Phytic acid protects porcine intestinal epithelial cells from deoxynivalenol (DON) cytotoxicity.

(1)

HAL Id: hal-02652261

https://hal.inrae.fr/hal-02652261

Submitted on 29 May 2020

HAL is a multi-disciplinary open access

archive for the deposit and dissemination of

sci-entific research documents, whether they are

pub-lished or not. The documents may come from

teaching and research institutions in France or

abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est

destinée au dépôt et à la diffusion de documents

scientifiques de niveau recherche, publiés ou non,

émanant des établissements d’enseignement et de

recherche français ou étrangers, des laboratoires

publics ou privés.

Phytic acid protects porcine intestinal epithelial cells

from deoxynivalenol (DON) cytotoxicity.

Graziela Drociunas Pacheco, Caio Abércio Da Silva, Philippe Pinton, Isabelle

P. Oswald, Ana-Paula Loureiro-Bracarense

To cite this version:

Graziela Drociunas Pacheco, Caio Abércio Da Silva, Philippe Pinton, Isabelle P. Oswald, Ana-Paula

Loureiro-Bracarense.

Phytic acid protects porcine intestinal epithelial cells from deoxynivalenol

(DON) cytotoxicity..

Experimental and Toxicologic Pathology, Elsevier, 2012, 64 (4), pp.345-7.

(2)

Author's personal copy

ExperimentalandToxicologicPathology64 (2012) 345–347

ContentslistsavailableatScienceDirect

Experimental

and

Toxicologic

Pathology

j our na l ho m e p a g e :w w w . e l s e v i e r . d e / e t p

Short

communication

Phytic

acid

protects

porcine

intestinal

epithelial

cells

from

deoxynivalenol

(DON)

cytotoxicity

Graziela

Drociunas

Pacheco

a

_,

_Caio

_Abércio

_da

_Silva

a

_,

_Philippe

_Pinton

b

_,

Isabelle

P. Oswald

b

_,

_Ana

_Paula

_Frederico

_Rodrigues

_Loureiro

_Bracarense

a,∗

a_Universidade_Estadual_de_Londrina,_Centro_de_Ciências_Agrárias,_Campus_{Universitário,}_Londrina,_CX_Postal_6001,_Brazil b_INRA,_Laboratoire_de_{Pharmacologie-Toxicologie,}₁₈₀_Chemin_de_{Tournefeuille}_BP3,₃₁₉₃₁_Toulouse,_France

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received12August2010 Accepted27September2010 Keywords: Intestinalcell Inositol IP6 Mycotoxin Toxicity

a

b

s

t

r

a

c

t

Thepurposeofthisstudywastoevaluatetheeffectsofphyticacid(IP6)asapossibleinhibitorofcellular

damageinducedbytoxicsubstancessuchasmycotoxinsonaporcineintestinalepithelialcellline (IPEC-1).Weﬁrstobservedthatadoseof5mMphyticaciddecreasescellviabilityandtransepithelialelectrical resistance(TEER)ofcellmonolayer.Wenextinvestigatetheeffectofnon-cytotoxicdoseofphyticacidon thedeoxinivalenol(DON)induceddecreasedTEER.Weshowedthattreatmentwith0.5mMor1.0mM phyticacidrestoresthedecreaseinTEERcausedby25␮MDON.Inconclusionthisstudydemonstrates thatphyticaciddecreasedthenegativeeffectsofdeoxynivalenolonthemembraneintegrityoftheIPEC-1 intestinalepithelialcellline.

1. Introduction

Phyticacidisahighlyphosphorylatedmoleculepresentincereal

grainsandseeds.Itservesasthemajorformofstorageof

phos-phorusin theseedaswellas beinganatural antioxidant(Graf

&Eaton,1993).Itisfoundinmostmammaliancells, whereinit

isimportantin regulatingvitalcellularfunctionssuchassignal

transduction,cellproliferation anddifferentiation(Szwergoldet

al.,1987;Shamsuddinetal.,1997).

Mycotoxinsaretoxicsecondarymetabolitesproducedbyfungi

thatmaycontaminateanimalandhumanfoodatallstagesofthe

foodchain(Oswaldetal.,2005).DONisatypeof␤-trichothecene

thatismainlyproducedbyFusariumgraminearumandFusarium

culmorum,whicharefoundnaturallyworldwide,andtheir

cyto-toxicityhasbeendemonstrated inanimalsandvariouskindsof

cells(Zhangetal.,2009).ItisknownthatDONdisruptsthe

func-tionsofcellularmembranes(Bunner&Morris,1988)andalters

theintercellularcommunication(Kheraetal.,1982).Adecreasein

trans-epithelialelectricalresistancewasobservedbyPintonetal.

