Rev. Bras. Anestesiol. vol.67 número1

REVISTA

BRASILEIRA

DE

ANESTESIOLOGIA

www.sba.com.br

SCIENTIFIC

ARTICLE

Comparisons

of

the

effects

of

the

sevoflurane

and

propofol

on

acute

ischemia

reperfusion

and

DNA

damages

in

rabbits

Sema

Oncul

_,

_Lale

_Karabiyik,

_Erdem

_Coskun,

_Ela

_Kadioglu,

_Ozlem

_Gulbahar

GaziUniversityFacultyofMedicine,DepartmentofAnesthesiologyandIntensiveCare,Ankara,Turkey

Received29May2015;accepted17August2015 Availableonline12April2016

KEYWORDS

Genotoxicity; Oxidativestress; Propofol; Sevoflurane;

Ischemiareperfusion injury

Abstract

Backgroundandobjectives: Theaimofthisstudywastocomparetheeffectsofsevoflurane

andpropofolanesthesiaonoxidativeDNAdamagethatoccursinlow-extremityischemiaandis

causedbytourniquetapplication.

Methods:FourteenNewZealandrabbitswererandomlyallocatedintotwoequalgroups.Group

S(n=7)receivedsevoflurane(2.5---4percent)inhalationandGroupP(n=7)receivedapropofol

infusion(1---2mg·kg−1_·min−1_{),afterwhichapneumatictourniquetwasplacedontherightlower}

extremity.Bloodsampleswerecollectedpriortotourniquetplacement(baseline),120minafter

ischemia,15minafterischemiaand120minutes(min)afterischemia.Malondialdehyde(MDA)

levelswereanalyzedtodeterminelipidperoxidation,andsinglecellgelelectrophoresis(SCGE)

wasusedtodetermineDNAdamage.

Results:At15minafterischemia,theMDAlevelsinGroupP(8.15±2.61␮M)werehigherthan

baseline(6.26±3.19␮M,p=0.026)andGroup S(4.98±0.77␮M,p=0.01).DNA damagewas

similarinbothgroups,althoughDNAdamagewashigherthanbaseline(tailmoment0.63±0.27,

tailintensity3.76±1.26)inGroupPatthe15thminuteofreperfusion(tailmoment1.05±0.45,

p=0.06; tail intensity5.33±1.56, p=0.01).The increase in tailmoment andtail intensity

returnedtonormallevelsinbothgroups2hoursaftertheterminationofischemia.

Conclusion: Giventhatoxidativestress andgenotoxiceffectdisappearinthelatestages of

reperfusion,weconcludethatneithersevofluranenorpropofolcanbeconsideredsuperiorto

theotherinanesthesiapracticesforextremitysurgeriesinvolvingtheuseofatourniquet.

©2016PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeAnestesiologia.

ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

∗_{Correspondingauthor.}

E-mail:[email protected](S.Oncul). http://dx.doi.org/10.1016/j.bjane.2015.08.013

36 S.Onculetal.

PALAVRAS-CHAVE

Genotoxicidade; Estresseoxidativo; Propofol;

Sevoflurano; Lesãodeisquemia reperfusão

Comparac¸õesdosefeitosdesevofluranoepropofolsobreisquemia-reperfusãoaguda edanosaoDNAemcoelhos

Resumo

Justificativaeobjetivos: Oobjetivodeste estudofoi compararos efeitos daanestesiacom

sevofluranoepropofolsobreodanooxidativoaoDNAqueocorrenaisquemiadeextremidade

inferioreécausadapelaaplicac¸ãodetorniquete.

Métodos: Quatorzecoelhosdarac¸aNovaZelândiaforamalocadosaleatoriamenteemdois

gru-posiguais.GrupoS(n=7)recebeuinalac¸ãodesevoflurano(2.5---4%)eGrupoP(n=7)recebeu

perfusãodepropofol(1---2mg·kg−1_·min−1_{),logoapósumtorniquetepneumáticofoicolocadona}

extremidadeinferiordireita.Amostrasdesangueforamcoletadasantesdacolocac¸ãodo

torni-quete(fasebasal),após120minutosdeisquemia,15minutosapósaisquemiae120minutosapós

aisquemia.Osníveisdemalondialdeído(MDA)foramanalisadosparadeterminaraperoxidac¸ão

delipídios,eelectroforeseemgeldecélulaúnica(EGCU)foiusadaparadeterminarodanoao

DNA.

