Ivermectin induces P-glycoprotein expression and function through mRNA stabilization in murine hepatocyte cell line

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Ivermectin induces P-glycoprotein expression and

function through mRNA stabilization in murine

hepatocyte cell line

Cécile Menez-Berlioz, Laila Mselli-Lakhal, Magali Vignault-Foucaud, Patrick

Balaguer, Roger Alvinerie, Anne Lespine

To cite this version:

Cécile Menez-Berlioz, Laila Mselli-Lakhal, Magali Vignault-Foucaud, Patrick Balaguer, Roger

Alvinerie, et al.. Ivermectin induces P-glycoprotein expression and function through mRNA

stabi-lization in murine hepatocyte cell line. Biochemical Pharmacology, Elsevier, 2012, 83 (2), pp.269-278.

�10.1016/j.bcp.2011.10.010�. �hal-01191347�

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Ivermectin

induces

P-glycoprotein

expression

and

function

through

mRNA

stabilization

in

murine

hepatocyte

cell

line

Ce´cile

Me´nez

a,

*

,

Laı¨la

Mselli-Lakhal

a

,

Magali

Foucaud-Vignault

a

,

Patrick

Balaguer

b,c,d,e

,

Michel

Alvinerie

a

_,

_Anne

_Lespine

a

INRAUMR1331,Universite´ deToulouse,INP,TOXALIM,Toulouse,F-31027,France

b

IRCM,InstitutdeRechercheenCance´rologiedeMontpellier,Montpellier,F-34298,France

c

INSERM,U896,Montpellier,F-34298,France

d_{Universite´ Montpellier1,}_Montpellier,_F-34298,_France e

CRLCVald’AurellePaulLamarque,Montpellier,F-34298,France

1. Introduction

Macrocyclic lactones (MLs), such as ivermectin, are a large family of broad spectrum antiparasitic drugs intensively used worldwideinlivestocktotreatdiseasescausedbygastrointestinal nematodesandexternalparasites.Inhumans,ivermectinisused throughmassdrugadministrationprogramsforthetreatmentof onchocerciasis,a tropicalparasiticdiseasecaused bytheﬁlarial nematode Onchocerca volvulus. MLs resistance has become a

worsening problem in veterinary medicine and recent reports suggestthathumanivermectin-resistantparasitesareemerging, thusthreateningthesustainableefﬁcacyofthisdrug[1].

P-glycoprotein (P-gp, MDR1/ABCB1) is a plasma membrane proteinbelongingtotheATP-bindingcassette(ABC)transporters family. Thistransporter is an ATP-dependent efﬂux pump that reduces the intracellular concentration of a broad range of hydrophobic compounds and is involved in the multidrug resistance(MDR)[2].TheactivityofefﬂuxABCtransporterscan besubjecttoregulationbyinhibitionorinductionand consider-able evidence has accumulated to indicate that MDR gene expressioncanberapidlyandtransientlyinducedfollowingacute exposure to some xenobiotics [3,4]. The regulation of ABC transporters occurs by transcriptional or post-transcriptional events. At transcriptional level, the constitutive androstane receptor (CAR, NR1I3)and the pregnaneX receptor(PXR, SXR, NR1I2),twonuclearreceptorsprimarilyexpressedintheliver,are

BiochemicalPharmacology83(2012)269–278

ARTICLE INFO

Articlehistory:

Received29August2011 Accepted11October2011 Availableonline18October2011

Keywords: Ivermectin Macrocycliclactones P-glycoprotein Mdr1 Hepatocytes ABSTRACT

Ivermectiniswidelyusedinhumanandveterinarymedicineforthecontrolofhelminthinfections. IvermectinisknowntointeractwithP-glycoprotein(P-gp/MDR1),beingagoodsubstrateandapotent inhibitor,however,theinfluenceofivermectin ontheexpressionofthetransporter hasnot been investigated.ExpressionofP-glycoproteinwas investigatedin culturedmouse hepatocytesacutely exposedtoivermectin.ThetwoP-glycoproteinmurineisoforms,Mdr1aandMdr1b,mRNAlevelswere assessed byreal-timeRT-PCR. Ivermectin induced acleartime- and concentration-dependent up-regulationofMdr1aandMdr1bmRNAlevels(asearlyasa12-hexposureandupto2.5-foldat10mM). Moreover,ivermectin-treatedcellsdisplayedenhancedcellulareffluxoftheP-glycoproteinsubstrate calcein that was inhibited by the P-glycoprotein blocker valspodar, providing evidence that the ivermectin-inducedP-glycoprotein was functional. Themechanisms underlyingthese effectswere investigated.Ivermectin-mediatedMdr1mRNAinductionwasindependentofthetwonuclearreceptors CARandPXR,whichareknowntobeinvolvedindrugtransportersregulation.Moreover,byusing reportercelllinesthatdetectsspecificligand-activatedtranscriptionfactors,weshowedthativermectin didnotdisplayedCAR,PXRorAhRligandactivities.However,studieswithactinomycinDrevealedthat thehalf-lifeofMdr1aandMdr1bmRNAweresignificantlyprolongedbytwo-foldinivermectin-treated cellssuggestingapost-transcriptionalmodeofivermectinregulation.Thisstudydemonstratesforthe firsttimethativermectininducesP-glycoproteinoverexpressionthroughpost-transcriptionalmRNA stabilization,thusofferinginsightintothemechanismofreducedtherapeuticefficacyanddevelopment ofivermectin-resistantparasites.

ß2011ElsevierInc.Allrightsreserved.

Abbreviations:ABC,ATP-bindingcassette;AhR,aryl-hydrocarbonreceptor;CAR, constitutiveandrostanereceptor;IVM,ivermectin;MLs,macrocycliclactones; P-gp,P-glycoprotein;PXR,pregnaneXreceptor.

*Correspondingauthorat:INRAToxAlim–UMR1331INRA/INP,180cheminde Tournefeuille–BP93173,31027ToulouseCedex3,France.Tel.:+33561285152; fax:+33561285310.

E-mailaddress:cecile.menez@toulouse.inra.fr(C.Me´nez).

ContentslistsavailableatSciVerseScienceDirect

Biochemical

Pharmacology

j our na l ho me p a ge : w ww . e l se v i e r . com / l oc a te / b i och e mph a rm

0006-2952/$–seefrontmatterß2011ElsevierInc.Allrightsreserved. doi:10.1016/j.bcp.2011.10.010

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nowbothwidelyrecognizedtoplaythemostimportantroleinthe regulationof MDR1 [5–7]. Other speciﬁcligand-activated tran-scription factors have been implicated in regulation of drug transportersexpressionsuchasaryl-hydrocarbonreceptor(AhR) [8].Apartfromtranscriptionalregulation,overexpressionofP-gp has also been shown to be controlled partially through post-transcriptional mechanisms, such as mRNA stability alteration [9,10].

