In vitro impact of five pesticides alone or in combination on human intestinal cell line Caco-2.

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In vitro impact of five pesticides alone or in combination

on human intestinal cell line Caco-2.

Sylvain Ilboudo, Edwin Fouché, Virginie Rizzati, Adama M. Toé, Laurence

Gamet Payrastre, Pierre I. Guissou

To cite this version:

Sylvain Ilboudo, Edwin Fouché, Virginie Rizzati, Adama M. Toé, Laurence Gamet Payrastre, et al.. In

vitro impact of five pesticides alone or in combination on human intestinal cell line Caco-2.. Toxicology

Reports, Elsevier, 2014, 1, pp.474-489. �10.1016/j.toxrep.2014.07.008�. �hal-01187880�

ContentslistsavailableatScienceDirect

Toxicology

Reports

jo u r n al hom e p ag e :w w w . e l s e v i e r . c o m / l o c a t e / t o x r e p

In

vitro

impact

of

five

pesticides

alone

or

in

combination

on

human

intestinal

cell

line

Caco-2

Sylvain

Ilboudo

_,

_Edwin

_Fouche

_,

_Virginie

_Rizzati

_,

_Adama

_M.

_Toé

_,

Laurence

Gamet-Payrastre

_,

_Pierre

_I.

_Guissou

a_{INRAUMR1331Toxalim(ResearchcentreinfoodToxicology),180ChemindeTournefeuille,F-31027Toulouse,France} b_{InstitutdeRechercheenSciencedelaSanté(IRSS/CNRST),03,BP7192,Ouagadougou,BurkinaFaso}

c_{LaboratoiredeToxicologie,EnvironnementetSanté;EcoleDoctoraledelaSanté,UniversitédeOuagadougou,03,BP7021,}

Ouagadougou,BurkinaFaso

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received13May2014

Receivedinrevisedform1July2014 Accepted14July2014

Availableonline25July2014 Keywords: Pesticide Mixture Caco-2 Oxidativestress Locustcontrol Antioxidant Cellsignaling

a

b

s

t

r

a

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InBurkinaFaso,asinmostSaheliancountries,thefailuretofollowgoodagricultural prac-ticescoupledwithpoorsoilandclimateconditionsinthelocustcontrolcontextleadto highenvironmentalcontaminationswithpesticideresidues.Thus,consumersbeingorally exposedtoacombinationofmultiplepesticideresiduesthroughfoodandwaterintake, thedigestivetractisatissuesusceptibletobedirectlyexposedtothesefood contami-nants.Theaimofourworkwastocompareinvitrotheimpactoffivedesertlocustcontrol pesticides(DeltamethrinDTM,FenitrothionFNT,FipronilFPN,Lambda-cyalothrineLCT, andTeflubenzuronTBZ)aloneandincombinationonthehumanintestinalCaco-2cells viabilityandfunction.Cellswereexposedto0.1–100␮Mpesticidesfor10daysaloneor inmixture(MIX).OurresultsshowedacytotoxiceffectofDTM,FNT,FPN,LCT,andTBZ aloneorincombinationinhumanintestinalCaco-2cells.Themostefficientwereshown tobeFPNandFNTimpactingthecelllayerintegrityand/orbarrierfunction,ALPactivity, antioxidantenzymeactivity,lipidperoxidation,Aktactivation,andapoptosis.The pres-enceofantioxidantreducedlipidperoxidationlevelandattenuatedthepesticides-induced celltoxicity,suggestingthatkeymechanismofpesticidescytotoxicitymaybelinkedto theirpro-oxidativepotential.Acomparativeanalysiswiththepredictedcytotoxiceffect ofpesticidesmixtureusingmathematicalmodelingshownthatthecombinationofthese pesticidesledtosynergisticeffectsratherthantoasimpleindependentordoseaddition effect.

©2014TheAuthors.PublishedbyElsevierIrelandLtd.Thisisanopenaccessarticleunder theCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction

Pesticidesareusefultoolsagainstdesertlocustin vari-ousAfricanandotherworldcountriesandseverallocust invasions need intensive useof these chemicals across

∗ Correspondingauthor.Tel.:+33561285383. E-mailaddress:laurence.payrastre@toulouse.inra.fr

(L.Gamet-Payrastre).

millionsofhectares[1].Astheresultsofthewidespreaduse andthelackofsafemanagementofpesticidesindeveloping countries[2–4],variouscompartmentsoftheenvironment arecontaminatedandexposuretopesticidesisaconcern towardthegeneralpopulation[5].

Epidemiological studies often established a positive correlation between occupational exposure to pesti-cides and the incidence of human chronic patholo-gies such as cancers, diabetes, neurodegenerative and reproductivedisorders, birthdefectsanddevelopmental

http://dx.doi.org/10.1016/j.toxrep.2014.07.008

2214-7500/© 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license

toxicity, nephropathies, and respiratory, cardiovascular and autoimmune diseases (fora review seeMostafalou andAbdollahi[6]).Moreover,pesticidesareknown indi-viduallytoinducetoxicityatthecellularlevelthroughout oxidant-mediatedresponsessuchasapoptoticornecrotic cellsdeath,membranelipidsperoxidation,metabolic per-turbation,deregulationofseveralsignalingpathways[7]

oralterationoftightjunctions[8,9].

Our previousinvestigations showed thepresence of pesticideresiduesinwaterandplantsamplesfromlocust area in Burkina Faso several years aftertheir use [10]. Elsewhere,pesticideresiduesindrinkingwaterandplant

[11–13]havebeenreportedtobepresentat

concentra-tionsaboveMaximumAllowableConcentration(MAC)and MaximumResidueLevels (MRLs)respectively.High lev-elsof pesticides inwater and plants assources offood commoditiesareaconcernforconsumersandmaybeat theonsetofhumanhealthperturbations.Moreover,due totheiruseinawidevarietyofconsumerproductsitis likely that humans are exposed toseveralpesticides at anyonetimeanditiswelladmittedthatconsumersare exposedthroughfoodstuffsandwaterintaketococktails offoodcontaminants.Althoughitwaswelladmittedthat themixtureisdevoidofhazardwhentheconcentration ofeachcompounddoesnotreachanhealthconcernlevel, Kortenkamp et al. [14] demonstrated significanteffects of combination of pesticides when present below their individualnoobservableadverse effectlevels(NOAELs). Predictingtheeffectsassociatedwithsimultaneous expo-suretodifferentfoodcontaminantsisoftenrelyingontwo suitable assessmentconcepts,theconcentrationordose addition(CA)andtheconceptofindependentaction(IA). CAisthoughttobeapplicabletomixtures ofchemicals thatactonthesametoxicologicalendpointbyacommon mechanism of action, while IA is commonly appliedto chemicalswithdissimilarmodesofaction[15].However, combinedeffectsofpesticidemixtureshavebeenassessed invariouscellandanimalmodelandresultssuggestedthat theeffectofpesticidemixturecannotalwaysbepredicted fromtheeffectofindividualcompounds[16–19].Itthus appearstheimportanceofassessingmixturesin toxicolog-icalstudies.Thishasstimulatedourinterestinassessing theeffectsassociatedwithsimultaneousexposureto pes-ticidesbothtroughtoxicologicalstudiesandmathematical modeling.

