Rev. bras. farmacogn. vol.27 número5

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w ww . e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Short-term

carcinogenesis

evaluation

of

Casearia

sylvestris

Cleide

A.S.

Tirloni

a

_,

_Giseli

_K.

_Traesel

a

_,

_Francislaine

_A.R.

_Lívero

c

_,

_Salvador

_D.V.

_Neto

a

_,

Ronaldo

de

Faria

Junior

a

_,

_Thaís

_C.

_Paim

a

_,

_Joyce

_A.

_Santos

b

_,

_Silvia

_A.

_Oesterreich

a

_,

_Ariany

_C.

_Santos

a

_,

Roosevelt

I.C.

Souza

a

_,

_Euclides

_L.

_Cardozo

_Junior

c

_,

_Arquimedes

_Gasparotto

_Junior

a,∗

a_Faculdade_de_Ciências_da_Saúde,_Universidade_Federal_da_Grande_Dourados,_Dourados,_MS,_Brazil

b_Faculdade_de_Ciências_Exatas_e_{Tecnológicas,}_Universidade_Federal_da_Grande_Dourados,_Dourados,_MS,_Brazil c_Laboratório_de_Farmacologia_e_Toxicologia_de_Produtos_Naturais,_Universidade_Paranaense,_Umuarama,_PR,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received31January2017

Accepted17May2017

Availableonline7August2017

Keywords:

Genotoxicity Mutagenesis

Short-termcarcinogenesis

Toxicity

Tumormarkers

a

b

s

t

r

a

c

t

CaseariasylvestrisSw.,Salicaceae,isanimportantmedicinalplantwidelyusedinBrazilforthetreatment ofvariouscardiovasculardisorders.ThisspecieswasincludedasofinterestbyBrazilianUnifiedHealth System.Althoughpreclinicalstudiesdescribedcardiovascularprotectiveeffectsandapparentabsenceof toxicity,nostudieshaveevaluateditscarcinogenicpotential.Inthisstudy,weproposedashort-term car-cinogenesisevaluationofC.sylvestrisinWistarrats,aimingtocheckthesafetyofthisspeciestouseitas proposedbyBrazilianUnifiedHealthSystem.C.sylvestrisleaveswereobtainedandthecrudeextractwas preparedbymacerationfrommethanol/water.Wistarratswereorallytreatedfor12weekswith50,250 or500mgkg−1_of_crude_extract_or_vehicle._Body_weight,_daily_morbidity_and_mortality_were_monitored. Bloodandbonemarrowsampleswerecollectformicronucleustest,cometassayandtumormarkers evaluation.Vitalorganswereremovedtomacroandhistopathologicalanalyses.Thecrudeextractdid notinducemutagenicandgenotoxiceffectsandnoalterationswereobservedinimportanttumor mark-ers.Finally,nodetectablesignsofinjurythroughgrosspathologyorhistopathologicalexaminations wereobserved.Ourresultscertifytheabsenceofthecrudeextracttoxicity,indicatingitssafety,evenat prolongedexposureasproposedbyBrazilianUnifiedHealthSystem.

Introduction

Itisestimatethatabout80%oftheworld’spopulationdepends ontraditional practicestoprimary health care,and 85%of this portionusesplants(WHO,2011).Consideringthedifficultaccessof thesepopulationstoconventionalformsoftreatment,theWorld HealthOrganization(WHO)suggeststheadoption oftraditional practicesasatoolformaintaininghealthandencouragesthe devel-opmentofpublicpoliciestoinsertthemintotheofficialhealth systemofits191membercountries,includingBrazil(WHO,2011). Inthiscontext,BrazilianMinistryofHealthproposedactionsfor developmentofpublicpoliciesfortheinclusionofmedicinalplants intheBrazilianUnifiedHealthSystem(SUS),allowingthe expan-sionofdiseaseprevention,maintenanceandrecoveryofhealth. Thepurposeistoexpandthetherapeuticoptionstousers, ensur-ingaccesstomedicinalplantsinviewofacompletehealthcare

∗ Correspondingauthor.