P-gphasbeenclearlyidentifiedasthemainfactorthatcontrols the concentration of ivermectin by affecting its absorption, distribution, and elimination in the host [11,12], but also its neurotoxicitybylimitingitspenetrationinthebrain[13].Despite thekeyroleofP-gpinmetabolism,toxicity,efficacyanddrug–drug interactions of many other xenobiotics [14], the question of whetherivermectincanaffecttheexpressionlevelsofP-gphasnot beenaddressed.Severalargumentsfromtheliteratureimplythat ivermectinmaymodulateP-gpexpression.(i)ManyP-gpfunction modulators are oftenfound to influence the expressionof the transporter in cells [15], and ivermectin is reported as good substrateandpotentinhibitorofP-gp[16,17].(ii)Ivermectinwas previously shown to induce the expression and activity of cytochrome P450 isoenzymes, including CYP1A, 2B and 3A subfamilies in vivo [18,19], suggesting that ivermectin could interactwithcellulartranscriptionfactorssuchasAhR,CARand PXR which are involved in chemical induction of these cyto-chromes [20]. Since several studies have shown that the biotransformation systems (phase I/II) and efflux proteins are regulatedbythesamenetworkoftranscriptionfactors,onecan speculatethativermectinmayalsoregulateABCefflux transpor-tersexpression.(iii)P-gphomologsexpressionlevelswereshown tobeincreasedinivermectin-resistantnematodes[21–23].

Inthiscontext,thisstudyaimedatinvestigatingP-gpregulation in response to acute ivermectin treatment. Hepatocytes were chosen as a suitable in vitro model system for studying the potentialofxenobioticstomodulateP-gpgeneexpression[24]and elucidating the role of specific transcription factors in the regulation of gene involved in the metabolism of xenobiotics [25,26].Theobjectivesweretoevaluatetheabilityofivermectinto modulate P-gp gene expression, to determine the functional significanceofthismodulationandtofurtherestablishmolecular mechanismsunderlyingthiseffect.Our findingdemonstratefor the first time that acute exposure to ivermectin led to over-expression of functional P-gp in mouse liver cells through increasedstabilityofmRNAinthecell.

2. Materialsandmethods 2.1. Materials

1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene(TCPOBOP), preg-nenolone 16

a

-carbonitrile (PCN), dimethylsulfoxide (DMSO), T0901317,geneticin(G418),TritonX-100,calcein-AM, actinomy-cinD(Act-D)andivermectinwerepurchasedfromSigma–Aldrich Chimie(StQuentinFallavier,France).LuciferinwasfromPromega (Charbonnie`res-les-bains,France).Valspodar(VP)wasagenerous gift from Novartis (Basel, Switzerland). 2,3,7,8-Tetrachlorodi-benzo-p-dioxin(TCDD,dioxin)waspurchased fromPromochem (Wesel,Germany).Dulbecco’smodiﬁedEagle’smedium(DMEM), phosphate buffered saline (PBS), fetal bovine serum (FBS), glutamine,penicillin–streptomycinsolution(10,000units/ml pen-icillinand10,000

m

g/mlofstreptomycin),trypsin–EDTAsolution (0.05% trypsin, 0.53mM EDTA) and lipofectamine 2000 were obtained from Invitrogen – Life Technologies (Cergy Pontoise, France). Tissue culture plastic ﬂasks and culture plates were supplied by Sarstedt (Orsay, France). All otherchemicals were obtainedfromSigma–Aldrich,unlessotherwisestated.

2.2. Cellcultureandtreatment

JWZmurinehepaticcells,alsoreferredtoasMuSH immortal-ized hepatocytes [27], were kindly provided by Dr. J.P. Gray (Pennsylvania, USA). These cells expressed standard levels of Retinoid X Receptor (RXR), the heterodimeric partner of the nuclearreceptorsCARandPXR,whileCARandPXRtranscriptare barely detectable, providing a useful tool for mechanistic examinationoftheroleofnuclearreceptorsin generegulation, aspreviouslydescribed[27,28].JWZcellswereculturedinDMEM supplementedwith10%fetalbovineserum,2mML-glutamineand

100U/mlpenicillin, 100

m

g/mlstreptomycin. Cellswerekept at 348Cin 5% CO2 and 95%humidity. Every 3–4days, cells were

trypsinizedandseededat5105_cells_per_6-well_cluster_trays_for

induction studies. The cells were grown at more than 80% conﬂuenceandexposedtoivermectinordifferentcompoundsof interestatvariousconcentrationsortothevehiclefor6,12,24,48 or72h.AlldrugsweredissolvedinDMSOataﬁnalconcentration of0.1%(v/v).

2.3. Cytotoxicityassays

2.3.1. Cellviability:theMTS-tetrazoliumsaltassay

Thecellviabilityinpresenceofivermectinwasevaluatedusing theCellTiter961

AQueousNon-RadioactiveCellProliferationAssay

(Promega, Charbonnie`res-les-bains, France). The MTS (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium)assayisbasedontheabilityofviablecells toconvertasolubletetrazoliumsalttoaformazanproduct.JWZ cellswereseededat5104_cells_in_96-well_culture_plates.₂₄_h

afterseeding,increasingconcentrationsofivermectinwereadded; mediaand DMSO wereused as negativeand positive controls, respectively.JWZcellswerefurtherincubatedat348Cfor24,48or 72h.Thereafter,20

m

loftheMTS/PMSmixture(MTS/PMSratio: 20:1)wasaddedtoeachwellandplateswereincubatedat348Cfor 4h.Attheendofincubationperiod,absorbancewasrecordedat 492nmusingamultiwell-scanningspectrophotometer.

2.3.2. Releaseoflactatedehydrogenase

Theeffectofivermectinuponcellintegritywasalsodetermined by measurement of lactate dehydrogenase (LDH)-release as a markerofcellmembranedamageusingtheCytotoxicityDetection Kit(RocheAppliedScience,Meylan,France).JWZcellsgrownon 96-welldisheswereincubatedwithincreasingconcentrationsof ivermectin.After24,48or72hofexposure100

m

lsampleswere withdrawnandanalyzedforLDHcontent.Incontrolexperiments, cellswereincubatedwithmediumor1%TritonX-100.Afterwards, resultswerenormalizedto0%and100%LDHrelease,respectively. 2.4. FunctionaldetectionoftheP-gpactivity(calceinassay)

P-glycoprotein transport activitywasassessed following the intracellular accumulation of the P-gp fluorescent substrate calcein,aspreviouslydescribed[29].Briefly,confluentJWZcells in 24-well cluster plates were cultured with (treated cells) or without(controlcells)10

m

Mivermectinfor48h.Cellswerethen incubatedwithorwithoutthereferenceP-gpinhibitorvalspodar (5

m

M) for 40min. After this pre-incubation period, cells were washedwithPBSbufferand 0.5

m

Mcalcein-AM(inDMSO)was added for a 2-hincubation period at 348C. Medium wasthen removed,cellswerewashed2timeswithice-coldPBSandalysis buffer (1%Triton X-100 and SDS 0.1% in PBS) was added. The intracellularﬂuorescencecorrespondingtocalceinaccumulation wasmeasuredincelllysatebyusingamicroplatereader(TECAN, InﬁniteTM_200,_Lyon,_France),_with_a₄₈₅_nm_excitation_wavelength

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contentperwelldeterminedbyusingaBio-Radproteinassaykit (Bio-RadLaboratories,Marnes-la-Coquette,France).