Weaimedtostudytheinvitrotoxicologicalproperties offivepesticidesaloneorincombination(Deltamethrin DTM,FenitrothionFNT,FipronilFPN,Lambda-cyalothrine LCT,andTeflubenzuronTBZ)andtocomparetheresults tothatpredictedfromamathematicalmodelinorderto assesstheroleofmodelingin predictingeffects of mix-ture.ThesecompoundsareintensivelyusedinBurkinaFaso againstthedesertlocustandhavebeenidentifiedasfood contaminantsfoundinedibleplantsanddrinkingwaterin thiscountry. Itisnoteworthythattheseinsecticidesare alsolargelyusedintheUEonvariouscrops,vineandfruit farmingexceptedfenithrotionanddiazinonwhicharenot approvedforutilizationintheUE.

Theoralroutebeingthemainwayofconsumers expo-sure,intestinaltractisadirecttargetorganofxenobiotics. Establishedcelllines,suchasCaco-2cellshaveprovedto

bethebestmodelforstudiesofintestinalabsorptionand toxicityofxenobiotics[20–22].Thesecells,despitetheir colonicorigin,expressedinculturethemain morpholog-icalandfunctionalcharacteristicsofintestinalcells[23]. ThehumancoloniccelllineCaco-2cellswasthususedas aninvitromodelinourstudy.

2. Materialsandmethods

2.1. Chemicals

The pesticides deltamethrin (CAS No. 52918-63-5), fenitrothion(CASNo.122-14-5),fipronil(CASNo. 120068-37-3), lambda-cyhalothrin (CAS No. 91465-08-6), and teflubenzuron (CAS No. 83121-18-0) were purchased from Fluka (Riedel-de-Haën®, France). Purity of each compound was ranged from 95.2 to 99.6% accord-ing to manufacturer specification. Dulbecco’s modified Eagle’smedium(DMEM),RoswellParkMemorialInstitute medium(RPMI),l-glutamine,Dimethylsulfoxide(DMSO), non essentialamino acids (NEAA),Penicillin and Strep-tomycin, Phosphate buffer saline (PBS), Trypsin-EDTA, p-nitrophenol, p-nitrophenyl-phosphate, hydrogen per-oxides(H2O2),Reducedglutathione,glutathionereductase,

␣-NADPH,2,7-dichlorodihydrofluoresceindiacetate(H2

-DCF-DA), Hank’sbalanced salt solution(HBSS), Hoechst 33342, propidiumiodide (PI), thiobarbituricacid (TBA), 1,1,3,3-tetramethoxypropane (TMOP), and Bicinchoninic Acid(BCA)werepurchasedfromSigma–Aldrich(France). Fetal bovineserum (FBS)and the sterilematerial used forculture(flasksandcultureplates)werefromDutscher (France).Whennot specifiedchemicalswerepurchased fromSigma–Aldrich(France).

2.2. Cellculture

Caco-2cellswereobtainedfromtheAmericanType Cul-tureCollection(ATCC,USA)andculturedinahumidified atmosphereof5%CO2and95%airat37◦C.Culturemedium

(DMEM)wassupplementedwith1%l-glutamine,1%NEAA, 1% Penicillin/Streptomycin and 10% of heat-inactivated FBS.Whenthecellculturereached80%confluence,cells weredispersedwith0.025Mtrypsin-EDTAandreseeded innewflask.Thepassagenumberofthecellsusedinthe experimentswasbetween25and39.Theculturemedium wasreplacedevery48h.

2.3. Pesticidetreatments

Pesticidesconcentrationsassessedin ourstudywere rangedfrom0.1to100␮M.Thechosendoseswerebased on the LMR values adapted to our in vitro study as follows: LMR values expressed in mg/kg of vegetables were converted into ␮g of active substance supposed to be present in the digestive tract by considering a totalassimilationoftheLMRamountofpesticideinvivo upon a dietary intake of 100g of vegetables. LMRs for DTM, FNT, TBZ, FNT, and LCT vary respectively from 0.01 to 2; 0.05 to 6; 0.05 to 1; 0.002 to 5; and 0.02 to 3mg/kg vegetables (according to Codex

of pesticides (100mM) were prepared in DMSO. One dayafter seeding, cell layers were washed withserum freemediumandthenincubatedwith4%FBScontaining mediumsupplementedwiththeindicatedconcentrations ofpesticides.Thefreshly mixturesolutionwasmadeby addingindividualpesticidesdirectlyinmediumcontaining 4%FBSatequimolarproportionrespectingthesamerange concentrationofsingleagents.Treatmentswithpesticides wererenewedevery48h.Controlcellswereexposedto 0.1%DMSO alone. Eachexperiment was repeated inde-pendently 3 times in quadruplicate (MTT assay) or in triplicate.

2.4. EvaluationofcellviabilitybyMTTassay

Theeffectsof pesticidesonCaco-2 cellsproliferation andviabilityweremonitoredduringa10days-periodof exposure.Reductionofthepermeanttetrazoliumsalt 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bro-mide(MTT)wasmonitoredasdescribedbyMosmann[24], withmodifications. Briefly cellswereseeded in24-well plates(5×104_{cells/well).After20hofincubation,thecells}

weretreatedwithvariousconcentrations(0.1–100␮M)of pesticides alone and in combination. At theend of the incubationtime (3,7 and10 days ofexposure),culture mediumwasdiscardedandthemonolayerwashedtwice withPBS.CellswerethenincubatedwithMTT(500␮lper wellat aconcentrationof0.5mg/ml)in RPMIfor4hat 37◦C.Thereactionwasstoppedusing500␮lofasolution containing10%SDSand1NNaOHat37◦C.Then, 200␮l offormazansolutionwastransferredinto96-wellplates andabsorbancesweremeasuredbyamicroplatereader (Tecan,Lyon,France)at570and690nm.Therelativecell viabilitywasexpressedastheratio(%)oftheabsorbance intheexperimentalwellstothatofthecontrolwells(cells treatedwithDMSO).TheEC50(cytotoxicconcentrationfor 50%celldeath)wasdeterminedfromthedose-response curve.

2.5. Observationofmorphologicchanges

Caco-2cells wereseededin12-wellplates ata den-sityof1.125×105_{cells/wellandallowedtogrowfor20h}

in500_␮lmedium.Thereafter,cellswereexposedto pes-ticides(25␮M)for72handmorphologicalchangeswere analyzedusingaphasecontrastmicroscope(Olympus). 2.6. Evaluationofpesticideeffectsoncellmonolayer integrity

2.6.1. Transepithelialelectricalresistance(TEER) measurement

Cellswereseededat2.5_×105_{cells/wellin6-well}

Tran-swellcultureplatecoatedwithcollagenandallowedto growfor4days.Then4%FBS-supplementedmedium con-taining pesticides (1, 5, 10 or 25␮M) or DMSO (0.1%) wereintroducedinbothapicalandbasolateral compart-ments.Treatmentwasrenewedevery2days.Monolayer integritywasmeasuredat day0,3,7 and 10using the MillicellElectricalResistanceSystem(Millipore,Molsheim,

France).Resultsareexpressedaselectricalresistanceofthe monolayer(cm2_{)andwerethemean±SEMof3}

inde-pendentexperimentsinwhicheachtreatmentwasdonein triplicate.

