Rev. bras. farmacogn. vol.27 número3

(1)

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

Original

Article

Reproductive

effects

of

the

psychoactive

beverage

ayahuasca

in

male

Wistar

rats

after

chronic

exposure

Alana

de

Fátima

Andrade

Santos

a

_,

_Ana

_Luiza

_Sarkis

_Vieira

b

_,

_Aline

_Pic-Taylor

c

_,

_Eloisa

_Dutra

_Caldas

a,∗

a_Laboratório_de_Toxicologia,_Departamento_de_Farmácia,_Universidade_de_Brasília,_Brasília,_DF,_Brazil b_Centro_de_Cirurgia_{Experimental,}_Faculdade_de_Medicina,_Universidade_de_Brasília,_Brasília,_DF,_Brazil c_Departamento_de_Genética_e_Morfologia,_Instituto_de_Biologia,_Universidade_de_Brasília,_Brasília,_DF,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received15November2016 Accepted9January2017 Availableonline9March2017

Keywords: Ayahuasca

N,N-dimethyltryptamine ␤-Carbolinesalkaloids Malereproductivetoxicity Wistarrats

Testosterone

a

b

s

t

r

a

c

t

Ayahuascaisapsychoactive beverageused ancestrallybyindigenousAmazonian tribesand,more recently,byChristianreligionsinBrazilandothercountries.Thisstudyaimedtoinvestigatethe repro-ductiveeffectsofthisbeverageinmaleWistarratsafterchronicexposure.Theratsweretreatedby gavageeveryotherdayfor70daysat0(control),1×,2×,4×and8×thedoseusedinareligiousritual

(12animalspergroup),andanimalseuthanizedonthe71st_day._Compared_to_controls,_there_was_a

sig-nificantdecreaseinfoodconsumptionandbodyweightgaininratsfromthe4×and8×groups,anda

significantincreaseinthebrainandstomachrelativeweightatthe8×group.Therewasasignificant

increaseintotalserumtestosterone,andadecreaseinspermatictransittimeandspermaticreserves intheepididymiscaudaeinthe4×group,butnotinthehighestdosegroup.Nosignificantchanges

werefoundintheotherreproductiveendpoints(spermatozoidmotilityandmorphology,total sperma-tozoidcountanddailyspermproduction),andhistologyoftestisandepididymis.Thisstudyidentifieda no-observed-adverse-effect-levelforchronicandreproductiveeffectsofayahuascainmaleWistarrats at2×theritualisticdose,whichcorrespondsinthisstudyto0.62mg/kgbwN,N-dimethyltryptamine, 6.6mg/kgbwharmineand0.52mg/kgbwharmaline.Apotentialtoxiceffectofayahuascainmalerats wasobservedatthe4×dose,withanon-monotonicdose–response.Studiesinvestigatingtheroleof

ayahuascacomponentsinregulatingtestosteronelevelsareneededtobetterunderstandthisaction. ©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Ayahuasca,which in Quechua means“wine ofthe souls”,is

a hallucinogenic plant concoction used ancestrally by Amazon

indigenousgroupsinxamanicritualsfordiagnosis,healing,and

spiritualdevelopment(Grobetal.,1996;McKenna,2004;Tupper,

2008).Sincethe1930s,Christianreligiouscommunitieshavealso usedthisconcoctionintheirrituals,includingSantoDaime,

Bar-quinhaandUniãodoVegetal(UDV)(Macrae,2004;Tupper,2008),

andthisuseislegalinBrazil(CONAD,2004).Theayahuasca reli-gionsalsohavecentersinothercountriesinSouthAmerica,Europe,

AsianandNorthAmerica(Halpern,2004;Tupper,2008;Labateand

Feeney,2012).Itsuse,however,goesbeyondreligiousrituals,being

alsousedforrecreationalpurposesby peopleseekingits

hallu-cinogeniceffects,withapotentialriskofintoxication(DosSantos,

2013;Winstocketal.,2014).Ontheotherhand,variousstudies

∗ _{Corresponding}_author.

E-mail:[email protected](E.D.Caldas).

havesuggestedthetherapeuticactionofayahuasca,includingfor

drugaddiction,anxietyanddepression(Pic-Tayloretal.,2015;Dos

Santosetal.,2016a;Domínguez-Clavéetal.,2016).

Ayahuascais generally produced withPsychotria viridisRuiz

& Pav., Rubiaceae, bush leavesand Banisteriopsiscaapi (Spruce

ex Griseb.) Morton, Malpighiaceae, vine, both native from the

Amazon. The B. caapi stem contains the ␤-carbolinesalkaloids

harmine,harmalineandtetrahydro-harmine,whicharereversible

inhibitorsofmitochondrialmonoamineoxidase(MAO)enzymes

(McKennaet al.,1984; Harvey et al.,1998)responsible for the

oxidationofneurotransmitters,suchasserotonin,dopamineand

noradrenalin. P.viridis contains N,N-dimethyltryptamine (DMT),

whichisalsofoundinotherplantsandanimals,includinghumans

(Callawayetal.,1996).Duetoitsstructuralsimilaritytoserotonin

(5-hydroxytryptamina,5HT),DMTbindswithserotonergic

recep-tors,mainlythe5-HT2Atype,producingitshallucinogeniceffects

(Smithetal.,1998;Halberstadt,2015).StudiesshowthatDMTalso

actsasasubstratefortheserotonintransporter(SERT)andforthe vesicularmonoaminetransporter(Nagaietal.,2007;Cozzietal.,

2009;Halberstadt,2015).

http://dx.doi.org/10.1016/j.bjp.2017.01.006

(2)

Whenadministeredorally,DMTisrapidlydegradedbytheMAO

present inthe liverand intestine. However, in thepresence of

␤-carbolines, it can reachthebrain and becomes orally active.

Therefore,thehallucinogeniceffectsofayahuascaconsumptionare

producedbythesynergicactionoftheactivecompoundspresent

intheplantspeciesusedinitspreparation(BuckholtzandBoggan,

1977;Callawayetal.,1996,1999).Theeffectsincludealterationsin

affectiveandemotionalstates,thoughts,memoryandbody

sensa-tions,synesthesiaandhallucinationwithalterationsinthevisual,

olfactoryand auditorysenses (Shanon,2003; Pireset al.,2010;

DosSantos et al., 2016b). Somaticeffects may include nausea,

vomiting,diarrhea,tremors,dizziness,tachycardia,mydriasisand hypertension(CallawayandGrob,1998;Ribaetal.,2003;Vivesand

García-albea,2012).