2.5. TotalRNAisolationandRT-PCRanalysis 2.5.1. IsolationofRNAandcDNAsynthesis

Aftertwowasheswithice-coldPBS,thecellswereharvested andtotalcellularRNAwasisolatedusingTrizolReagent (Invitro-gen, Cergy Pontoise, France) according to the manufacturer’s instructions.TotalRNAwasquantiﬁedusingnanodropND-1000 spectrophotometer(NanodropTechnologiesInc.,Wilmington,DE, USA).RNApuritywascheckedbymeasurementoftheA260/280nm

ratio, which was routinely in the range of 1.8–2.0, and RNA integrity was checked upon migration in 1% agarose gel and visualizationwithethidiumbromidestainingandUVirradiation. cDNAwas synthesizedfrom2

m

gof totalRNAusing the High-CapacitycDNA Reverse Transcriptionkit (AppliedBiosystems – LifeTechnologies,Courtaboeuf,France).

2.5.2. QuantiﬁcationofmRNAexpressionbyRT-PCR

Real-timequantitative PCR (RT-qPCR)was performed using anABIPrism 7300SequenceDetectionSysteminstrumentand software (Applied Biosystems, Courtaboeuf, France). Gene-specificprimers for SYBRGreen assays are described in Table 1. All primers were designed using Primer Express software version2.0(AppliedBiosystems)andsynthesizedbyInvitrogen (CergyPontoise,France).AllprimerswereenteredintotheNCBI Blastprogramtoensurespecificity.Resultswereexpressedusing the comparative Ct method as described in User Bulletin 2 (AppliedBiosystem).Briefly,the

D

Ct valueswerecalculatedin

every sample for each gene of interest as following: Ctgene o-f interestCt reporter gene,withTATA-box bindingprotein(TBP)as

thereportergene.Thefoldchange intheleveloftargetmRNA betweentreatedanduntreatedcellswasthenexpressedas2DCt with

D

CtS.D.whereS.D.isthestandarddeviationofthemeanof

the

D

Ct value. A dissociation curve allowed us to verify the

speciﬁcityoftheampliﬁcation.

2.6. TransienttransfectionassayandDNAconstructs

The pCR3-mCARconstruct expressing the murineCARwas kindlyprovidedbyDr.M.Negishi(NorthCarolina,USA)andthe pSG5-mPXR construct expressing the murine PXR was kindly provided by Dr. J.M. Pascussi (Montpellier, France). These plasmids were described previously [28,30]. JWZ cells (1.5106₎ _were_plated _onto_10-cm _culture _dishes _and_grown

inDMEMsupplementedwith10%fetalbovine serum,2mML

-glutamine,100U/mlpenicillin,100

m

g/mlstreptomycinina5% CO2atmosphereat348C.24hafterseeding,cellsweretransiently

transfectedwith8

m

g/plateofplasmidconstructcontainingthe cDNAformouseCAR(pCR3-mCAR),mousePXR(pSG5-mPXR),or theircontrolexpressionvector(pCR3orpSG5),using lipofecta-mine2000accordingtothemanufacturer’sprotocol.Cellswere transferredto6-wellplatesatadensityof5105_cells/well₁₆_h

after transfection. Drugtreatments were performed24hafter transfection.

2.7. StudyoftheCAR-,PXR-andAhR-activatingcapacityof ivermectin

2.7.1. Stablereportercelllines

HG5LN parental cells, obtained by integration of GAL4RE5

-b

Glob-Luc-SVNeoinHeLacellsandcontainingastablyintegrated GAL4-responsivegene,havealreadybeendescribed[31]. mCAR-andmPXR-expressingcellswereobtained,aspreviouslydescribed [32,33], by transfecting HG5LN cells with

pSG5-GAL4(DBD)-mPXR(LBD)-puro or pSG5-GAL4(DBD)-mCAR(LBD)-puro, which enables theexpressionoftheDNA bindingdomainof theDNA bindingdomainoftheyeastactivatorGAL4(Met1–Ser147)fused to the ligand binding domain of mCAR or mPXR and confers resistancetopuromycin.TherodentAhR-activatingcapacitywas assessedwiththeH4IIEXRE-LUCcellline,whichwasobtainedby transfecting rat H4IIE hepatocytes cells with CYP1A1-Luc and pSG5-puro plasmids.A similardioxin reporter cellline, HAhLP, usedtoassessinteractionswithhumanAhR,haspreviouslybeen described[34].

2.7.2. Cellcultureconditions

HG5LN-mCAR,HG5LN-mPXRandH4IIEXRE-LUCcelllineswere

culturedinDMEMF12,supplementedwith5%offetalcalfserum (FCS)and1%antibiotic(penicillin/streptomycin)ina5%CO2and

95%air-humidiﬁedatmosphereat378C.HG5LNcellmediumwas

supplementedwith1mg/mlgeneticin(G418)andHG5LN-mPXR

and -mCARcellmediumwith1mg/ml geneticinand0.5

m

g/ml puromycin. Totest ivermectinforitsmCAR-,mPXR- and rAhR-activatingcapacity,cellsweregrowninDMEMF12supplemented with5%dextran-coated,charcoal-treatedfetal calfserum (DCC-FCS).

2.7.3. Livingcellluciferaseassay

Reportercellswereseededatadensityof2.5104_cells_per

well in 96-well white opaque tissue culture plates (Greiner, France) and grown in 150

m

l DCC-FCS. Ivermectin or the referenceeffector(agonistorantagonist) wasadded24hafter seeding and the cells were then incubated for 24h with the compounds.Variousconcentrationsweretestedthroughserial dilutionusinganautomatedworkstation(biomek1

2000, Beck-manCoulter).Toensure100%cellviability,ivermectin concen-tration rangeused wassetfrom 108_M_to_3.3

106_M._Each

concentration was tested in quadruplicate in at least two experimentswhichincludedbothnegative(solvent)andpositive (reference agonist or antagonist) controls. At the end of incubation, medium was removed and replaced with 0.3mM luciferin-containingculturemedium.Intactlivingcell lumines-cence was measured for 2s usinga Microbeta Wallac lumin-ometer.Resultswereexpressedasafold-inductionofthebasal luciferaseactivitymeasuredinuntreatedcells,whichwassetto 1.ReferenceagonistligandwasusedinHG5LN-mPXRandH4IIE

XRE-LUCcells(PCN10

m

MandTCDD(dioxin)10nM, respective-ly).DuetothehighconstitutiveactivityofCARinHG5LN-mCAR

cells, a reference antagonist ligand was used as control (T0901317 10

m

M). All effectors were dissolved in DMSO at 10mMandthendilutedinculturemedium.

