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Which exposure stage (gestation or lactation) is more vulnerable to atrazine toxicity? Studies on mouse dams and their pups

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ToxicologyReports1(2014)53–68

ContentslistsavailableatScienceDirect

Toxicology

Reports

j o u r n al ho me 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

Which

exposure

stage

(gestation

or

lactation)

is

more

vulnerable

to

atrazine

toxicity?

Studies

on

mouse

dams

and

their

pups

Sameeh

A.

Mansour

a,∗

,

Doha

A.

Mohamed

b

,

Jean

F.

Sutra

c

aEnvironmentalToxicologyResearchUnit(ETRU),PesticideChemistryDepartment,NationalResearchCentre,Dokki,Cairo,Egypt bFoodScience&NutritionDepartment,NationalResearchCentre,Dokki,Cairo,Egypt

cTOXALIM(ResearchCentreinFoodToxicology),UMR1331INRA/INP/UPS,EquipeTMR,BP93173,180chemindeTournefeuille,31027 ToulouseCedex3,France

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received11March2014

Receivedinrevisedform14April2014 Accepted14April2014

Availableonline2May2014 Keywords: Atrazine VitaminE Mice Oxidativestress Gestation Lactation

a

b

s

t

r

a

c

t

Eitherduringgestationorlactation,theexperimentalmousedamsreceivedoneofthe followingtreatments:(a)dietfreeofpesticide;(b)dietenriched withatrazine(ATZ); 31.0␮gkg−1;(c)dietfreeofpesticide+oral vitaminE(␣-tocopherol; 200mgkg−1 per

mouse);and(d)dietenrichedwithATZ(31.0␮gkg−1)+oralvitaminE(200mgkg−1per mouse).Attheweaning,pupsanddamswerekilledandselectedorgansandbloodsamples werecollectedforanalyses.Comparedwiththecontrolresults,ATZinducedalterationin anumberofbiochemicalandhistopathologicalparameterseitherinthedamsortheir off-spring.TheameliorativeeffectofvitaminE,basedonestimatingthe“AmeliorativeIndex; AI”tomalondialdehyde(MDA)andsuperoxidedismutase(SOD)rangedbetween0.95and 1.06(≈1.0)forthedamsandthepupseitheringestationalorlactationalexposureroutes. Ingeneral,themousepupsweremorevulnerabletoATZtoxicitythantheirmothersand exposureduringgestationwassuggestedtobemoreeffectivethanduringlactation.The findingsmaysupporttheneedtofurtherinvestigatingtheadverseeffectsofexposureto lowdosesofcommonlyusedpesticides,especiallyduringpregnancyandbreast-feedingas wellaseffectsonnewbornchild.

©2014PublishedbyElsevierIrelandLtd.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction

Atrazine, ATZ (6-chloro-N-ethyl-N-isopropyl-1,3,5-triazinediyl-2,4-diamine; IUPAC) is a chloro-S-triazine compound usedasa selectivepre-emergence and post-emergenceherbicideforthecontrolofweedsinavariety ofagriculturalcrops,aswellasinforestryandfor non-selectiveweedcontrolonnon-cropareas[1].In1991,the US-EPAestablishedaMaximumContaminantLevel(MCL)

∗ Correspondingauthor.Tel.:+20233371211;fax:+20233370931. E-mailaddress:samansour@hotmail.com(S.A.Mansour).

of3.0␮g/lforATZindrinkingwater.Themostsignificant regulatory decision in recent years occurred in 2003, aftertheEU,US,andothernationsdecidedtore-examine thesafetyofATZinlightofnewevidence.Atthattime, researchhad expandedbeyondtoxicity studiesandwas beginningtoinvestigatelong-term,low-doseexposuresin animals,particularlyfemaleandfetalanimals[2].

Some European countries have included ATZ in the listof pesticideresiduestobecontrolledbecauseitisa potentialcontaminantduetoitschemicalcharacteristics, includinglipophilicity,slowhydrolysis,andmoderateto lowwatersolubility,andhighsolubilityinorganicsolvents withhighabsorptionbyorganicmatter,clayandfattissues http://dx.doi.org/10.1016/j.toxrep.2014.04.001

2214-7500/© 2014 Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

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[3].Duetoinabilitytokeepwater contaminationbelow 0.1ppb,whichisauniformlimitfortheresidueofany pes-ticideindrinkingandgroundwaterintheEuropeanUnion (EU),theEUregulatorsannouncedthebanofATZuse. How-ever,intherestoftheworldtheuseofATZcontinueswith few,ifany,restrictions,anditiseventhemostfrequently detectedpesticideinground-andsurfacewateroftheUSA [4].

PossibleexposuretoATZcanbeassumed duetothe applicationtoagriculturallandandcontaminationof sur-faceandgroundwater[5].ATZisreadilyabsorbedthrough gastrointestinaltract[6].Occupationalexposuremayoccur through both inhalation and dermal absorption during its manufacture, its formulation and its application by spraying.Itisfoundwidely,togetherwithitsdealkylated degradationproducts,inrivers,lakes,estuaries, ground-waterandreservoirs.Indrinkingwater,thelevelsrarely exceed1␮g/l.Surveysofvariousfoodsandfeedshave gen-erallyindicatednodetectableATZresidue.AlthoughATZ generallyhaslowlevelofbioaccumulationinfish,itdoes accumulateinbrain,gallbladder,liverandgutof some fishes[7].Therefore,consumptionofcontaminatedfishcan alsocontributetohumanexposure.TheresiduesofATZ werefoundinthefarmers’bloodandurine[8].

ThemajorsiteforATZdetoxificationisliver,whereit isundergoingbiotransformationthroughoutphaseIand phaseIIreactions.Inrodentspecies,twocytochromeP450 (CYP)enzymes(CYP1A&CYP2B)aredistinctivelyinvolved inATZbiotransformation[9].ATZhasalotofadverseeffect onhealthsuchastumors,breast,ovarian,anduterine can-cersaswellasleukemiaandlymphoma.Itisanendocrine disruptingchemical interrupting regularhormone func-tionandaltersreproductivefunctionnotonlyinhuman [10],butalsoin manyotherspeciessuchasdeveloping alligators[11],birds[12],goat[13],andasmost vulnera-bleamphibians[14,15]andfish[16],causingbirthdefects, reproductivetumors,andweightlossinamphibiansaswell ashumans.Italsocausesinductionofthedetoxifying hep-aticmicrosomaloxidativeenzymes,continualsynthesisof esterases,physiologicaladaptationtodecreasedesterase levels,andadaptationofcholinergicreceptors[17].Thus, interfering withthebiochemical pathways mechanisms includingreproductivefunctionsasawholeandalsogene expressionchanges,ATZinevitablyaffectsbiodiversityand causesenvironmentalhavoc[18].

Withregardtofetalandchildhoodexposures,theUS DepartmentofHealthandHumanServicesarrivedata con-clusionthatitisnotknownwhetherATZoritsmetabolites canbetransferredfromapregnantmothertoa develop-ingfetusthroughtheplacentaorfromanursingmother toheroffspringthroughbreastmilk[19].Intheirarticle, PathakandDikshit[17]reportedthesameATSDR’s aware-ness.However,Stokeretal.[20]foundthat3hfollowing administrationof14C-ATZtotheWistarrat,therewasa distributionof14C-chlorotriazines(14C-ClTRI)[refertoall residuesoftheinitialdoseof14Catrazine],totheorgans ofthedam,withthehighestamountsintheliverand kid-ney(1.1and0.3%oftheadministereddose,respectively). Recently,Fraitesetal.[21]quantifiedthedistributionof ATZanditschlorinatedmetabolitesinmaternal,fetal,and neonatal fluid and tissue samples following gestational

and/or lactationalexposureof SpragueDawley damsto ATZatdifferentdoses.Dose-dependentlevelsof chloro-triazines, primarily diaminochlorotriazine (DACT), were present in most samples analyzed, including fetal tis-sue.

Thereisgrowingevidencethatexposuretoxenobiotics (eitherpharmaceuticalsorenvironmentalchemicals) dur-inggestationmayresultinavarietyofadverseoutcomes when the developing organismreaches adulthood.Oral exposuretoATZisknowntoalterendocrine and repro-ductivefunctioninadultandperipubertalrodentsofboth sexes[22–25],butfewstudieshaveinvestigatedtheeffects ofATZexposureinuteroorduringtheperinatalperiod. A numberofstudieshave shownthatATZ canpromote oxidativestress,byincreasingtheconcentrationofreactive oxygenspecies(ROS) andproductsofoxidativedamage suchaslipidperoxides,andthereforeinfluencingthe activ-ityofantioxidantenzymes(AOE)[6,26–28].Liveristhefirst targetofingestedoxidantsandalsoveryimportanttissuein defenseagainstoxidativestress.Testesalsopossessproper antioxidantdefense,andchangesintheactivityof antiox-idantenzymeshavebeenrecordedinxenobiotic-exposed animals[29].