(2009)andVanDeWalleetal.(2010).

Infact,studieshavedemonstratedthattrichothecenesstimulate

lipidperoxidation,howeverthedietaryuseofantioxidantscan

pro-∗ Correspondingauthorat:UniversidadeEstadualdeLondrina,CentrodeCiências Agrárias,DepartamentodeMedicinaVeterináriaPreventiva,CampusUniversitário, Londrina,CXPostal6001,Brazil.Tel.:+554333714062;fax:+554333714714.

E-mail addresses: anauel02@yahoo.com.br, ana.bracarense@pq.cnpq.br (A.P.F.R.L.Bracarense).

videprotectionagainstcelldamage.Inthisstudy,weusedaporcine

intestinalepithelialcelllinetodeterminetheeffectsofphyticacid

andDONonintestinalepithelialintegrity.

2. Materialsandmethods

2.1. Cellcultureandreagents

TheIntestinalPorcineEpithelialCells(IPEC-1)celllineisa

new-bornswineintestinalepithelialcelllinethatwasderivedfromthe

smallintestineofanewbornunsuckledpiglet.Thecelllineswere

maintainedbyserialpassagesaspreviouslydescribed(Bouhetet

al.,2004).PuriﬁedDON(Sigma,StQuentinFallavier,France)was

dissolved in DMSOand stored at −20◦_C _before_dilution _in _cell

culture media.Inositol hexaphosphate(phytic acidsodium salt

hydratepurchasedfromSigma–AldrichCo.)wasdissolvedin

ster-ilizedwater(0.4g/mL)andstoredat−20◦_C_before_dilution_in_cell

culturemedia.

2.2. MTSbioassay

The effects of phytic acid on the viability of IPEC-1 cells

were studied in a colorimetric assay using the CellTiter 96®

Aqueous Non-Radioactive Cell Proliferation Assay Kit (Promega

Charbonnières, France). IPEC-1 cells were seeded at a

con-centration of 5×103_cells/well _in ₁₀₀_␮L _of _adequate _medium

in ﬂat-bottomed 96-well plates (Polylabo-Nunc). After

cul-turing for 24h, various concentrations of phytic acid (0;

(3)

Author's personal copy

346 G.D.Pachecoetal./ExperimentalandToxicologicPathology64 (2012) 345–347

0.5; 1.0; 2.5 and 5.0mM) were prepared, pH-adjusted with

NaOH to 7.0 and sterilized by passing through a 0.22-␮m

membrane ﬁlter. Subsequently, the solutions were added to

the cells. After 48h, 20␮L of a freshly prepared MTS/PMS

[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium/phenazine methosulfate] solution

was added to the wells, and the cells were further incubated

for2–4h. Theamountofsolubleformazanproduced bycellular

reductionoftheMTSwasmeasuredbytheabsorbanceat492nm

onanELISAplatereader(Spectrathermo,Tecan,Trappes,France).

2.3. Measurementoftransepithelialelectricalresistance(TEER)

DifferentiatedIPEC-1cellswerethentreatedwith0;0.5,1.0and

5.0mMofphyticacid.Afterfurtherculturingfor24h,theTEERwas

measured.Aftermeasuring,thecellsweretreatedwith25␮Mof

DON.After24hofculture,theTEERwasmeasuredagain.

Experi-mentalTEERvalueswereexpressedaskOhms× cm2_.

2.4. Statisticalanalysis

Data wereanalyzedwithStatviewsoftware,version5.0(SAS

InstituteInc.,Cary,NC),usingANOVAandPLSDFishertest.P

val-ues<0.05wereconsideredsigniﬁcant.