Table1

PrimersequencesusedinquantiﬁcationofthegeneexpressionbyqRT-PCR.

Targetedgene Forwardprimer(50_to₃0₎ _Reverse_primer₍₅0_to₃0₎

Mdr1a(Abcb1a) CATGACAGATAGCTTTGCAAGTGTAG GGCAAACATGGCTCTTTTATCG

Mdr1b(Abcb1b) AAGCCAGTATTCTGCCAAGCAT CTCCAGACTGCTGTTGCTGATG

Cyp3a11 TCACACACACAGTTGTAGGCAGAA GTTTACGAGTCCCATATCGGTAGAG

Cyp2b10 TTTCTGCCCTTCTCAACAGGAA ATGGACGTGAAGAAAAGGAACAAC

TBP ACTTCGTGCAAGAAATGCTGAA GCAGTTGTCCGTGGCTCTCT

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2.8. DeterminationofMdr1mRNAsstability

Thehalf-livesofMdr1aandMdr1bmRNAweredeterminedby an Act-D chase assay, as previously described [35]. JWZ cells were pre-treatedwith ivermectin (10

m

M) or DMSO for 24h. Thereafter,thetranscriptioninhibitoractinomycinDwasadded to inhibit further RNA synthesis. Act-D was used at a ﬁnal concentrationof5

m

g/mlfor1h,then,toavoidAct-D toxicity, mediumwasreplacedandAct-D,inthepresenceorabsenceof ivermectin10

m

M,wasaddedataconcentrationof1

m

g/mlfor thefollowing24h.Cellswereharvested,atvarioustimes(0,3,6, 10and24h)aftertheadditionof1

m

g/mlAct-D,totalRNAwas isolatedandlevelsofMdr1aandMdr1bmRNAswerequantiﬁed by RT-qPCR as described above. TBP mRNA levels were also monitoredascontrol.Thedatawererepresentedaspercentage decreasein mRNA levelsversus timeusing a semilogarithmic scale.ThemRNAhalf-lifevaluesweredeterminedwiththeuseof regressioncurves.

2.9. Statisticalanalysis

All experiments were conducted at least in triplicate and resultsareexpressedasmeanstandarddeviation(S.D.). Statisti-cal analysis was performed using one-way analysis of variance (ANOVA)withaTukeypost-testtocomparetheeffectofivermectin overcontrolcells,whileindividualcomparisonsbetweenpairsof data were performed using the Mann–Whitney test (GraphPad Instat,SanDiego,CA,USA).Statisticalsigniﬁcancewasacceptedas p<0.05.

3. Results

3.1. Effectsofivermectinoncellviability

Theaimofthesestudieswastodeterminesubtoxic concentra-tionsofivermectinwhichcouldbeusedinthesubsequentstudies onhepatocytesJWZcells.Thecytotoxicityofivermectintowards JWZcellswasexaminedusingtheMTSassayandLDHrelease.The dose-dependentviabilityofJWZcellstreatedwithivermectinfor 24,48 or72his presentedinFig.1.Theseresultsshowedthat ivermectin had cytotoxic effects in a concentration- and time-dependentmanner.A24-htreatmentwithivermectinupto25

m

M induced no signiﬁcant mitochondrial toxicity or cell-growth inhibitiontowardsJWZcells,asrevealedbytheMTSassaywith morethan90%cellviability;howeverLDHreleasearound35%was observed.WhenJWZcellswereexposedtoivermectinfor72hat concentrationbelow12.5

m

M,nocytotoxiceffectswereobserved. TheeffectiveLD50values(concentrationthatcauses50%lethality)

at 24, 48 and 72h were determined graphically and were approximately75,30and20

m

M,respectively.Subsequentstudies wereperformedfollowinganivermectinincubationperiodupto 72handwithconcentrationsupto10

m

M,atimeand concentra-tionexposureofivermectinthatgavemorethan80%cellviability andmoderateLDHrelease(lessthan20%).

3.2. Time-dependenteffectsofivermectinonP-glycoproteinmRNA expression

We ﬁrst analyzed the effects of ivermectin treatment as a functionoftime ontheexpressionofthe2genesencodingthe murineP-glycoprotein,Mdr1aandMdr1b(Abcb1aandAbcb1b)in JWZ mouse cultured hepatocytes. JWZ cells were exposed to 10

m

Mivermectinfor6,12,24,48,or72handmRNAlevelswere determined by RT-qPCR. Fig. 2 shows a time-dependent up-regulationinMdr1aandMdr1bmRNAwhencellswereexposedto 10

m

M ivermectin. A signiﬁcant increase in mRNA level was

0 20 40 60 80 100 120 100 10 1 0 Ivermectin (µM) LDH release (% of control) 24h 48h 72h 0 20 40 60 80 100 120 100 10 1 0 Ivermectin (µM)

Cell viability (% of control)

24h 48h 72h

A

B

Fig.1. Cytotoxiceffectsofivermectin onJWZcellsasafunctionoftimeand concentration.CytotoxicitywasmeasuredbytheMTSassay(A)andLDHrelease(B) following24,48or72hincubationat348C,5%CO2and95%relativehumidity.For

theMTSassay,mediumandDMSOwereusedaspositive(100%cellviability)and negative(0%cellviability)controls,respectively.ForLDHassay,mediumandTriton 1%wereusedasnegative(0%LDHrelease)andpositive(100%LDHrelease)controls, respectively,and results were normalized to 0% and 100% LDH-release. All measurementswereexpressedasmeanS.D(n=8).

0 1 2 3 72 60 48 36 24 12 0 Time (h) G e n e exp ressi o n l e vel s Fold induc tion r e la ti v e t o c o nt ro l Mdr1a Mdr1b ** *** ** *** *** *** 6

Fig.2.Time-dependenteffectsofivermectinonMdr1aandMdr1bmRNAlevels. MouseJWZhepatocytesweretreatedwith10mMivermectin(IVM)orwiththe vehiclealone(DMSO)for6,12,24,48or72h.Mdr1aandMdr1bmRNAexpression wasthendeterminedbyreal-timeqPCRusinggene-speciﬁcprimersasdescribedin Section2.ThemRNAexpressionlevelswereexpressedas-foldincreaseoverthe correspondingcontrol(untreatedcells)giventhearbitraryvalueof1.Dataare reported as the meanS.D. of three independent experiments. **p<0.01; ***p<0.001versusuntreatedcontrol.

