In Vitro Activity of the Antifungal Azoles Itraconazole and Posaconazole against Leishmania ...

14 Pages·2013·5.42 MB·English

InVitroActivity of the Antifungal Azoles Itraconazole
and Posaconazole against Leishmaniaamazonensis
Sara Teixeira de Macedo-Silva1,2, Julio A. Urbina3, Wanderley de Souza1,2,4, Juliany Cola
Fernandes Rodrigues1,2,4,5*
1Laborato´riodeUltraestruturaCelularHerthaMeyer,InstitutodeBiof´ısicaCarlosChagasFilho,UniversidadeFederaldoRiodeJaneiro,RiodeJaneiro,Brazil,2Instituto
NacionaldeCieˆnciaeTecnologiadeBiologiaEstruturaleBioimagem,RiodeJaneiro,Brazil,3InstitutoVenezolanodeInvestigacionesCient´ıficas,CentrodeBioqu´ımicay
Biof´ısica,Caracas,Venezuela,4InstitutoNacionaldeMetrologia,QualidadeeTecnologia,Inmetro,RiodeJaneiro,Brazil,5Nu´cleoMultidisciplinardePesquisaemBiologia
(NUMPEX-BIO),PoloAvanc¸adodeXere´m,UniversidadeFederaldoRiodeJaneiro,DuquedeCaxias,Brazil
Abstract
Leishmaniasis, caused by protozoan parasites of the Leishmania genus, is one of the most prevalent neglected tropical
diseases.Itisendemicin98countries,causingconsiderablemorbidityandmortality.Pentavalentantimonialsarethefirst
lineoftreatmentforleishmaniasisexceptinIndia.Inresistantcases,miltefosine,amphotericinBandpentamidineareused.
Thesetreatmentsareunsatisfactoryduetotoxicity,limitedefficacy,highcostanddifficultadministration.Thus,thereisan
urgent need to develop drugs that are efficacious, safe, and more accessible to patients. Trypanosomatids, including
Leishmaniaspp.andTrypanosomacruzi,haveanessentialrequirementforergosterolandother24-alkylsterols,whichare
absent in mammalian cells. Inhibition of ergosterol biosynthesis is increasingly recognized as a promising target for the
developmentofnewchemotherapeuticagents.Theaimofthisworkwastoinvestigatetheantiproliferative,physiological
and ultrastructural effects against Leishmania amazonensis of itraconazole (ITZ) and posaconazole (POSA), two azole
antifungal agents that inhibit sterol C14a-demethylase (CYP51). Antiproliferative studies demonstrated potent activity of
POSA and ITZ: for promastigotes, the IC values were 2.74 mM and 0.44 mM for POSA and ITZ, respectively, and for
50
intracellular amastigotes, the corresponding values were 1.63mM and 0.08 mM, for both stages after 72h of treatment.
Physiological studies revealed that both inhibitors induced a collapse of the mitochondrial membrane potential (DYm),
whichwasconsistentwithultrastructuralalterationsinthemitochondrion.Intensemitochondrialswelling,disorganization
andruptureofmitochondrialmembraneswereobservedbytransmissionelectronmicroscopy.Inaddition,accumulationof
lipid bodies, appearance of autophagosome-like structures and alterations in the kinetoplast were also observed. In
conclusion, our results indicate that ITZ and POSA are potent inhibitors of L. amazonensis and suggest that these drugs
couldrepresent noveltherapies forthe treatment ofleishmaniasis,eitheralone orincombination with otheragents.