Numerous foodsprovide vitamin E.Nuts, seeds, and vegetable oils are among the best sources of alpha-tocopherol,andsignificantamountsareavailableingreen leafy vegetablesand fortified cereals [30]. Vitamin E is thecollectivenameforagroupoffat-solublecompounds withdistinctiveantioxidantactivities[31].Naturally occur-ring vitamin E exists in eight chemical forms (alpha-, beta-, gamma-, and delta-tocopherol and alpha-, beta-, gamma-,and delta-tocotrienol) thathave varyinglevels of biological activity [31]. Alpha- (or ␣-) tocopherol is theonlyformthatisrecognizedtomeethuman require-ments. The research hasnot foundany adverse effects fromconsumingvitaminEinfood.However,highdoses of␣-tocopherolsupplementscancausehemorrhageand interruptbloodcoagulationinanimals,andinvitrodata suggestthathighdosesinhibitplateletaggregation[32]. VitaminEisafat-solubleantioxidantthatstopsthe pro-ductionofreactiveoxygenspecies(ROS)formedwhenfat undergoesoxidation.Scientistsareinvestigatingwhether, bylimitingfree-radicalproductionandpossiblythrough othermechanisms,vitaminEmighthelppreventordelay thechronicdiseasesassociatedwithfreeradicals.In addi-tiontoitsactivitiesasanantioxidant,vitaminEisinvolved in immunefunctionand,asshown primarilybyinvitro studiesofcells,cellsignaling,regulationofgeneexpression, andothermetabolicprocesses[31].VitaminEisan impor-tantbiologicalfreeradicalscavengerinthecellmembranes [33].AdministrationofvitaminEat100mgkg−1bwto Wis-tarratstreatedwithATZ(300mgkg−1bw)amelioratedthe effectsofATZsuggestingitaspotentialantioxidantagainst ATZ-inducedoxidativestress[6].

Most studies onATZ toxicity to rodentsduring ges-tation/lactationwere carriedout ondoses much higher thanthedosecharacterizedasthe“acceptabledailyintake, ADI”(0.005mgkg−1foodperday).Examplesforthedoses used in this respectwere1, 5, 20and 100mgkg−1/day [20,21,34,35];100mgkg−1/day[36];50–100mgkg−1/day [37];and50–200mgkg−1/day[18].

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S.A.Mansouretal./ToxicologyReports1(2014)53–68 55 Women,duringpregnancyandlactation,mayexposeto

lowdosesofATZfromdifferentenvironmentalsources.In thesameprotocolofthepresentstudywerecently inves-tigatedtheeffectofexposureofmousedamstoATZduring theoverallperiodofgestationandlactation,andthe reflec-tion ofsuchexposureonthe offspring. Itwas foundof interest,inthepresentstudy,toelucidateandcompareATZ toxicitytoeachexposurestage,andtoassessthe amelio-rativeeffectofvitaminEsupplementation.Inthecourse ofthepresentstudy,wewillbaseourinvestigationonthe ADIofATZ,whichmaybethelowestdosetobetestedon pregnantandlactatingmousedams.

2. Materialsandmethods

2.1. Reagents

HighpurityPESTANAL®AnalyticalStandardofAtrazine (97.4%)waspurchasedfromFluka(Riedel-deHaën,France) and addedas acomponent ofrodent nuggetsat adose of25␮g/kgoffood.Thisdoseallowedthemicetoingest theequivalentof0.005mgkg−1foodperday(ADI)defined for humans by Food& Agricultural Organization/World HealthOrganization[38]thatwasextrapolatedformice on thebasis of mean body weight. Information onthe toxicity ofATZwasobtainedfromExToxNet(ExToxNet: http://extoxnet.orst.edu/).

d-l ␣-tocopherol acetate was purchased from Fluka (Riedel-deHaën,France).Thevitaminwasdissolvedincorn oil(120mg/3mL).Eachmouse(withameanbodyweight of nearlyequal to20g)received100␮lbyoral gavages twiceaweekonthefirstandfourthday.Thisdoseequals 200mgkg/mousetwiceaweekbasedonthedosage fol-lowedbyKalenderetal.[39].

2.2. Preparationofpesticideenrichedfeed

ATZ (0.125mg) wasdissolvedin 1ml methanol. The resultingsolutionwasmixedwithacetone(1/9;v/v)and dispersedon10gofthevitaminmixturepowder(vitaminic mixture 200, Scientific Animal Food Engineering, Safe, France).Thevitaminpowdercontaining125␮gATZwas thenhomogenizedinarotavapor(Laborota4000Fischer Bioblock,France)for30minat45◦Ctoevaporatethe sol-ventandthenmixedfor50minatroomtemperaturewith theremainingquantityofvitaminmixture(40g)needed tomake therodentnuggets (5kg). Thevitamin powder enrichedwithATZwasthensenttotheNationalInstituteof AgriculturalResearch(INRA)animalfeedpreparationunit (UPAEINRA,Paris,France)wherevitamins(1%)andmineral components(7%)weremixedwiththeothercomponents oftherodentnuggets.ThepresenceoftheATZwas quan-tifiedbyEurofins(Nantes,France).Thefinalquantitywas aroundthatweexpected(i.e.,0.031mgkg−1;LC/MS/MS). Thecontrolfeedwaspreparedasdescribedabovewitha mixtureofmethanol/acetone(1/9,v/v)butlacking pesti-cide.Itwasanalyzedforthepresenceoftheendogenous pesticide (ATZ) and also for the presence of the most commonpesticidesfoundintheenvironment (including theorganophosphorus,OCandpyrethroidpesticides,and polychlorobiphenyls).Theresultsshowedthatnuggetsin

controlfooddo notcontainpesticides atthedetectable level(0.01mgkg−1).

2.3. Animals

Ten-week-oldfemaleand male C57BL/6J micewere purchasedfromCharlesRiverLaboratories(Domainedes Oncins-BP 109, 69592 L’arbresle, Cedex, France). Mean bodyweightswere20±2gforfemalesandmales.The ani-malswereacclimatizedfor2weeksbeforestartingdosing. Thirty-two(32)virginfemalemiceweredistributedinto 16cages.Ineachcage,onemalewasplacedovernightand thepresenceofspermatozoawascheckedinthevaginal smearinthefollowingmorning.Thisdaywasconnotedas gestationday0(GD0).Atthattime,pregnantfemaleswere dividedinto2majorgroups,eachof16animals, individu-allyhousedincleanplasticcagesinthelaboratoryanimal room(23±2◦C;40%RH),fedontheprepareddietandtap waterwasallowedadlibitum.Oneofthetwomajorgroups wasspecifiedasa“GestationGroup(G)”,whiletheothera “LactationGroup(L).TheGgroupreceivedthetested treat-mentsduringgestationonly(20days),whiletheLgroup wastreatedduringlactationonly(20days).Thedayof par-turitionwasconsideredday0oflactation,postnatalday0 (PND0).Theoffspringofeachlitterwerecounted,sexed andeachlitterwasrandomlyreducedto6pupsofequal numberofsexestomaximizethelactationperformance [40].Theexperimentalworkonanimalswasperformedin ToxalimUnit,INRA,Toulouse(France),andinaccordanceto itsinstitutionalethicalcommitteesinanaccreditedanimal house.

2.4. Experimentaldesign

Duringgestationperiod,theexperimentalmousedams (D)receivedoneofthefollowingtreatments:(a)dietfree of pesticide; control (CD); (b) diet enriched with ATZ; 31.0␮gkg−1 (AD); (c) diet free of pesticide+oral vita-minE(␣-tocopherol);200mg/kg−1permouse(VD);and (d)dietenrichedwithATZ;31.0␮gkg−1+oralvitaminE; 200mgkg−1permouse(AVD).ThedoseofvitaminEwas dividedinto2equalportionsandgiventwiceaweek.Pups oftheexperimentaldamsweredesignatedasCP,AP,VP andAVP,tosigncontrol,ATZ,vitEandATZ+vitEtreated groups,respectively.

Similarly,thegroupoflactation(L)constitutedoffour subgroupsofmousedamswiththeirpupsandreceivedthe sametreatments,mentionedabove,duringlactationperiod only.