Resultados: Aos 15minutos apósa isquemia, osníveis deMDA no Grupo P (8,15±2,61␮M)

foramsuperioresaosdafasebasal (6,26±3,19␮M,p=0,026)edpGrupo S(4,98±0,77␮M,

p=0,01).Odanocausadoao DNAfoi semelhantenosdoisgrupos,emboratenha sidomaior

quenafasebasal(momentodacauda0,63±0,27,intensidadedacauda3,76±1,26)noGrupo

Pno15minutodereperfusão(momentodacauda1,05±0,45,p=0,06;intensidadedacauda

5,33±1,56,p=0,01). Oaumentonomomentodacaudae intensidadedacaudavoltouaos

níveisnormaisnosdoisgruposduashorasapósotérminodaisquemia.

Conclusão:Comooestresseoxidativoeoefeitogenotóxicodesaparecemnosestágiosfinaisda

reperfusão,concluímosquenãohásuperioridadetantodesevofluranoquantodepropofolem

práticasdeanestesiaparaprocedimentoscirúrgicosdeextremidadesqueenvolvemousode

torniquete.

©2016PublicadoporElsevierEditoraLtda.emnomedeSociedadeBrasileiradeAnestesiologia.

Este ´e um artigo Open Access sobuma licenc¸a CC BY-NC-ND(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Proximal tourniquets are often used in limb surgeries to provideabloodlessoperatingfield.Whenoxygenis reintro-duced intoischemic tissueafter tourniquetdeflation,the existence of massive free oxygen radicals causes peroxi-dationofmembrane lipids.Thisprocesstriggersoxidation of the polyunsaturated fatty acids, destroying membrane structuresandproducingtoxicmetabolitessuchas malondi-aldehyde(MDA).1,2_{Thisprocessplaysasignificantroleinthe} pathogenesisofreperfusioninjuries.MDAisalowmolecular weightaldehydeandanintermediateproductalipid perox-idation.Ithasoftenbeenusedasamarkeroffreeradical formation.

˙Ischemia---reperfusion (I/R) induced oxygen radical for-mationcausesoxidativeDNAdamageandplaysasignificant roleinthepathogenesisofreperfusioninjuries.3,4_Alimited amount of information exists on oxidative DNA damage intourniquet-induced reperfusioninjuries, although anes-theticdrugsandsurgery-relatedstressareknowntocause cellulardamage.5

Sevoflurane and propofol are volatile and intravenous agentsthatareincommonuseinthefieldanesthesia.Some clinical andexperimental studies without I/R have shown thatsevofluranecausesreversibleDNAdamage,5,6_although

differentinvivoandinvitrostudieshaveshownthat propo-foldecreasesDNAdamage,orcausesnoDNAdamage.7---9

Genotoxic assays are commonly used to monitor DNA damage.The SCGE(cometassay)hasbeenwidely usedin bothinvitroandinvivostudies,becauseitisasensitiveand simplemethodologyformeasuringsingle-strandand double-strandbreaks,alkalilabilesites,oxidativelesionsandDNA repair.10,11

Thisstudycomparestheeffectsofsevofluraneand propo-folonDNAdamageinatourniquet-inducedlowerextremity ischemiamodel.Forthepurposeofthestudy,thesurgical stressfactorwaseliminated,MDAlevelsweremeasuredto determinelipidperoxidation,andSCGEwasusedto deter-mineDNAdamage.

Methods

Figure1 Infusionofpropofol,intravenousandintraarterial catheterization.

Figure2 ApplyingthemaskofthegasmixtureandBIS probe.

Arterialandvenouslineswereinsertedthroughtheearin alltestsubjectsduringsamplingandforisotonicsaline infu-sion,respectively. Baselinebloodsamples weretakenand theinvasivearterialbloodpressurewasmonitored(Fig.1). Hemodynamicparametersofinvasivebloodpressure,heart rate,respiratoryrate,periphericoxygensaturation(SpO2) (NihonCohden,Life ScopeBSM-5135K, Japan)and Bispec-tralIndex(BIS)Monitoring(BispectralIndexMonitorModel 2000,AspectMedicalSystems,Inc.,Newton,MA,USA)were recorded at 15min intervals during the procedure. After hairremoval,pediatricprobeswereusedforBISmonitoring (AspectMedical Systems,Inc.,Norwood,USA).During the study,theBISwaskeptat50±10percentand5mL·kg−1_·h−1 isotonicsalinewasinfusedduringtheprocedure.