Citation:deMacedo-SilvaST,UrbinaJA,deSouzaW,RodriguesJCF(2013)InVitroActivityoftheAntifungalAzolesItraconazoleandPosaconazoleagainst
Leishmaniaamazonensis.PLoSONE8(12):e83247.doi:10.1371/journal.pone.0083247
Editor:HenkD.F.H.Schallig,RoyalTropicalInstitute,TheNetherlands
ReceivedJune17,2013;AcceptedOctober31,2013;PublishedDecember23,2013
Copyright:(cid:2)2013deMacedo-Silvaetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:ThisworkwassupportedbyConselhoNacionaldeDesenvolvimentoCient´ıfico(CNPq),Fundac¸a˜oCarlosChagasFilhodeAmparoa`PesquisadoEstado
doRiodeJaneiro(FAPERJ),FinanciadoradeEstudoseProjetos(FINEP),andCoordenac¸a˜odeAperfeic¸oamentodePessoaldeN´ıvelSuperior(CAPES).Thefunders
hadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
Introduction epidemiologyoftheleishmaniasisintheAmazonregion[6].When
theimmunesystemfailstomountanappropriateresponseagainst
The leishmaniases, which are among the most prevalent the parasite, L. amazonensis can cause clinical manifestations of
neglected tropical diseases, are caused by protozoan parasites of diffuse cutaneous leishmaniasis [5]. It is a serious public health
the Leishmania genus. The disease is endemic in 98 countries problem in Brazil, because the lesions cover a large part of the
worldwide, and more than 2 million new cases occur annually, body, sometimes producing mutilated lesions, and is devastating
with high levels of morbidity and mortality [1]. There are three for the patients, because it is incurable using currently available
majorclinicalmanifestationsofthedisease:visceral,mucocutane- treatments.
ousandcutaneousleishmaniases.Othercutaneousmanifestations Pentavalentantimonialcompounds(e.g.,sodiumstibogluconate
include diffuse cutaneous leishmaniasis, recidivans leishmaniasis andmeglumineantimoniate)havebeenthedrugsofchoiceforthe
andpost-kala-azardermalleishmaniasis[2].Thepathologycaused treatment of leishmaniasis for decades worldwide despite their
by Leishmania depends on several factors, which include the severe side effects [7,8]. However, they have been recently
infecting species andthehostimmune response[3,4]. Morethan discontinued in the India. In addition, antimonials are associated
90%ofcasesofvisceralleishmaniasisandcutaneousleishmaniases with significant failure and relapse rates, especially in immuno-
occur in India, Sudan, Bangladesh, Nepal, Brazil, Afghanistan, compromised hosts [9–11]. Pentamidine and amphotericin B are
Saudi Arabia, Algeria, Iran, Iraq and Syria [1]. In Brazil, otherparenteralalternativesthatcancausesignificantsideeffects
Leishmania amazonensis is one of the species responsible for the [8,12]. Miltefosine (Impavido) is the first oral drug available for
cutaneous form of the disease [5] and it is important for the treatment of visceral leishmaniasis in India [12–13], but it is
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EffectsofTwoAzolesinLeishmaniaamazonensis
teratogenic,andthereareindicationsthatresistancetothedrugis
appearinginendemicareas[13].Inaddition,miltefosinealsohas
significant effects against cutaneous leishmaniasis in human [12]
andinmurinemodelsofcutaneousleishmaniasisbyinfectionwith
L.amazonensis[14].Thus,thereisanurgentneedtodevelop new
drugs that are efficacious,safe, and moreaccessible topatients.
Sterolsareconstituentsofcellularmembranesthatareessential
fortheirnormalstructureandfunction.Trypanosomatidshavean
essential requirement for ergosterol and other 24-alkyl sterols,
which are absent in mammalian cells [15–16], and ergosterol
biosynthesis inhibitors (EBIs) have proved to be potential
candidates for the treatment of leishmaniasis and other diseases
caused by protozoan parasites, such as Chagas disease (17–21).
Itraconazole (ITZ) and posaconazole (POSA) are two known
azoles that inhibit sterolC14a-demethylase (CYP51),an essential
enzyme in the sterol biosynthesis pathway, with potent effects
against fungi and trypanosomatids [22–28]. In particular, POSA
hasshown potentactivityinmurinemodelsofacute andchronic
Chagas’ disease [17,29]. In addition, POSA and ITZ have also
been studied in murine models of cutaneous and visceral
leishmaniases by infection with L. amazonensis, L. donovani [18]
and L. infantum [30]. Furthermore, there are some studies
describingtheeffectofITZonpatientswithcutaneousleishman-
iasis[31–33].Thus,wedecidedtoinvestigatetheinvitroeffectsof
POSA and ITZ on the proliferation and ultrastructure of L.
amazonensis.We foundthat bothcompounds are potentinhibitors
ofL.amazonensisgrowthandinducedmultipleseverealterationsin
the ultrastructure of promastigotes and intracellular amastigotes.