3. Results

3.1. PhyticaciddecreasesthecellviabilityandTEERina

dose-dependentmanner

Theeffectofphyticacidoncellviabilitywasinvestigatedvia

MTSassay.Theconversionoftetrazoliumsalttocoloredformazan

bysuccinatedehydrogenase,themainmitochondrialenzyme,is

proportionaltothenumberoflivingcells.Inthepresentstudy,we

searchedforanon-cytotoxicdoseofphyticacid.Theresults

pre-sentedinFig.1demonstratethatphyticaciddecreasedtheviability

oftheIPEC-1celllineinadose-dependentmanner.Asigniﬁcant

decreaseincell viabilitywasnotedat aconcentrationof 5mM

phyticacid.Theeffectofphyticacidtreatmentwasalsodetermined

ondifferentiatedcells.IPEC-1cellswereseedingintranswell

sys-temandallowtodifferentiatefor14daysbeforephyticacidwas

addedontheapicalpartofthemonolayer.ChangesinTEERare

indicativeofalterationsinepithelialbarrierfunctionorinthe

tran-Fig.1. EffectsofphyticacidoncellviabilityofIPEC-1celllineasmeasuredbythe MTSassay.IPEC-1cellswereseededatlowdensityin96-multiwellplates,allowed toadhere,andthentreatedfor48hwithincreasingconcentrationsofphyticacid. TheamountofsolubleformazanproducedbycellularreductionoftheMTSwas thenmeasuredbyabsorbanceat492nm.Valuesareexpressedasthepercentageof themaximalabsorbanceobservedinuntreatedcultures,mean±SDofseven inde-pendentwells.Statisticalsigniﬁcancebetweentreatedcultureandcontrolisshown with*_p_<_0.05.

Fig.2. Effectofphyticacidontrans-epithelialelectricalresistance(TEER)in polar-izedintestinalepithelialcellmonolayers.IPEC-1cellsweregrownanddifferentiated oninsertsthenatday0concentrationsofphyticacid(0.5,1.0and5.0mM)were addedintheapicalcompartiment.After24htheTEERwasmeasured.Thevalues areexpressedinkohms×cm2_as_the_mean_±_SD_of₁₀_independent_experiments. ***_p_<_0.001.

scellularpermeabilityofions.After24hoftreatment(Fig.2),we

observedthattheTEERvaluesinthecontrolandthetreatments

with0.5mMand1mMphyticaciddidnotdiffer.Ontheotherhand,

IPEC-1cellstreatedwith5mMofphyticacidexhibiteda

signiﬁ-cantly(P<0.001)lowerTEERvaluewhencomparedwiththeother

treatments.Thisconﬁrmsthatthephyticacidatthis

concentra-tionaffectedtheintegrityofintestinalepithelialcellmonolayers,a

ﬁndingalsoobservedinthecellviabilitytest.

3.2. PhyticacidrestoresthenegativeeffectsofDONonthe

membraneintegrityofIPEC-1

PreviousstudiesdemonstratedthattheIPEC-1cellstreatedwith

DON(25␮M)for24hexhibitedasigniﬁcantdecreaseintheTEER

values(Pintonetal.,2009;VanDeWalleetal.,2010).The

pretreat-mentsofcellmonolayerfor24hwith0.5mMor1.0mMofphytic

acidwereabletopartiallyrestore(P<0.001)thedecreasedTEER

valuesinducedbyDON(Fig.3).Thisresultindicatesthatphytic

acidmaypreventthetoxiceffectinducedbyDON.

Fig.3. ProtectiveeffectofphyticacidonDON-induceddecreasedtrans-epithelial electricalresistance(TEER)inpolarizedintestinalepithelialcellmonolayers.IPEC-1 cellsweregrownanddifferentiatedoninsertsthenatday0variousconcentrations ofphyticacid(0.5and1.0mM)wereaddedintheapicalcompartiment.After24h, 25␮MofDONwasaddedintheapicalcompartimentandtheTEERwasmeasured againafterothers24h.Thevaluesareexpressedinkohms× cm2_as_the_mean_±_SD

(4)

Author's personal copy

G.D.Pachecoetal./ExperimentalandToxicologicPathology64 (2012) 345–347 347

4. Discussion

Animportantfunctionofgastrointestinalepitheliais to

pro-videabarrieragainstthepenetrationoffoodcontaminantsand

pathogenspresent intheintestinal lumen(Arrietaet al.,1996;

Oswald,2006).Severalcomponentspresentinplantsarecapableof

interferingwithbothbacterialecologyandtheimmuneresponse

oftheguttofoodantigens.Beneﬁcialnonnutrients,suchas

antioxi-dants,occurinfoodsofplantorigin(Weglarzetal.,2007).Thus,the

intakeoffoodshighinphyticacidmayindirectlyinﬂuencehealth.

Inthepresentstudy,phyticaciddecreasedtheviabilityofthe

IPEC-1celllineinadose-dependentmanner.Asigniﬁcantdecreaseincell

viabilitywasnoticedataconcentrationof5mMphyticacid.This

resultagreeswithWeglarzetal.(2007),whoevaluatedtheeffect

ofphyticacidonthegrowthofCaco-2cells.

Inordertoevaluatetheeffectofanon-cytotoxicdoseofphytic

acidonDONexposedcells,weusedaconcentrationof25␮MDON.