A studyconducted by this research group in female Wistar

ratsfoundthattheacutelethaloraldoseofanayahuasca

infu-sionpreparedbytheUDVwasover50×theritualdose.Thissame

studyidentifiedgreaterneuronalactivityinregionsofthebrain

richinserotoninergicreceptors,suchastheamygdala,theraphe

nucleusandthehippocampus,inanimalsexposedtoasingledose

ofayahuasca,equivalentto30×theritualdose(Pic-Tayloretal.,

2015).AstudyconductedbyMotta(2013)showedthatfemalerats

exposeddailytoayahuascaduringpregnancyatdoseshigherthan

2×theritualdosehadreductioninreproductionrates,increase

inreabsortions,lowerbodyweightandlowerrelativefetusorgan

weights and fetus visceral malformations. Similarresults were

foundinapreviousstudyconductedbyOliveiraetal.(2010).

Several studies have addressed the relation between

psy-choactivedrugsandmaleinfertility.Testsonanimalsshowthat

substancessuchastetra-hydrocanabinol,foundin plantspecies

ofthegenusCannabis,reducetestosteronelevels,affectingsperm

productionandmotility,andconsequentlymalefertility(Morgan

etal.,2011;Onyije,2012).Drugssuchasalcohol,tobacco,cocaine,

andandrogenicanabolicsteroidsamong othersarealso

consid-eredpotentialinfertilityagents(Onyije,2012;Vigneraetal.,2013;

Kulkarnietal.,2014).Furthermore,Alvarengaetal.(2014)found

that a single dose of ayahuasca significantly decreased sexual

performanceofmalerats,buthigherperformancewasobserved

in sleep deprived rats treated at the lowest dose(250␮g/ml).

However,studiesevaluatingtoxicityaspectsregardingmale

repro-duction in animals exposed chronically to ayahuasca are still

needed.

Theaimofthisstudyistoinvestigatethepotentialtoxicological effectofanayahuascainfusiononreproductioninWistarrats,after

chronictreatment.

Materialsandmethods

Ayahuascamaterial

The ayahuasca used in this study was prepared in April,

2011 by the Núcleo Luz do Oriente of the UDV, Federal

Dis-trict,Brazil,andstoredina −20◦_C_freezer_before_{lyophilization.}

Theinfusion wasprepared with Banisteriopsiscaapi (Spruce ex

Griseb.)Morton,Malpighiaceae,collectedinÁguasLindasdeGoiás (15◦₄₆′₁₇′′_S, ₄₈◦₁₄′₅₆′′_W) _and _the _leaves _of _Psychotria _viridis Ruiz&Pav.,Rubiaceae,collectedin Sobradinho, FederalDistrict (15◦₇₅′₂₃′′_S,₄₇◦₇₂′₉₂′′_W)._Samples_of_both_species_were_deposited

in the University of Brasilia Herbarium under the references

AzevedoEP149880BrahmsandTrietoB149879Brahms,

respec-tively.ThelevelsofDMT,harmalineandharminepresentinthe

ayahuascainfusion,determined prior totheexperiment bygas

chromatography-tandemmass spectrometry(GC–MS/MS; Trace

UltracoupledwithaTSQQuantumXLSTripleQuadrupole;Thermo

Scientific),were0.146mg/mlofDMT,0.12mg/mlofharmaline,and

100 RT: 18.00 - 28.00

21.21 21.21

DMT

21.31

25.83

25.96

26.24 Harmaline

Harmine 25.54 26.22

26.23 26.26

26.24 NL: 2.70E8 TIC MS AYAL

NL: 6.71E4 TIC F: + c EI SRM ms2 130 000 [76 999-77 001] MS AYAL

NL: 1.69E6 TIC F: + c EI SM ms2 213 000 [169 999-170 001] MS AYAL

NL: 2.70E8 TIC F: + c EI SRM ms2 212 000 [168 999-169 001, 211 999-212 001] MS AYAL

26.27 26.29

50

0 100

50

0 100

50

0 100

50

0

18 20 22 24

Time (min)

Relativ

e ab

undance

26 28

Fig.1. GC–MS/MStotalionchromatogram(TIC)oftheayahuascasample,and theextractedionchromatogramsinselectedreactionmonitoring(SRM)modeof DMT(m/z130→77;RT=21.3min),harmaline(m/z213→170;RT=25.8min)and harmine(m/z212→169;RT=26.2min).

1.56mg/mlofharmine(Pic-Tayloretal.,2015).Fig.1showsthe

GC–MS/MStotalionchromatogramoftheayahuascaprovidedby

theUDV.Onlytheharminepeakcanbeseen,asitispresentata

concentrationovertentimeshigherthanDMTandharmaline.Fig.1

alsoshowsthechromatograms,inselectedreaction monitoring

mode, ofthe threeayahuasca components(abundance

normal-izedto100%ineachcase).Tetrahydroharminewasnotanalyzed

inthisstudy.Thelyophilizedmaterialwasappropriatelyweighed

accordingtothedosesselectedbeforetreatment,consideringbody weight,anddilutedinfilteredwater,maintainingafinalvolumeof 2ml.

Experimentalprotocol

Thestudywasconductedwith60maleratsofthespecies

Rat-tusnovergicus,Wistarlineage,providedbyGranjaRG(SãoPaulo,

Brazil)aged4weeks,andofuniformweight(210±10g).The ani-malswerekeptattheFacultyofHealthSciencesoftheUniversity ofBrasilia(UnB)animalhouseinAlesco®

polypropylenezincbar

cagesinventilatedshelves.Theyunderwenta15-day

acclimatiza-tionperiodbeforethetreatmentwasinitiated,weremaintained

undercontrolledtemperatureconditions(23±2◦C)anddark/light

cyclesof12h/12hduringtheexperiment,andweregiven

commer-cialrodentfeed(Purina®

)andfilteredwateradlibitum.Thisproject

wasapprovedbytheAnimalUseEthicsCommissionoftheUnB(n◦

107766/2010).

Clinicalassessment oftheanimalswasperformeddaily,and

bodyweightandfeedconsumptionverifiedevery3days.Onthe

71stday,theanimalswereeuthanizedbyexposuretoCO2anda

4mlbloodsamplewasimmediatelycollectedbycardiacpuncture

andsubjectedtocentrifugationtocollecttheserum.Thestudywas

carriedoutaccordingtoprotocolEPA/630/R-96/009/1996

(Guide-linesforReproductiveToxicityRiskAssessment).

Experimentaldoses

Theselecteddosesusedinthestudyweredeterminedaccording

tothe ritualdoseconsumed during a UDVceremony,

approxi-mately150ml for anindividual weighing 70kg(1×). Based on

thelevelsfoundintheinfusionbyGC–MS/MS,the1×dose

cor-respondsto3.3mg/kgbwofharmine,0.26mg/kgbwofharmaline

and0.31mg/kgbwofDMT.Theanimalswererandomlydistributed

(3)

waterandfourtreatedgroupsthatreceived1×,2×,4×and 8×

theusualdosefor70days,bygavage.Thesedosescoincidewith

thoseusedin thefemalereproductivetoxicitystudyconducted

previously(Motta,2013)thatdemonstratedthatthedaily inges-tionofayahuascaatdosesequalorgreaterto4×theusualdoseis lethalforfemalepregnantratsafterthe5thdayofadministration,

withanimalspresentingsigns ofpiloerection,convulsion,

chro-modacryorrhea,lordosisandcyanosis.Similarresultswerefound

inapre-testconductedaspartofthepresentstudywithmalerats

administereddailywith4×dosesofayahuasca.Thus,inorderto

ensurethesurvivaloftheanimalsduringthetreatment,thepresent

studywasconductedwithadministrationtakingplaceon

alternat-ingdays.