Attheendofweaning,bloodsamplesweretakenfrom thefacialarteryofeachanimal(damsandpups)andadded to heparinized centrifuge tubes (Multivette®, SARSTED, Germany)forseparatingplasma.Centrifugationwas per-formedonSigma1K15BioblockScientific(Subra,France) for 10minat 4◦C and 4000rpm (1400×g). Theplasma werekeptineppendorftubesandstoredat(−80◦C).Then theanimalsweresacrificedbycervicaldislocation,andthe heart,spleen,liver,kidneys,testesorovarieswereremoved andweighted.Smallpiecesoforganswerekeptin10% for-malinforhistopathologicalstudies.Otherpiecesofliver werepackedin aluminium sheets and keptin nitrogen

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(−80◦C)forcertainbiochemicalestimations.The fraction-atedbloodsamplesandspecimensofinternalorganswere shippedfrozen, in additiontoformaldehyde – reserved specimens,totheNationalResearchCentre(NRC),Cairo, Egyptforbiochemicalanalysesandhistopathological stud-ies.

2.5. Biochemicalanalyses

Theseincluded butyryl cholinesterase (BuChE), alka-linephosphatase(ALP),urea,Malondialdehyde(MDA)and super oxidedismutase (SOD). Measurements were per-formedspectrophotometricallyusingaShimadzuUV-VIS Recording2401PC(Japan), and in accordance withthe manufacturer’sinstructionsgivenin thepamphlets.The activityofplasmacholinesterase(BuChE;EC3.1.1.8)was measuredat405nmandexpressedinunitsperliterusing themethodofKnedelandBöttger[41].ALP(EC3.1.1)was measuredinplasmaat510nmin termsofinternational units per liter according to Belfield and Goldberg [42]. Concentrationofurea(milligramperdeciliter)was deter-minedinplasmaat550nmusingthemethodofFawcett andScott[43].MDAandSODactivitiesweredetermined inlivertissues,basedonmethodspublishedbySatoh[44] andNishikimietal.[45],respectively.Spectrophotometric measurementswerecarriedoutat534nmforlipid perox-ide,intermsofMDA,andexpressedinnanomolepergram tissue.SODmeasurementsat560nmwerecalculatedin termsofunitspergramlivertissue.

2.6. Histologystudies

Liver,kidneys,ovariesandtestesfromthe experimen-talmiceweredissectedandfixedin10%neutralformalin, dehydrated in ascending grades of alcohol and imbed-dedinparaffinwax.Paraffinsections(5mmthick)were stainedforroutinehistologicalstudyusinghaematoxylin andeosin(H&E).Twoslideswerepreparedforeachmouse; eachslidecontainedtwosectionsforeachorgan.Tenfield areas for each section were selected and examinedfor histopathologicalchangesunderlightmicroscope(400× magnifications). The histopathology was carried out in thePathologyDepartment,FacultyofVeterinaryMedicine, CairoUniversity,Cairo,Egypt.Tissueinjuryintheexamined organswasscoredindifferentratingsaccordingtoÖnder etal.[46].

2.7. Statisticalanalysis

The data were analyzed by using GraphPad Prism 5 Demo (www.graphpad.com/downloads/docs/ Prism5Regression.pdf), and expressed as means±S.E. Pairedsamples(t)testwasusedtocomparethedataof thecontrolwiththoseoftreatments,whereP<0.05and P<0.01wereconsideredforsignificantandhighsignificant differences,respectively.WhenP>0.05,valueswerenot significantlydifferentfromoneanother.

3. Results

3.1. Signsoftoxicityandobservations

Allanimalswerecarefully observedduringgestation andlactationperiods.Generally,theexperimentalanimals showednormal behaviorexceptsignsofrestless onthe damsduringgestationperiod.Thenumberofpupspereach femalerangedbetween6and8ofnearly1:1sexratio. 3.2. Bodyandrelativeorgansweights

The initialstartingbody weight ofmouse damswas approximately20g/animal.IntheGexperimentalgroup, themeanbodyweightofcontrolmothers(CD),attheendof experimentalperiod,recorded29.70gwhichdidnotdiffer significantlythanbodyweightsoftheothertested treat-ments.ThebodyweightofmalepupsinATZtreatment(AP; 11.84g)andfemalepupsinvitamintreatment(VP;11.48g) wassignificantly(P<0.05)lower thanthatofthe corre-spondingcontrolvalues(Table1).Generally,theinternal organsweights relativetobody weightsshowedno sig-nificantdifferencesbetweenthetestedtreatments,except forliverandheartofATZ-treateddamswhichshowed val-uessignificantlydifferedthanthoseofthecorresponding controlvalues.Also,significantdifferencewasrecordedfor therelativeweightofkidneysfromATZ-femalepups(1.47% comparedto1.296%forthecontroltreatment;Table1).

Inthelactationexperiment,L(Table2),themeanbody weightofcontrolmothers(CD)ormaleandfemalepups (CP) didnot differsignificantly among theother tested treatments. The relative weight of liver from ATZ (AP; 6.342%) or ATZ+vitamin (AVP; 6.982%) – female pups showedsignificant(P<0.05)andhighsignificant(P<0.01) elevationscompared tocontrolvalue(5.256%).The rela-tiveweightofheartfrommaleandfemalepupsexposed toATZ+vitamintreatmentshowedsignificantdecreases, whileovariesfromfemalepupsexposedtoATZtreatment (AP;0.748%)showedhighsignificantelevationcompared tothecorrespondingcontrolvalue(CP;0.080%).

The relative weights of liver, heart, spleen and ovary/testesintheGexperimentalgroup,aswellas kid-neys, spleenandtestes intheLexperimentalgroupdid notshowsignificantdifferencesamongthepupsofthese treatments(Tables1and2).

3.3. Biochemicalanalyses

Due to the little amount of blood obtainable from thetestedmouse,theestimatedbiomarkerswerecarried outonalkalinephosphatase(ALP),butyrylcholinesterase (BuChE)andureainplasma,aswellasmalondialdehyde (MDA)andsuperoxidedismutase(SOD)inlivertissues.The resultsoftheseparametersarerepresentedbycolumn his-togramsdesignatedbyasterisksforthevaluesofsignificant differencesthanthecontrolvalues.

ControlvaluesofALPrecorded75.3,56.0and56.3IU/l forthemousedams(CD),malepupsCP(M)andfemale pupsCP(F),respectively,ingestationalexposure experi-ment(Fig.1a).ThevaluesrecordedforATZ-treateddams, as wellas maleand female pupswere higher thanthe

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S.A. Mansour et al. / Toxicology Reports 1 (2014) 53–68 57 Table1

Bodyandrelativeorgansweightsofmousedamsandtheirpupsexposedtoatrazine,withandwithoutvitaminE,duringgestation. Treatmentgroups B.Wt.(g) Relativeorgansweights(%)

Liver Kidneys Heart Spleen Ovary/testes

CD 29.70±0.87 5.353±0.33 1.387±0.09 0.8733±0.003 0.3900±0.03 0.1200±0.01 AD 30.43±0.77 6.733±0.33* 1.180±0.07 0.7700±0.03* 0.3300±0.02 0.1133±0.03 VD 28.00±0.61 5.613±0.59 1.330±0.07 0.6833±0.07 0.3433±0.01 0.07667±0.02 AVD 27.70±0.47 5.953±0.60 1.370±0.08 0.7467±0.13 0.4000±0.03 0.1133±0.01 Pups M F M F M F M F M F M F CP 14.10±0.62 12.78±0.38 5.312±0.51 5.298±0.13 1.248±0.05 1.296±0.04 0.6240±0.04 0.6960±0.04 0.4820±0.03 0.5060±0.03 0.5600±0.04 0.1300±0.01 AP 11.84±0.60* 12.68±0.50 5.108±0.71 5.150±0.45 1.346±0.03 1.470±0.07* 0.6340±0.03 0.6140±0.04 0.5900±0.05 0.5960±0.07 0.5780±0.02 0.1100±0.01 VP 14.16±0.71 11.48±0.32* 4.992±0.23 4.870±0.19 1.192±0.03 1.356±0.03 0.5940±0.02 0.7100±0.09 0.4840±0.04 0.5660±0.04 0.6160±0.05 0.1000±0.02 AVP 12.44±0.83 12.74±0.81 4.956±0.71 5.016±0.39 1.262±0.07 1.386±0.10 0.6340±0.03 0.6840±0.04 0.5400±0.05 0.5160±0.05 0.5260±0.06 0.1240±0.02 Groupsymbols:C=control;D=dams;P=pups(M:maleandF:female);A=atrazine;V=vitaminE;AV=atrazine+vitaminE.

Statisticalanalysis:

Bodywt.CP(M)versusAP(M):*significantdifferenceat<0.05;CP(F)versusVP(F):*significantdifferenceat<0.05. Relativewt.ofliver:CDversusAD:*significantdifferenceat<0.05.