2.6.2. Phenolreddiffusion

Caco-2 cells culture conditions were the same as described above. At last day of exposure (day 10), the culture medium was discardedand apical and basolat-eral sides were rinsed twice with 2ml of PBS (37◦C). Two milliliters of phenol red containing medium were introducedinapicalsideofmonolayer.Inthebasolateral side,phenolredcontainingmediumwasreplacedby equiv-alentmediumwithoutphenolred.Diffusionofphenolred fromapicaltobasolateral(A–B)compartmentwas eval-uated for 4h. Quantification of phenolred in the basal sidewasperformedbymeasuringmediumabsorbanceat 560nmandpercentdiffusionwascalculated.

2.7. Alkalinephosphataseessayforcelldifferentiation Caco-2 cells were seeded into 12-well plates (1.125×105_{cells/well). Next day cells were treated}

withpesticides (1,5,10, and 25␮M) orvehicle(DMSO 0.1%)for10days.Thencelllayerswerewashedtwicewith 1ml ofcold NaCl9‰and harvested byscraping witha rubberpoliceman in500␮lof cold TRISbuffer(50mM, pH=7.5).Cellsuspensionswerethenhomogenizedbyfive passagesthrougha25Gneedlefittedwitha1mlsyringe. Alkalinephosphataseactivity,markerofenterocyticcells differentiation[25],wasmeasuredin wholecell lysates accordingtoWalterand Schütt[26]withmodifications. Briefly, ALP splits paranitrophenyl-phosphate (pNPP), liberatingyellowcoloredp-nitrophenol(pNP)underbasic condition.Quantificationofp-nitrophenol(␮molespermg ofproteins)wasperformedat405nmuponanincubation timeof30minatpH10.4and37◦C.Resultsareexpressedas percentofALPactivityintreatedcellscomparedtocontrol. Thetotal proteinconcentrationwasdeterminedbyBCA protein assay according to the manufacturer-suggested protocol.

2.8. Pesticide-inducedoxidativestressassessment 2.8.1. Lipidsperoxidationmeasurement

Caco-2 cells were seeded in 24-well plates (1.25×105_{cells/well) and were treated as described}

aboveupona48h-periodofexposurewiththeindicated concentrationofpesticides.Lipidperoxidationwas quan-tified in cell medium by using the thiobarbituric acid reactivesubstances (TBARs)assay[27],whichmeasures malondialdehyde (MDA) equivalents. Briefly, 50_␮l of cell mediumwere mixedwith5␮lof 0.5N chlorhydric acid (HCl) and 50␮l of TBA buffer (sodium hydroxide 0.12MTBA,pH7).Sampleswerethenboiledfor10minat 95◦C.Afterice-coolingindark,200␮lofn-butanolwere added,samplesweremixedandcentrifugedfor10minat 2000rpmat4◦C,150␮lofsupernatantfromeachsample were transferredinto96-well plateand theabsorbance

wasrecordedat532nm.TheTBARscontentwascalculated fromacalibrationcurvebyusingTMOPasMDAprecursor. 2.8.2. Antioxidantenzymeassays

Caco-2 cells were seeded in 6-well plates (1.5×105_{cells/well) and were exposed next day to}

pesticides (1,5,10, and 25␮M)or vehicleas described above. At day 10, cells were harvested by scraping on ice.Aftercentrifugation(900rpm,4◦C),cellpelletswere washedtwicewithicecoldPBSandsuspendedin phos-phate buffer (50mM, pH=7) supplemented with 1mM EDTA.Cellmembranesweredisruptedusingultrasounds three times for 10son ice. After ultracentrifugation at 105,000×g for 1h at 4◦C, supernatants were used to determine the catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) activities. The total protein concentration of individual sample was determined by BCA protein assay kit according to the manufacturer-suggestedprotocol.

CATactivitywasmonitoredaccordingtothemethod describedbyAebi[28].Briefly,hydrogenperoxide enzy-matic decomposition by sample catalasewasmeasured for30sat240nmonaspectrophotometer.TheGPx activ-ity wasdeterminedaccordingtothemethoddeveloped byLawrenceandBurk[29].Briefly,GPxreactswithH2O2

togenerateglutathionedisulfide(GSSG)whichisreduced byglutathionereductase(GR)inthepresenceofNADPH. ThedisappearanceofNADPHwasmonitoredat340nmon aspectrophotometerasindirectmeasureofGPxactivity. TheSODactivitywasdeterminedfollowingthemethodof OberleyandSpitz[30].Briefly,xanthine–xanthineoxidase systeminthepresenceofwatergeneratesthe superox-ideanion(O2•

¯

(NBT)reducingittoFormazandye.SampleSODandNBT competeforO2•

¯

for differentproteinconcentrations ofeach samplewas measuredastheamountofSODpresentinsample.One unitofSODwasdefinedastheamountofenzymewhich inhibitsby50%theformationofformazanblueobservedat 560nm.InhibitionofNBTreductionwaskinetically mon-itoredfor5minusingTecan®microplatereader.Specific enzymaticactivitieswerereportedaspercentofactivityin treatedcellscomparedtocontrol.

2.8.3. IntracellularROSmeasurement

Intracellular ROS were measured according to the method described by Wang and Joseph [31] with or withoutpretreatmentwithseveralantioxidants(vitamin E, vitamin C or Trolox). Cells plated in 48-well dishes (5_×104_{cells/well)wereallowedtogrowfor48hin}

com-plete medium supplemented with 10% FBS. Then cell layers werewashed twicewithPBS and treatedfor 6h at 37◦C with 25 or 100␮M of pesticides (0.025% of DMSOascontrolcells)inHBSSmediumcontaining2,7 -dichlorodihydrofluorescein diacetate (H2-DCFDA 5␮M).

Attheend ofthe6hincubation,mediumwasremoved and thecelllayers washedtwicewithPBS.Cell fluores-cencewasmeasuredwithTecan®microplatereaderatthe excitation and emissionwavelength of485and 530nm respectively.

2.9. Determinationofpesticides-inducedcelldeath mechanism

2.9.1. DetectionofapoptosisandnecrosisbyHoechst 33342andPIstaining

Caco-2cellswereseededin12-wellplatesatadensityof 1.25×105_{cells/well.After20hincubation,thecellswere}

treatedwith25␮Mofpesticidesaloneorincombination for48h.Attheendofthetreatmenttheculturemedium wasdiscardedandthemonolayerwasstainedwith5_␮g/ml ofHoechst33342and1␮g/mlofPIsolutioninthedarkfor 30minat37◦C.ThecelllayerwasthenwashedwithPBS. Themorphologicalfeaturesofapoptosis,suchascellular nucleusshrinkage,chromatincondensation,and nuclear fragmentation, were showedvia Hoechst stainingin PI negativecellsusingfluorescentmicroscopewithstandard excitationfilters.NecroticcellswerestainedinredbyIP. Eachexperimentwasrepeatedindependentlythreetimes intriplicate.Torecordthenumberofnormal,apoptoticand necroticcellsbytreatment,fourrandommicroscopicfields perwellwereconsidered.Eachexperimentwasrepeated threetimesintriplicate.