Inparticular,thesedrugsaffectedthestructureandfunctionofthe
single giant mitochondrion present in these cells and induced an
accumulation of lipidbodies and autophagosomes.
Materials and Methods
Ethics Statement
The experiments using animal models to obtain macrophages
andLeishmaniawereapprovedbytheEthicsCommitteeforAnimal
Experimentation of the Health Sciences Centre, Federal Univer-
sity of Rio de Janeiro (Protocols n. IBCCF 096/097/106),
according to the Brazilian federal law (11.794/2008, Decreto no
6.899/2009). All animals received humane care in compliance
with the ‘‘Principles of Laboratory Animal Care’’ formulated by
theNationalSocietyforMedicalResearchandthe‘‘Guideforthe
Care and Use of Laboratory Animals’’ prepared by the National
Academy ofSciences, USA.
Parasites
TheMHOM/BR/75/JosefastrainofL.amazonensisusedinthis
study was isolated in 1975 by Dr. Cesar A. Cuba-Cuba (Brasilia
University, Brazil) from a patient with diffuse cutaneous
leishmaniasis and kindly provided by the Leishmania Collection of
theInstitutoOswaldoCruz(CodeIOCL0071-FIOCRUZ).The
strain was maintained by inoculation into the base of the tails of Figure 1. Antiproliferative effects of posaconazole and itra-
Balb/C mice. Axenic promastigotes were cultured at 25uC in conazole on Leishmaniaamazonensis.L.amazonensispromasti-
Warren’smedium(brainheartinfusionplusheminandfolicacid) gotes(A, B) andintracellular amastigotes (C,D) were treated
[34]supplementedwith10%fetalbovineserum(Cultilab,Brazil). withposaconazole(POSA)(A,C)oritraconazole(ITZ)(B,D)to
evaluatetheparasitegrowth.Thearrowsindicatethetimeofthe
InfectivemetacyclicpromastigotesoftheJosefastrainwereusedto
additionofthedrugsattheindicatedconcentrations.Theresultswere
obtain intracellular amastigotes inmacrophage cultures.
plotted as the mean of three independent experiments and the bars
representthestandarddeviation.
Drugs and Reagents doi:10.1371/journal.pone.0083247.g001
Posaconazole (POSA) was provided by the Schering Plough
Research Institute (United States). Itraconazole (ITZ) was (DMSO) and stored at 220uC. For experiments, new dilutions
purchasedfromJanssenPharmaceuticalCompanies(Brazil).Both were prepared in culture medium to ensure that the DMSO
drugs were dissolved in a 10mM stock of dimethyl sulfoxide concentrationintheculturemediumdidnotexceed0.1%.Allthe
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Table1.IC ,CC andselectiveindexes(SI)forthetreatmentofL.amazonensispromastigotesandintracellularamastigoteswith
50 50
posaconazole(POSA) and itraconazole (ITZ).
Intracellularamastigotes
Promastigotes(IC50-mM) (IC50-mM) CC50(mM)* SelectivityIndex(SI)**
Timeofincubation 48h 72h 48h 72h 72h 72h
Posaconazole 3.94 2.74 1.95 1.63 20 12.2
Itraconazole 0.88 0.44 0.64 0.08 15 187.5
*CC wasobtainedafter72hoftreatmentwithbothcompounds.
