxidant, in heart tissue. Additionally, they observed that eugenol administeredorallyat5mg/kgincreasedtissueantioxidant levels, regulated membrane peroxidation and normalized heartrate.Atthesametime,theyreportedthatMDA lev-els decreased as a result of eugenol therapy, while GSH andGPxlevelsincreased.Withtheseproperties,they sug-gestedthateugenolisacytoprotectiveagentthatprevents lipid peroxidation and that protects enzymatic and non-enzymatic antioxidant systems.19 Said et al. investigated
the protective efficacy of eugenol in aluminum-induced braindamageanddescribedeugenol asapotential neuro-protectiveagentdue toitsantioxidantandanti-apoptotic characteristics.20 Herreraetal.investigatedthe
effective-nessofeugenolagainstgingivalinflammationandreported anti-inflammatoryeffectswhenadministeredatlowdoses.21
In the present study, we observed an increase in MDA levels in cochlear tissue in cisplatin-induced ototoxicity and a decrease in GPx and SOD activities. Eugenol also reversedthiseffect,preventingoxidativestressand exhibit-ingotoprotectiveactivity.Wealsodeterminednosignificant variationfollowingtheDPOAEtestbetweentheSNRvalues ofratsreceivingeugenoltogetherwithcisplatinandcontrol groupSNRvaluesandday1SNRvalues.Thesefindingsshow thateugenolpreventscisplatin-relatedcochleardamage.
Conclusion
Inconclusion, ourfindingsdemonstrate, for thefirst time inthecurrentliterature,thateugenolmayprotectagainst ototoxicity by raising levels of antioxidant enzymes and
lowering those of oxidant parameters. We suggest that eugenolmaybeasuitabledrugoptionagainstcisplatin oto-toxicity.
Ethical
approval
Thisstudy wasperformedinaccordancewiththePHS Pol-icyonHumaneCareandUseofLaboratoryAnimals,theNIH GuidefortheCareandUseofLaboratoryAnimals,andthe AnimalWelfareAct(7U.S.C.etseq.);theanimaluse proto-colwasapprovedbytheInstitutionalAnimalCareandUse Committee(IACUC)ofAtaturkUniversity.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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