2.9.2. WesternblotanalysisofAkt

Cellswere seededat a density of 1.5×105_cells/well

into6-well plates and were exposed 20h after incuba-tionto25␮Mofeachindividualpesticideormixturefor 10days.Attheend ofpesticidetreatments,Caco-2cells were washed several times in cold phosphate-buffered saline, were scraped off the plate and lysed in modi-fiedRIPAlysis buffer(50mM Tris–HCl,pH7.4, 150mM NaCl,1%tritonX-100,5mMEDTApH8.0)containing1× proteaseinhibitorcocktail(HALT,Thermoscientific),and phosphataseinhibitorcocktail(1mMNaVO4,50nMNaF), for30minonice.Celllysateswereclearedby centrifuga-tion(10,000rpm,15minat4◦C).Proteinconcentrations weredeterminedusingBradfordreagent(sigma).Twenty fivemicrograms(25␮g)samplesofextractedproteinwere resolvedonSDS-polyacrylamidegelsand transferred to nitrocellulose membranes. The membranes were incu-batedinthepresenceof1/2000phospho-Aktser473(Cell signaling,4060)or1/1000Akt1/2/3(santacruz biotech-nology, sc-8312) antibodiesat 4◦C overnight. Antibody bindingwas detectedusing aninfrared fluorescent dye conjugatedantibodiesabsorbingat 800nm(Biotium, CF 770 conjugated antibodies, 20078). Immunoblots were visualizedusinganOdisseyInfraredimagingscanner (Li-Cor,Science Tec, lesUlis, France). Relative fluorescence unitsallowedaquantitativeanalysis.Eachexperimentwas doneindependently3times.

2.10. Dataanalysisofcombinedcytotoxiceffects

Combinedeffectsofpesticideswereanalyzedaccording toTakakura etal. [32].The total concentrationof mix-tureatwhichaneffectisgeneratedcanbecalculatedon thebasis of theconcentration–response curves of indi-vidualpesticidesusingtheCAconcept accordingtothe

Fig.1.Pesticides-inducedmorphologicalchangesinCaco-2cells.Caco-2cellswereseededin12-wellplatesandallowedtogrowfor20h.Thereafter, cellswereexposedtopesticides(25␮M)for72handmorphologicalchangeswereanalyzedusingOlympusinvertedmicroscope.Imagesselectedare representativeofthreeindependentexperiments.Photographswereperformedwithamagnificationof×20.

followequation:ECxmix=







whereECxmixisthe

totalconcentrationofthecocktailprovokingx%effect,ECxi theconcentrationofcomponentiprovokingthex%effect whenappliedsingly,andPidenotestherelative propor-tionsofthetotalmixtureconcentrations.

TheIApredictionconceptis usedtoexplicitly calcu-late combined effects according to:E(Cmix)=1−

1



i=1

[1− E(PICmix)]

E(Cmix)denotestheeffectprovokedbythetotalmixture

atconcentrationCmix,whileE(Picmix)aretheeffectsthat

theindividualcocktailcomponentiwouldcauseifapplied singlyatthesameconcentrationatwhichitispresentin thecocktail.

2.11. Statisticalanalysis

Valuesarerepresentativeofthemeans±SEM(standard error of the mean) of three independent experiments performedatleastintriplicate.Statisticalanalyseswere performedwithGraphPadPRISM 5(GraphPadSoftware Inc.,SanDiego,CA,USA).Comparisonshaveusedone-way ortwo-wayanalysisofvariance(ANOVA)followed respec-tivelybyDunnett’sand Bonferronimultiplecomparison posttests.Thelevelofsignificancewassetatp<0.05.

3. Results

3.1. Effectofpesticidesoncellproliferationandviability Cell proliferation and viability were assessed by measuring MTT reduction assay. Overall, microscopy observationsofCaco-2cellslayerrevealedthatexposureto pesticidesfor3,7and10daysledtomorphologicalchanges

comparedtocontrolcells(Fig.1).Furthermore,asshown in Fig.2,eachpesticidealone orinmixture,exertedan impactoncellviabilityinatimeandconcentration depend-entmanner.Theirobservedtoxicity(EC50valuesofTable1)

inCaco-2cellscouldberankedinthefollowingdecreasing order:FPN>MIX>FNT>LCT>DTM>TBZ.FNTandTBZ tox-icitywasobservedupona 3days-periodofexposureto 1and 5␮Mrespectively.TheimpactofexposuretoLCT (1␮M),DTM(8␮M)orFPN(5␮M)appearedlater(J7). 3.2. Effectofpesticidesoncellmonolayerintegrity

Impactofpesticidesaloneorincombinationonthecell monolayerintegritywasassessedbyanalyzing,alonga10 days-periodoftreatment, theTEER(_cm2_)(Fig.4)and

thepassageofphenolredacrossthecellmonolayer(Fig.4, insetfigures).Controlcells,treatedwiththevehiclealone, showedacontinuouslyincreaseofTEERuntiltheendofthe treatment(from420.00±15.14to1010.30±39.96cm2

at day10, Fig.4).Our resultsshowedthat exposureto FNTorTBZledtoanetdecreaseofmembraneresistance associated withan increase of theapparent permeabil-ityofthecelllayer(Fig.4).Ontheotherhand,exposure of Caco-2cells toLCT,DTM,and MIXinduceda signifi-cantincrease ofmembrane resistanceassociated witha decreaseofmembranepermeabilitysuggestinganet rein-forcementofcellularintegrity.TreatmentofCaco-2cells withFPNfailedtoproduceaclearandnoticeablechangein TEERvaluesrelativelytocontrol.Wenextcheckedwhether theobservedchangesinviabilityandcelllayerintegrity uponexposuretopesticidescouldbelinkedtoachangein thecellulardifferentiationprocessofCaco-2cells. 3.3. Effectofpesticidesonalkalinephosphataseactivity

Caco-2cellsdifferentiationwascharacterizedbydomes formationandcellspolarizationwithapicalbrushborder

Fig.2. DoseresponsecurvesoftheimpactofpesticidesonCaco-2cellsviability.Cellsweretreated20hafterseedingwiththeindicatedpesticidealoneor incombinationatincreasingdosesof0.1–100␮M.Treatmentswererepeatedeach48h.MTTtestwasperformedon3,7and10daysoldcellsasdescribed inSection2.Thedataarenormalizedtotheviabilityofcontrolcells(100%).Resultsarethemean±SEMofquadrupletsfromthreerepeatedindependent experiments.Asteriskindicateslevelofsignificanceatp≤0.05.

Fig.3.Comparisonbetweenthemeasuredandpredictedeffectsofthemixtureofpesticides.Themeasuredeffectsofthemixturewereassessedbyanalyzing thedose–responsecurvesofitscytotoxicitydeterminedbytheMTTassayaftera3,7,and10days-periodoftreatmentinCaco-2cellsasdescribedinSection

2.Cytotoxicityisexpressedasthemean±SEMofthreeindependentexperimentsinquadruplet.Thecombinedeffectsofpesticideswerealsopredicted usinganindependentaction(IA)andaconcentrationaddition(CA)modelsasdescribedinSection2.