The rabbitswere randomly allocated to twogroups of seven.Anesthesia wasinduced withthe inhalation of7---8 percent sevoflurane in Group S, and with the infusion of 1---2mg·kg−1_·min−1 _{propofolin GroupP(Figs.1 and2).An} adequate levelofinduction wasdeterminedbythe disap-pearanceofeyemovementsandcorneareflexinadditionto maintainingBIS<60percent.Theanesthesiawasmaintained inGroupSthroughtheinhalationof2.5---4percent sevoflu-raneandthroughtheinfusionof1---2mg·kg−1_·min−1_propofol inGroupP.Agasmixturewasadministeredbyusingaface

Table1 MDAlevels(␮M)[mean±SD(min---max)].

Time(min) GroupS

(n=7)

GroupP

(n=7)

p-value

Baseline 5.10±1.94

(1.53---7.24)

6.26±3.19

(3.32---13.05)

0.425

BTD 5.40±1.36

(3.43---7.12)

8.32±3.86

(4.55---14.28)

0.083

ATD15 4.98±0.77b

(4.22---6.12)

8.15±2.61a

(5.45---13.39)

0.01

ATD120 6.40±3.00 (2.76---12.16)

7.09±2.91 (3.88---11.37)

0.0672

BTD, beforetourniquetdeflation;ATD,aftertourniquet defla-tion.

a _{p=0.026,comparisonwiththebaseline.} b _{p=0.01,comparisonwithGroupP.}

mask at the flow rate of 4L·min−1 _{(Dameca, Denmark).} Spontaneousrespiration wasmaintained toachieve an O2 saturation of ≥95 percent. Both groups were completely expressedbyrubberbandagefromtherightextremity,anda tourniquetwasplacedbyapplying200mmHgpressure (Pedi-atriccuffno2,DeRoyal,Powell,TN,USA).Tourniquetwas appliedfor120min.

Bloodsampleswerecollected asthebaselinesamples, 1minbeforetourniquetdeflation(BTD),and15and120min after tourniquet deflation (ATD 15 and ATD 120, respec-tively).

Malondialdehydedetermination

One of the most common methods used to indicate the presence of free oxygen radicals is the determination of MDA as an index of lipid peroxidation. During the study, thedecreaseinthe MDAlevelsassessedoverthe thiobar-bituric acid (TBA) reactions was found to be an indirect indicationofadecreaseoffreeoxygenradicals.Theplasma MDAlevelwasmeasuredbyimplementingthecolorimetric methodbasedontheTBAreactionwiththeMDA.14

SCGE(cometassay)method

ThecometassayfollowedtheprotocolsdescribedbySingh etal.andTiceetal.withsomemodifications.15,16_TheSCGE methodexaminestheDNAdamageandrepairmechanisms under different experimental conditions. Normal lympho-cytes have around, bright-lookingnucleus in the middle, whichislessdenseonthesides.Itgainsanirregularlyedged appearancealongwiththebeginningofthemigrationofDNA fragmentsoutsidethenucleus.Lymphocytestaketheshape ofacometasthedamageincreases,hencethename‘comet assay’.Dependingontheseverityofthedamage,the micro-scopicappearanceofthecellextendsfromthecentertothe periphery.Thetailintensity,intermsoffluorescence,tail

38 S.Onculetal.

0 2 4 6 8 10 12 14

BTD

Baseline ATD 15 ATD 120

Group S Group P ∗

MDA

levels

(

µ

M)

Figure3 MDAlevels(mean±SD)(*p=0.026,comparisonwith thebaseline;†_{p=0.01,comparisonwithGroupP.BTD,before}

tourniquetdeflation;ATD,aftertourniquetdeflation).

Statisticalevaluation

Data analysis was performed by using Statistical Package for the Social Sciences (SPSS) for Windows, version 15.0. Time-relatedchangeswithingroupsandinaccordancewith controlwerecomparedbyPairedttestandWilcoxon-Xtest, andcomparisonsbetweengroupswereperformedby imple-mentingtheStudent’sttestandtheMann---WhitneyUtest. Datawasexpressedasmean±SDandp<0.05wasaccepted assignificant.

Results

MDA

MDAlevelswerenotfoundtobestatisticallysignificant dif-ferent compared with baseline samples in Group S. The increase in the MDA levels in Group P at ATD 15 was foundtobehigherthanthebaselinevalues(8.15±2.61␮M; p=0.026). At the same time, this value was significantly higherthaninGroupS(4.98±0.77␮M)atATD15(p=0.01)

(Table1,Fig.3).