E-mail:arquimedesjunior@ufgd.edu.br(A.GasparottoJunior).

(Carvalhoet al.,2011).Oneofthestrategies toattendthis was

theelaborationofFormHerbalMedicinesoftheBrazilian Phar-macopoeia,whichsupportsthehandlingpracticesanddispensing intheSUS,contemplating47speciesofnaturalplants,including theCaseariasylvestrisSw.,Salicaceae(Anvisa,2011).

Caseariasylvestrisisdistributedintropicalregionsandoccurs in almost all Brazilian territory, where is popularly known as ‘guac¸atonga’(TorresandYamamoto,1986).Traditionally,itsleaves and barks are used for diarrhea, fever, hyperlipidemia, inflam-mation, obesity, skin diseases and tonic health (Ferreira et al., 2011).Betweenpharmacologicalpropertiesthereareanti-cancer

(Ferreira et al., 2016), antihyperlipidemic (Schoenfelder et al.,

2008), antiinflammatory and antioxidant (Albano et al., 2013), antiatherogenic(Brantetal.,2014),besidesnoacuteorprolonged toxicologicaleffects(Amenietal.,2015).

Despite theimportance of medicinalplants forhumancare, theirtoxicologicalpotentialisfrequentlyevaluatedslightly and not as priority (Ferreira et al., 2007).Most of phyto-derivative commercializedmedications,called“natural”,areusedbythe pop-ulationwithouthavingcarriedoutadetailedstudyofthechemical

http://dx.doi.org/10.1016/j.bjp.2017.05.009

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composition,efficacyandsafetyofuse.Thisresultislimited knowl-edgeaboutsideeffects,especiallyonthegeneticmaterial(Varanda,

2006).

Carcinogenicevaluationofthemedicinalplantsisessentialto establishcontrolmeasuresinwidespreaduse,beforetheplants beingconsideredtherapeuticagents.Carcinogenesistestarepart of the regulatory process of new drug development, and life-spanstudiesinrodentshavebeenconsideredthegoldstandard since early in the 20th century. However, they require many financialresources,nearly thousandanimals,and take approxi-mately2–3 years from initiation toreport (Cohen and Arnold, 2016).Currently,short-termassays,(1–3monthsofduration),have beenproposedbyregulatoryauthoritiesasinexpensiveandrapid alternative toevaluate carcinogens substances (Bajpayee et al.,

2005).

So,despitetheC.sylvestrispresentimportantpreclinical phar-macological effects no data on its carcinogenic potential have beenpublished.Then,weproposedapreclinicalshort-term car-cinogenesisevaluationofC.sylvestris,aimingtocheckthesafety of this species to use it in herbal medicines as proposed by SUS.

Materialandmethods

Plantmaterialandextractpreparation

LeavesofCaseariasylvestrisSw.,Salicaceae,wereharvestedin abotanicalgardenof VeraCruz do Oeste,Brazil(620–650mof altitude,S25103028′′_–W_53152037′′₎_during_the_summer_of_2013.

Thespecimenwasidentifiedbytaxonomistsanddepositedinthe HerbariumofBotanicalMuseumofCuritiba,Brazil(no.359503). Leavesweredriedinaforceddraftoven(45◦_C,₄₈_h)._The_crude

extractwaspreparedbymacerationfrommethanol/water(70:30 v/v)atroomtemperaturefor15days(Brantetal.,2014).Thesolvent waseliminatedbyarotaryevaporatorandlyophilized(yielding 17.14%).Thefreeze-driedextract(MECS)wasdissolvedinfiltered waterbeforetheexperiments.

Liquidchromatography–massspectrometry(LC–MS)

MECSwasexaminedbyultra-high performanceliquid chro-matography.Samplesat2mgml−1_{concentration}_were_prepared_in H2O–MeOH(7:3v/v)andtheanalysiswascarriedoutona reversed-phaseHSS-C18column.Thebinarysolventwascomposedof0.1% aqueousformicacidandmethanol(v/v).Thelinearsolventgradient ataflowrateof0.4mlmin−1_was_developed_by_increasing_methanol percentagefrom0%to38%(10min), thento60%at13minand returningtoinitialconditionat14min,re-equilibratedfor3min. Thecolumnwasheatedat60◦_C_and_samples_kept_at_room

tempera-ture.2␮lwasinjectedandcompoundsweredetectedat200–400

byhigh-resolutionmassspectrometry(HR-MS).