Fig.4. Effectofpesticidesoncelllayersintegrity.Caco-2cellswereallowedtogrowonpermeablemembranefiltersupportsinpresenceofpesticidesat concentrationsrangingfrom1to25␮Mfor10days.Transepithelialelectricalresistance(TEER)wasmeasuredatdays0,3,7,and10followingproceeding describedinSection2anditsvalueisexpressedaspercentofcontrol.Inparallel,phenolredpassagethroughcelllayerwasassessedatday10(inset)as describedbySection2.BothTEERandPRdiffusionvaluesareexpressedinmeans±SEMof3independentexperiencesdoneintriplicate.(Forinterpretation ofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthearticle.)

Table1

EC50values(␮M)ofpesticidesaloneonCaco-2cellsupon3,7,and10days-periodofexposure.Caco-2cellswereexposedtoDTM,FNT,FPN,LCT,andTBZ

eitheraloneorasamixtureofthefivefor3,7and10daysatconcentrationsrangingfrom0.1to100␮M.Afterexposure,cellviabilitywasassessedbyMTT assayandeffectconcentrations50%(EC50)werecalculatedaccordingtothedoseresponsecurvesinFig.1asdescribedinSection2.

Time(days) EC50(␮M)a

DTM FNT FPN LCT TBZ

3 191.0±5.5 31.6±0.7 24.1±0.4 72.9±2.7 571.1±51.0 7 136.4±10.7 25.6±0.8 21.6±0.5 74.3±4.8 471.3±37.6 10 111.9±3.3 21.3±0.5 18.1±0.5 59.5±2.3 429.4±24.5

a_{Mean±SEMofthreeindependentexperiments.}

bearing specific enzymes.Differentiation ofnon-treated Caco-2 cells wasaccompaniedwithagraduate increase of ALPactivity(Fig.5)culminantatday10. Ourresults showed a clearly dose dependent perturbation of the enzymeactivityuponexposuretopesticidesaloneorin combination.FNTandFPNexposureofCaco-2cellsledto adecreasedALPactivitycomparedtocontrolwhereasthe oppositewasobserveduponexposuretoDTM,LCT,TBZ,or MIX.

3.4. Pesticides-inducedoxidativestressinCaco-2cells OxidativestatusinCaco-2cellsuponexposureto pes-ticideswasinvestigatedbymeasuringtheactivitiesofthe mainenzymesinvolvedinthecontrolofROSlevel(CAT, GPx,andSOD).AsshowninFig.6,a10days-periodof treat-mentofCaco-2cellswithpesticidesaloneorinmixture(1, 5,10,and 25␮M)inducedinadosedependentmanner anincrease in CAT(DTM/LCT/MIX),GPx(FNT/FPN/MIX), andSOD(FNT)activitiessuggestingachangeinoxidative statusofCaco-2cellsuponexposuretothesecompounds (Fig.6A–C).TheincreasedSODactivityuponexposureto FNTsuggestsadownstreamproductionofO2•−.The

acti-vation of GPx or CAT observed upon exposure to FNT, FPN,DTM,LCT,andMixsuggestedanincreaseinlevelof

H2O2. On theotherhandTBZ didnot appear toinduce

noticeable changes in oxidative status since no change inenzymeactivitywasobserved.Inordertoconfirmthe prooxidative activities of pesticides in Caco-2 cells we next evaluated H2O2 production in cells by using

spe-cific fluorescentprobe. Asshowed inFig. 7A,FPN, FNT, DTM,andMIXtreatmentsinducedthestrongesteffecton intracellularH2O2generationevidencedbytheincreased

fluorescencestaining.Theseresultswerewellcorrelated withtheincreasedactivityofCATandGPxasdescribed above.We alsodeterminedthelipidperoxidationstatus byexaminingtheTBARsconcentrationsuponashortterm periodofexposure(48h)topesticides(25and100␮M). Resultsclearly indicated asignificantincrease of TBARS level upon exposuretoeach pesticidealone orin mix-ture.FPNandFNTwerefoundtobethemostefficientwith respectively192.45and167.66%withrespecttocontrolat 25␮M(Fig.7B).Furthermore,pretreatmentwith100␮M ofVitaminCfor4hor10␮MofVitaminEorTroloxfor 24hbeforeexposureofCaco-2cellstopesticidespartially reversedtheproductionofH2O2(Fig.8A)andledtoanet

decreaseofpesticidecytotoxicityspeciallyinCaco-2cells exposedtothemostcytotoxiccompoundsDTM,FPN,FNT, LCT,andMIX(Fig.8B).Theseresultssuggestthatoxidative stresscouldbeatleastinpartinvolvedinCaco-2cellsdeath inducedbythesepesticidestreatment.

Fig.5.EffectofpesticidesonCaco-2cellsdifferentiation.Cellswereexposedtosingleormixtureofpesticidesatconcentrationsrangingfrom1to25␮M for10days.CelldifferentiationwasevaluatedbymeasuringALPactivityasdescribedinSection2.DataareexpressedasthepercentageofALPactivityin treatedcomparedtocontrolcells(exposedtothevehicleonlyDMSO)andarethemean±SEMofthreeindependentexperiencesinwhicheachtreatment wasperformedintriplicate.Asteriskindicatelevelofsignificanceatp<0.05(*),p<0.01(**),p<0.001(***).

Fig.6. EffectsofpesticidesontheactivityofantioxidantenzymesinCaco-2.Cellswereexposedfor10daystopesticidesindividuallyandinmixtureat concentrationsrangingfrom1to25␮M.Catalase(A),GPx(B),andCu/ZnSOD(C)activitiesweremeasuredincytosolicfractionsofcelllysatesasdescribed inSection2.Dataareexpressedastheratiooftheactivityintreatedcellscomparedtocontrolcells(DMSO).Resultsarethemean±SEMfor3independent experimentswhereeachtreatmentwasdoneintriplicate.Statisticaldifferencesareshownas*p<0.05,**p<0.01,***p<0.001.

3.5. Pesticides-inducedcelldeathmechanismonCaco-2 cells

Toelucidate themechanismofpesticide-inducedcell deathinCaco-2cells,weperformedadoublestainingof celllayerwithHoechst33342andPIfollowedbya micro-scopicobservation.Cellsexhibitingacondensednucleiand noPIstainingcouldbeclassifiedasapoptoticwhereascells stainedwithbothHoechstandPIcouldbeconsideredas necroticcells(Fig.9A).Resultsshowedanincreasedofcell deaththroughboth apoptotic and necrotic mechanisms (Fig.9BandC).ItisnoteworthythatonlyFPNandmixture treatmentsledtoahugeincreasedofapoptoticwhereasthe percentageofnecroticcellsincreasedtoalesserextent.In ordertoconfirmapoptosisincellsexposedtopesticideswe

nextexaminethelevelofexpressionoftheactiveformof Akt(phospho-Akt),akeycellsignalingenzymeinvolvedin theinhibitionofapoptosisandinductionofcell prolifera-tion.Resultsshowedareducedlevelofphospho-Aktincells exposedtopesticidesespeciallyinFPNandMIXexposed cellscomparedtocontrol(Fig.10AandB)confirmingthe roleofapoptosisatleastinpartinthemechanismof pes-ticideinducedcelldeathinourmodel.