Bloodandorgananalysis

Twomlofserumofeachanimalwasstoredat−20◦_C_for_later serologicdosageoftotaltestosterone,luteinizing(LH)andfollicle

stimulating(FSH)hormones,andbiochemicalanalyseswere

per-formedtoassesspancreatic(amylaseandlipase),hepatic(total,

direct,andindirectbilirubin),renal(creatinineandurea),

hepa-tobiliary,andpancreatic(glutamicoxaloacetictransaminase,and

glutamicpiruvictransaminase)functions,andtriacylglyceridesto assess lipidmetabolization. Spleen, liver,stomach, brain,heart, kidneys,testicles,epididymis,prostrateandseminalbladderwere

removedandwashedwithNaCl0.9%salinesolution,then

submit-tedtomacroscopicevaluationandweighing.

Reproductiveparameters

During the necropsy, the content of the deferent duct was

extractedbydiffusionin1mlofDulbecco’sModifiedEagleMedium (GIBCOTM₎_previously_heated_at₃₄◦_C_for_sperm_motility_analysis, withoutsurpassingthe10-minperiodaftereuthanasia.The

result-ingsolution(100␮l)wereinsertedinahemacytometer(Neubauer

chamber)andreadingsweretakenwithanopticmicroscope(Leica

GalenIII),andmagnifiedto400×.Fiftyspermatozoidswere

ana-lyzedperanimal, andthenumber ofprogressive(rapidorslow

progression)andnon-progressive(noprogressionormotionless)

counted(Seedetal.,1996).

Therighttestisofeach animal wasperforatedat oneofthe

extremitiestoremovethetunicaalbuginea,andtheparenchyma

washomogenizedfor1mininUltra-Turrax(IKA®

T10basic) in

10mlof0.05%TritonX-100todeterminetotalspermatozoidcount

(no.×106),dailyspermproduction,andspermtransitperiod(days). TherightepididymistailofeachanimalwascutupinaPetridish

andhomogenizedtodeterminethespermaticreserve.

Two samples of each testis and epididymis homogenates

(100␮l)wereanalyzedintheNeubauerchamberusinganoptic

microscope(ZeissPrimoStar;400×).Thespermatids(testis)and

spermatozoids(testisandepididymis)resistanttohomogenization

werecountedinfivechamberfieldsandthemeanofthetotalcount reportedforeachanimal(Straderetal.,1996).

Inrats,maturespermatozoidsinstages17–19represent48%

of seminiferous epithelium cycle length, which has a duration

periodof 12.75days(Robbet al.,1978).Hence,6.1days isthe

periodintheseminiferouscycleduringwhichmature

spermato-zoidsarepresent(Kempinasetal.,1998).Dailyspermproduction

wasobtaineddividingthenumberofresistantspermatozoidsin

thetesticleby6.1days(Robbetal.,1978;Blazaketal.,1985).The

spermtransittimewasobtaineddividingthenumberof

spermato-zoidsinthetailoftheepididymisbythedailyproductionofsperm

(Amannetal.,1976;Robbetal.,1978).

ThetailoftheleftepididymisofeachanimalwascutupinaPetri dishcontaining2mlofaphosphate-salinebuffersolution(PBS;7.4 pH)tocollectthespermatozoids.Theresultingsolutionwasdiluted

in5mlofPBSandstoredunderrefrigerationforupto10daysfor furtheranalysis.Twoslidesperanimalwerepreparedbysmearand

allowedtodryatroomtemperature,thenfixedwithmethanol(PA

grade)for10minandstainedwith1%eosinefor45min(Wyrobek

andBruce,1975).

Two hundred spermatozoids of each animal were analyzed

under microscopeand the percentage ofnormal and abnormal

spermatozoids(head, tailandmultiple) quantified(IRDG,2000;

SharmaandSingh,2010).

Testisandepididymishistology

ThelefttestisofeachanimalwaspreviouslyfixedinBouin solu-tionfor 3hat4◦_C, _cut_{transversally}_in _half_and_maintained_for anadditional4hatthesametemperatureandsolution.Afterthis period,theorganswerewashedin50%ethanol(proanalysisgrade)

overnightatroomtemperaturetoremovetheexcessofBouin,and

maintainedin70% ethanoluntil thehistologicalprocedurewas

performedusingclassicaltechniques(dehydration,diaphanization

andinclusioninparaffin).Non-consecutive5␮mthicksliceswere

obtainedusingmicrotome(Leica),thenstainedwithhematoxiline

andeosin(H&E).Threeslidesofeachanimalwereanalyzedunder

microscope.Ineachslide,tenseminiferoustubuleswereanalyzed

forthepresenceofcompletespermatogenesis,byJohnsen’s

Tubu-larBiopsyScore(JTBS)(Johnsen’s,1970).Foreachanimal,thefinal scorewasthemeanscoreobservedin10tubulesofthethreeslides (n=30)(MJTBS).

Thehead/bodysegmentsoftheepididymisofeachanimalwere

fixedinBouinfor7hat4◦_C,_following_the_same_histological proce-duredescribedabove.Longitudinalsectionsoftheepididymiswere

analyzedtodeterminethespermdensity,andclassifiedas

mod-eratehypospermia(+++),discretehypospermia(++),andnormal

spermdensity(+).Thepresenceofalterationsintheepididymis

andtesticle suchasexfoliationofgermcells, tubular

vacuoliza-tion,inflammatoryinfiltrates,granulomas, necrosis,edemasand

tubulesofreduceddiameter,andcellulardisorganizationwerealso investigated(Lanningetal.,2002).

Statisticalanalysis

Thestatisticalanalysisofthespermmorphologyandmotility

wasconductedbytheKruskal–Wallistest,andtheother

parame-terswereevaluatedusingone-wayanalysisofvariance(ANOVA),

withposthoccomparisonsbetweengroupsusingTukeyorDunnett

(homogeneous variance)or DunnettT3 (non-homogenous

vari-ance).TheanalyseswereperformedusingIBMSPSSStatisticsV.20

software,andthedifferenceswereconsideredstatistically signifi-cantatp≤0.05.

Results

Feedconsumption,bodyandorganweightandbiochemical

evaluations

Theanimalstreatedwithayahuascaat1×,2×and4×dosesdid

notshowadverseclinicalsignsduringtreatment.However,

ani-malstreated with8×dosesshowedevidentsigns ofstressand

constantvocalizationduringthegavageprocedure,and

piloerec-tionandtremorsafterayahuascaadministration.Twoanimalsof

the8×groupdiedduringtheexperiment,thefirstonthe16thday

oftreatmentwithsignsoftremorsandconvulsionminutesafter

gavage,andthesecondonthe37th_day_due_to_faulty_{administration,}

withalltheadministeredmaterialbeingfoundinthelung.