50
**SelectivityIndex(SI)wascalculateddividingtheCC50bytheIC50valuesobtainedaftertreatmentfor72h.
doi:10.1371/journal.pone.0083247.t001
reagents for electron microscopy were from Electron Microscopy Cytotoxicity Assay
Sciences (England). Cytotoxicity effects of ITZ and POSA against murine macro-
phages were evaluated using the CellTiter 96H Aqueous MTS
In vitro Antiproliferative Effects Reagent Powder (Promega, United States). Murine macrophages
Promastigote cultures were initiated at a cell density of obtainedasexplainedabovewerecultivatedina96-wellplatewith
1.06106cells/ml.After24hofgrowth,POSAorITZwasadded RPMImediumcontaining10%fetalbovineserum(Gibco,Brazil)
atdifferentconcentrations(0.05;0.1;0.5;1;3;5;8mMforPOSA, and maintained at 37uC in 5% CO2. After 24h of cultivation,
and 0.05; 0.5; 1; 3; 5; 8 mM for ITZ) from concentrated stock different concentrations of the drugs (5, 15, 25 and 35mM for
solutions. Celldensities wereevaluated daily over96h ofgrowth POSA and ITZ) were added every 24h until 72h of treatment,
usingaNeubauerchamber.ToevaluatetheeffectsofPOSAand whenthecytotoxicitywasmeasured.Cellviabilitywasassessedby
ITZonL.amazonensisintracellularamastigotes,macrophagesfrom the MTS/PMS assay reaction and results were expressed as
theperitonealcavityofCF1micewereharvestedbywashingwith optical density measured at 492nm in a microplate reader and
Hank’s solution, plated in 24-well tissue culture chamber slides spectrofluorometer SpectraMax M2/M2e (Molecular Devices,
andallowedtoadheretotheslidesfor24hat37uC,5%CO ,in United States) [35]. The cytotoxicity concentration to reduce
2
RPMI medium (Gibco, Brazil) supplemented with 10% fetal 50%of viablemacrophages (CC50)was determined.
bovine serum. Adherent macrophages were infected with meta-
cyclic promastigotes at a macrophage-to-parasite ratio of 1:10 at Electron Microscopy
35uC, 5% CO , for 2 h and then washed two times with RPMI Control,ITZ-orPOSA-treatedpromastigotesandintracellular
2
medium toremove non-ingested parasites. Infected cultures were amastigotes inside macrophages were fixed in 2.5% glutaralde-
incubatedinRPMImediumsupplementedwith10%fetalbovine hyde in 0.1M cacodylate buffer (pH7.2) and postfixed in a
serumwithoutdrugs.After24hofinfectionwhenthenumberof solutioncontaining1%OsO4,1.25%potassiumferrocyanideand
amastigotes per macrophage was in the range of two to four, 0.1 M cacodylate buffer, pH7.2. For transmission electron
differentconcentrationsofPOSA(1;3;4;5;6 mM)orITZ(0.01; microscopy, cells were dehydrated in acetone and embedded in
0.05;0.09;0.5;1 mM)wereadded.FreshmediumwithPOSAor epoxy resin. Ultrathin sections were stained with uranyl acetate
ITZwasaddeddailyfor3days(72 hoftreatment). Thecultures and lead citrate and observed under a Zeiss 900 electron
werefixedinBouin’ssolution(70%picricacid,5%aceticacidand microscope. For scanning electron microscopy, promastigotes
25% formaldehyde in aqueous solution), washed in 70% ethanol weredehydratedinethanol,criticalpoint-driedinCO2,mounted
andindistilledwater.ThecultureswerethenstainedwithGiemsa on stubs, sputtered with a thin gold layer and observed under a
for 1 h. To determine the percentage of infected cells, 600 FEIQuanta 250scanning electron microscope.