Relativewt.ofkidneys:CP(F)versusAP(F):*significantdifferenceat<0.05. Relativewt.ofheart:CDversusAD:*significantdifferenceat<0.05.

Table2

Bodyandrelativeorgansweightsofmousedamsandtheirpupsexposedtoatrazine,withandwithoutvitaminE,duringlactation. Treatmentgroups B.wt.(g) Relativeorgansweights(%)

Liver Kidneys Heart Spleen Ovary/testes

CD 27.63±1.13 8.303±0.84 1.470±0.05 0.8067±0.09 0.3633±0.04 0.0800±0.03 AD 29.43±0.54 9.127±0.22 1.560±0.006 0.8000±0.15 0.2767±0.01 0.1067±0.06 VD 27.20±1.51 7.440±0.90 1.377±0.06 0.7200±0.03 0.3833±0.03 0.05333±0.02 AVD 28.03±0.18 9.653±0.12 1.473±0.024 0.8233±0.14 0.2667±0.05 0.08333±0.05 Pups M F M F M F M F M F M F CP 7.00±0.58 7.88±0.68 5.542±0.36 5.256±0.40 1.436±0.17 1.486±0.13 0.8360±0.11 0.8740±0.10 0.5700±0.07 0.5840±0.05 0.5760±0.08 0.0800±0.03 AP 8.16±0.89 6.78±0.28 6.180±0.94 6.342±0.19* 1.422±0.15 1.450±0.06 0.6840±0.05 0.7200±0.03 0.5300±0.07 0.6060±0.12 0.4780±0.07 0.7480±0.05*** VP 8.30±0.41 8.16±0.25 6.352±0.34 6.198±0.14 1.498±0.08 1.524±0.02 0.7740±0.02 0.7700±0.02 0.5960±0.8 0.5420±0.06 0.4680±0.08 0.0980±0.01 AVP 6.48±0.58 6.76±0.38 5.834±1.02 6.982±0.31** 1.462±0.13 1.524±0.08 0.6200±0.05* 0.6660±0.05* 0.3040±0.04 0.3900±0.05 0.4820±0.08 0.1040±0.01 Groupsymbols:C=control;D=dams;P=pups(M:maleandF:female);A=atrazine;V=vitaminE;AV=atrazine+vitaminE.

Statisticalanalysis:

–Nosignificantdifferencesinbodyweights.

–Liver:highsignificantdifference(<0.001):CP(F)versusAVP(F).

–Heart:significantdifference(<0.05):CP(M)versusAVP(M)andCP(F)versusAVP(F). –Ovary:highsignificantdifference(<0.001):CP(F)versusAP(F).

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ALP CD AD VD AVD CP ( M) CP (F ) AP (M) AP (F)VP ( M) VP (F) AVP (M) AVP (F) 0 50 100 150 * *** *** * *** ***

Treatment Groups

Ac ti vi ty ( IU /l) [a]: CD = 75.3; CP (M) = 56.0; CP (F) = 56.3 IU/l ALP CD AD VD AVD CP(M ) CP (F) AP (M ) AP (F) VP (M ) VP (F ) AVP (M) AVP ( F) 0 50 100 150 ** ** *** *** *** *** Treatment Groups A ctiv ity ( IU /l) [b]: CD = 73.3; CP (M) = 54.7; CP (F) = 54.3 IU/l

Fig.1. ALPactivityinplasmaofmousedamsandtheirpupsexposedto

atrazine,withandwithoutvitaminE,duringgestation(a)andlactation(b)

periods.Statisticalanalysis:Nosignificantdifference;*significant

differ-enceatP<0.05;**highlysignificantdifferenceatP<0.01.Groupsymbols:

C=control; D=dams;P=pups (M:maleandF:female);A=atrazine;

V=vitaminE;AV=atrazine+vitaminE.

correspondingcontrolvalues.Thedifferenceswere

statisti-callysignificantatP<0.05incaseofdams,andwerehighly

significantatP<0.01incaseofpups.SimilarpatternofALP

elevationwasobservedonmiceofATZ+vitaminEgroups.

Inlactational exposureexperiment,controlvalues of

ALPrecorded73.3,54.7and54.3IU/lforthemousedams

(CD),malepupsCP(M)andfemalepupsCP(F),

respec-tively(Fig.1b).ThevaluesrecordedforATZ-treateddams

andtheirpupswerehighlysignificantthanthe correspond-ingcontrolvalues. Similarpatternof ALP elevationwas observedonmiceofATZ+vitaminEgroups.

It seemed that theelevation of ALP activity in ATZ-treatedgroupwashigherinlactationalthaningestational exposureroute,andthemousepupsweremuch vulner-ableto alterationin ALP activitythan theirmothers in bothcasescompared.Ontheotherhand,thevitamin treat-mentdidnotcausedetectablealterationinALPactivity, eitherinmousedamsortheiroffspring,whiletreatment ofATZ+vitaminEinducedhighelevationofALPactivityin thedamsandtheirpups(Fig.1).

In gestationalexposureexperiment (Fig. 2a),control values of urea recorded 43.0, 34.3 and 34.7mg/dl for the mouse dams (CD), male pups CP (M) and female pups CP (F), respectively. ATZ treatment induced high

Urea CD AD VD AVD CP (M ) CP (F ) AP (M ) AP (F ) VP (M ) VP (F ) AVP ( M) AVP ( F) 0 20 40 60 80 *** ** *** *** ** *** Treatment Groups C onc entr at ion (m g/ l) [a]: CD = 43.0; CP (M) = 34.3; CP (F) = 34.7 mg/dl Urea CD AD VD AVD CP(M)CP (F) AP (M ) AP (F ) VP (M)VP (F ) AVP (M) AVP (F) 0 20 40 60 80 ** ** *** *** *** *** Treatment Groups Co n ce nt ra tion (m g /l) [b]: CD = 42.0; CP (M) = 34.7; CP (F) = 35.0 mg/dl

Fig.2.Ureaconcentrationinplasmaofmousedamsandtheirpups exposedtoatrazine,withandwithoutvitaminE,duringgestation(a)and lactation(b)periods.Statisticalanalysis:Nosignificantdifference; *sig-nificantdifferenceatP<0.05;**highlysignificantdifferenceatP<0.01. Groupsymbols:C=control;D=dams;P=pups(M:maleandF:female); A=atrazine;V=vitaminE;AV=atrazine+vitaminE.

significantincrease(P<0.01)accountingto64.0,57.3and 58.7mg/dl,respectively.Similarpatternofureaelevation wasobservedonmiceofATZ+vitaminEgroups.

In lactationalexposureexperiment, controlvalues of urearecorded42.0,34.7and35.0mg/dlforthemousedams (CD),malepupsCP(M)andfemalepupsCP(F),respectively (Fig.2b).ATZtreatmentinducedhighsignificantincrease accountedto62.7,57.7and57.7mg/dl,respectively.

Theresultsobtainedindicatethatureaelevationafter ATZ treatment of mouse dams was somewhat higher after gestational exposure than after lactational expo-sure. Mouse pups exhibited a similar pattern of urea alterationsastheirmothers,bothaftergestationaland lac-tationalexposure.WhilevitaminEtreatmentdidnotcause detectablealterationinureaconcentration,eitherinmouse damsortheiroffspring,thetreatmentofATZ+vitaminE inducedhighelevationofureainthedamsandtheirpups (Fig.2).

In gestational exposure experiment, the butyryl cholinesterase(BuChE)recorded3419,1503and1548U/l, respectively for control mouse dams, male offspring and female offspring. In ATZ-treated groups, the above mentioned valuesweregenerallydeclinedandrecorded

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S.A.Mansouretal./ToxicologyReports1(2014)53–68 59 BuChE CD AD VD AVD CP (M) CP (F)AP (M)AP ( F) VP (M)VP (F ) AVP (M) AVP (F) 0 1000 2000 3000 4000 **

Treatment Groups

A cti vi ty (U /l ) [a]: CD = 3419; CP (M) = 1503; CP (F) = 1548 U/l BuChE CD AD VD AVD CP (M) CP (F)AP (M ) AP (F) VP (M)VP (F) AVP (M) AVP (F) 0 1000 2000 3000 4000 * *

Treatment Groups

A cti vi ty (U /l ) [b]: CD = 3379; CP (M) = 1487; CP (F) = 1544 U/l

Fig. 3.ActivityofBuChEin plasmaof mousedams andtheir pups exposedtoatrazine,withandwithoutvitaminE,duringgestation(a)and lactation(b)periods.Statisticalanalysis:Nosignificantdifference; *sig-nificantdifferenceatP<0.05;**highlysignificantdifferenceatP<0.01. Groupsymbols:C=control;D=dams;P=pups(M:maleandF:female); A=atrazine;V=vitaminE;AV=atrazine+vitaminE.