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observed as early as 12h after treatment for Mdr1a (1.5-fold, p<0.01) and 24h for Mdr1b (1.6-fold, p<0.01). The two P-glycoproteinmRNAsweremaximallyup-regulatedafter48h (1.9-and2.4-foldovercontrolforMdr1aandMdr1b,respectively).The up-regulationofMdr1expressionappearedtobetransientwitha lowerinductionaftera longerexposuretoivermectin(1.2-and 2.0-fold of control levels with a 72hexposure for Mdr1a and Mdr1b, respectively). TBP mRNA, which wasused as a loading control,remainedstableoverthetimeperiodofthestudy(datanot shown).

3.3. Concentration-dependenteffectsofivermectinonP-glycoprotein mRNAexpression

Thedose-responserelationshipforivermectineffectstowards P-glycoproteinexpression wasthen characterized. JWZ hepato-cyteswereeitheruntreatedorexposedtovariousconcentrationof ivermectinrangingfrom0.5to10

m

Mfor48h.Resultsareshown inFig.3.Evenatlowconcentrationsuchas0.5

m

M,ivermectinwas abletoaltertheexpressionofMdr1awithaslightbutsignificant 1.3-fold increase (p<0.05). When higher concentrations were used,Mdr1aandMdr1bmRNAlevelsweresignificantlyincreased by ivermectin treatment in a clear concentration-dependence mannerupto2.1-and2.5-foldforMdr1aandMdr1b,respectively. 3.4. P-glycoproteinfunctionalactivityinJWZhepatocytesunder influenceofivermectin

We then determined whether the induction of Mdr1 gene expressionbyivermectinresultedinamodulationof P-glycopro-teintransportfunction.TomeasureP-gptransportactivityinJWZ cells,weusedafluorometricassaypreviouslydescribed[29].This assayutilizeslipophilicnonfluorescentcalcein-AMwhich pene-tratesintothecell,whereitisimmediatelycleavedbyesterasesto highlyfluorescentcalcein.Becausecalcein-AMisa high-affinity substrate for P-gp, intracellular calcein fluorescence inversely

correlateswithP-gpactivityand,therefore,isameasureof P-gp-mediatedtransport. Thus,anincreaseinP-gpactivityresultsin reducedintracellularcalceinﬂuorescenceandviceversa.

As expected, the presence of valspodar, the P-glycoprotein-selective blocker, in untreated cells resulted in a signiﬁcant increaseincalceinaccumulationofmorethat1.6-foldcompared with control (Fig. 4). This result indicates that P-gp actively extrudedcalceinfromJWZcellsandthatvalspodarclearlyinhibit P-gp transport function in these cells. When JWZ cells were cultured with10

m

M ivermectinfor 48hbefore measuringthe intracellularcalceinaccumulation,atreatmentwhichwasshown to increase Mdr1 gene expression, intracellular calcein ﬂuores-cencewasreducedbymorethan30%comparedwithuntreated cells, indicating that the P-gp-mediated efﬂux of calcein was increased.TheseresultsdemonstratethattheinductionofMdr1 mRNAexpressionobservedwith10

m

Mivermectinfollowinga 48-h exposure is associated with an increase in P-gp transport function.Itisimportanttonotethatthiseffectwasabolishedwith valspodar (Fig. 4), indicating that the reduction in intracellular calceinﬂuorescencewasspeciﬁcallymediatedbyP-gp.

3.5. Roleoftranscriptionfactorsintheivermectin-induced modulationofP-glycoproteinmRNAexpression

BecauseCAR,PXRandAhRhavebeenrecognizedas transcrip-tionfactorsplayinganimportantroleinthexenobiotic-induced regulationoftheexpressionofefﬂuxABCtransporterssuchasP-gp [6,36,37],weinvestigatedtheirroleintheivermectin-induced up-regulation of Mdr1 mRNA expression through two different approaches.

3.5.1. InductionofMdr1aandMdr1bmRNAbyivermectinin CAR-andPXR-overexpressingcells

Nuclearreceptors-overexpressinghepatocyteshaveprovedto beusefulmodelsinelucidatingtheroleoftranscriptionfactorsin the induction of ABC efﬂux transporters or cytochromes by xenobiotics[25,26].We thereforeﬁrstlyevaluatedtheeffectsof ivermectin in JWZ hepatocytes overexpressing the functional mouse CAR (Fig.5A)or PXR(Fig. 5B). Aspreviously described,

0 1 2 3 Mdr1b Mdr1a

Gene expression levels

Fold induction relative to control

CTR IVM 0.5 µM IVM 1 µM IVM 5 µM IVM 10 µM * *** *** *

Fig.3.Concentration-dependenteffectsofivermectinonMdr1aandMdr1bmRNA levels. Mouse JWZ hepatocytes were either untreated (CTR) or exposed to increasingconcentrationsofivermectin(IVM),rangingfrom0.5to10mM,for 48h.ChangesinMdr1aandMdr1bmRNAlevels,normalizedwithrespecttoTBP mRNAlevels,weredeterminedbyreal-timeqPCRusinggene-speciﬁcprimersas describedin Section 2. ThemRNAexpression levels wereexpressedas-fold inductionrelativetocontrol(DMSO)andarereportedasthemeanS.D.ofsix independentexperiments.*p<0.05;***p<0.001versuscontrol.

Fig. 4. P-glycoprotein transportactivity in JWZ cells exposed to ivermectin. Intracellularcalceinfluorescencewasassessedinuntreatedcellsorfollowinga 48-htreatmentwithivermectin(IVM,10mM)withorwithouta40-minpre-exposure to the P-gp-specific inhibitor valspodar (VP, 5mM). Data are expressed as percentage of control; each data represents meanS.D. (n=4). ***p<0.001, significantlygreaterthancontrol;###

p<0.001,signiﬁcantlylowerthancontrol. C.Me´nezetal./BiochemicalPharmacology83(2012)269–278 273

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Cyp2b10andCyp3a11, theprototypicaltargetgenesofCAR [38] andPXR[39],wereusedasa positiveindicatorforCARorPXR activation,respectively;whileTCPOBOP,asyntheticdirectagonist ofmouseCAR,andPCN,asyntheticagonistofmousePXR,were usedaspositivecontrols.