HR-MSanalysesweredevelopedonanelectrosprayionization massspectrometryoperatinginthenegativeionizationat atmo-sphericpressure.Thesourcetemperaturewas350◦_C_and_N

2stream wasusedfor sampledesolvation withsheath gasand auxiliary flowrateat60and20arbitraryunits,respectively.Theionization parameterswere:sprayvoltageof3.5kV,capillaryof−20Vand tubelensof−130V.Formassaccuracy,externalcalibrationwas performedandresolutionwassetat30,000FWHM(atm/z400)in LC–MSmode.Acquisitionwasobtainedintotalioncurrentmode, withmassrangeofm/z100–1000.Compoundfragmentationwas obtainedbycollision-induceddissociationusingheliumandenergy

Carcinogenesisandmutagenesisevaluation

Animals

Femaleand maleWistar rats,12weeks old,werehousedat 22±2◦_C,₁₂_h_light_dark_cycle,₅₅_±_10%_humidity_conditions,_with

adlibitumaccesstowater andchow. Theethicalcommittee on animaluseoftheFederalUniversityofGrandeDourados(UFGD) approvedalltheprocedures(no.11/2015).

Experimentaldesignandsamplecollection

Ratsweredistributedintoeightgroups(n=8–10)fortreatment withMECS(50,250and500mgkg−1_,_p.o._,_though_gavage,₁_ml_kg−1₎ orvehicle(filteredwater,1mlkg−1_,_control_group)_daily,_during₁₂ weeks.Thedoseswereselectedbasedonpreviousdatareporting cardiovasculareffectsofMECS(Brantetal.,2014).Aten-timelower dosefromthehighestdosewasalsotested.

Rats morbidity and mortality, aggressiveness, eyes and ear pallidness,convulsions,salivation,motoractivity,breathing, heart-beat,diarrhea,comaandinjuryweremonitored.Bodyweight(BW), consumeofchowandwaterwereweeklycontrolled.

Attheendoftheexperiment,theanimalswerefood-deprived for12handanesthetizedwithisoflurane.Bloodsampleswere col-lectedfromcaudalveinforgenotoxicitytestandafterdecapitation fortumormarkersanalyses.Liver,spleen,kidneysandlungswere removed for determining therelative weights, gross pathology andhistopathologicalanalyses.Therightfemurwascollectedfor micronucleusassay.

Grosspathologyandhistopathologicalevaluation

Sampleswereharvested,fixedin10%formalin,dehydratedwith alcoholandxylene,embeddedinparaffinwax,sectioned(4␮m)

andstainedwithhematoxylin/eosin.Thesliceswereanalyzedbya veterinarypathologist,withaslicescanner,at10×magnification.

Micronucleustest

The proximal epiphyses were cut off from femurs and the spinalcordwasremovedbydrainwithfetalbovineserum.After centrifugation (5min, 1000×g) the pelletwas homogenized, a dropofthiswasplacedonaslideandthesmearwasperformed. Slices were dried at room temperature and fixed in absolute methylalcohol(10min)and24hlatterwerestainedwithgiemsa (15min),washedindistilledwaterandblindanalyzed.Two thou-sandpolychromaticerythrocytes(MN/PCE)/animalwerecounted using an optical microscope (1000× magnification). The poly-chromatic/normochromaticerythrocytesratio(PCE/NCEratio)was calculatedbyanalyzing100randomerythrocytes/animal(OECD,

1997).

Cometassay

Bloodofrats (20␮l) treatedwithMECSduring 12weeks or

cyclophosphamide (20mgkg−1_, _p.o._, _through _gavage, ₁_ml_kg−1_, 24hbeforesamplecollection)washomogenizedin120␮lof1.5%

low-melting-pointagarosegeland transferredtosliceswith5% agarosegel.Afterimmersioninalysisbuffer(2.5MNaCl,100mM EDTA,and10mMTris[pH 10.0]with1%Triton X-100and10% dimethylsulfoxide)for4h, sliceswereincubatedinanalkaline buffer(300mMNaOHand1mMEDTA,pH>13,4◦_C)_for₂₀_min.