3305U/lindamswithhighsignificantdifferenceatP<0.01 (Fig.3a).

In lactational exposure experiment, the butyryl cholinesterase(BuChE)recorded3378,1487and1544U/l, respectively for control mouse dams, male offspring and female offspring. In ATZ-treated groups, the above mentioned valuesweregenerallydeclinedandrecorded 3298U/lindams,a valuewhichwassignificantlylower (P<0.05)thanthecorrespondingcontrolvalue(Fig.3b).

Theoffspring(maleandfemale)eitheringestationalor lactationalexposures,incomparisonwiththe correspond-ingcontrolvalues,didnotshowsignificantalterationin BuChEactivity(Fig.3).

Control values of malondialdehyde (MDA) concen-tration in the gestational exposure experiment were accountedto82.0,52.3and51.3nmol/gtissueformouse dams,malepupsandfemalepups,respectively.In com-parison, ATZ-treated groups recorded 93.3, 58.0 and 57.7nmol/gtissue,respectively.Only,thevaluerecorded formousedamswassignificantly(P<0.05)higherthanthat recordedforcontrol(Fig.4a).

ConcentrationofMDAforcontrolanimalsinthe lac-tational exposureexperiment equaled to81.3, 53.0 and 51.0nmol/gtissueformousedams,malepupsandfemale pups, respectively. In comparison, ATZ-treated groups recorded94.7, 58.3and59.0nmol/gtissue, respectively.

MDA CD AD VD AVD CP ( M) CP(F ) AP (M)AP (F)VP(M ) VP (F) AVP ( M) AVP (F) 0 50 100 150 * Treatment Groups

Co

nc

en

tr

at

ion

(n

mo

l/g)

[a]: CD = 82.0; CP (M) = 52.3; CP (F) = 51.3 nmol/g tissue MDA CD AD VD AVD CP (M ) CP (F ) AP (M ) AP (F ) VP (M ) VP (F ) AVP (M) AVP (F) 0 50 100 150 * * Treatment Groups C on ce nt ra tio n (n m o l/g) [b]: CD = 81.3; CP (M) = 53.0; CP (F) = 51.0 nmol/g tissue

Fig.4.ConcentrationofMDAinlivertissueofmousedamsandtheirpups exposedtoatrazine,withandwithoutvitaminE,duringgestation(a)and lactation(b)periods.Statisticalanalysis:Nosignificantdifference; *sig-nificantdifferenceatP<0.05;**highlysignificantdifferenceatP<0.01. Groupsymbols:C=control;D=dams;P=pups(M:maleandF:female); A=atrazine;V=vitaminE;AV=atrazine+vitaminE.

Onlythevaluesrecordedformousedamsandfemalepups weresignificantly(P<0.05)higherthanthoserecordedfor controlvalues(Fig.4b).

Alterationsofsuperoxidedismutase(SOD)activityin mousedamsandtheiroffspringfollowingadministration ofatrazine,withandwithoutvitaminE,tothemothers duringgestationaland lactationalperiodsseparatelyare presentedinFig.5.Thegeneralpatternofalterationwas adeclineofactivityduetoadministrationofATZ.In ges-tationalexposure,SODactivityinmousedamsrecorded 174.7U/gtissuecomparedtocontrolvalueof210.0U/g tis-sue,achievinghighsignificantdifferenceatP<0.01. The pups, either males or females, behaved similarly with respecttohighsignificantdecline ofSOD activity.Also, femaleand malepupsfrommothergroupstreatedwith ATZ+vitaminEshowedlowerenzymeactivitycompared tocontrol(Fig.5a).

Inlactationalexposureexperiment,controlSOD activ-ityrecorded210.7,190.7and190.3U/gtissueformouse dams,maleoffspring and femaleoffspring,respectively. In ATZ-treated groups, these values were 180.7, 167.0 and 163.3U/g tissue, achieving high significant decline (P<0.01)formousedams,significantdecline(P<0.05)for

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SOD CD AD VD AVD CP ( M) CP (F ) AP ( M) AP (F ) VP (M ) VP ( F) AVP (M) AVP (F ) 0 50 100 150 200 250 *** ** ** ** * **

Treatment Groups

Ac tiv ity (U /g ) [a]: CD = 210.0; CP (M) = 190.0; CP (F) = 190.7 µg/g tissue SOD CD AD VD AVD CP(M ) CP (F ) AP (M ) AP (F)VP (M ) VP ( F) AVP (M) AVP (F) 0 50 100 150 200 250 ** ** * ** *

Treatment Groups

A ct iv ity (U /g ) [b]: CD = 210.7; CP (M) = 190.7; CP (F) = 190.3 µg/g tissue

Fig.5. ActivityofSODinlivertissueofmousedamsandtheirpups exposedtoatrazine,withandwithoutvitaminE,duringgestation(a)and lactation(b)periods.Statisticalanalysis:Nosignificantdifference; *sig-nificantdifferenceatP<0.05;**highlysignificantdifferenceatP<0.01. Groupsymbols:C=control;D=dams;P=pups(M:maleandF:female); A=atrazine;V=vitaminE;AV=atrazine+vitaminE.

malepups,andhighsignificantdecline(P<0.01)forfemale pups(Fig.5b).

3.4. Histopathologyexamination

Microscopicexaminationofsectionsfromliver,kidneys, ovariesand testes preparedfromtheanimalssubjected to different treatments in the present study was per-formed.Fig.6illustratesthehistopathologicstructureof thestudied organs prepared from the normal (control) groups.Thelatterwasconsideredasabaseforcomparing thehistopathologicalterationsinATZandATZ+vitaminE groups.Suchalterations werescoredintermsof degree ofchangeincellsshowingdamageoralterations,as fol-lows:(0)=nochange;(1)=mildchange(e.g.,<25%ofcells showingdamage);(2)=moderatechange(e.g.,25–50%cell damage);and(3)=severechange(e.g.,>50%celldamage). TheresultsarepresentedinTables4and5.

Sectionsofcontrolorgans(Fig.6)werecharacterized bynormalhistopathologicstructure,observedonhepatic lobules,centralveinsandappearanceofhepatocytes.Renal paranchymaandtubulesshowednormalhistopathological structureofkidneys. Graffianfollicleand corpusluteum

werecharacterizedinnormalovary.Sectionsoftesteswere characterizedbynormalseminiferoustubules,andovaries showednormalcorpusluteum(Fig.6a–i).

In ATZ-treatedgroupsduringgestation(Table4),the followinghistopathologiceffectswereobserved:

-Liver:mildhepaticandmoderatevacuolardegeneration ofhepatocytes(dams,maleandfemalepups);moderate perivascularmononuclearcellsinfiltrationaccompanied withmildKupffercellsactivation(dams)andmoderate fibroblastproliferationaroundbileduct(femalepups). -Kidneys:moderatevacuolationofepithelialliningrenal

tubules and glomerular tuft (dams, male and female pups);moderatefocalrenalhemorrhage(dams); mod-eratethickeningofparietallayerofBowman’scapsule (dams)andmildcongestionofintertubularbloodvessels (femalepups).

-Ovary: moderate degeneration of corpus luteum and moderatecongestionofbloodvessels(dams).

-Testis:moderatedegenerationofspermatogonealcells; moderate thickeningof basement membrane of sem-iniferous tubules, as well as mild small diameter seminiferoustubules.

In ATZ+vitamin E-treated groups during gestation (Table4), most(if not all) ofthe abovehistopathologic changesdisappeared,exceptforsomemildchanges rep-resented by hepatic degeneration of hepatocytes (male pups);mildvacuolardegenerationofhepatocytes,aswell askupffercellsactivation(dams).

In ATZ-treatedgroups duringlactation(Table5), the followinghistopathologiceffectswereobserved:

-Liver:mildhepaticdegenerationofhepatocytes(female pups);severe(dams)tomoderate(pups)vacuolar degen-erationofhepatocytes;mildperivascularmononuclear cells infiltration(malepups);mildfibroblast prolifera-tionaroundbileduct(malepups)andseverefocalhepatic hemorrhage(damsandmalepups).

-Kidneys: severe vacuolation of epithelial lining renal tubules(damsandoffspring);moderatevacuolationof epithelialliningglomerulartuft(damsandmalepups), butmildforfemalepups;severefocalrenalhemorrhage (dams)and moderatecongestionofintertubularblood vessels(dams).

-Ovary:moderate(dams)andmild(femalepups) degen-erationofcorpusluteumandseverecongestionofblood vessels(dams).