macrophages(infectedandnon-infected)werecountedinabright
field optical microscope using a 1006immersion oil objective. Estimation of Mitochondrial Transmembrane Electric
Association indexes (mean number of parasites internalized per Potential (DYm)
cell, multiplied by the number of infected macrophages and Themitochondrialmembranepotential(DYm)ofL.amazonensis
dividedbythetotalnumberofmacrophages)weredeterminedand promastigoteswasanalyzedafter48hoftreatmentwithPOSAor
used to calculate the percentage of infection in each study ITZ (1 and 5 mM), using the JC-1 fluorochrome (Molecular
condition. The concentration that inhibited 50% of the growth Probes, United States) [36]. This fluorochrome is a lipophilic,
(IC value) was calculated and the control infection (without cationic, mitochondrial vital dye that accumulates in the
50
treatment) was used as reference parameter (100% of infection). mitochondria in response to DYm. At low concentrations, the
The results are expressed as the mean of three independent dye exists as a monomer, which emits at 530nm (green
experiments. fluorescence); at higher concentrations the dye accumulates in
themitochondrionandformsJ-aggregates,whichemitat590nm
IC Calculations (redfluorescence).Control,POSAandITZ-treatedpromastigotes
50
For the calculation of the IC values, percentage of growth were harvested, washed in PBS, pH 7.2, added to the reaction
50
inhibitionwasplottedasafunctionofdrugconcentrationbyfitting medium containing 125mM sucrose, 65mM KCl, 10mM
thevaluestoanon-linearcurveanalysiswheref = min+(max2 HEPES/K+pH 7.2,2 mMPi,1 mMMgCl2and500mMEGTA
min) /(1 +(x/EC )Hillslope),where theIC isthe concentration andcountedusingaNeubauerchamber.ToevaluatetheDYmfor
50 50 each experimental condition, 1.06107 parasites were incubated
that inhibits 50% of the growth. The regression analyses were
with10mg/mLJC-1for40min,withreadingsmadeeveryminute
performed with GraphpadPrism4 Software (UnitedStates).
using a microplate reader and spectrofluorometer SpectraMax
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EffectsofTwoAzolesinLeishmaniaamazonensis
Figure2.LightmicroscopyofmurinemacrophagesinfectedwithL.amazonensisamastigotes.(A)Controlculturewithmanyamastigotes
insideparasitophorousvacuoles.(B–H)After72hoftreatmentwithdifferentconcentrationsofPOSAandITZ,asignificantreductioninthenumber
ofparasitesandthepresenceofseveralemptyparasitophorousvacuoleswasobserved.
doi:10.1371/journal.pone.0083247.g002
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EffectsofTwoAzolesinLeishmaniaamazonensis
Figure3.Scanningelectronmicroscopy(SEM)ofL.amazonensispromastigotes.Controlparasites(A)andpromastigotesthatweretreated
withdifferentconcentrationsofPOSAandITZfor48h(B–I)wereobservedbySEM.(B,C)1mMITZ;(D–F)1mMPOSA;(G)5mMITZ;(H,I)5mMPOSA.
The images show dramatic alterations in promastigote shape (B–I), a promastigote with four flagella (E), and profound changes in the plasma
membrane(B,C,F).
doi:10.1371/journal.pone.0083247.g003
M2/M2e. After 36min of readings, 2mM FCCP was added to (1.06107) were then incubated with 10mg/mL Nile Red (Sigma,
abolishtheDYm.Forthepositivecontrol,cellswereincubatedin Brazil) for 20min and 1 mM Sytox Blue for 20min. The
thepresenceof2 mMFCCP,amitochondrialprotonophore.The experiments were performed in triplicate, using a black 96-well
relativeDYmvaluewasobtainedbycalculatingtheratiobetween plate. The cells were washed twice before analysis. The final
thereadingat590nmandthereadingat530nm(590:530ratio). volume in each well was 200ml of cell suspension in PBS.