Nucleoidswereelectrophoresed(20min,25V,300mA,inthedark at 4◦_C), _whereupon _the _alkali _was _neutralized _with _0.4_M _Tris

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nucleoiddiameter).Readingswereusedtocalculatedamagedcell (sumof0–3classesdamage)andscoreofdamage(thevalueofthe damagemultipliedbytheclassnumberandthenthesumofthe threeclasses)(OECD,2014).

Tumormarkers

PlasmaticlevelsoftumormarkersCA15-3(breast),CA19-9 (pancreas,biliaryanddigestivetract),CA125(ovariesandliver),CA 27-29(breast),CA72-4(stomachandovarian),alpha-fetoprotein (AFP;livercancer),squamouscellcarcinomaantigen(SCC;cervix squamouscell,head,neck,esophagusandlung);and thyrocalci-tonin(TC;thyroid)weredeterminedbyelectrochemiluminescence usinganautomatedanalyzer.ResultsareexpressedasUl−1 _(CA 15-3,CA19-9,CA125,CA27-29),ngl−1_(AFP_and_SCC)_or_pg_ml−1 (TC).

Statisticalanalyses

Differencesweredeterminedbyone-wayanalysisofvariance (ANOVA)orKruskalWallisfollowedbyBonferroni’sorDunn’spost hoc. Weighgain of rats was analyzedby two-way ANOVA fol-lowedbyBonferroni’sposthoc.Thelevelofsignificancewas95% (p<0.05).Thedataareexpressedasmean±standarderrorofthe mean(S.E.M.).Graphsweredrawnandstatisticalanalysiswas car-riedoutusingtheGraphPadPrismsoftwareversion5.0forMacOS X(GraphPad®Software,SanDiego,CA,USA).

Results

Phytochemicalcharacterization

BioactiveclerodanediterpenestypicalofgenusCaseariawere identified,highlightingthecasearvestrinsA–CandcasearinsBand G. Gallic acidderivatives such as isobutyl gallate-3,5-dimethyl etherandmethylgallate-3,5-dimethyletherhavealsobeen identi-fied.Furthermore,thefingerprintobtainedfromMECSshowedfive peakswithabsorbanceat335nmcompatiblewithflavonoid glyco-sides.ThepeakRt6.093issimilartorutinanditsaveragecontent wasfoundtobe20.2mgg−1₍_Fig.₁_).

Bodyweight,relativeorgansweightandclinicalevaluation

TreatmentwithMECSdidnotalter theBW (g)of femaleor malerats(Fig.2AandB,respectively).Therelativeweightofliver, spleenandkidneyswerethesamebetweenallgroupsfound (Sup-plementary Table). Moreover, no alterations in chow or water consumption,andbehavioralwerefound.

100

80

60

40

20

0 2 4 6 8 10 12 14 16

0

Minutes

mA

U

Rutin - 6.093

Fig.1.UPLCanalysisofMECSobtainedfromCaseariasylvestris.

Grosspathologyandhistopathologicalanalyses

MECSdidnotinducelung,kidneyorlivermacroscopic alter-ations(datanotshown).Thehistologicalexaminationconfirmed theabsenceofdamageinlung(A),kidney(B)orliver(C)infemale or male rats treated with vehicle (Figs. 3 and 4, respectively),

MECS50mgkg−1₍_Figs.₃_and₄_,_{respectively),}_MECS₂₅₀_mg_kg−1

(Figs.3and4,respectively)orMECS500mgkg−1 ₍_Figs.₃_and₄_,

respectively).