-Testis:moderatedegenerationofspermatogonealcells, mildthickeningofbasementmembraneofseminiferous tubules,moderatesmalldiameterseminiferoustubules andsevereintratubularodema.

In ATZ+vitamin E-treated groups during lactation (Table5), most(if not all) ofthe abovehistopathologic changesdisappearedordeclined,exceptforsomeeffects representedbymildkupffercellsactivation(malepups); mildvacuolationofepithelialandendothelialliningrenal tubules(dams)andmilddegenerationofspermatogoneal

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S.A.Mansouretal./ToxicologyReports1(2014)53–68 61

Fig.6.Histopathologicalsectionsfororgans(liver,kidney,ovaryandtestis)preparedfromnormalmiceforcomparisonwithatrazine-treatedmice,with andwithoutvitaminE,duringgestationandlactationperiods(H&Estain400×).(a)Liverofcontrolmousedamshowingthenormalhistologicalstructure ofhepaticlobulefromcentralvein(CV)andnormalhepatocytes(H)(H&Estain400×).(b)Liverofcontrolmaleoffspringshowingthenormalhistological structureofhepaticlobulefromcentralvein(CV)andnormalhepatocytes(H)(H&Estain400×).(c)Liverofcontrolfemaleoffspringshowingthenormal histologicalstructureofhepaticlobulefromcentralvein(CV)andnormalhepatocytes(H)(H&Estain400×).(d)Kidneyofcontrolmousedamshowing thenormalhistologicalstructureofrenalparenchyma.Notenormalglomerulus(G)andnormalrenaltubules(T)(H&E400×).(e)Kidneyofcontrolmale offspringshowingthenormalhistologicalstructureofrenalparenchyma.Notenormalglomerulus(G)andnormalrenaltubules(T)(H&E400×).(f)Kidney ofcontrolfemaleoffspringshowingthenormalhistologicalstructureofrenalparenchyma.Notenormalglomerulus(G)andnormalrenaltubules(T)(H&E 400×).(g)Ovaryofcontrolmousedamshowingnormalgraffianfollicle(GF)andnormalcorpusluteum(CL).(H&Estain400×).(h)Ovaryoffemaleoffspring showingnormalgraffianfollicle(GF)(H&Estain400×).(i)Testisofcontrolmaleoffspringshowingnormalseminiferoustubules(ST)(H&Estain400×).

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cells(malepups).Numerousfollicleswereseeninasection ofovaryfromthepups.

4. Discussion

In toxicological studies, organ and relative organs weightsareimportantcriteriaforevaluationoforgan tox-icity[47],andincreaseordecreaseoforgansweightsthan normalmaybeconsideredasasignoftoxicity[48].Several investigators(e.g.,[36,37,49,50])havereportedtheeffect ofATZ onbodyand internal organsweights, aswellas onthefunctionofliverandkidney.Therefore,our find-ingsmayaddfurtherawarenesstopossibleeffectsofthe tested dose in the present study which was extremely lowerthanthedosesusedbythepreviousinvestigators. Inlactationalexposureexperiment,therelativeweightsof liverandheartfromtheATZ+vitaminEgroupofpups(AVP; Table2)haveexercisedsignificantdifferencescompared tothecorrespondingcontrolvalues.Suchresultmayrefer tointeractionbetweenthepesticideandvitaminEwhich appearedas“potentiatingeffect”forliverand“antagonistic effect”forheart,aresultwhichmayneedfurther investi-gationinfuturestudies.

HepaticeffectsofATZintermsofenzymaticalteration hadbeenstudiedindifferentanimalspecies.Inthepresent study,elevation(P<0.05)ofALPactivitywasobservedin ATZ-treateddamsduringgestation(groupAD;Fig.1a),and highelevation(P<0.01)forthosetreatedduringlactation (groupAD; Fig.1b),resultswhich coincidewiththatof SantaMariaetal.[49]onWisterratstreatedwithATZat 100mgkg−1/dayfor14days.Eitheringestationalor lacta-tionalexposure,themousepupsshowedelevationofALP activityveryhigherthanthatinthedams,aresultreflecting highvulnerabilityoftheyoungmicetoindirectexposure toATZviatheirmothers.

Alkalinephosphatasesarea groupofenzymesfound primarilyintheliver(isoenzymeALP-1)andbone (isoen-zymeALP-2).Therearealsosmallamountsproducedby cellsliningtheintestines(isoenzymeALP-3),theplacenta, andthekidney(intheproximalconvolutedtubules).What ismeasuredinthebloodisthetotalamountofALPreleased fromthesetissuesintotheblood.Theprimaryimportance ofmeasuringALPistocheckthepossibilityofbonedisease or liverdisease. Whenthe liver,bile ducts or gallblad-dersystemarenotfunctioning properlyorare blocked, thisenzymeisnotexcretedthroughthebile andALPis releasedintothebloodstream[51].Ontheotherside,the observedhighelevation (P<0.01) of ureaconcentration maybe consideredas an indication of kidney dysfunc-tionduetoexposuretoATZatsuchverylowdoseused inthepresentstudy.Ourresultscoincidewiththose previ-ouslyreportedregardingtheprofoundeffectofATZonliver andkidneyfunctionsinavarietyofexperimentalanimals [36,37,49,50].

Cholinesterase(ChE,3.1.1.8), also knownas pseudo-cholinesterase, hasbeen recognized as an enzyme that hydrolyzescholineesters.Itissynthesizedmainlyin hep-atocytes and secreted into the blood stream [52]. ChE activityis reduced in liver dysfunction due to reduced synthesis, in contrast to other serum enzymes associ-atedwiththeclinicalassessmentofliverfunctionwhose

activities increase as a result of increased release from their cellular sources following cell membrane damage [53].Cholinesteraseshadbeenlongrecognizedas biomark-ers of effect induced mainly by organophosphorus and carbamatepesticides[54];so,investigationsonother anti-cholinesterases were being limited. To the best of our knowledge, the literature offers very little information abouttheeffectofatrazineonmammalsChEesactivity, whilemuchstudiesonaquaticvertebrateorganismsare available in the literature withspecial concern tojoint actionofATZandotherpesticides[55–58].Thisled Boon-thai et al.[55] tosuggestthat AChE asa biomarkerfor organophosphorusandcarbamatepesticidesmaybevalid touseforotherchemicalgroups.

Inasinglepaper,itwasreportedthatadministration ofATZtoratsat300mgkg−1 affectederythrocyte mem-branescausingsignificantinhibitionofAChEactivityand induction ofoxidativestress interms ofincreased mal-ondialdehyde (MDA) levels.However, administration of vitaminE(100mgkg−1bwdaily)amelioratedtheoxidative stressandchangesintheerythrocytemembranesinduced byATZ[59].Suchresultscorroboratedourfindings regard-inghighsignificant(P<0.01)declineofBuChEactivityin ATZ-treatedmousedams duringgestation(Fig.3a), and significant(P<0.05)decline inATZ-treatedmousedams duringlactation(Fig.3b).InbothcasesofexposuretoATZ, co-administration of vitamin E normalized the enzyme activitytothatofthecontrolvalues.Itseemedthatthere werenosignificantalteration inBuChE activitiesinthe pupswhosemothersweretreatedwithATZeitherduring gestationorlactationstages(Fig.3).Thismaybereferred totheadministeredlowdoseofATZwhichisnotstrong inhibitortocholinesteraseenzymes[58].

Infact,thetoxicityofmanyxenobioticsisassociated withtheproductionofoxygen-freeradicals,more gener-allyknownas“reactiveoxygenspecies”(ROS),whichare notonlytoxicthemselvesbutarealsoimplicatedinthe pathophysiologyofmanydiseases[60].Theharmfuleffects ofROSarebalancedbytheantioxidantactionof nonenzy-maticantioxidantsinadditiontoantioxidantenzymes[61]. Antioxidantsprotectcellsfromthedamagingeffectsoffree radicals, which are molecules thatcontain anunshared electron[62].Ithasbeenreportedthat manypesticides mayinduceoxidativestressfollowingacuteexposurein humans[63]andanimals[64–67].Increasedlipid peroxi-dation(LPO)invarioustissuesmaybeoneofthemolecular mechanisms involved in the ATZ-induced toxicity. SOD providesthefirstlineofdefenseagainstoxygenderived freeradicalsanddecreasesoxidativestressbydismutation ofO2−[68].Previousstudiesinourlaboratoryonrat(Rattus norvegicus),usingdifferentinsecticides,revealedincrease ofLPOanddecreaseofSODlevelsfollowingtheinsecticidal treatments[64–67,69–71],andcorroborateourfindingson ATZherbicide.Also,oraladministrationofATZtoratsat 120mgkg−1wasfoundtoimpairtheantioxidantdefense andincreasedLPO(intermsofMDAconcentration)inthe liverandsignificantlydecreasedSODactivity[72].