Table1showstheaveragefeedconsumption,measuredevery

(4)

Table1

Totalweightgain,feedconsumptionandbodyweight(g)attheendofthetreatmentofthecontrolandayahuascatreatedanimals(mean±standarderror).

Variable Control (n=12)

1× (n=12)

2× (n=12)

4× (n=12)

8× (n=10) Feedconsumption 72.1±0.7* 70.9±1.4* 74.6±1.1* 66.9±0.7# 64.2±1.0# Finalbodyweigh 405.4±11.9* 401.1±13.7*# 409.7±11.4* 381.1±12.3*# 355.3±10.5# Totalweightgain 204.1±7.9* 194.8±7.6* 197.5±8.9* 167.3±9.2# 158.5±7.5# Differentsymbolsbetweengroupsindicatesignificanceatp<0.05;ANOVAfollowedbyTukeyorDunnett.

treatmentforthecontrolandayahuascatreatedgroups.A signifi-cantdropinfeedconsumptionwasobservedinanimalstreatedat 4×and8×doses,whichhadasignificantimpactonthefinalbody weightforthe8×groupandonthetotalbodyweightgainforboth groups.

Table2showstherelative(%)weightsoftheanimalorgansin

thecontrolandtreatedgroups,andtheabsoluteweightsforthe

brainandreproductiveorgans.Animalsofthe8×groupshowedan

increaseintherelativeweightofthebrainandstomachin

rela-tiontothecontrol,however nodifferencewasobserved inthe

absolutebrainweights.Asignificantdecreaseintherelative

epi-didymisweightwasobservedinthe8×groupcomparedwiththe

4×group,aswellasasignificantincreaseintherelativeseminal vesicleweightcomparedwiththe2×group.Theabsoluteweightof thetesticleswassignificantlylowerfortheanimalsofthe8×group

incomparisonwiththeothergroups.Nomacroscopicalterations

wereobservedintheevaluatedorgans.

Thebiochemicalevaluationsconductedtoassessthehepatic,

pancreatic,renalandlipidmetabolizingfunctionsdidnotindicate

significantdifferencesamongthegroups(datanotshown).

Reproductiveendpoints

Asignificantincreasewasobservedintotaltestosteroneofthe

animalstreatedwith4×dosesofayahuascacomparedwiththe

controlgroupandwiththe8×group(Fig.2A).Therewereno

sig-nificantdifferencesinLHandFSHlevelsbetweenthetreatedand

controlgroups(datanotshown).

Thepercentagesofprogressive(spermmotility)and

morpho-logicallynormal (spermnormality)spermatozoidsareshown in

Fig.3.Onaverage,morethan75%ofthespermatozoidspresented

normalmotility(progressive),withnosignificantdifferenceamong

the groups of the study (Fig. 3A). Although all treated groups

showedlowerpercentagesofmorphologicallynormal

spermato-zoidsthanthecontrol,downto46.9%inthe4×group,thisreduction wasnotstatisticallysignificant(p=0.29)(Fig.3B).

Table3showsthepercentageofabnormalspermatozoidsfound

in the animals of the experimental groups. The percentage of

spermatozoidswithheadlesstailswasrelativelyhigheveninthe

animalsof thecontrol group,possibly asa consequence ofthe

smearingprocesswhenpreparingtheslides.Therewasanincrease

inthepercentageofspermatozoidswithaflattenedheadinthe ani-malsinthe8×groupinrelationtothe2×group,whichisprobably alsoanartifactduetothecellosmolaritychangesduringthe refrig-eratedstorageperiod,whichoccurredforupto10days.Therewas anincreaseinthepercentageofspermatozoidswithabenttailin thegroupstreatedwith2×and4×dosesinrelationtothe1×group.

Overall,nosignificantdifferencesinthetotalnumberof

abnor-malitieswerefoundbetweenthecontrolandtreatedanimals.A

significantreductionwasobservedinthespermreserveinthe

epi-didymistailandinthespermtransittimeoftheanimalstreated

withthe4×doseinrelationtothecontrolgroupandthe8×group (Fig.2Band2C).Nosignificantdifferenceswereobservedinthe totaldailyspermcountsinthetesticleamongthegroups(p=0.55),

althoughthecountwaslowerforthe8×doseincomparisonwith

thecontrolandtheothertreatedgroups(Fig.2D).

NosignificantdifferenceswereobservedinthemeanJTBSscore

(MJTBS)among thegroups, which ranged from9.43±0.12 (2×

group)to9.5±0.10(control).Allanimalspresentedscore of10

forat leastoneof thethirtytubulesanalyzed. Furthermore,no

morphologicalchangeswereobservedinthetestisofcontroland

treatedanimals.Smallnumberoflymphocytesandmacrophages

wereseenintheinterstitialtissueofepididymisheadsegmentsof

someanimalsfromallexperimentalgroups.

Discussion

Thisstudyshowedthatmaleratstreatedwithayahuascaat4×

and8×dosesevery2daysfor70dayshadlowerbodyweightgain

andlowerfeedconsumptionattheendofthetreatmentcompared

withtheothergroups,withsignificantlylowerfinalbodyweight

observedonlyinthehighestdosegroup.Therelativeweightsofthe

stomachandbrainweregreaterinthehighestdosegroupin

rela-tiontothecontrolgroup.Theseresultssuggestchronictoxicityof ayahuascainmaleratsathigherdoses.AstudyconductedbyMotta

(2013)demonstratedthatayahuascaadministereddailybetween

the6thand21stdayofgestationinducedtoxicity,withasignificant reductioninfeedconsumptionat1×,4×and8×doses,increases intherelativeweightsofthestomachofthe1×,2×and8×groups, anddilationofthestomachsandintestinesoftheanimalsofthe8× group.

Therewerenosignificantdifferencesobservedbetweenthe

rel-ative weights of thereproductiveorgans ofthe animals inthe

treatedgroupsand thoseofthecontrolgroup,buttheabsolute

weightofthetestisofthe8×groupwassignificantlylowerthan thecontrol.Nosignificantmorphologicalchangeswereobservedin thetestisorepididymisoftreatedanimals.Exposuretoayahuasca didnotleadtosignificantchangesinspermmotilityand

morphol-ogy,norinthenumberofabnormalspermatozoids,andtherewere

nosignificantalterationsindailyspermproduction,althoughthe

8×grouppresentedareductioninthisparameter.

Inthetestis,theMJTBSscorewasusedtoevaluate spermatoge-nesisaccordingtothepresenceorabsenceofdifferentcelltypesin theseminiferousepithelium(Johnsen,1970).Thisstudyidentified anaveragescorehigherthan9(outof10)forallgroups,

indicat-ingthatthespermatogenesiswascompleted,andthatayahuasca

didnotaffectspermproduction.Inadditiontospermatozoids,cells

fromall stages of spermatogenesis (spermatogonias,

spermato-cytes,roundandlatespermatids)werepresentinthetestis.