Each experiment was repeated at least three times in triplicate Readings were taken with a microplate reader and spectrofluo-
using a black 96-well plate, and the figures shown are represen- rometerSpectraMaxM2/M2eusingthefollowingwavelengthsfor
tative ofthese experiments. excitation and emission, respectively: 485 and 538nm for Nile
Red, and 444 and 560nm for Sytox Blue. Each experiment was
Evaluation of Membrane Integrity and the Presence of repeatedatleastthreetimesintriplicate,andthefiguresshownare
Lipid Bodies via Nile Red Accumulation representative of these experiments. After the readings, control
andtreated-parasitesincubatedwithNileRedwerefixedwith4%
Control and treated promastigotes were harvested, washed in
nascent formaldehyde in0.1 Mphosphate buffer, pH 7.2,before
PBS, pH 7.2, and counted using a Neubauer chamber. Cells
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EffectsofTwoAzolesinLeishmaniaamazonensis
Figure4.AnalysisoflipidbodyaccumulationandplasmamembraneintegrityinL.amazonensispromastigotes.(A–B)Quantitative
fluorimetricanalysisusingNileRed(A)andSytoxBlue(B).Fluorescenceintensityisexpressedasarbitraryunits(A.U.).Theresultswereplottedas
mean of three independent experiments and the bars represent the standard deviation. *p,0.01; **p,0.05; ***p,0.0001. (C–H) Differential
interferencecontrast(DIC)microscopy(C,E,G)andfluorescencemicroscopyusingNileRed(D,F,H)ofcontrolL.amazonensispromastigotesand
promastigotes treated with 1mM POSA or ITZ for 48h. The images demonstrate an accumulationof lipid bodies that are randomly distributed
throughoutthecytoplasm,confirmingtheincreaseinthefluorescenceintensityobservedinFig.4A.
doi:10.1371/journal.pone.0083247.g004
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EffectsofTwoAzolesinLeishmaniaamazonensis
Figure 5. Ultrathin sections of control L.amazonensispromastigotes (A) and promastigotes treated with ITZ (B–D). (A) Control
promastigotes; (B–C) 5mM ITZ; (D) 1mM ITZ. The images show the presence of several electron-dense lipid bodies (asterisks), which sometimes
appear near the plasma membrane, the endoplasmic reticulum and mitochondrion profiles (arrow), and autophagosomes (arrowhead). A,
autophagosome;f,flagellum;k,kinetoplast;m,mitochondrion;N,nucleus.
doi:10.1371/journal.pone.0083247.g005
observation under a Zeiss Axioplan epifluorescence microscope Results
using an optical filter set with 450–490nm for excitation and
528nm foremission. Susceptibility of Leishmania amazonensis to POSA and
ITZ
Statistical Analysis Figure1showstheeffectsofPOSAandITZontheproliferation
All the graphics were made using the mean of three of L. amazonensis promastigotes and intracellular amastigotes
independent experiments and the bars represent the standard invitro. Both drugs were effective against promastigotes, causing
deviationofthose.Thestatisticalsignificanceofdifferencesamong concentration- and time-dependent inhibition of growth. IC
50
the groups was assessed using the one-way analysis of variance values of 3.94 mM and 0.88mM were observed for POSA and
(ANOVA) test followed by Bonferroni’s multiple comparison test ITZ,respectively,after48hoftreatment.After72hoftreatment,
in the GraphPad Prisma 4 Software. Results were considered the IC values were 2.74 mM and 0.44mM for POSA and ITZ,
50
statistically significant whenp,0.01. respectively. When incubated with intracellular amastigotes, the
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EffectsofTwoAzolesinLeishmaniaamazonensis
Figure 6. Evaluation of mitochondrial transmembrane electric potential (DY ) in L.amazonensispromastigotes using the JC-1
m
fluorochrome. (A) Values of DYm were evaluatedover 36min, before the addition of 2mM FCCP to abolish the mitochondrial potential. Two
concentrationsofPOSAandITZwereused(1and5mM)for48hoftreatment.TheDYmvaluesareexpressedastheratioofthereadingat590nm
(aggregate)tothereadingat530nm(monomer).(B)AnalysisofDYmatthelastminutebeforetheadditionof2mMFCCP.Thedatasuggestthat
similaralterationsinDY areinducedbyPOSA,ITZ,andFCCP.Theexperimentswereperformedthreetimes,eachtimeintriplicate,andthefigures
m
shownarerepresentativeoftheseexperiments.**p,0.05;***p,0.0001.