DNAdamageassessment

Therewasnosignificantdifferenceinthebaseline,BTD,ATD 15and ATD120 levelsbetween the groups (Table2). The increaseinthetailmoment(p=0.031)andthetailintensity (p=0.029)inGroupSatBTDwerefoundtobestatistically significantcomparedwithbaselinesamples(Table 2).The increasein thetailmoment(p=0.006)andthe tail inten-sity(p=0.01)inGroupPatATD15werefoundtobehigher thanthebaselinevalues(Table2).Therewasnosignificant differencebetweenGroupSandGroupPatalltimeperiods.

Discussion

Ourfindingsindicatethatneithersevofluranenorpropofol issuperiortotheotherintermsofpreventingtheoxidative stressandgenotoxicitythatemerge intourniquet-induced I/Rinjuries.

A proximaltourniquet is often used in limb surgery to provideabloodlessoperatingfield.Tourniquetapplication

causes metabolic changesthat depend ontheduration of tourniquet inflation and anesthetic technique.17 _{The safe} rangeofinflationtimeisacceptedtobebetween1and3h. Forthisreasonweoptedforatourniquetperiodof2hinthe presentstudy.

˙Ischemia---reperfusionresults in thegenerationof toxic reactive oxygen species (ROS) in the organs. ˙Ischemia reduces the activity of cellular defence enzymes against ROS,andreperfusionortheintroductionofoxygenfurther disturbs the delicate balance of oxidants/antioxidants.18 MeasurementofMDAlevelsisthemostsensitivemarkerto determine thedegreeoflipidperoxidation.19,20 _Sensitivity reaches its highest level between the fifth and twentieth minutes following tourniquet deflation.2 _{In our study the} increase in the MDAlevels wasfound to behigher in the propofol group 15min following tourniquet deflation than in the sevoflurane group. It was observed that the lipid peroxidationcouldbebettersuppressedinthesevoflurane group duringthe early reperfusionperiodascomparedto thepropofolgroup.

Budic etal. have demonstrated that total intravenous anesthesia with propofol and regional anesthesia tech-niques provide better antioxidant defence and reduce endothelialdysfunctionthangeneralinhalationalanesthesia withsevofluraneduringtourniquetapplicationinpediatric extremity surgery.19,20 _{Arnaoutoglou et al. have reported} thatpropofolmayhave antioxidantpropertiesin orthope-dic surgery requiring tourniquet application, but the use of sevoflurane needs further study.21 _{Some authors have} demonstrated that plasma concentrations of propofol are abletoinhibitlipidperoxidationintherangethatis clini-callyusedinanesthesia.22_{Brazetal.,however,usedclinical} doses of propofol (plasma concentration: 2---4␮g·mL−1) in elective surgeries without I/R,and showed that propofol does not decrease lipid peroxidation.7 _{These and several} other authors believe that only higher concentrations of propofolcouldshowfreeradicalscavengingactivity.7,23 Sim-ilartothestudybyBrazetal.,wefoundthatpropofoldid notdecreaselipidperoxidation;contrarytoother studies, however, we did notperform any surgery, andperformed ischemiafor120min.Althoughplasmapropofollevelswere not measured in our study, increased MDA levels were found at the 15th minute of reperfusion in the propofol group.

˙Ischemia---reperfusioninduced oxygenradicalformation causes oxidativeDNAstrandbreaksand playsasignificant roleinthepathogenesisofreperfusioninjuries.Thesingle cellgelelectrophoresis(cometassay)isasimple,sensitive, andrapidmethodforestimatingDNAdamage.10 _Numerous studies have shown that I/R-induced DNAdamage can be investigatedinhumanperipheralleucocytesusingthe mod-ifiedalkalinecometassay.3,24_{Afterischemia,thegenotoxic} damagewasdetectedinhumanperipheralleucocytes,and itleadsespeciallytothereleaseofsubstantialamountsof oxygen radicalsand other reactive agents.10 _{In ourstudy,} weusedthismethodtoanalyzetheDNAdamagecausedby anestheticagents.

reperfusion

and

DNA

damage

during

anesthesia

39

Table2 Tailmoment,taillengthandtailmoment,in50cells[mean±SD(min---max)].