Thespermdensitiesintheepididymisheadandbodyregions

wereassessedduringthehistologicalanalysisinasemi-qualitative

manner, and no significant differences were found among the

groups.However,thespermcountinthetailoftheepididymisof

animalsfromthe4×groupwassignificantlylowerthanthecontrol andthe8×group.Indeed,KempinasandKlinefelter(2014)pointed outthatthetoxiceffectsonspermquantityandqualitymayoccur

intheabsenceofhistopathologicalalterationsintheepididymis

(5)

Table2

Relative(%)andabsolute(g)organweightsofthecontrolandayahuascatreatedanimals(mean±standarderror).

Control (n=12)

1× (n=12)

2× (n=12)

4× (n=12)

8× (n=10) Liver,% 3.56±0.33 3.47±0.20 3.43±0.25 3.49±0.20 3.52±0.26 Spleen,% 0.20±0.04 0.19±0.01 0.18±0.03 0.18±0.02 0.18±0.03 Rightkidney,% 0.35±0.02 0.34±0.02 0.36±0.04 0.35±0.01 0.37±0.04 Leftkidney,% 0.34±0.02 0.32±0.02 0.34±0.03 0.34±0.02 0.35±0.04 Stomach,% 0.46±0.06* 0.50±0.02*# 0.48±0.03*# 0.51±0.02*# 0.52±0.04# Heart,% 0.32±0.03 0.32±0.04 0.31±0.02 0.33±0.02 0.33±0.03 Brain,% 0.52±0.06* 0.53±0.06*# 0.51±0.06a 0.55±0.04*# 0.59±0.05# Brain,g 2.10±0.03 2.10±0.02 2.08±0.06 2.08±0.03 2.07±0.03 Testis,% 0.48±0.06 0.47±0.03 0.47±0.04 0.51±0.04 0.49±0.04 Testis,g 1.93±0.07* 1.86±0.04*# 1.93±0.04* 1.93±0.05* 1.73±0.05# Epididymis,% 0.20±0.04*# 0.21±0.05*# 0.22±0.07*# 0.25±0.05* 0.18±0.05# Epididymis,g 0.82±0.05*# 0.85±0.05*# 0.92±0.09* 0.93±0.05* 0.65±0.05# Prostate,% 0.20±0.09 0.23±0.06 0.20±0.06 0.23±0.07 0.17±0.03 Prostate,g 0.80±0.11*# 0.92±0.09* 0.82±0.07*# 0.87±0.07*# 0.59±0.04# Seminalvesicle,% 0.37±0.06* 0.34±0.08*# 0.29±0.05# 0.34±0.05*# 0.40±0.09* Seminalvesicle,g 1.48±0.06* 1.32±0.07*# 1.19±0.05# 1.30±0.05*# 1.42±0.07*# Differentsymbolsbetweengroupsindicatesignificanceatp<0.05;ANOVAfollowedbyTukeyorDunnett.

700

A

B

D

C

450

400

350

300

250

200

150

100

50

0

25

20

15

10

5

0 600

500

400

300

200

100

0

14

12

10

8

6

4

2

0 Control

T

estosterone ng/dl

N

×

10

6

N

×

10

6 /g testis

Da

ys

1X 2X 4X 8X

Control 1X 2X 4X 8X

Fig.2.Totaltestosterone(A);spermaticreserveintheepididymistail(B);spermatictransittime(C);dailyspermaticproduction(D)ofthecontrolandtreatedgroups(n=12 foreachgroup,exceptforthe8×,n=10)(mean±standarderror).Differentsymbolsbetweengroupsindicatesignificanceatp<0.05;ANOVAfollowedbyTukeyorDunnett.

85 ₆₅

60

55

50

45

40

35

30

25

20 80

A

B

75

70

65

60

55

50

Control 1X 2X 4X 8X Control

%

1X 2X 4X 8X

(6)

Table3

Percentageofabnormalspermatozoidsofthecontrolandayahuascatreatedanimals(mean±standarderror).

Parameter Control (n=12)

1× (n=12)

2× (n=12)

4× (n=12)

8× (n=10) Headlesstail 18.2±3.7 28.7±7.6 14.3±2.3 17.2±4.2 17.3±1.7 Flattenedhead 0.3±0.1*# 0.3±0.1*# 0.1±0.1* 0.3±0.1*# 0.9±0.4# Pinhead 1.4±0.5 0.7±0.2 0.7±0.3 1.3±0.2 1.2±0.5 Bentneck 1.4±0.3 2.1±0.7 2.0±0.8 0.8±0.3 0.9±0.3 Benttail 18.7±3.6*# 14.6±2.8* 27.3±3.4# 29.1±3.7# 24.2±4.3*# Coiltail 0.2±0.1 0.7±0.3 0.7±0.2 0.7±0.3 0.7±0.4 Multiplesabnormalities 0.0±0.0 0.2±0.2 0.2±0.1 0.4±0.2 0.2±0.1 Total 41.9±3.0 49.9±6.6 48.1±3.1 53.1±5.8 47.7±4.7 Differentsymbolsbetweengroupsindicatesignificanceatp<0.05;Kruskal–Wallistest.

2weeksbeforetheirarrivalinthatregion,whilethosefoundinthe headwerereleasedbythetesticlejustafewdaysprior(Lanning

etal.,2002).

Studieshave shown that delays in sperm transport time in

theepididymisdo notalter thefertilityofthegametes(Billups

et al., 1990; Kempinas et al., 1998), but a lower transit time

mayaffectspermdevelopment,maturityandfertility(Klinefelter,

2002;Fernandezetal.,2008).Meistrich(1975)demonstratedthat

treatmentofWistarratswithestradiolderivativestogetherwith

testosterone didnot affecttesticular functions,but accelerated

spermtransporttimeandconsequentlyreducedthespermreserve

intheepididymis,similartowhatwasfoundinthisstudywith

ayahuasca.However,other authorshave reportedtheaction of

toxicagentsonspermreservesinthetailoftheepididymis,but

nochangesinspermproduction(Goyaletal.,2001;Klinefelterand

Suarez,1997;Bellentanietal.,2011).

Themostrelevantresultfoundinthisstudywasthesignificant increaseintotaltestosteronelevelsinanimalstreatedchronically

withayahuascaatthe4×doseincomparisonwiththe8×group

andthecontrol,whichwasinparallelwithasignificantdecrease inspermreserveoftheepididymistailandinspermtransittime. Theseresultsindicatedthattheeffectsobservedat4×dosewasnot

aleatory,andreflectsanon-monotonicdose–response.Alvarenga

etal.(2014)didnotfindanyeffectontestosteronelevelsofmale

ratsafterasingleayahuascadose,butadecreaseinsexual

perfor-mancewasobservedatalldoses(250–1000␮g/ml).Theincrease

intestosteroneinthe4×groupmaybeassociatedtoanincrease

intheabsoluteweightsoftheepididymisofthisgroup,sinceitis

anandrogynous-dependentorgan.Fernandezetal.(2008)founda

potentialrelationbetweenlowerreproductiveorganweightsand

lowertestosteroneplasmalevelsinmaleWistarratstreatedwith

diethylstilbestrol.