In untransfected JWZcells,mouse CARand PXRconstitutive expression levels were extremely low under basal conditions, which correlated with the impaired induction of Cyp2b10 by TCPOBOPorCyp3a11by PCN,respectively (Fig. 5). InJWZ cells transientlytransfectedwithpmCARorpmPXR,TCPOBOPorPCN wereabletoincreaseeitherCyp2b10(3.4-fold,p<0.001,Fig.5A)or Cyp3a11(4.1-fold, p<0.001,Fig.5B)expression, demonstrating that these nuclear receptors could be further activated in our model by an agonist. Fig. 5 shows that a 48-h exposure of ivermectinmodiﬁedtheexpressionlevelofMdr1aandMdr1bin the exact same manner in CAR- or PXR-overexpressing cells comparedwithJWZcellstransfectedwiththecontrolplasmid:the overexpression of these nuclear receptors did not further up-regulate Mdr1 in ivermectin-treated cells and no signiﬁcant differencesinfold-inductionlevelswereobservedcomparedwith controlJWZcells.Theseresultsprovidesupportforthehypothesis thattheivermectin-inducedregulationswereindependentofthe activationofCARorPXRnuclearreceptors.

3.5.2. ActivationofPXR,CARandAhRbyivermectin

Inordertoprovidemoredataonthecapacityofivermectinto activate speciﬁc transcription factors, we used bioluminescent reporter cell lineswhich have already proved to bea valuable model to detect nuclear receptor ligands [31,40]. These cells expressed the GAL4-DNA binding domain fused to different nuclearreceptorligandbindingdomainsandallowthedetection ofcompoundsthatactivatesomespeciﬁctranscriptionfactors.

Ivermectin was tested for its ability to activate rodent transcriptionfactors (murineCAR, murinePXR andrat AhR)by using three different reporter celllines (HG5LN-mCAR, HG5

LN-mPXRandH4IIEXRE-LUC)inwhichPXR,CARandAhRagonistsor antagonists induced or repressed luciferase gene expression. Activationwasmeasuredinthesecellsinthepresenceofincreased concentrations ofivermectin from108_M_to_3.3

106_M. _The

vehicle alone (DMSO) was used as negative control (luciferase activitybaselinesetto1)andknownreferenceeffectorswereused aspositivecontrol,asalreadydescribed[34].Resultsarepresented inTable2.Asexpected,thebasalluciferaseactivityofthemPXR andrAhRcelllinesaftertreatmentwiththeirreferenceagonistwas increased (2.3- and 10.1-fold, respectively) and the luciferase activityofthemCARcelllinewassigniﬁcantlydecreasedformore thanfourfoldaftertreatmentwiththeantagonistligandT0901317

Fig.5.InﬂuenceofthenuclearreceptorsCAR(A)andPXR(B)ontheivermectin-inducedmodulationofMdr1aandMdr1bgeneexpression.JWZcellsweretransiently transfectedwithmouseCAR(pCR3-mCAR),mousePXR(pSG5-mPXR),ortheirrespectivecontrolexpressionvector(pCR3andpSG5).24haftertransfection,cellsweretreated withthevehiclealone(0.1%DMSO;CTR),TCPOBOP(250nM),PCN(1mM)orincreasingconcentrationsofivermectin(IVM)for48h.ChangesinMdr1a,Mdr1b,Cyp2b10and Cyp3a11mRNAlevels,normalizedwithrespecttoTBPmRNAlevels,weredeterminedbyreal-timeqPCRusinggene-speciﬁcprimersasdescribedinSection2.ThemRNA expressionlevelswereexpressedas-foldinductionrelativetocontrolandarereportedasthemeanS.D.(n=6).*p0.05;**p0.01;***p0.001versuscontrol.

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(Table 2). Ivermectin, at all concentrations tested, did not modulatedluciferase gene expressionin HG5LN-mCAR,HG5

LN-mPXR or H4IIE XRE-LUC cell lines (Table 2): the activation in ivermectin-treated cells was not statistically different from untreatedcells.Theseresultsshowedthativermectinwasnota CAR,PXRorAhRligand.

3.6. EffectofivermectinonMdr1aandMdr1bmRNAstability The level of mRNA expression is the result of the balance between the transcription and the elimination rate, through processinganddegradation.TheabsenceofinﬂuenceofCARand PXR on ivermectin-induced up-regulation of P-glycoprotein encoding genes prompted us to study theeffect of ivermectin onMdr1aandMdr1bmRNAstability.Therefore,thehalf-livesof Mdr1mRNAincontrolandivermectin-treatedcells were exam-ined,usinganAct-Dchaseexperiment.JWZcellswerestimulated withivermectinfor24htoinduceMdr1mRNAexpression,after whichthetranscriptioninhibitorAct-Dwasaddedinthepresence ofivermectinforanother24h.Steady-statelevelsofMdr1aand Mdr1bmRNAweredeterminedatvarioustimepointsafteradding Act-D. There was a signiﬁcantly slower rate of decline for ivermectin-treated cells versus control (Fig. 6). The half-life of Mdr1a and Mdr1b mRNA under control conditions were 8.6h

(Fig. 6A) and 10.4h (Fig. 6B), respectively. By contrast, in ivermectin-inducedcells,Mdr1aandMdr1bmRNAhalf-liveswere 17.3h(Fig. 6A) and 17.2h(Fig. 6B), respectively. Thistwofold increase of the half-life of the P-gp mRNA demonstrates that ivermectintreatmentleadstothestabilizationoftheP-gpmRNA.It hastobenoted that thedegradationproﬁlesfor TBP mRNAin untreated and ivermectin-treated cells were similar (data not shown).Taken together,theseresultsclearlyindicatedthat up-regulationofMdr1mRNAfollowingexposuretoivermectinoccurs, atleastpartially,atpost-transcriptional levelthroughaspeciﬁc increaseinMdr1mRNAstability.

4. Discussion

Despite the well documented interactions of ivermectin, a commonly used anthelmintic, withP-gp, thereis no dataon a possible modulation of the expression of this transporter by ivermectin.Wehaveusedmurineculturedhepatocytestostudy the effects of short-term exposure to ivermectin on P-gp expressionandfunction.

In this study, ivermectinwas found toup-regulate the two mouseP-gpisoformsMdr1aandMdr1bgeneexpressioninbotha time-anddose-dependentmanner,demonstratingthespeciﬁcity oftheivermectin-inducedeffect.Mdr1mRNAlevelswererapidly

Table2

Modulationofluciferaseexpressionbyivermectininstabletransfectedcells. Celllines Transcriptionfactor Luciferaseactivity(foldinductiontoCTR)

CTR(DMSO) IVMconcentration Referenceeffector 108_M _3.3 108_M ₁₀7_M _3.3 107_M ₁₀6_M _3.3 106_M HG5LN-mCAR mCAR 1.000.05 1.030.07 nd 1.050.05 nd 1.040.05 nd T090131710mM (referenceantagoniste) 0.240.02*** HG5LN-mPXR mPXR 1.000.03 1.020.03 nd 1.040.03 nd 1.030.03 nd PCN10mM (referenceagoniste) 2.330.05*** H42EXRE rAhR 1.000.15 1.090.07 1.030.01 1.120.20 1.150.11 1.050.05 1.070.12 TCDD(dioxin)10nM

(referenceagoniste) 10.830.86*** TheluciferaseactivityofHG5LN-mCAR,HG5LN-mPXRandH42EXREcellswasmeasuredinuntreated,ivermectin-treatedandreferenceligand-treatedcells.Concentration

rangeofivermectinwasfrom108

Mto3.3106

M.ReferenceeffectorsusedwereT0901317(10mM),PCN(10mM)andTCDD(dioxin,10nM)formCAR,mPXRandrAhR, respectively.Theincubationperiodwithcompoundswas24h.Resultsareexpressedasafold-inductionofthebasalluciferaseactivitymeasuredinuntreatedcells(CTR), whichwassetto1,andarepresentedasmeanS.D.(quadruplet).***p<0.001versuscontrol.nd:notdetermined;m:murine;andr:rat.