3.6. Comparisonoftheobservedeffectversusthe predictedeffectofpesticidemixture

Theexperimentallymeasuredeffectofpesticides mix-ture on cell viability (Fig. 1) was compared with the predictedeffectcalculatedusingCAandIAmodelsfromthe

Fig.7.EffectsofpesticidesexposureonlipidperoxidationandonthelevelofhydroxideperoxideinCaco-2.Cellswereexposedfor6and48h(lipid peroxidationandH2O2dosagerespectively)toFPN,FNT,LCT,DTM,andTBZeitheraloneorasamixtureofthefiveat25and100␮M.(A)H2O2production

wasmeasuredusingH2-DCF-DA.(B)LipidperoxidationwasassessedbyTBARsquantificationintheincubationmedium.Dataareexpressedastheratio

offluorescenceintreatedcellscomparedtountreatedcells(DMSOexposed).Resultsarethemean±SEMfor3independentexperimentsinwhicheach treatmentweredoneintriplicate.Differenceisstatisticallysignificantforp≤0.05(*),p≤0.01(**),andp≤0.001(***).

impactofsinglepesticide.ResultsarepresentedinTable2. The95%CIfromthemeasuredEC50failedtooverlapthe

EC50obtainedinthepredictedmodelateachtesteddose

(Table2andFig.3).Theseresultssuggestthatthe

com-binedtoxicologicaleffectsofthe5pesticidesresultedfrom complexinteractionsdifferentfromthatobservedin inde-pendentordoseadditionsituation.

4. Discussion

InBurkinaFaso,asinmostSaheliancountries,the fail-ure tofollow good agriculturalpractices (GAP) coupled withpoorsoilandclimateconditionsinthelocustcontrol contextleadtohighenvironmentalcontaminationswith pesticideresidues[10].Consumersbeingorallyexposed

Table2

Statisticalcomparisonbetweenmeasuredandpredictedcombinedeffectconcentrations(EC50).Caco-2cellswereexposedtothemixtureofthefivefor3,

7and10daysatconcentrationsrangingfrom0.1to100␮M.Afterexposure,cellviabilitywasassessedbyMTTassayandpredictedeffectconcentrations 50%(EC50)werecalculatedaccordingtoSection2.IA,independentactionmodel;CA,concentrationaddition;CI,confidenceinterval.

Time(days) Measured IA CA

Mean 95%CI Mean 95%CI Mean 95%CI

3 27.5 26.1–29.0 7.7 7.4–8.0 51.3 51.0–51.5

7 22.6 21.5–23.8 7.5 7.2–7.8 43.3 42.8–43.8

Fig.8. EffectsofantioxidantonH2O2productionandpromotionofcellviability.Twentyhoursafterseeding,Caco-2cellswerepretreatedwithVitamin

C(4hat100␮M)andVitaminEorTrolox(10␮Mfor24h).Cellswerethenexposedtopesticidesat100␮Mfor6hinthepresenceofH2-DCF-DA(H2O2

dosage)or72h(cellviabilityassay).H2-DCF-DAfluorescence(A)andcellsviability(B)wereassessedasdescribedinSection2.Datarepresenttheratio ofthevalueintreatedcellscomparedtountreatedone(DMSO).Resultsarethemean±SEMfor3independentexperimentsinwhicheachtreatmentwas doneintriplicate.Asteriskindicatelevelofsignificanceatp<0.05(*),p<0.01(**),p<0.001(***)forcomparisonbetweenpesticide-treatedresultsand untreatedcontrolone.Statisticaldifferencesbetweenpesticide-treatedresultsinpresenceandwithoutantioxidantareshownas+_forp<0.05,++_forp<0.01,

and+++_forp<0.001).

toa combination ofmultiplepesticideresiduesthrough foodandwaterintake,thedigestivetractisthusatissue susceptibletobedirectlyexposedtothesefood contam-inants mixtures. The aimof our work was tocompare invitrotheimpactoffivedesertlocustcontrolpesticides (DTM,FNT, FPN,LCT,andTBZ)commonlyfoundinfood andwaterin BurkinaFaso aloneandincombination on thehumanintestinalCaco-2cellsviabilityandfunction. Pesticidesconcentrationsrangeswerechosenaccordingto theirLMRandadaptedtotheinvitrostudyasdescribedin materialandmethod.

Ourresultswhichare summarizedin Table3clearly showedacytotoxiceffectofDTM,FNT,LCT,andTBZalone or in combination in human intestinal Caco-2 cells at doserangingfrom1to8␮M(1␮Mbeingequivalentto

0.7–1.3␮g/well accordingtothepesticide).These doses couldbeconsideredasrelevanttohumanexposure.Indeed based ontheLMR values of each compound, pesticides couldbepresentinthedigestivetractatdoserangingfrom 0.2to300␮g,whenconsideringatotalassimilationofthe LMRamountofpesticideinvivouponadietaryintakeof 100gofvegetables.

ThemostcytotoxiccompoundswereFPNandFNTthat impactedthe celllayerintegrity, ALP activity,oxidative status, Akt activation, and apoptosis (data summarized

inTable3).Theseresultsareobservedforthefirsttime

in a human intestinal cell line (exceptedFPN) and are in agreementwith their observed cytotoxicity in other invitromodels.CytotoxicityofDTMwasindeedobserved in corticalneurons[33],in ratspermatozoa[34] and in

Fig.9. ImpactofpesticidesaloneorincombinationonCaco-2cellsdeath.Cellsweretreatedwithpesticides(25␮M)for48h.Attheendoftheincubation period,cellswerelabeledwithHoechst33342andIPfor30min.Celllayerwasthenobservedusingfluorescencemicroscopetorecordnormalviable (uncondensednucleiandnoPIstaining),apoptotic(condensednucleiandnoPIstaining),andnecroticcells(uncondensednucleiandPIstaining).(A) Showstheimageofstainedcellsindicatingnecrotic(encircled)andapoptoticcells.Photographswereperformedwithamagnificationof×20.(B)Shows thepercentageofnonapoptoticnornecroticcellsintreatedanduntreatedassays,and(C)showsthepercentageofapoptoticandnecroticcells.Statistical differenceswithuntreatedresultareshownas***p<0.001(normal/apoptoticcells)and+++_{p<0.001(necroticcells).}

SH-SY5Ycells[35].FNTwasshowntoexertcytotoxiceffect in rathepatocytes [36].FPN hasbeenshown todisplay toxiceffectinSH-SY5Ycells[37–39],inmouseN2a neu-roblastoma [40]. LCTexertedcytotoxic effectin human lymphocytes[41].