doi:10.1371/journal.pone.0083247.g006
clinically relevant form of the parasite, the effect of the drugs on with a compromised plasma membrane; thus, it is an efficient
parasitegrowthwasmorepotent,withIC valuesof1.95mMand dead-cell indicator. Quantitative fluorimetric analysis indicated
50
0.64 mMforPOSAandITZafter48hoftreatment,respectively, that treatment with 1 mM POSA and ITZ for 48h induced
and 1.63mM for POSA and 0.08mM for ITZ after 72h of significanteffectsintheaccumulationoflipidbodies(Fig.4A)and
treatment.AlltheIC valuesaresummarizedintable1.Figure2 in plasma membrane integrity (Fig. 4B). For both analyses, the
50
shows several images from bright field optical microscopy of the effectsinducedbyITZweregreaterthanthoseinducedbyPOSA
infectionsduringthetreatment.Parasiteswerenotobservedafter (Figs. 4A, B), congruent with the relative effects of the drugs on
72h of treatment with concentrations of POSA .4mM and promastigote proliferation. Fluorescence images indicated that
concentrationsofITZ.1 mM(Figs.2C–H).Thecytotoxiceffects Nile Red accumulated inside lipid bodies that were randomly
of POSA and ITZ were evaluated against murine macrophages distributedthroughoutthecytoplasmandthatthenumberoflipid
using the MTS assay, and the CC values are summarized in bodiesmarkedlyincreasedindrug-treatedpromastigotes(Figs.4F,
50
Table 1. POSA was less toxic than ITZ, with CC values of H). Transmission electron microscopy confirmed the presence of
50
20mM and 15mM, respectively. Selectivity indexes were calcu- lipid bodies after treatment with ITZ and POSA. Lipid bodies
lated using the IC values obtained after 72h of treatment, and were observed in control parasites (Fig. 5A), but in treated
50
ITZ wasmore selectivethan POSA invitro(Table 1). promastigotes, several osmiophilic lipid-storage bodies appeared
Scanningelectronmicroscopyrevealedadramaticalterationin close to the plasma membrane (Fig. 5B–D, asterisks), the
theshapeofpromastigotesaftertreatmentwithPOSAorITZfor endoplasmic reticulum, autophagosomes (Fig. 5B–C, asterisks
just48h(Figs.3B–I).Promastigotesappearedrounded(Figs.3D, andarrowhead), andthemitochondrion(Fig.5B, arrow).
G, H, and I), swollen (Figs. 3B, C, and E) or with cytoplasmic
shrinkage(Fig.3F).Inaddition,cellswithmorethanoneflagellum EffectsofPOSAandITZonMitochondrialPhysiologyand
(Fig.3E)andchangesinthecellsurface(Figs.3B,C,andI)were Ultrastructure in L. amazonensis
also observed.
The effects of POSA and ITZ on mitochondrial function and
ultrastructure were analyzed using two criteria: mitochondrial
Effects of ITZ and POSA on Plasma Membrane Integrity transmembrane electric potential (DYm), indicated by the JC-1
and Nile Red Accumulation fluorochrome, andtransmissionelectron microscopy.
To evaluate the effects of EBIs on plasma membrane integrity Promastigotes were treated with 1 and 5 mM POSA and ITZ,
and Nile Red accumulation, control and treated promastigotes respectively, for 48h prior to the analysis of DYm with JC-1, a
were incubated with Sytox Blue and Nile Red, respectively. Nile cell-permeant cationic, lipophilic fluorochrome. The classic
Redisafluorescencemarkerwithspecialaffinityforneutrallipids protonophore uncoupler FCCP was used as a positive control to
thatbecomeconcentratedinthelipidbodies.SytoxBlueisavital dissipate the mitochondrial electrochemical H+ gradient. Simul-
dye with high affinity for nucleic acid that easily penetrates cells taneous measurements of J-aggregates (red fluorescence), which
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Figure7.UltrathinsectionsofL.amazonensispromastigotestreatedwithdifferentconcentrationsofITZandPOSA.(A,B)1mMITZ;(C,
D)1mMPOSA;(E)3mMPOSAfor48h;(F)5mMPOSAfor72h.Severalalterationswereobservedinthemitochondrion-kinetoplastcomplexsuchas:
intensedisorganizationandswelling(A,B,D);alterationsinthemitochondrionmembranesandtheappearanceofcircularcristae(B,C,arrows);
changesinthestructureofthekinetoplast(B,D,E,F);andthepresenceofautophagosomes(A,C,D).InFig.7E,twolargevacuolescontaining
membranesandportionsofthecytoplasmwereobserved(asterisks).FP,flagellarpocket;GC,Golgicomplex;k:kinetoplast;m,mitochondrion;N:
nucleus,A:autophagosome.