Time(min) GroupS(n=7) GroupP(n=7) p-value

Tailmoment Taillength Tail

intensity

Tailmoment Taillength Tail

intensity

Tail moment

Tail length

Tail intensity

Baseline 0.75±0.27

(0.36---1.17)

25.19±1.88

(22.93---28.81)

4.55±1.37

(2.43---6.76)

0.63±0.27

(0.29---1.17)

25.25±3.55

(22.10---32.87)

3.76±1.26

(1.73---6.07)

0.422 0.971 0.285

BTD 1.12±0.38a

(0.77---1.86)

30.34±9.79 (24.98---52.23)

6.14±1.48a

(4.41---8.62)

1.80±1.72 (0.58---5.32)

35.10±18.05 (23.66---71.39)

7.53±4.23 (3.53---14.43)

0.328 0.551 0.427

ATD15 1.51±1.70 (0.51---5.34)

34.46±24.90 (23.61---90.89)

7.47±5.96 (3.52---20.76)

1.05±0.45a

(0.50---1.90)

27.69±7.73 (21.77---44.36)

5.33±1.56a

(3.33---8.42)

0.506 0.505 0.377

ATD120 1.12±0.68 (0.65---2.63)

29.13±9.51 (21.99---49.89)

6.15±3.35 (3.83---13.53)

1.06±0.56 (0.51---1.89)

26.22±5.28 (21.11---36.68)

5.45±2.09 (3.38---8.85)

0.881 0.492 0.646

BTD,beforetourniquetdeflation;ATD,aftertourniquetdeflation. Therewasnosignificantdifferencebetweenthegroups.

40 S.Onculetal.

5---30minaftertourniquetrelease,andthendeclined over a 2h period,but did not return to pre-ischemic baseline values; this suggests that I/R induces genotoxic effects inhuman leucocytes,presumablyin responsetooxidative stressduringreperfusion.3

Some studieshave demonstrated thatgeneticmaterial canbedamaged withthehalogenatedanesthetics, evalu-atedbysinglecellgelelectrophoresisassay.5,6,25 _Karabiyik etal.usedthismethodtoevaluateDNAdamageinpatients who received sevoflurane and isoflurane anesthesia. The authorshave concludedthatboth agents causereversible DNA damage, but that there is no significant difference betweentheminthisregard.6_{Propofoldiffersfromvolatile} anesthetics because it does not show mutagenic effects in vitro or in vivo.7,9 _{Braz et al. reported that propofol} does notinduce DNA damagein white blood cells (WBCs) by using an alkaline version of the single cell gel elec-trophoresisassay.7_{Inaddition,propofoldidnotinducesister} chromatidexchanges(SCEs)inchildren’slymphocytes8_and didnotincreasechromosomalaberrationsincardiacsurgery patients.9

AccordingtoAllevaetal.itisdifficult toassessinvivo whetheranestheticsor surgicalstressisthemaincauseof thecell damage.These authors evaluatedgenotoxicity in patientssubjectedtosevofluraneanesthesiaanddidnotfind significantalterationsinDNAafter15minanesthesia induc-tionbeforesurgery.5_{Inarecentstudy,Brazetal.reported} thatpatientswhounderwentminorsurgeryundergeneral anesthesiawithinhaledsevofluraneandisofluraneor with intravenouspropofol didnot induceDNAstrand breaks or alkali-labilesitesinhumanperipheral lymphocytes.26 Sim-ilarlyinourstudy,weeliminatedthesurgicalstressfactor andourresultsalsocontradictedthoseofotherstudies.In ourstudy,tailmomentandtailintensitylevels(indicators of DNAdamage) increasedgradually in both groups when comparedtothebaselinelevels.Inthesevofluranegroup, DNA damage increased at the 120th minute of ischemia; incontrastDNA damagewasmoreprominent in theearly periodofreperfusion,andthisincreasewasparalleltothe increase in MDA levels. These findings may indicate the inabilityofpropofoltosuppressoxidativestressefficiently. In the sevoflurane group, increased DNA damage prior to reperfusionsupportsthegenotoxicpropertyofsevoflurane. Thedifferencebetweenthegroupsdisappearedatthe120th minuteofreperfusion.

Conclusions

ThisexperimentalI/Rstudyhasshownthatsevofluranecan control lipidperoxidation better than propofol. Propofol, mayrequire higherdoses (compared tothe clinical dose) topreventlipidperoxidation.Giventhelack ofdifference betweenthetwoanestheticsinlatestagesofreperfusion, we believe that neither sevoflurane nor propofol can be consideredsuperiorintermsofpreventingoxidative stress-relatedgenotoxicityinextremitysurgeriesinvolvingtheuse ofatourniquet.Itisknownthatallanestheticshaveharmful side-effects,andsowhatisimportantistochoosethe appro-priateagentwiththeleasttoxicside-effect.Inthisregard, it canbe said thatthere is a need for more genotoxicity studiesonoxidativestress.

Limitations

The evaluationof DNAdamagefromthe localtissue sam-ple(muscle,etc.)andthemeasurementofplasmapropofol levelswouldallowmoreinformativestudies.Thismightbe consideredasthelimitationsofourstudy.

Funding

GaziUniversity---ScienceResearchProjects.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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