Fig.6.EffectofivermectinonP-glycoproteinmRNAdecaypatternafteractinomycinDaddition.JWZcellsweretreatedwithDMSO(0.1%,CTR,whitesquare)orivermectin (10mM,IVM,blacksquare)for24h,afterwhichthetranscriptioninhibitoractinomycinD(Act-D5mg/mlfor1hand1mg/mlforthefollowing24h)wasaddedtoinhibit nascentRNAsynthesis.Cellswereharvestedatvarioustime-points(3,6,10or24hpostAct-Dtreatment),totalRNAwasisolatedandMdr1a(A)andMdr1b(B)mRNAlevels weredeterminedbyqRT-PCR,asdescribedinSection2.ThedatawereplottedasthepercentageofmRNAremainingusingasemilogarithmicscaleandwerereportedasthe meanS.D.ThemRNAlevelatzerotimepoint(beforetheAct-Dtreatment)wasconsideredas100%.Thehalf-livesofmRNA(t1/2)weredeterminedwiththeuseofregression

curves.

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induced,asearlyas12hafterivermectinadditionforMdr1a,with concentrationaslowas0.5

m

M,andthemaximaleffectforboth Mdr1aandMdr1bwasobserved48hafterincubation. Further-more,theinductionofMdr1wasfoundtobetransient:therapid increaseinMdr1mRNAlevelreachedaplateauat24hor48hand thendecreased afterlonger exposuretimes,suchas72h.This kindoftransientinductionofMDR1hasalreadybeendescribedin hepatocytesafterexposuretohepatocytegrowthfactor[41],or with two PXR activators, rifampicin and hyperforin [42]. The time- and concentration-dependent effect suggests that in-creased levels of P-gp in ivermectin-treatedcells likely corre-spondtoanacuteinductioninresponsetotheanthelminticdrug. Thisis,toourknowledge,theﬁrstreportshowingthativermectin isabletoup-regulatetheexpressionofthiscrucialtransporter. KnowingthativermectinisagoodsubstrateofP-gp,ourresults are in agreement with thefact that,among other factors,the ability of drugs tointeract with ABC transporters can lead to overexpressionoftheseproteins[15].TheexpressionoftheMDR1 genecanberapidlyandtransientlyinducedafterashort-term exposuretoavarietyofxenobioticsin manycelllines,suchas rodent cultured hepatocytes [3,43], human cancer cell lines [44,45], but also after acute in vivo exposure of a tumor to cytotoxicchemotherapy[4].Ourresultsdealtwiththeexpression ofmurineP-gp anditwould behazardous toextrapolatethis conclusiontootherspeciessuchashumans.Additionaldataare neededinordertoexploretheabilityofivermectintoregulatethe P-gpgeneexpressioninhumanhepatocytes.Nevertheless,ithas beendemonstrated thatmdr1a andmdr1b geneexpression in rodentandMDR1inhumansare,atleastpartially,controlledby the same negative regulatory element [46]. In addition, P-gp mRNA levels were increased in a dose- and time-dependent mannerupontransientexposuretoavarietyofcytotoxicdrugs includingdoxorubicin[44,45]anddaunorubicin[45,47]inhuman cellsbutalsoinrodentcells[3,48],demonstratingthatsimilar regulatoryeffectofxenobioticonP-gpexpressioninhumanand rodentcellscanbeobserved.

Thepossiblemechanismsinvolvedinivermectinup-regulation of Mdr1a and Mdr1b gene expression were investigated. The rapidityoftheresponseuponivermectintreatmentexcludesgene ampliﬁcation, gene rearrangement or mutations and supports transcriptionalactivationorpost-transcriptionalregulationasthe underlyingmechanism.

Weﬁrstlyexaminedtheroleofsometranscriptionfactorssince transcriptional activation is the most common mechanism describedforup-regulationofMDR1geneexpression,andseveral regulatoryelementshavebeenidentiﬁedandcharacterizedinthe MDR1promoterregion.SincethenuclearreceptorsCARandPXR arecellularsensorscapableofrespondingtochemicalexposure [49–51]andhavebeenimplicatedinMDR1regulation[37,52],we investigated their potential role in the ivermectin-induced modulation of P-gp. We therefore used mouse hepatocytes transiently overexpressing the functional mouse CAR or PXR. Similarpatternsandextentofthedose-dependentmodulationsof P-gp induced by ivermectin were observed in CAR- or PXR-overexpressingcellsandincontrolcells,whichexpressCARand PXRataverylowlevel.Thisstronglysuggeststhativermectinmay have CAR- and PXR-independent effects on gene expression. Furthermore,usingbioluminescentcelllinesexpressing transcrip-tionfactors,wehavedemonstratedthativermectinwasnotaCAR, PXRorAhRligand.Inaccordancewithourresults,itwaspreviously shownthattoxicinsultincreasedMDR1expressionthrough CAR-independent mechanisms [53]. However, the role of other transcriptionfactors,includingnuclearreceptors,inthe ivermec-tin-inducedregulationscannotbediscardedsincenumberofthem suchasPPAR

g

,aswellasNrf2, aredescribedtobeinvolvedin efﬂuxABCtransporterstranscriptionalregulation[8,37].