doi:10.1371/journal.pone.0083247.g007
accumulate in intact and energized mitochondria, and of J- classicalinhibitorofmitochondrialmetabolism,control(untreated)
monomers(greenfluorescence)thatareamarkerforde-energized promastigotes were incubated with 2 mM FCCP during the
mitochondria, were used to calculate the DYm, expressed as a evaluation of DYm (Fig. 6B), and the observed alteration in the
ratio of fluorescence intensity obtained at 590 and 530nm. A mitochondrial electrochemical H+ gradient was similar to that
decrease in the red:green fluorescence intensity ratio indicates a observed incells pre-treated with5 mM EBIs.
collapseinthemitochondrialtransmembrane potential[36].Pre- Alterationsinmitochondrialultrastructurewerealsoinvestigat-
treatment of promastigotes for 48h with POSA and ITZ at the edbytransmissionelectronmicroscopy.Figure5Ashowsacontrol
indicated concentrations led to a marked reduction in the DYm promastigote presenting a normal ramified mitochondrion near
(Figs. 6A, B), which was concentration-dependent (Fig. 6B). To the plasma membrane. Treatment with POSA and ITZ induced
compare the effects of EBIs on the DYm with the effects of a dramaticalterationsofthemitochondrionultrastructure(Figs.7A–
PLOSONE | www.plosone.org 9 December2013 | Volume 8 | Issue 12 | e83247
EffectsofTwoAzolesinLeishmaniaamazonensis
Figure8.UltrathinsectionsofL.amazonensispromastigotes.Promastigotesweretreatedwith1mMPOSA(A,B),and3mMPOSA(C,D)for
48h. All images show the presence of small and large vacuoles containing several vesicles, membrane profiles and portions of the cytoplasm
(asterisks).Theendoplasmicreticulumappearsincloseassociationwiththenucleus,themitochondrionandautophagosomes(B–D,arrowheads).In
Fig. 8A, changes in kinetoplast structure and vesiculation of the inner mitochondrial membrane were observed. N: nucleus; k: kinetoplast; m:
mitochondrion;f:flagellum;A:autophagosome;FP;flagellarpocket.
doi:10.1371/journal.pone.0083247.g008
F). The main alteration observed was a profound mitochondrial Effects of POSA and ITZ on the General Ultrastructure of
swelling, which was followed by remarkable changes in the L. amazonensis Promastigotes and Intracellular
mitochondrion morphology (Fig.7A) and itsmembranes, leading
Amastigotes
totheappearanceofseveralcircularcristae(Figs.7B–C,arrows).
Alterations in the Golgi complex of promastigotes were
Significant alterations in kinetoplast structure, suggesting a
observed after treatment with 1 mM ITZ for 48h (Fig. 7B). In
decompaction of the kDNA, were also observed after treatment
addition, autophagosomes were observed after treatment with
with both EBIs (Figs. 7B, D–F). In addition, structures similar to
different concentrations of POSA for 48h (Figs. 8A–D). These
autophagosomes (represented by letter A and asterisks in the
promastigotes presented a total disorganization of the cytoplasm,
images)sometimesappearednearthemitochondrion(Figs.7A,C,
withtheendoplasmicreticulumappearingincloseassociationwith
E).
thenucleus,themitochondrionandautophagosomes(Figs.8B–D,
arrowheads).Severallargevacuolescontainingmanysmallvesicles
and membrane profiles were found in treated promastigotes
(Figs. 8B–D, asterisks). In addition, some treated parasites also
presented with vesicles leaving the flagellar pocket (Figs. 8A, C,
arrows).
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