DMTbindsstronglytothe5-HT2Atypeserotonergicreceptors,

andactsasasubstratefortheSERT,thusinhibiting neurotransmit-terreuptake,whichremainlongerinthesynapticcleft(Smithetal.,

1998;Nagaietal.,2007;Cozzietal.,2009;Halberstadt,2015).

Addi-tionally,the␤-carbolinealkaloidspresentintheayahuascainfusion mayalsoinhibitthisreuptake(Callawayetal.,1999;Cozzietal.,

2009).McKenna(2004)statedthattheregularuseofayahuasca

apparentlyproducesserotonergicalterationsincreasingSERT

den-sityinthebrain,withanimpactonthepositivechangesinbehavior reportedbyayahuascausers(Callawayetal.,1994).

Theincreases in testosterone levels in animalstreated with

ayahuascaat the4× dosesuggests anendocrine compensation

mechanism,whichmayberespondingtotheincreasesin

sero-toninlevelsinthesynapticcleftsandinserotonergicactivitydue toDMT.Thisincreasewasnotobservedatthe8×dose,indicating

anon-monotonicdose–response,whichiscommonforendocrine

disruptors(Lagardeetal.,2015).Thisobservationmaybeexplained

bytheinvolvementoftestosteroneinthenegativefeedback

mech-anism,inhibiting thereleaseof GnRHinthehypothalamus and

consequentlyinhibitingthereleaseofgonadotropins(LHandFSH)

bythepituitarygland(SpritzeranddosReis,2008),althoughno

changesinthesehormoneswereobservedinayahuascatreated

animalsinthepresentstudy.Anotherhypothesisisthatthegreater

serotonergicactivity at the 8× dose due to higher DMT levels

(Pic-Tayloretal.,2015)maybedesensitizingreceptorsand

con-sequentlyreducing the SERTdensity. The 5HT2B receptors play

aregulatoryroleinthehomeostasisofsynapticserotoninlevels,

possiblyparticipatinginthecontrolofSERTintheraphenuclei

neuronswhich,onceactivated,inhibittheextracellular accumula-tionofserotonininducedbyselectiveserotoninreuptakeinhibitors (SSRI)(Diazetal.,2012).Furthermore,Kranzetal.(2015)showed

adirectrelationbetweenincreasesinplasmatestosteronelevels

inducedbytreatmentintranssexualsandincreasesinthedensityof serotoninreuptakesitesinvariousbrainregions.Studieswith

ani-malshaveshownthattreatmentwithtestosteroneincreasedthe

densityof5-HT2Areceptorsincertainareasofthebrain(Sumner

andFink,1998;Mcqueenetal.,1999;Herrera-Pérezetal.,2013;

Kranzetal.,2015).

Thisstudyallowedtheestablishmentofa

no-observed-adverse-effect-levelofayahuascaformaleWistarratstreatedevery2days for70days,at2×thedoseusedataUDVritual,whichcorresponds

to0.62mg/kgbwofDMT,6.6mg/kgbwofharmine,and0.52mg/kg

bw of harmaline. The effects observed at higher doses include

decreasesinfeedconsumptionandbodyweightgains,andincrease

ofrelative stomachand brainweights.A potentialreproductive

toxiceffectwasobservedatthe4×dose,withanon-monotonic

dose–response.Studiesinvestigatingtheroleofayahuasca

com-ponentsinregulatingtestosteronelevels,aswellasofSERTinthe brainsofmaleratsareneededtobetterunderstandthistoxicaction. Thisisthefirsttimethatthepotentialeffectofayahuascaon

maleanimalreproductionparametersafterchronicexposurewas

investigated.Theresultsofthisstudyareimportantasthe

world-wideuseofthisbeveragehasincreasedsubstantiallyinthelast

decades.Additionally,asthetherapeuticuseofayahuascahasbeen considered,thesafetyofitsuseneedstobestablished.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare

thattheproceduresfollowedwereinaccordancewiththe

regula-tionsoftheresponsibleClinicalResearchEthicsCommitteeandin

accordancewiththoseoftheWorldMedicalAssociationandthe

HelsinkiDeclaration.

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearsinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat

(7)

Authors’contributions

AFASconductedtheexperimentsandanalyzedmostofthedata,

performedthestatisticsandpreparedthedraftmanuscript.ALSV

performedthehistologyanalysis.APTandEDCconceived,designed

andcoordinatedthestudy.Allauthorsreadandapprovedthefinal manuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorsthankLucianaNolli,Karina Castro,JulianaAlves

deMorais,NágelavonZuben,CamilaFerreiraandStefanyAlves

for helping during the different phases of theexperiment. We

alsothanktheSabin Laboratoryforperformingthebiochemical

and hormone analyses. This work received the financial

sup-portof theFederal DistrictResearchFoundation (FAP-DF;grant

193000358/2010).AFASantoswasgrantedaMasterscholarship

byCAPES.

References

Alvarenga,T.A.,Polesel,D.N.,Matos,G.,Garcia,V.A.,Costa,J.L.,Tufik,S.,Andersen, M.L.,2014.Canayahuascaandsleeplosschangesexualperformanceinmale rats?Behav.Process.108,110–116.

Amann,R.P.,Johnson,L.,ThompsonJR.,D.L.,Pickett,B.W.,1976.Dailyspermatozoal production,epididymalspermatozoalreservesandtransittimeofspermatozoa throughtheepididymisoftherhesusmonkey.Biol.Reprod.15,586–592. Bellentani,F.F.,Fernandes,G.S.A.,Perobelli,J.E.,Pacini,E.S.A.,Kiguti,L.R.A.,Pupo,

A.S.,Kempinas,W.D.G.,2011.Accelerationofspermtransittimeandreduction ofspermreservesintheepididymisofratsexposedtosibutramine.J.Androl. 32,718–724.

Billups,K.L.,Tillman,S.,Chang,T.S.,1990.Ablationoftheinferiormesentericplexus intherat:alterationofspermstorageintheepididymisandvasdeferens.J.Urol. 143,625–629.

Blazak,W.F.,Ernst,T.L.,Stewart,B.E.,1985.Potentialindicatorsofreproductive tox-icity:testicularspermproductionandepididymalspermnumber,transittime, andmotilityinfischer344rats.Fundam.Appl.Toxicol.5,1097–1103. Buckholtz,N.S.,Boggan,W.O.,1977.Monoamineoxidaseinhibitioninbrainand

liverproducedby␤-carbolines:structure–activityrelationshipsandsubstrate specificity.Biochem.Pharmacol.26,1991–1996.