WefoundthatDONdecreasedtheTEERvalues,whichisagood

indicatoroftheepitheliumintegrity.Similarresultwasobserved

byPintonetal.(2009)whousedvariousconcentrationsofDON

rangingbetween5.0and50␮Minapolarizedintestinalepithelial

cellmonolayer.TheprotectiveeffectofIP6wassuggestedbasedon

itsantioxidanteffectanditsabilitytoaltercellsignalingpathways

orantioxidantenzymesthatdetoxifythereactiveoxygenspecies

(ROS)(Shamsuddinetal.,1997).Phyticacidanditshydrolysis

prod-ucts(IP5,IP4,IP3,IP2 eIP1)arestillabletoprotectthebiological

membranesagainstlipidperoxidation(Miyamotoetal.,2000).

Inthepresentstudy,phyticacidataconcentrationof0.5mMor

1.0mMprotectedthemembranesoftheIPEC-1intestinalepithelial

celllineagainstcelldamageinducedbythemycotoxinDON.The

administrationofproductsrichinphyticacidinfoodorfeed,such

asco-productsderivedfrommaizeandrice,mayactendogenously,

protectingcellularsystemsagainsttheeffectscausedbysubstances

thatinducecellulardamages,suchasmycotoxins,reducingthe

negativeeffectscausedbytoxins.Thus,lossesinanimalproduction

andhealthproblemswilldecreaseconsiderably.However,further

studiesareneededtoconﬁrmtheeffectsofcellularoxidativestress

producedbyDONintheintestinalIPEC-1cellsandhowphyticacid

caneffectivelyminimizetheseeffects.

Inconclusion,ourresultsindicatethatphyticacidaffectscell

viabilityataconcentrationof5mM,but0.5mMand1mMphytic

aciddecreasedthenegativeeffectsofdeoxynivalenolonthe

mem-braneintegrityoftheIPEC-1intestinalepithelialcellline.

Acknowledgement

This study was supported in part by Grant 593/08 from

CAPES/COFECUB,Brazil/France.

References

ArrietaMC,BistritzL,Meddings JB.Alterationsinintestinalpermeability.Gut 1996;55:1512–20.

BouhetS,HourcadeE,LoiseauN,FikryA,MartinezS,RoselliM,GaltierP,MengueriE, OswaldIP.ThemycotoxinfumonisinB1alterstheproliferationandthebarrier functionofporcineintestinalepithelialcells.ToxicolSci2004;77:165–71. Bunner DL, MorrisER.Alterationof multiplecellmembrane functionsinL-6

myoblastsbyT-2toxin:animportantmechanismofaction.ToxicolApplPharm 1988;92:113–21.

GrafE,EatonJW.Suppressionofcoloniccancerbydietaryphyticacid.NutrCancer 1993;19:11–9.

KheraKS,WhalenC,AngersG,VesonderRF,Kuiper-GoodmanT.Embryotoxicityof 4-deoxynivalenol(vomitoxin)inmice.BEnvironContamTox1982;29:487–549. MiyamotoS,KuwataG,ImaiM.Protectiveeffectofphyticacidhydrolisis prod-ucts on iron-induced lipid peroxidation of liposomal membranes. Lipids 2000;35:1411–4.

OswaldIP,MarinDE,BouhetS,PintonP,TaranuI,AccensiF.Immunotoxicological riskofmycotoxinsfordomesticanimals.FoodAdditContam2005;22:354–60. OswaldIP.Roleofintestinalepithelialcellsintheinnateimmunedefenceofthepig

intestine.VetRes2006;37:359–68.

PintonP,NougayrèdeJP,DelRioJC,MorenoC,MarinDE,FerrierL,Bracarense AP,Kolf-ClauwM,OswaldIP.Thefoodcontaminantdeoxynivalenol,decreases intestinalbarrierpermeabilityandreducesclaudinexpression.ToxicolAppl Pharm2009;237:41–8.

ShamsuddinAM,VucenikI,ColeK.Minireview-IP6:anovelanticanceragent.Life Sci1997;61:343–54.

Szwergold BS, Graham RA, Brown TR. Observation of inositol pentakis- and hexakis-phosphatesinmammaliantissuesby31PNMR.BiochemBiophResCo 1987;264:874–81.

VanDe WalleJ,Sergent T,PirontN,ToussaintO, SchneiderYJ,LarondelleY. Deoxynivalenolaffectsinvitrointestinalepithelialcellbarrierintegritythrough inhibitionofproteinsynthesis.ToxicolApplPharm2010;245:291–8. WeglarzL,Wawszczykj,OrchelA.PhyticacidmodulatesinvitroIL-8andrelease

from colonicepithelialcell stimulatedwith LPSandIL-1␤. DigestDisSci 2007;52:93–102.

ZhangX,LipingJ,ChengyanG,etal.Theroleofoxidativestressin deoxynivalenol-inducedDNAdamageinHepG2cells.Toxicon2009;54:513–8.