Micronucleustest

CyclophosphamideincreasedthenumberofMN-PCEinfemale andmaleratsby283.9%(Fig.5AandB,respectively);anddecreased thePCE/NCEratioby99%infemaleandmale(Fig.5CandD, respec-tively)when compared withcontrol group.MECSdidnot alter the values of MN-PCE or PCE/NCEratio in female (Fig. 5A and C, respectively)ormale rats(Fig.5Band D,respectively)when comparedwithC-.Illustrativeimageofnormochromatic erythro-cyte,polychromaticerythrocyteandmicronucleusarepresentedin

Fig.5E(C-),F(MECS50mgkg−1_),_G_(MECS₂₅₀_mg_kg−1₎_or_H_(MECS 500mgkg−1_).

Cometassay

Classesandnumberofdamagedcellsfoundinthecometassay offemaleandmalearepresentedinTables1and2,respectively.No alterationswereobservedinthenegativecontrolgroup. Cyclophos-phamideinducedanintensedamageinallsamples.In contrast, MECSdidnotinducedalterations.

Tumormarkers

MECSdidnotaltertheplasmaticlevelsofCA15-3,CA19-9, CA125, CA27-29,CA72-4,SCCand TC offemale ormale rats

Table1

Numberofdamagedcellsandclassesofdamageoffemaleratstreatedwithvehicle(C−,negativecontrol),cyclophosphamide(C+,positivecontrol)orfreeze-driedextract

ofCaseariasylvestris50,250and500mgkg−1_.

Groups Damagecells(%) Classesofdamage Score

1 2 3 4

C− 10.2±0.64 89.8±1.22 7.1±0.70 2.7±0.63 0.4±0.16 13.7±1.84

C+ 87.0±3.75a _8.5

±2.21a _20.1

±8.01a _63.1

±7.51a _8.52

±4.52a _194.1

±8.91a

MECS50mgkg−1 _8.7

±3.01 91.3±0.94 6.5±0.87 1.9±0.31 0.3±0.21 11.20±2.61

MECS250mgkg−1 _7.8_±_0.55 _92.1_±_1.41 _5.5_±_0.79 _1.6_±_0.43 _0.7_±_0.33 _11.67_±_2.34

MECS500mgkg−1 _7.2_±_0.49 _93.0_±_0.88 _5.2_±_0.63 _1.8_±_0.33 _0.2_±_0.13 _9.40_±_2.75

Valuesexpressedasmean±S.E.M.,n=8–10.Class0,nodamage;class1,tailofcometshorterthanthediameterofnucleoid;class2,tailofcometonceortwicethediameter

ofnucleoid;class3,tailofcometmorethantwicethediameterofnucleoid.C−,negativecontrol,C+,cyclophosphamide,positivecontrol.Differencesbetweengroupswere

evaluatedbyone-wayANOVAfollowedbyBonferroni’stest.

a_p_<_0.05_when_compared_with_C

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A

B

350

350 300

300 250

200

250 400 450

150

1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12

Weeks Weeks

g g

Control MECS 50 mg.kg-1

MECS 250 mg.kg-1

MECS 500 mg.kg-1

Fig.2.EffectofCaseariasylvestrisextract(MECS)onbodyweightgain(g)offemale(A)ormale(B)rats.Animalsreceived(orally)vehicle(controlgroup)or50,250or

500mgkg−1_MECS._Treatments_were_performed_once_a_day,_during₁₂_weeks._Values_are_expressed_as_means_±_S.E.M.₍_n₌_8–10)._Statistical_comparison_was_performed_using

two-wayANOVAfollowedbyBonferroni’stest.

1

3

4 A

_B

C

A

B

A

_B

A

B

2

Fig.3.Lung(A),kidney(B)orliver(C)histologyoffemaleratstreatedwithvehicleorC.sylvestrisextract(MECS).Animalsreceived(1)vehicle(controlgroup),(2)50mgkg−1

MECS,(3)250mgkg−1_MECS_or₍₄₎₅₀₀_mg_kg−1_MECS._Treatments_were_performed_once_a_day,_during₁₂_weeks._Slides_were_stained_with_Hematoxylin_and_Eosin.₄₀_×

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1

2

3

4 A

B

C

A

B

C

A

_B

C

A

B

_C

Fig.4. Lung(A),kidney(B)orliver(C)histologyofmaleratstreatedwithvehicleorC.sylvestrisextract(MECS).Animalsreceived(1)vehicle(controlgroup),(2)50mgkg−1

MECS,(3)250mgkg−1_MECS_or₍₄₎₅₀₀_mg_kg−1_MECS._Treatments_were_performed_once_a_day,_during₁₂_weeks._Slides_were_stained_with_Hematoxylin_and_Eosin.₄₀

×

magnification.