Callaway,J.C.,Grob,C.S.,1998.Ayahuascapreparationsandserotoninreuptake inhibitors:apotentialcombinationforsevereadverseinteractions.J.Psychoact. Drugs30,367–369.

Callaway,J.C.,Airaksinen,M.M.,Mckenna,D.J.,Brito,G.S.,Grob,C.S.,1994.Platelet serotoninuptakesitesincreasedindrinkersofayahuasca.Psychopharmacology (Berl.)116,385–387.

Callaway,J.C.,Raymon,L.P.,Hearn,W.L.,Mckenna,D.J.,Grob,C.S.,Brito,G.S.,Mash, D.C.,1996.QuantitationofN,N-dimethyltryptamineandharmalaalkaloidsin humanplasmaafteroraldosingwithayahuasca.J.Anal.Toxicol.20,492–497. Callaway,J.,McKenna,D.,Grob,C.,Brito,G.,Raymon,L.,Poland,R.,Mash,D.,1999.

PharmacokineticsofHoascaalkaloidsinhealthyhumans.J.Ethnopharmacol.65, 243–256.

CONAD,2004.ConselhoNacionaldePolíticassobreDrogas,ftp://ftp.saude.sp.gov. br/ftpsessp/bibliote/informeeletronico/2010/iels.jan.10/iels16/URS-CONAD-1 250110.pdf.

Cozzi,N.V.,Gopalakrishnan,A.,Anderson,L.L.,Feih,J.T.,Shulgin,A.T.,Daley,P.F., Ruoho,A.E.,2009.Dimethyltryptamineandotherhallucinogenictryptamines exhibitsubstratebehaviorattheserotoninuptaketransporterandthevesicle monoaminetransporter.J.Neural.Transm.(Vienna)116,1591–1599. Diaz,S.L.,Doly,S.,Narboux-Nême,N.,Fernandez,S.,Mazot,P.,Banas,S.M.,

Bou-tourlinsky,K.,Moutikine,I.,Belmer,A.,Roumier,A.,Maroteaux,L.,2012.5-HT2B receptorsarerequiredforserotonin-selectiveantidepressantactions.Mol. Psy-chiatry17,154–163.

Domínguez-Clavé,E.,Soler, J.,Elices, M.,Pascual, J.C.,Álvarez,E.,delaFuente Revenga,M.,Friedlander,P.,Feilding,A.,Riba,J.,2016.Ayahuasca: pharmacol-ogy,neuroscienceandtherapeuticpotential.BrainRes.Bull.126,89–101. DosSantos,R.G.,2013.Acriticalevaluationofreportsassociatingayahuascawith

life-threateningadversereactions.J.Psychoact.Drugs45,179–188.

DosSantos,R.G.,Osório,F.L.,Crippa,J.A.,Hallak,J.E.,2016a.Antidepressiveand anx-iolyticeffectsofayahuasca:asystematicliteraturereviewofanimalandhuman studies.Rev.Bras.Psiquiatr.38,65–72.

DosSantos,R.G.,Balthazar,F.M.,Bouso,J.C.,Hallak,J.E.,2016b.Thecurrentstate ofresearchonayahuasca:asystematicreview ofhumanstudiesassessing

psychiatricsymptoms,neuropsychologicalfunctioning,andneuroimaging.J. Psychopharmacol.30,1230–1247.

EPA, 1996. Guidelines for Reproductive Toxicity Risk Assessment 61., pp. 56274–56322.

Fernandez,C.D.B.,Porto,E.M.,Arena,A.C.,Kempinas,W.D.G.,2008.Effectsofaltered epididymalspermtransittimeonspermquality.Int.J.Androl.31,427–437. Goyal,H.O.,Braden,T.D.,Mansour,M.,Williams,C.S.,Kamaleldin,A.,Srivastava,

K.K.,2001.Diethylstilbestrol-treatedadultratswithalteredepididymalsperm numbersandspermmotilityparameters,butwithoutalterationsinsperm pro-ductionandspermmorphology.Biol.Reprod.64,927–934.

Grob,C.S.,McKenna,D.J.,Callaway,J.C.,Brito,G.S.,Neves,E.S.,Oberlaender,G.,Boone, K.B.,1996.Humanpsychopharmacologyofhoasca,aplanthallucinogenusedin ritualcontextinBrazil.J.Nerv.Ment.Dis.184,86–94.

Halberstadt,A.L.,2015.Recentadvancesintheneuropsychopharmacologyof sero-tonergichallucinogens.Behav.BrainRes.277,99–120.

Halpern,J.H.,2004.Hallucinogensanddissociativeagentsnaturallygrowinginthe UnitedStates.Pharmacol.Ther.102,131–138.

Harvey,R.,Champe,P.C.,Mycek,M.J.,1998.Lippincott’sIllustratedReviews Phar-macology.Artimed,PortoAlegre.

Herrera-Pérez,J.J.,Fernández-Guasti,A.,Martínez-Mota,L.,2013.BrainSERT expres-sionofmaleratsisreducedbyagingandincreasedbytestosteronerestitution. Neurosci.J.,http://dx.doi.org/10.1155/2013/201909.

IRDG,2000.ComputerAssistedSpermAnalysis(CASA)Group,RatSperm Mor-phologicalAssessment,1sted.IndustrialReproductiveToxicologyDiscussion Group,InvereskResearch,HuntingdonLifeSciences,Sequani,GlaxoWellcome (conts.),pp.8–13.

Johnsen,S.G.,1970.Testicularbiopsyscorecount–amethodforregistrationof spermatogenesisinhumantestes:normalvaluesandresultsin335hypogonadal Males.Hormones1,2–25.

Kempinas,W.D.G.,Klinefelter,G.R.,2014.Interpretinghistopathologyinthe epi-didymis.Spermatogenesis4,1–12.

Kempinas,W.D.G.,Suarez,J.D.,Roberts,N.L.,Strader,L.,Ferrell,J.,Goldman,J.M., Klinefelter,G.R.,1998.Ratepididymalspermquantity,quality,andtransittime afterguanethidine.Biol.Reprod.59,890–896.

Klinefelter,G.R.,2002.Actionsoftoxicantsonthestructureandfunctionofthe epi-didymis.In:Robaire,B.,Hinton,B.T.(Eds.),TheEpididymis–FromMolecules toClinicalPractice.KluwerAcademic/PlenumPublisher,NewYork,pp.353– 369.

Klinefelter,G.R.,Suarez,J.D.,1997.Toxicant-inducedaccelerationofepididymal spermtransit:androgen-dependentproteinsmaybeinvolved.Reprod.Toxicol. 11,511–519.

Kranz,G.S.,Wadsak,W.,Kaufmann,U.,Savli,M.,Baldinger,P.,Gryglewski,G., Haeusler,D.,Spies,M.,Mitterhauser,M.,Kasper,S.,Lanzenberger,R.,2015. High-dosetestosteronetreatmentincreasesserotonintransporterbindingin transgenderpeople.Biol.Psychiatry78,525–533.