Thealterationofthecelllayerintegritywhichincreased withthetimeofexposuretopesticides,wascharacterized byeitheradecreaseinTEERwithaproportionalincrease ofthepassageofPR(FNTandTBZ),orariseinTEERwith areducedpassageofPRfromtheapicaltothebasalside oftheepithelium(DTMandLCT).Itiswelladmittedthat freeradicalsmaydirectlydamagecellmembranethrough theoxidationofPolyUnsaturatedFattyacidswithinthe phospholipidsstructureofthemembraneitselfandthus alterthemembraneresistanceorpermeability.However, thedifferentialeffectofDTM/LCTononehandandofFPN and TBZ ontheother handonTEERin ourmodel can-notbeexplainedbytheirpro-oxidativepropertiesbecause it isanalmostuniversalpropertyofpesticides.The dif-ferentialimpactbetweenDTMandFNTonTEERmaybe due totheirspecificactivityoncytoskeleton and mem-branefluidityrespectively.IndeedFTNisknowntoalter membrane fluidity and as suggested by Gonzales-Baro

etal.[42] itis possiblethatthetoxic propertiesofFNT couldbe related toan increased permeability of mem-branes.DTMeffectonTEERmaybelinkedtoitsimpact ontheexpressionofa transcriptionalfactor involvedin theexpressionofgenesrelatedtothecytoskeleton organi-zation(NFATC1/cathepsin/c-SRC)[43].FPNtreatmentthat inducedoxidativestressinourmodelandthehighest cyto-toxicity(EC50=18.06␮M)ledtochangesintheapparent

permeabilitytophenolredwithoutalteringtheTEER.This isinagreementwiththehypothesisformulatedby Kar-czewskiandGroot[44]accordingtowhichanalterationof permeabilitycanoccurwithoutnoticeablechangein elec-tricalresistance.

AssuggestedbySambuyet al.[22],thealterationof thepermeability andtheintegrity ofthecellmonolayer suggest perturbation of the intestinal barrier function. Modificationoftheepitheliumintegrityisconsideredas acriticaltoxicokineticparameterthatinfluencesthefate andconsequentlythesystemictoxicityofchemicalsinthe organism.Previousfindingshaveshowntheinvolvement of tight junction’s failure and cytoskeleton disorganiza-tion in chemicals induced-cell monolayer permeability alteration[45].Oxidativestressisalsoknowntodisturb

Fig.10. Mechanismofpesticideinducedcelldeath.Onedayafterseeding,cellswereexposedfor48htopesticide(DTM,FNT,FPN,LCT,andTBZ)alone orincombination(MIX)(25␮M).Controlcorrespondstocellstreatedwiththevehicleonly(DMSO0.1%).(A)Showsthewesternblotanalysisoftotaland phospho-Akt(Ser473).(B)RepresentstheratioPhospho-Akt/Total-Aktexpressedasthepercentageofthecontrol.Resultsarethemean±SEMofthree independentexperimentsintriplicate.Statisticaldifferencesareshownas*p<0.05,**p<0.01,***p<0.001.

intestinal and renal epithelium or blood brain barrier integritybyalteringtightjunctionmolecules[8,9,46].In thepresentstudy,pesticides-inducedfunctionalalteration oftheepithelialCaco-2celllayerwaswellcorrelatedwith theprooxidativeactivityofthesechemicals.

Inparallelourresultsshowedadecreased activityof ALPincellsexposed toFPN andFNT,whileexposureto DTM,LCT,andTBZ,increaseditsactivity.ALPisamarker of intestinal cell differentiation and is alsoinvolved in lipidabsorption acrosstheapical membrane of entero-cytes,in regulationof duodenalsurfacepH,in bacterial transepithelial passage limitation, and in detoxification ofintestinal lipopolysaccharide[47]. Thedecreased ALP activityinCaco-2cellsexposedtoFPNandFNTcouldbe correlatedwiththeircytoxicity.Theinvolvementof oxida-tivestressintheregulationofALPactivityhasalsobeen demonstrated [48,49].It maybe indeed suggested that pesticideinducedCaco-2cellstoxicityandALPactivity dis-turbancecouldbelinkedtotheirprooxidativeproperties.

ExposureofCaco-2cellstoindividualpesticidesinour modelalsoledtoadecreasedadaptivecapacityofcells sug-gestedbyatimedependentdiminutionoftheEC50values

ofindividualcompound.Moreovermorphologicalchanges aswellasareductionofcelldensityuponexposuretoFNT, FPN,andMIXsuggestedthatcellsdidnotreachconfluence. Althoughtheinductionofcellcyclearrestorincreased sus-ceptibilityofdividing cellsupon pesticideexposurehas beenshowninvariouscellmodels[50–52],theincreased numberofapoptoticandnecroticcellsuponexposureto pesticidesalone orin combination in ourmodel (sum-marizedinTable3)supportedthehypothesisthatthese compoundsaffectedratherthecellviabilityprocessesthan thecycleprogression.Ourresultsareinagreementwith previousstudiesshowingproapoptoticpropertyofFPNand

DTM[33,37,39].Herewealsoshowedthatexposuretoeach

individualpesticideisassociated withadecreased level ofphosphorylatedAkt(Ser473),FPNandLCTleadingto thegreatestimpact,confirmingthestart-upofan apop-toticmechanism[53,54]uponexposuretoeachcompound. Howeverfromourresultsitappearsthatapoptosiswasnot theonemechanisminvolvedincelldeathinducedby pesti-cidessincethepercentofnecroticcellswasalsoincreased. MoreoverourdatashowedthatFPNandtoalesserextent DTMandFNTwerestrongerapoptoticcompoundsthanLCT andTBZsuggestingdissimilarmechanismofactionofthese pesticides.

FromouroveralldatasummarizedinTable3,DTMand LCTwhichbelongtothesamechemicalfamily(pyrethroid), appearedtosharethesamemechanismofactioninthe inducedCaco-2cellstoxicity,althoughDTMwasmorepro apoptoticthanLCT(Table3).OntheoppositeFPN,FNTand TBZshoweddifferentialimpactsonCaco-2cellscompared fromeachother.FNTandTBZdifferentiallyaffectedALP activity,andFPNdidnotaffectTEERandexertedclearlythe strongestapoptoticeffectwithminorchangesofnecrotic cellnumbercomparedtocontrol.