Table2

Numberofdamagedcellsandclassesofdamageofmaleratstreatedwithvehicle(C−,negativecontrol),cyclophosphamide(C+,positivecontrol)orfreeze-driedextractof

Caseariasylvestris50,250and500mgkg−1_.

Groups Damagecells(%) Classesofdamage Score

1 2 3 4

C− 11.99±0.74 87.9±0.75 9.39±0.56 1.8±0.33 0.8±0.25 15.39±3.62

C+ 91.0±2.91a _9.0_±_2.91a _19.6_±_7.82a _62.0_±_9.6a _9.40_±_3.57a _171.8_±_2.91a

MECS50mgkg−1 _10.59_±_0.61 _89.5_±_1.08 _7.19_±_0.91 _2.5_±_0.52 _0.9_±_0.40 _14.89_±_3.14

MECS250mgkg−1 _10.49_±_0.76 _89.6_±_1.16 _8.39_±_1.22 _1.8_±_0.36 _0.3_±_0.15 _12.89_±_3.45

MECS500mgkg−1 _9.73

±0.55 91.7±0.76 6.79±0.52 2.44±0.90 0.5±0.22 13.17±3.20

Valuesexpressedasmean±S.E.M.,n=8–10.Class0,nodamage;class1,tailofcometshorterthanthediameterofnucleoid;class2,tailofcometonceortwicethediameter

ofnucleoid;class3,tailofcometmorethantwicethediameterofnucleoid.C−,negativecontrol,C+,cyclophosphamide,positivecontrol.Differencesbetweengroupswere

evaluatedbyone-wayANOVAfollowedbyBonferroni’stest.

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A

B

E

F

G

H

C

D

15

10

5

0

C- C+

C+ 50 250 500

MECS(mg.kg-1)

C- 50 250 500 MECS(mg.kg-1)

C+ C- 50 250 500

MECS(mg.kg-1)

MN-PCEs

15

10

5

0

C- 50 250 500 C+ MECS(mg.kg-1)

MN-PCEs

2.5 2.0 1.5 1.0 0.5 0.0

2.0 1.5

1.0 0.5

0.0

∗ ∗

∗ PCEs/NCEs r ∗

atio

PCEs/NCEs r

atio

Fig.5.EffectofCaseariasylvestrisextract(MECS)onthecountsof(AandC)femaleand(BandD)maleMN-PCEsandPCE/NCEratio,respectively.Cytotoxicityofvehicle

and50,250or500mgkg−1_MECS_is_presented_in_panels_E,_F,_G_and_H,_{respectively.}_Arrows_indicate_{normochromatic}_erythrocyte;_angled_arrows_indicate_{polychromatic}

erythrocytes;andsquareindicatemicronucleusinpolychromaticerythrocyte.SlideswerestainedwithGiemsaandshownwith1000×magnification.Statisticalcomparison

wasperformedusingone-wayANOVAfollowedbyBonferroni’stest.Valuesareexpressedasmean±S.E.M.(n=8–10).*p<0.05whencomparedwithcontrolgroup.

MN-PCE,micronucleatedpolychromaticerythrocytes;PCE/NCE,polychromatictonormochromaticerythrocytesratio.C−,controlgroup;C+,cyclophosphamide,positivecontrol

group.

Table3

Tumormarkersoffemaleratstreatedwithvehicle(controlgroup)orfreeze-driedextractofCaseariasylvestris50,250and500mgkg−1_during₁₂_weeks.