Kulkarni,M.,Hayden,C.,Kayes,O.,Hayden,C.,2014.Recreationaldrugsandmale fertility.Trends.Urol.MensHealth5,19–23.

Labate,B.C.,Feeney,K.,2012.AyahuascaandtheprocessofregulationinBraziland internationally:implicationsandchallenges.Int.J.DrugPolicy23,154–161. Lagarde, F., Beausoleil, C., Belcher, S.M., Belzunces, L.P.,Emond, C., Guerbet,

M., Rousselle, C., 2015. Non-monotonic dose–response relationships and endocrinedisruptors:aqualitativemethodofassessment.Environ.Health 14:13,http://dx.doi.org/10.1186/1476-069X-14-13.

Lanning,L.L.,Creasy,D.M.,Chapin,R.E.,Mann,P.C.,Barlow,N.J.,Regan,K.S., Good-man,D.G.,2002.Recommendedapproachesfortheevaluationoftesticularand epididymaltoxicity.Toxicol.Pathol.30,507–520.

Macrae,E., 2004.Theritualuseofayahuascaby threeBrazilian religions.In: Coomber,R.,South,N.(Eds.),DrugUseandCulturalContextsBeyondetheWest. FreeAssociationBooks,London,pp.27–45.

McKenna, D.J., 2004. Clinical investigations of the therapeutic potential of ayahuasca:rationaleandregulatorychallenges.Pharmacol.Ther.102,111–129. McKenna,D.J.,Towers,G.H.N.,Abbott, F.,1984. Monoamineoxidaseinhibitors insouthAmericanhallucinogenicplants:tryptamineand␤-carboline con-stituentesofayahuasca.J.Ethnopharmacol.10,195–223.

Mcqueen,J.K.,Wilson,H.,Sumner,B.E.H.,Fink,G.,1999.Serotonintransporter(SERT) mRNAandbindingsitedensitiesinmaleratbrainaffectedbysexsteroids.Brain Res.Mol.BrainRes.63,241–247.

Meistrich,M.L.,1975.Alterationofepididymalspermtransportandmaturationin micebyoestrogenandtestosterona.Nature258,145–147.

Morgan, D.J., Muller,C.H., Murataeva, N.A., Davis, B.J., Mackie, K.,2011. 9-Tetrahydrocannabinol(9-THC)attenuatesmousespermmotilityandmale fecundity.Br.J.Pharmacol.165,2575–2583.

Motta,L.S.G.,(Dissertac¸ãodeMestrado)2013.Toxicidadeaguda,neurotoxicidade, toxicidadereprodutivaeembriotoxicidadedocháayahuasca(Banisteriopsis caapiePsychotriaviridis)emratasWistar.ProgramadePós-graduac¸ãoem Ciên-ciasdaSaúde,UniversidadedeBrasília,Brasília,pp.82.

Nagai,F.,Nonaka,R.,Satoh,H.K.K.,2007.Theeffectsofnon-medicallyused psychoac-tivedrugsonmonoamineneurotransmissioninratbrain.Eur.J.Pharmacol.559, 132–137.

Oliveira,C.D.R.,Moreira,C.Q.,deSá,L.R.M.,deSouza,S.H.,Yonamine,M.,2010. MaternalanddevelopmentaltoxicityofayahuascainWistarrats.BirthDefects Res.B:Dev.Reprod.Toxicol.89,207–212.

Onyije,F.M.,2012.Drug:amajorcauseofinfertilityinmale.AsianJ.Med.Pharm. Res.2,30–37.

(8)

effectsofayahuascainfusion(BanisteriopsiscaapiandPsychotriaviridis)infemale Wistarrat.Behav.Process.118,102–110.

Pires,A.P.S.,Oliveira,C.D.R.,Yonamine,M.,2010.Ayahuasca:umarevisãodos aspéc-tosfarmacológicosetoxicológicos.Rev.Cienc.Farm.BásicaApl.31,15–23. Riba,J.,Valle,M.,Urbano,G.,Yritia,M.,Morte,A.,Barbanoj,M.J.,2003.Human

pharmacologyofayahuasca:subjectiveandcardiovasculareffects,monoamine metaboliteexcretion,andpharmacokinetics.J.Pharmacol.Exp.Ther.306,73–83. Robb,G.W.,Amann,R.P.,Killian,G.J.,1978.Dailyspermproductionandepididymal

spermreservesofpubertalandadultrats.J.Reprod.Fertil.54,103–107. Seed,J.,Chapin,E.,Clegg,E.D.,Dostal,L.A.,Foote,H.,Hurtt,M.E.,Klinefelter,G.R.,

Makris,S.L.,Perreault,S.D.,Schrader,S.,Seyler,D.,Sprando,R.,Treinen,K.A., Veeramachaneni,D.N.,Wise,L.D.,1996.Methodsforassessingspermmotilit, morphology,andcountsintherat,rabbit,anddog:aconsensusreport.Reprod. Toxicol.10,237–244.

Shanon,B.,2003.Osconteúdosdasvisõesdaayahuasca.Mana9,109–152. Sharma,P.,Singh,R.,2010.Protectiveroleofcurcuminonlindaneinduced

repro-ductivetoxicityinmaleWistarrats.Bull.Environ.Contam.Toxicol.84,378–384. Smith,R.L.,Canton,H.,Barrett,R.J.,Sanders-Bush,E.,1998.Agonistpropertiesof N,N-dimethyltryptamineatserotonin5-HT2Aand5-HT2Creceptors.Pharmacol. Biochem.Behav.61,323–330.

Spritzer,P.M.,dosReis,F.M.,2008.Gonads.In:Aires,M.M.(Ed.),Physiology. Guan-abaraKoogan,RiodeJaneiro,pp.1115–1119.

Strader,L.F.,Linder,R.E.,Perrbault,S.D.,1996.Comparisonofratepididymalsperm countsbyivosHTM-IDENTandhemacytometer.Reprod.Toxicol.10,529–533. Sumner,B.E.H.,Fink,G.,1998.Testosteroneaswellasestrogenincreasesserotonin 2AreceptormRNAandbindingsitedensitiesinthemaleratbrain.BrainRes. Mol.BrainRes.59,205–214.

Tupper,K.W.,2008.Theglobalizationofayahuasca:harmreductionorbenefit max-imization?Int.J.DrugPolicy19,297–303.

Vignera,S.L.,Condorelli,R.A.,Barlecia,G.,Vicari,E.,Calogero,A.E.,2013.Doesalcohol haveanyeffectonmalereproductivefunction?Areviewofliterature.AsianJ. Androl.15,221–225.

Vives,S.G.,García-albea,J.,2012.Exceptionalstatesofconsciousnessand psy-chotropicsubstanceconsumption.ActasEsp.Psiquiatr.40,96–103.

Winstock,A.R.,Kaar,S.,Borschmann,R.,2014.Dimethyltryptamine(DMT): preva-lence,usercharacteristics and abuseliability in a largeglobal sample. J. Psychopharmacol.28,49–54.