Vitamin E is an important biological free radical scavenger in the cell membranes [33]. In the present investigationwestudiedwhethervitaminEhasthe poten-tialto attenuateATZ-induced oxidative stress,either in

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S.A.Mansouretal./ToxicologyReports1(2014)53–68 63 mousedamsexposedduringgestationorduringlactation

aswellasontheiroffspring.Inmousedams,the signifi-cant(P<0.05)elevationofMDAinducedbyATZandthe highsignificantdecline(P<0.01)ofSODwerenormalized withco-administrationofvitaminE(Figs.4and5). More-over,thefemalepupsfromlactationexperiment(Fig.4b) showedsignificantelevationofMDAactivitywhichwas returnedtoitsnormalvaluebyco-administrationof vita-min E. Such resultscoincide with Singh et al. [6] who foundasignificantincreaseinhepaticLPOfollowingATZ administrationtomaleWistarratsat300mgkg−1bw,but administrationofvitaminE(100mgkg−1bw)attenuated ATZ-inducedLPOinliver.AccordingtoPascoeetal.[73], vitaminEallowsfreeradicalstoabstractahydrogenatom fromtheantioxidantmoleculeratherthanfrom polyun-saturated fattyacids(PUFA),thus breakingthechain of freeradicalreactions.Theresultingantioxidantradicalis relativelyanon-reactivespecies.

To assess the effect of ATZ on antioxidative stress enzymes(e.g.,MDAandSOD)ina“quantitativemanner”, we calculated the percentage of change in ATZ-treated groupsrelativetocontroluntreatedgroups,eitherforthe damsortheiroffspring,both incasesofgestationaland lactationalexposures.Accordingtothedatapresentedin Table3,theaverageofchangeinMDAandSODinthepups ofgestationalexposurewasaccountedto11.6%and15.1%, respectively,comparedto12.8%and13.3%,respectivelyfor thepupsoflactationalexposure(basedoncalculatingthe averageofthevaluesof maleandfemalepupsgiven in thetable).Forthedams,thechangeaccountedto13.8% and16.8%ingestationand16.4%and14.3%inlactation. ThismeansthattheeffectonMDAwasmorepronounced inATZexposureduringlactation,whiletheopposite for SODwhich wasmorepronouncedinATZexposure dur-inggestation.Interestingly,suchtrendwasobtainedeither for thedams or theiroffspring (Table3). Such findings clearly demonstratethat pupshave exerciseddegree of effectsnearlyequaledtothattheirmotherswereaffected. Indeed,thedifferencesbetweenthetwocasescompared weresmall,maybeduetothelowdoseofATZusedinthe presentstudy(31.0␮gkg−1).Ontheotherhand,we com-paredtheresultsofMDAandSODinATZ/vit.Egroupswith theresultsofcontrol groups,toassess theameliorative effectof vitamin E,based oncalculating the “Ameliora-tiveIndex;AI”.AsAIapproaching“1”,astheamelioration reachinghighdegreeofnormalizationtothecontrolvalue. TheobtainedresultsrevealedthattheAIofMDAandSOD rangedbetween0.95 and 1.06 (≈1.0)for thedams and pupseitheringestationalorlactationalexposureroutes. SimilarassessmentcouldbecarriedoutonBuChEchanges due toexposuretoATZ,and theameliorativeefficiency ofco-administrationofvitaminE.Generally,changesdue toATZwerelittleandaccountedto1.5–3.4%ingestation and1.9–4.0%inlactation.TheAIwasaccountedto approx-imately1.0forthetwostudiedcases(Table3).

Pregnantwomenareaspecialriskgroup,giventhe find-ingsshowingincreasedriskofchildhoodacutelymphocytic leukemiawhen women usepesticidesin thehomeand gardenduringpregnancy[74].Ontheotherhand, lacta-tionmayposestresstothemothersandsuchmotherswill bemorevulnerable,thannon-lactatingmothers,toother

chemicalstressors[75].Itisdocumentedthatthefetusis morevulnerabletothetoxiceffectsofanumberof environ-mentalexposuresthanarechildrenoradults[76].Highly lipophiliccompoundsareexpectedtocrosstheplacenta andreachthefetusduringpregnancy[77,78].Also,there aremanyfactorsinvolvedinthetransferofchemicalsinto themilkandsubsequentlytothesucklingneonate, includ-inglipophilicity,ionizationandmaternalplasmaprotein binding,whichcanallinfluencetransportofthecompound duringlactation[79].ATZisalipophiliccompoundandhas alowmolecularweightcompatiblewithpassivediffusion andtransferfromthedamtothepup[80,81].Intheir stud-iesongestationaland/orlactationalexposureofSprague DawleydamstoATZ(0,1,5,20,or100mgkg−1/day),Fraites etal.[21]concludedthatplacentadoesnotappeartolimit thetransferofATZanditsmetabolitesfromthematernal circulationtothedevelopingfetus.Also,levelsofATZand themetaboliteswerefoundinneonatalmilk,plasmaand tissues.Itworthytomentionthatchlorinatedmetabolites ofATZ [e.g.,diethylatrazine(DEA), di-isopropyl-atrazine (DIA),anddiaminochlorotriazine(DACT)]areconsidered equivalentintoxicitytoATZ,andexposuretometabolites arealsoof concern[19,82].Atrazineanditsmetabolites werefoundtocontaminate humanbloodin Japan[83], womenmammaryfattissueinArgentina[84]andmilkin Britishmothers(around0.5.2.3␮g/g)[85].

In thepresentstudy, thepupswereexposed toATZ intoxicationviaplacentaandbreastmilkintheexperiment ofgestationalexposure,andviabreastmilkonlyinthe sec-ondexperiment(lactationalexposure).In anattemptto elucidatewhichstageofexposure(gestationorlactation) toATZwasmoreaffected,weestimatedalterationofMDA andSOD(asexamples)followingATZtreatments,based onthedatapresentedinTable3.Theaverageofchange inMDAandSODinthepupsofgestationalexposurewas accountedto11.6%and15.1%,respectively,comparedto 12.8%and13.3%,respectively forthepupsoflactational exposure(basedoncalculatingaverageof thevaluesof maleandfemalepupsgiveninthetable).Forthedams, thechangeaccountedto13.8%and16.8%ingestationand 16.4%and 14.3%in lactation.Thismeansthat theeffect ofATZ onMDAwasmore pronouncedduring lactation, whiletheoppositeforSODwhichwasmorepronounced duringgestation.Interestingly,suchtrendwasobtained eitherforthedamsortheiroffspring(Table3).Performing similarcalculationswithALPmaygiveadditionalpicture. Ingestationalexposureexperiment,ALPactivityequaled 75.3and56.1IU/lforcontroldamsandtheirpups (with-outsexdifferentiation),respectively.ATZ-treatedgroups showed101.7and125.3IU/l,respectively(Fig.1a).In lac-tationalexposureexperiment,thesevaluesequaled73.3 and54.5IU/l,respectivelyforcontrolanimals,and95and 122.3IU/l for ATZ-treated groups (Fig. 1b). This means that,ingestationexperiment,thechangeinALPactivity wasaccountedto 35.1%and 123.4% for damsand their pups,respectively,whilechangesinALPactivityin lacta-tionexperimentreached29.6%and124.4%,respectivelyfor damsandtheirpups.ThismeansthattheeffectofATZon ALPinmousedamswashigherduringgestationthan dur-inglactation.Theeffectonpups-ALPwasnearlyequalin bothgestationalandlactationalexposure.

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Table3

AssessmentofoxidativestressofatrazineandtheameliorativeeffectofvitaminE,basedonmeasuredbiochemicalparametersfromgestationandlactation experimentalresults.