Tumormarker Groups

Control MECS50mgkg−1 _MECS₂₅₀_mg_kg−1 _MECS₅₀₀_mg_kg−1

CA15-3 1.08±0.07 1.07±0.05 0.96±0.05 1.01±0.06

CA19-9 10.01±0.29 9.12±0.35 9.55±0.45 9.34±0.33

CA125 13.34±0.55 13.91±0.41 14.40±0.69 14.33±0.62

CA27-29 6.20±0.49 5.23±0.37 5.09±0.28 5.22±0.43

CA72-4 1.34±0.12 1.43±0.19 1.42±0.13 1.21±0.12

AFP 1.73±0.15 1.26±0.07a _1.21_±_0.07a _1.18_±_0.07a

SCC 0.35±0.05 0.30±0.04 0.37±0.04 0.42±0.04

TC 4.36±0.37 4.00±0.25 4.35±0.39 4.05±0.26

Valuesexpressedasmean±S.E.M.,n=8–10.CA,cancerantigen;AFP,alpha-fetoprotein;SCC,squamouscellcarcinomaantigen;TC,thyrocalcitonin.Unit:CA15-3,CA19-9,

CA125,CA27.29andCA72-4:Ul−1_;_AFP:_ng_ml−1_;_SCC:_ng_ml−1_;_TC:_pg_ml−1_._Differences_between_groups_were_evaluated_by_one-way_ANOVA_or_Kruskal_Wallis_followed_by

Bonferroni’sorDunn’stest.

a_p_<_0.05_when_compared_with_control.

Table4

Tumormarkersofmaleratstreatedwithvehicle(controlgroup)orfreeze-driedextractofCaseariasylvestris50,250and500mgkg−1_during₁₂_weeks.

Tumormarker Groups

Control MECS50mgkg−1 _MECS₂₅₀_mg_kg−1 _MECS₅₀₀_mg_kg−1

CA15-3 1.01±0.05 1.02±0.12 0.94±0.03 1.04±0.06

CA19-9 10.17±0.31 9.82±0.37 9.74±0.46 9.80±0.37

CA125 14.18±0.43 13.71±0.42 13.12±0.40 13.09±0.49

CA27-29 6.00±0.46 5.37±0.42 5.37±0.39 5.67±0.27

CA72-4 1.26±0.09 1.36±0.13 1.38±0.10 1.39±0.09

AFP 1.10±0.06 1.13±0.08 1.35±0.08 1.21±0.06

SCC 0.35±0.03 0.35±0.04 0.45±0.04 0.40±0.05

TC 4.00±0.25 4.53±0.37 4.55±0.38 4.30±0.30

Valuesexpressedasmean±S.E.M.,n=8–10.CA,cancerantigen;AFP,alpha-fetoprotein;SCC,squamouscellcarcinomaantigen;TC,thyrocalcitonin.Unit:CA15-3,CA19-9, CA125,CA27.29andCA72-4:Ul−1_;_AFP:_ng_ml−1_;_SCC:_ng_ml−1_;_TC:_pg_ml−1_._Differences_between_groups_were_evaluated_by_one-way_ANOVA_or_Kruskal_Wallis_followed_by Bonferroni’sorDunn’stest.

(Tables 3and4,respectively).Theplasmatic levelsof AFPwere

significantlyreducedinfemaleratstreatedwithalldosesofMECS.

Discussion

Inthiswork,ashort-termcarcinogenesisstudywasconducted with a popular herbal preparation obtained from C. sylvestris. Throughaseriesofdetailedprotocols,weobservedthattheMECS

oftreatment.Furthermore,nosignificantincreaseintumor mark-erslevels, signsof tumorormalignant cells ingross pathology norhistopathologicalchangesinvitalorgansofWistarratswere detected.

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reportedin28.4%ofthem(Sponchiadoetal.,2016).Thishigh inci-denceofpositiveresultsshouldalerttotheimportanceofassessing thegenotoxicandmutagenicpotentialofherbalmedicinesbefore applyingthemastherapeuticagents.