Wealsoinvestigatedthejointeffectsofthefive pes-ticides on various cell functions by testing a mixture of pesticides in which each compound was present at equimolarconcentration.Althoughthemixturedilutedthe mosttoxiccompoundswhencombined,pesticidesledtoa highereffectthanthatexpectedregardingtotheeffectof eachsinglecompoundsuggestingasynergisticinteraction ofthecompounds.Themixtureinducedadosedependent cytotoxicitycharacterizedbycellsdeathaccordingto apop-totic andnecrotic mechanisms,thedisturbanceofTEER values, the increase of PAL activity, an alteration of oxidativestressenzymesactivity,andanincreasedlipid

Table 3 Summary of the effect of pesticides alone or in combination on cell viability and transepithelial membrane resistance (TEER), apparent permeability (phenol red diffusion) and alkaline phosphatase activity (ALP) at J10, on lipid peroxidation and H2 O2 production (upon 48 and 6 h, respectively) and on the ration of the percentage of apoptotic or necrotic cells in treated versus untreated cells (48 h). In control cells (DMSO treated cells) the ratio of the apototic cell percentage versus necrotic cell percentage was equal to 0.5. Cell viability (J10) * TEER (J10) Apparent permeability (J10) ALP activity (J10) ** Lipid peroxidation ** H2 O2 production * Percent of apoptosis in treated vs control cells Percent of necrosis in treated vs control cells Deltamethrin       4.3 2.7 8 ␮ M 10 ␮ M 25 ␮ M 1 ␮ M 100 ␮ M 25 ␮ M Fenitrothion       6.3 4.4 5 ␮ M 10 ␮ M 25 ␮ M 5 ␮ M 25 ␮ M 25 ␮ M Fipronil  ↔     8.6 3.2 5 ␮ M 5 ␮ M 10 ␮ M 25 ␮ M 100 ␮ M Teflubenzuron       3.9 3.8 5 ␮ M 5 ␮ M 25 ␮ M 5 ␮ M 25 ␮ M 100 ␮ M Lambda cyalothrin       3.0 2.7 1 ␮ M2 5 ␮ M2 5 ␮ M1 ␮ M 100 ␮ M 100 ␮ M Mixture       8.9 2.7 1 ␮ M 10 ␮ M 5 ␮ M 1 ␮ M 100 ␮ M 25 ␮ M * p < 0.05. ** p < 0.01.

peroxidation.Whencomparingtheeffectofthemixture withitspredictedtheoreticaleffectourresultspointedout adifferencebetweenthemeasuredimpactofthemixture andthetheoreticallycalculatedone.Intheanalysisof com-binedeffectstworeferenceconceptsarecurrentlyapplied for the assessment of mixture toxicities, i.e. the Loewe additivity(basedonthedoseaddition(CA)concept)and theBlissindependence(basedontheindependentaction (IA)concept)[55,56].Theyhavebeenwidelyappliedto predictthejointtoxicityofmixtures[19,32,57,58]. Evalu-ationoftheeffectsassociatedwithsimultaneousexposure tochemicalcontaminantshastorelyonsuitable assess-mentconcepts,but itisnotimmediatelyobviouswhich ofthetwomodels,CAorIA,shouldbeemployed (accord-ing toErmler et al. [15]). TheCA model is suitable for predictingthecombinedtoxicity ofmixtureswith com-poundssharingthesamemechanismofaction(inhibition ofcholinesterasesbyorganophosphorusforexample).For this type of mixture, the prediction is reliable. The IA modelismeanwhileindicatedinpredictingthecombined toxicityofmixtureswhoseconstituentsdonotsharethe samemechanismofaction(strictlydistinctmechanisms ofaction).AsalreadyunderlinedbyRajapakseetal.[59], thedifferencefoundinourmodelbetweenthepredicted andtheobservableeffectofthemixtureis materialized onone handbytheshiftoftheobservedand predicted concentration–effectrelationshipcurves,andontheother handbythelackofanoverlapbetweenthemeasuredand thepredicted95%CIofthedoseresponse.Suchdeviations arecurrentlyobservedsincethepredictedtoxicitybytheIA andCAconceptcandeviatebyafactornfromtheobserved values,nbeingthenumberofmixturecomponents[60]. Furthermore,adeviationlessthantwofoldhasbeen gen-erallyappliedas athreshold whichshows a correlation betweenpredictedandobservedmixturetoxicity[61,62]. InourstudyusingtheCAconceptweobservedadeviation factorof1.87,1.92,and1.75atday3,7,and10, respec-tively.Whereasafactorof3.57,3.01,and3.94wasfoundat thesametimeapplyingtheIAconcept(datanotshow).We canthuspresumethatCAconceptcouldbeconsideredas amoreaccuratemodelthantheIAconcepttopredictthe effectofthecombinedpesticidesinourstudy.However althoughthesimilarityordissimilarityofthemechanisms ofactionofthemixturecomponentsarethegoverning fac-torforthepredictionqualityofbothCAandIAconcepts itisassumedthatCAoverestimatesthemixturetoxicityof strictlydissimilaractingsubstances,andIAunderestimates thetoxicityofstrictlysimilaractingsubstances[60].Ermler etal.[15]alsoreportedthattheassessmentofchemical mixturescomposedofsubstancesthatdonotactviathe samemechanismismorecomplicated.Rideretal.[63]have recentlypresentedananalysisoftheirownmixturestudies withanti-androgenswheretheyinvestigatedtheeffectsof inuteroexposuresonmalereproductivetractdevelopment inrats.CApredictedthemixtureeffectsbetterthanIA,even withmixturescomposedofanti-androgenswithdiverse mechanisms of action. IA consistently underestimated the effects of these anti-androgen mixtures. Pesti-cidesfroma specificchemical class(organophosphorus, organochlorine,etc.)couldinducesimilar phenomenolo-gicaleffects,andsuchsimilarityofactionwouldsupport

thechoiceofCAas anassessmentconcept, irrespective ofthefinerdetailsofmolecularmechanismsofthe com-pounds.InthiscasethesimilarityassumptionofCAcould notentirelybemet,becauseofsubtledifferencesinthe waysinwhichthechemicalsinthecomponentmixtures interactedat thecellularand molecularlevel. Moreover fewercompounds canbe expectedto qualifyfor inclu-sioninacommonmixture/effectsassessmentandthismay leadtounderestimationoftherisk[15].Inthelightofour resultsandinagreementwiththeconclusionsofJunghans etal.[60]andErmleretal.[15],wecanmakethe follow-ingassumptions:(i) themixtureeffect couldbedue to dissimilarlyactingcomponentssincethetoxicityof mix-tureassessedusingIAconceptishigherthanthemeasured toxicity;(ii)inthemixture,compoundsinteracttogether leadingtoamixturetoxicitythatishigherthanthe pre-dictedeffectbyCA.

In conclusion, locust control that generates environ-mental and subsequent food commodities and water contaminationwithpesticidesmixtureisapublichealth concern.Inourstudy,weclearlyshowedthatpesticides oftenfoundinwaterandvegetablesinBurkinaFaso dam-agehuman intestinal cells in culture individually or in mixtureatdoseclosedtotherealexposure,bymodifying cellulargrowth,survivalandhomeostasissuggesting sub-sequentdisordersoftheintestinalepithelium.Moreover, fromthecomparisonofourmeasuredandpredictedresults itappearsclearlythatCAmodelisthemostsuitablefor predictinginteractionofpesticidemixtureinourmodel; howeveritdoesnotcorrelatewiththeobserved synergis-ticeffectwhichsuggestsdissimilaractingsubstance.Our invitroexperimentsservethepurposetohighlighttheneed forfurtherevaluation oftheinvivotoxicity ofchemical mixture.

Conflictofinterest

Theauthorsdeclarethattheydonothaveanyconflict ofinterest.

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canbefoundintheonlineversion.

Acknowledgment

ThisstudywassupportedbyagrantfromtheFrench MinistryofForeignAffairs(GrantNo.745249G)through theSCAC(ServicedeCoopérationetd’ActionCulturelle)of FrenchEmbassyinBurkinaFaso.

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