Mice/parameter Control(a) ATZ(b) ATZ+E(c) %ofchangea AmeliorativeIndex(AI)b

Gestationc

MDA(nmol/gtissue)

Dams 82.00 93.33 87.33 13.8 1.06

Pups(M) 52.33 58.00 55.33 10.8 0.95

Pups(F) 51.33 57.70 54.70 12.4 0.95

SOD(U/gtissue)

Dams 210.00 174.60 183.33 −16.8 1.05 Pups(M) 190.00 162.70 171.00 −14.4 1.05 Pups(F) 190.70 160.60 168.60 −15.8 1.05 BuChE(U/l) Dams 3419.0 3305 3358 −3.3 1.02 Pups(M) 1503.3 1452 1477 −3.4 1.02 Pups(F) 1548.0 1525 1540 −1.5 1.00 Lactationc

MDA(nmol/gtissue)

Dams 81.33 94.7 86.0 16.4 0.91

Pups(M) 53.00 58.3 54.7 10.0 0.94

Pups(F) 51.00 59.0 55.0 15.7 0.93

SOD(U/gtissue)

Dams 210.7 180.6 187.0 −14.3 1.03 Pups(M) 190.6 167.0 177.6 −12.4 1.06 Pups(F) 190.3 163.3 173.3 −14.2 1.06 BuChE(U/l) Dams 3379 3298 3341 −2.4 1.01 Pups(M) 1487 1427 1448 −4.0 1.01 Pups(F) 1544 1514 1527 −1.9 1.00

aPercentofchange=(ba)/a×100. b AmeliorativeIndex(AI)=c/a.

c Dataundergestationandlactationaremeanvaluesfortherespectivebiochemicalparameters.

Table4

Histopathologicchangesbasedonscoringseverityofinjuryindifferentorgansfrommousedamsandtheirpupsfollowingexposuretoatrazinewithand withoutvitaminEduringgestation.

Histopathologicalchanges Treatments

ATZ ATZ+vit.E

Dams Pups(M) Pups(F) Dams Pups(M) Pups(F)

Liver

Hepaticdegenerationofhepatocytes 1 1 1 0 1 0

Vacuolardegenerationofhepatocytes 2 2 2 1 0 0

Perivascularmononuclearcellsinfiltration 2 0 0 0 0 0

Fibroblastproliferationaroundbileduct 0 0 2 0 0 0

Focalhepatichaemorrhage 0 0 0 0 0 0

Kupffercellsactivation 1 0 0 1 0 0

Kidneys

Vacuolationofepithelialliningrenaltubules 2 3 3 0 0 0

Vacuolationofendothelialliningglomerulartuft 2 2 2 0 0 0

Focalrenalhaemorrhage 2 0 0 0 0 0

ThickeningofparietallayerofBowman’scapsule 2 0 0 0 0 0

Congestionofintertubularbloodvessels 0 0 1 0 0 0

Ovary

Degenerationofcorpusluteum 2 0 0 0

Congestionofbloodvessels 2 0 0 0

Numerousfollicles 0 0 0 2

Testis

Degenerationofspermatogonealcells 2 0

Thickeningofbasementmembraneofseminiferoustubules 2 0

Smalldiameterseminiferoustubules 1 0

Intratubularodema 0 0

Degreeofchanges:(0)=nochange;(1)=mildchange(e.g.,<25%ofcellsshowingdamage);(2)=moderatechange(e.g.,25–50%celldamage);and(3)=severe

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S.A.Mansouretal./ToxicologyReports1(2014)53–68 65 Table5

Histopathologicchangesbasedonscoringseverityofinjuryindifferentorgansfrommousedamsandtheirpupsfollowingexposuretoatrazinewithand

withoutvitaminEduringlactation.

Histopathologicalchanges Treatments

ATZ ATZ+vit.E

Dams Pups(M) Pups(F) Dams Pups(M) Pups(F)

Liver

Hepaticdegenerationofhepatocytes 0 0 1 0 0 0

Vacuolardegenerationofhepatocytes 3 2 2 0 0 0

Perivascularmononuclearcellsinfiltration 0 1 0 0 0 0

Fibroblastproliferationaroundbileduct 0 1 0 0 0 0

Focalhepatichaemorrhage 3 3 0 0 0 0

Kupffercellsactivation 0 0 0 0 1 0

Kidneys

Vacuolationofepithelialliningrenaltubules 3 3 3 1 0 0

Vacuolationofendothelialliningglomerulartuft 2 2 1 1 0 0

Focalrenalhaemorrhage 3 0 0 0 0 0

ThickeningofparietallayerofBowman’scapsule 0 0 0 0 0 0

Congestionofintertubularbloodvessels 2 0 0 0 0 0

Ovary

Degenerationofcorpusluteum 2 1 0 0

Congestionofbloodvessels 3 0 0 0

Numerousfollicles 0 0 0 3

Testis

Degenerationofspermatogonealcells 2 1

Thickeningofbasementmembraneofseminiferoustubules 1 0

Smalldiameterseminiferoustubules 2 0

Intratubularodema 3 0

Degreeofchanges:(0)=nochange;(1)=mildchange(e.g.,<25%ofcellsshowingdamage);(2)=moderatechange(e.g.,25–50%celldamage);and(3)=severe

change(e.g.,>50%celldamage).

Indeed,theresponseoftheantioxidantenzymes(e.g.,

MDAandSOD)toATZwaslowerthantheresponseofALP,

and that mayrefer tothe low doseofATZ usedin the

presentstudy(31.0␮gkg−1),andthedifferentresponseof

suchbiomarkerstoatoxicantsuchasATZ.

ExposureofmicedamstoATZattheequivalentdose

toitsADI(ca.0.005mgkg−1bw/d)hasresultedin

notice-ablehistopathologicaleffects onliver,kidney,ovaryand

testisasillustratedinFig.6andTables4and5.The

off-springfromthetreateddamsgenerallyexercisednearly similar effects. Histologically, it hasbeen reported that ATZ caused liver degeneration represented by chronic interstitialinflammation,lymphocyteandeosinophil infil-tration,andnarrowingandirregularformsofbilecanaliculi [49,86].Also,it inducedmultipleovarianfollicularcysts and persistence of corpusluteum [87], as well as kid-neydysfunctionrepresentedbyanincreaseintheprotein content of the urine in rats treated for 14 days with 10mgkg−1ofATZperday[88].Inthepresentstudy, bio-chemical alterations observed in ATZ-treatedmice and normalizationlevelsofthesebiochemicalparametersafter administration of vitamin E corroborated theresults of histopathological findings,where vitamin E supplemen-tation repaired the impairment of ATZ to the studied organs.

5. Conclusion

Inlightoftheresultsofthepresentstudy,itcanbe con-cludedthatexposureofadultfemalemicetocontaminated foodwithATZatitsacceptabledailyintake(ADI)levelcan

inducehepaticandrenaloxidativestressand histopatho-logicaleffects. Sincemousepupsfromdamsexposed to ATZduringlactationweredependentonlyonbreast feed-ing,thefindings ofthepresent investigationrevealthat toxiceffectsofATZ,aswellasameliorativeeffectof vita-minE,occurredvialactationprocess.Inpupsfromdams exposedtoATZduringgestation,thetoxiceffectsofATZ, aswellasameliorativeeffectofvitaminE,occurredmainly viagestationandprobablythroughthemother’smilkalso. Inlightoftheresultsofthepresentstudy,itmaybe dif-ficulttoconclude,inageneralstatement,whichexposure stagewasmuchaffectedfollowingexposuretoATZ.But wecansuggestwhichstagewasmorevulnerableto alter-ationofa specificparameter(e.g.,MDA,SOD, ALP,etc.). Forinstance,theeffectofATZonALPinmousedamswas higherduringgestationthanduringlactation.Theeffect onMDAwasmorepronounced inATZ exposureduring lactation,whiletheoppositeforSODwhichwasmore pro-nouncedinATZexposureduringgestation.Theresponse ofMDAandSODwassimilareitherforthedamsortheir offspring.Histologically,ATZinducedimpairmentin tis-suesoftheexperimentaldamsandtheiroffspring,bothin thoseexposedduringgestationandlactation.Takinginto considerationthatexposureofdamstoATZduring gesta-tionwasfollowedwithawithdrawalperiod(ca.21days), thismayleadustoconsiderthatgestationstagewasmore affectedthanlactationstage.Thefindingsmaysupportthe needtofurtherinvestigatingtheadverseeffectsof expo-suretolowdosesofcommonlyusedpesticides,especially duringpregnancyandbreast-feedingaswellaseffectson newbornchild.

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Funding

Thiswork wascarried out withinthe framework of IMHOTEPscientificcollaborationprogrambetweenEgypt andFrancewhichsupportedtravelmobilitybetweenthe twocountries.

Conflictofinterest

Theauthorsdeclarethatnoconflictsofinterest. Acknowledgements

The authors thank the NationalInstitute of Agricul-turalResearch (INRA,France) and AcademyofScientific Research&Technology(ASRT,Egypt)forsupportingthis studywithintheREFBHCIMHOTEP2011ProjectNo.25382 YG.SpecialthanksareduetoDr.L.Gamet-Payrastrefor providing facilitiesand following uptheanimal rearing and dosing in TOXALIM Unit. We also thank Prof. Dr. KawkabAbdel-Aziz,PathologyDepartment,Facultyof Vet-erinaryMedicine,CairoUniversity,Egyptforreadingthe histopathologicalslides.

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