Importantmarkersoftheactionofgenotoxiccompoundsarethe presenceofmicronucleiorthedecompressionofpartsofcellular DNA.Themicronucleiareoriginatedfromchromosomefragments thatareacentricorthataredelayedinrelationtotheothersintheir migrationtowardthepolesofthemitoticspindle.Thesechanges maybeinducedbyagentsthatarecapableofbreakingDNAor inter-feringwithspindle formation.Similarly,thepresenceof simple breaks,alkalinelabilesitesandcrosslinksresultingfromtheaction ofgenotoxiccompounds,altersthecellstructureoftheDNA,which isnormallysupercoiledandstronglycompacted,causingrelaxation inpartsofthemolecule(Tafazolietal.,2017).Inviewofthe above-mentioneddata,theOrganizationforEconomicCo-Operationand Development(OECD)recommendsthemicronucleustestandthe cometassayasanimportanttoolcapableofidentifyingpotentially genotoxicagents(OECD,1997,2014).Therefore,theabsenceof sig-nificantchangesintheseassaysprovidedveryconsistentdataon thelowgenotoxicityoftheMECS.

Anotherimportantaspectofthisstudywastheinvestigationof thecarcinogenicpotentialofMECS.Forthis,weinitiallyoptedfor thedosagesofdifferenttumormarkers,andlater,foradetailed histopathologicalanalysisofdifferentvitalorgans.Tumormarkers (orbiologicalmarkers)areproteinsorpiecesofproteinpresentin tumors,bloodorotherbiologicalfluidstowhichchangesintheir concentrationsarerelatedtothegenesisandgrowthofneoplastic cells.Ingeneral,tumormarkerscanbeproducedbynormalcells aswellasbycancercells,althoughareproducedatveryhigh lev-elsbytumorcells(Duff,2001).Thesemarkersmaybeusefulin thediagnosisandprognosisofdifferenttypesoftumors,aswell asassistinthedevelopmentofnewtreatmentmodalities(Touitou

andBogdan,1998).

Inourstudy,noneoftheanimalsreceivingMECSshoweda sig-nificantincreaseindifferenttumormarkerslevelswhencompared toanimalstreatedonlywiththevehicle.Ontheotherhand,female ratstreatedwithMECS(atalldoses)showedasignificant reduc-tioninAFPlevels.AFPisanimportantfetalserumprotein,whichis normallysynthesizedintheliver,yolksacandfetalgutwith func-tionsofplasmatransportandmaintenanceofoncoticpressure.In general,levelsabove500ng/mlarehighlysuggestiveofliver malig-nancyandvalueswellbelowthatleveldonot,ontheirown,have considerableclinicalsignificance(Sauzayetal.,2016).Generally, tumormarkersarecomplementarytestsandshouldalwaysbeused accompaniedbyothermethodsfordiagnosisortherapeutic mod-ification.Inourcase,inacomplementarywaytotumormarkers, allthevitalorgansofanimalstreatedwiththeMECSwere exam-inedthroughdetailedhistopathologicalanalysis,and,innoneof thecases,morphoanatomicalterations suggestiveof tumorand malignancywereobserved.

Takentogether,ourstudyindicatesthatthemethanolicextract fromC.sylvestrisissafeatthetesteddoses,withnomutagenic, genotoxicorcarcinogeniceffects.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare

thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).

Confidentialityofdata. Theauthorsdeclarethatnopatientdata

appearinthisarticle.

Righttoprivacyandinformedconsent.Theauthorsdeclarethat

nopatientdataappearinthisarticle.

Funding

ThisworkwassupportedbyFundac¸ãodeApoioao Desenvolvi-mentodeEnsino,Ciência,TecnologiadoEstadodoMatoGrossodo Sul(FUNDECT,59/300.046/2015),andConselhoNacionalde Desen-volvimentoCientíficoeTecnológico(CNPq,449464/2014-8).

Authors’contributions

CAST:invivoexperimentsanddataanalyses.ELCJ:LC–MS anal-yses.SDVN,RDFJandTCP:invivoexperiments.JAS,GKTandSAO: mutagenicityanalyses.ACSandRICS:histopathologicalanalyses. FARL:dataanalysis,datadiscussionandmanuscriptpreparation. AGJ:datadiscussionandmanuscriptcorrection.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2017.05.009.

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