Besidestranscriptionalactivation,post-transcriptional regula-tionatthelevelofmRNAstabilitywasshowntobeanimportant mechanism for P-gp overexpression in vitro [54] and in vivo [10,55].In ourstudy,Mdr1 mRNAdecaywasexamined. mRNA half-lifeinuntreatedcellswasfoundtobearound9hwhichisin therangeofvaluesreportedintheliterature[35,56].Interestingly, stabilityofMdr1aandMdr1b mRNAwasmarkedlyincreasedby ivermectintreatment,demonstratingthatthisdrugreducedthe degradationrateofMdr1mRNAthuscontributing,atleastinpart, totheincreaseofP-gpgeneexpression.Interestingly,varietyof drugs–suchascycloheximide[57],doxorubicin[58],vinblastine [58],colchicine[58], daunorubicin[47]oretoposide[47],were previouslyshowntoinduceMDR1geneexpressioninmammalian cellsuponacutetreatment,asshortasa24-hexposure,through enhancedmRNAstabilityratherthanincreasedtranscription.In furtheragreementwithourresults,atorvastatin,aP-gpsubstrate [59]andinhibitor[60],asivermectinis,wasabletoinduceP-gp mRNA expression[61]. More interestingly, regulation of MDR1 mRNA expression in human hepatocytes by atorvastatin was recentlyassociatedwithamodulationinmRNAdegradation[9]. ThedetailedmechanismthatcausesstabilizationofP-gpmRNAin alloftheabovestudiesiscurrentlyunknown.Thestabilityofa particular mRNA is partially controlled by speciﬁc interactions betweenitsstructuralelements andtrans-actingfactors:mRNA bindingproteinsandsomemicroRNAs.Recently,miRNAs,which contribute to post-transcriptional processing through 30

-UTR-interference,havebeeninvolvedintheregulationofABCB1mRNA degradation[62,63].Sinceseveralcompoundshavebeenshownto inﬂuence P-gp mRNA stability, it is reasonable to think that ivermectinmayinteractwitha putativetrans-actingstabilizing factor which is activated upon drug exposure, resulting in increasedmRNAstability.However,themRNAstabilizing mecha-nism which is responsiblefor the differentialstability of P-gp mRNAinivermectin-treatedcellsversusuntreatedcellsremainsto beelucidated.

In our conditions, when JWZ hepatocytes were exposed to 10

m

Mivermectin,Mdr1mRNAlevelwasincreasedupto2.5-fold, duetoatwofoldincreaseinMdr1mRNAhalf-life,comparedwith untreated cells. It is now recognized that a two to fourfold fluctuationinmRNAhalf-lifecanhavesignificanteffectsonmRNA andproteinabundance[64].Therefore,toinvestigatewhetherthe inductionoftheP-gpmRNAlevelbyivermectinwasreflectedat theproteinandtransportactivitylevel,anassaypreviouslyusedto screenforP-gp–druginteractions[29]wasperformed.Whencells weretreatedwith10

m

Mivermectinfor48h,conditionswhich caused a 2.5-fold increase in Mdr1 gene expression, the P-gp transportfunctionwasincreasedbymorethan30%.Consistently with our observation, an important correlation between P-gp modulation at the level of mRNA and the transport activity [3,65,66],butalsobetweenP-gpproteinlevelsandefﬂuxactivity followingtreatment withvariousdrugs[67]hasbeenreported. Interestingly,manycompoundsdeﬁnedasP-gpsubstratesactedto induceP-gpgeneexpressionandactivity,butrarelydisplayedan inhibitoryactivityonthetransporter[61].Ourresultsdemonstrate thativermectin,acompetitivesubstratedisplayingbothsubstrate and inhibitory activity, was also able to up-regulated mRNA expressionandconsequentlytransportactivityoftheP-gp.

P-gpisexpressedinmultiplekeyorgansindrugdisposition such as smallintestine, blood–brain barrier, kidneyand liver and it is well established that induction of the P-gp activity cancausereduceddrugsbiodispositionanddecreased therapeu-tic efﬁcacy [68]. It was recently shown that even moderate increasesintheexpressionofMdr1genesaresufﬁcienttocause doxorubicinresistanceinvivo[69],suggestingapossibleimpact onthepharmaco-andtoxicokineticofmanydrugsand endoge-nousP-gpsubstrates.Thepotentialinvolvementofivermectinin

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P-gp-mediateddrug–druginteractionswhenadministeredwith otherdrugsthatareP-gpsubstratesshouldthereforebetakeninto account in human andveterinary therapy. Furthermore, since ivermectindispositioninhostismainlycontrolledbyP-gpefflux attheintestinal[11]andhepaticlevel[12,13,70],up-regulationof itsexpressionmaycontributetotheautoinductionofivermectin elimination,resultinginadecreaseofthebodydispositionandthe intracellular levels of this anthelmintic agent in the host organism.Thistransportregulationmaybeconsideredasafirst stepinloweringthedrugdispositionandthereforedrugefficacy. Acquiredresistancetoivermectin isbecominga majorclinical concernintheprevention/treatmentofparasitesandeventhough the factors responsible for the development of resistance to antiparasitic compounds are not perfectly understood, one of them is the exposure of parasites to subtherapeutic drug concentrations[71].Therefore,thisstudyoffersinsightintothe potential for resistance development through modulation of ivermectin pharmacokinetic in the mammalian host. Further-more,P-gphomologsarethoughttoplayanimportantroleinthe resistanceofnematodeparasitesagainstMLs[21],anddatafrom the literature suggests that induction of P-gp expression in responsetoivermectintreatmentmayalsoexistinnematodes. Indeed, P-gp homologs were shown to be overexpressed in ivermectin-resistantwormssuchasHaemonchuscontortus[22] andCaenorhabditiselegans[23],andafterexposureofthesealice LepeophtheirussalmonistotheMLemamectin[72].Up-regulation oftheseproteinswouldservetoeliminateMLsfromtheparasite, limitingtheaccumulationofatoxicconcentrationatthetarget receptors,thereforeofferingprotectionoftheparasite. Interest-ingly,multidrugresistancephenotypehasbeenassociatedtoan overexpressionofsomeP-gphomologsthrough post-transcrip-tionalregulation,includingincreaseinmRNAstabilityinBacillus subtilis [73], but also in the protozoan parasites Entamoeba histolytica[74].Altogether,thesedatademonstratethatexposure toMLs,ivermectininparticular,potentlymodulatesP-gpsinboth mammals and parasites and that partial similarities in the mechanisms of ABC efflux transporters regulation may exist betweenmammalsandparasites.Theelucidationofthe molecu-larmechanismsofoverexpressionofABCeffluxtransportersin nematodes could provide useful markers for monitoring MLs resistanceandpossibletargetsforinterventionindrugresistant parasites.

Insummary, this reportdemonstrates fortheﬁrsttime that acute ivermectin treatment induces P-gp gene expression and activity in mouse hepatocytes through post-transcriptional regulationaffectingP-gp mRNAdegradation.Theseresultshave signiﬁcant clinical implications regarding alteration of drug dispositionanddrug–druginteractionsinhostandofferinsight intothepotentialforemergenceofmultidrugresistanceduring anthelmintictreatmentinparasites.

Acknowledgments

We gratefully acknowledge Abdel Boulahtouf and Virginie Belletforreportercelllinesstudies.WealsothankArnaudPolizzi (Integrative Toxicology and Metabolism, INRA UMR1331) for helpfuladvicesinqPCRstudies.

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