Sub-chronic exposure to fipronil induced oxidative stress, biochemical and histopathological changes in the liver and kidney of male albino rats

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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

Sub-chronic

exposure

to

ﬁpronil

induced

oxidative

stress,

biochemical

and

histopathological

changes

in

the

liver

and

kidney

of

male

albino

rats

Abdel-Tawab

H. Mossa

a,∗

_,

_Eman

_S.

_Swelam

b

_,

_Samia

_M.M.

_Mohafrash

a a_{Environmental}_Toxicology_Research_Unit_(ETRU),_Pesticide_Chemistry_Department,_National_Research_Centre,

33BohouthStreet,Dokki,Giza,Egypt

b_Economic_Entomology_and_Pesticides_Department,_Faculty_of_Agriculture,_Cairo_University,_Egypt

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received1October2014

Receivedinrevisedform25January2015 Accepted11February2015

Availableonline19February2015 Keywords: Fipronil Rats Liver Kidney Oxidativestress Histopathology

a

b

s

t

r

a

c

t

Fipronil(FPN)isabroad-spectrumN-phenylpyrazoleinsecticideandhasbeenusedin

agricultureandpublichealthsincethemid-1990s.Thepresentstudywasdesignedto

inves-tigatetheadverseeffectsofsub-chronicexposuretotheFPNontheliverandkidneyofmale

ratsatthreeconcentrations0.1,1and10mg/Lindrinkingwaterfor45days.Serum

aspar-tateaminotransferases(AST),alanineaminotransferases(ALT),alkalinephosphatase(ALP),

andlactatedehydrogenase(LDH)activityandlevelsofuricacid,creatinineandtotal

pro-teinweresigniﬁcantlyincreasedinFPN-treatedrats.Oxidativestressbiomarkerssuchas

superoxidedismutase(SOD),catalase(CAT),glutathioneperoxidase(GPx),

glutathione-S-transferase(GST)andglutathionereduced(GSH)weresigniﬁcantlydecreased,whilelipid

peroxidation(LPO)wassigniﬁcantlyincreasedintreatingratsinaconcentrationdependent

manner.FPNcausedhistopathologicalalterationsinliverandkidneyofmalerats.Fromour

results,itcanbeconcludedthatFPNinducedlipidperoxidation,oxidativestress,liver,and

kidneyinjuryinrats.Thesepathophysiologicalchangesinliverandkidneytissuescouldbe

duetothetoxiceffectofFPNthatassociatedwithagenerationoffreeradicals.

theCCBYlicense(http://creativecommons.org/licenses/by/4.0/).

1. Introduction

Fipronil (FPN,

5-amino-1-(2,6-dichloro-4-(trifluoro-methylphenyl)-4-(trifluoro-methylsulfinyl)

pyrazole-3-carbonitrile)isaphenylpyrazoleinsecticidethatis

exten-sivelyusedtocontrolinsectsindifferentcerealcropsand

inpublichealthmanagement[1].Itismoreeffectivethan

organophosphate,carbamateandpyrethroidsinsecticides

against several species of Lepidoptera, Orthoptera and

Coleopteran[2,3].Currently,exposuretophenylpyrazole

pesticidesisaglobalpublichealthissueandconcernsare

∗ Correspondingauthor.Tel.:+20233371211;fax:+20233370931. E-mailaddress:abdeltawab.mossa@yahoo.com(A.-T.H.Mossa).

increasedregardingtherelativesafetyofthesepesticide

groups because of widespread use, their toxicity, and

releasesintotheenvironment.

FPNisneurotoxictoinsectsandtheprimarymechanism

ofactionreferstoblocksiontropicgamma-aminobutyric

acidreceptor(GABAR)ofthecentralnervoussystemthat

causeshyper-excitationatlowdosesandconvulsions

lead-ingtoinsectdeathathighdoses[4].FPNismoretoxicto

insectsthanmammals[5]andhasmoderatelyacuteoral

toxicityLD50’srangingfrom40to100mg/kgbodyweight

inratsandmice[3,6].Therefore, completeselectivityof

pesticidesisdifﬁcultandmostofthepesticidesaretoxicto

non-targetorganisms,includinghumans[7].

FPN is a stronguncoupler of oxidative

phosphoryla-tionatrelativelylowconcentrations inSH-SY5Yhuman

http://dx.doi.org/10.1016/j.toxrep.2015.02.009

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neuroblastomacellsinvitroandinducedneuronal

apopto-sis,mediatedbyincreasedgenerationofreactiveoxygen

species(ROS)[8].BothHepG2cellsand primaryhuman

hepatocytesaresensitivetothecytotoxiceffectsofFPN[9].

FPNanditsmetabolitesinducedcytotoxicityinepithelial

model Caco-2 cells at micromolar concentration

expo-sure[8]. FPN inhibitsDNA and protein synthesis in rat

neuronotypicpheochromocytomaPC12cellsandinduced

oxidativestressmorethanchlorpyrifos[10].FPNcauses

endocrinedisruptionandadversereproductiveeffectsin

femalerats[11],elevationinlipidperoxidation(LPO)and

decreaseinthelevelsofglutathione(GSH)atthedosageof

0.5mg/kg/dayfor98daystobuffalocalves[12]andaltered

SODandCATactivitiesintheliverofCyprinuscarpio[13].It

decreasedtotalthyroxine(T4),increasedhepaticenzymes

inplasmaoffemalerat[14],andcausedacutehuman

poi-soning[15,16].

Liverandkidneyarethemostsensitiveandmain

tar-getorgansofpesticidetoxicityanddamage[17],theyplay

amajor rolein thebiotransformation ofpesticides.The

sensitivityof thesetissuestothis stresstopesticidesis

afunctionof thedisturbedbalancebetweenthedegree

ofoxidativestressandtheantioxidantcapability[17,18].

Previousstudiesshowthatpesticidesalterenzymaticand

non-enzymaticantioxidantandinducedoxidativestressin

animalsthatwasinvestigatedasapotentialmechanismof

pesticidetoxicity[16,18,19].Ithasbeenreportedthat

pro-longedexposuretolowdosesofﬁpronilleadstooxidative

stressinserumofpregnantratsandtheiroffspring[20].

Pesticide formulations are complex mixtures that

contain, besides the active ingredient(s), several other

components,suchassolvent,wetting,emulsifyingagents,

andadditives;thereforethetoxicityinformationonactive

ingredientsaloneisnotsufﬁcienttoevaluatetheadverse

health effects of commercial pesticides. Therefore, the

WHOemphasizedthenecessityofevaluatingtoxichazard

oftheformulatedpesticides[21].Overthelastdecade,the

usageofFPNhasincreasedconsiderablyandinformation

onadversehealtheffectsisverylimited.Tothebestofour

knowledge,therearenopublishedstudiesthathave

exam-inedtheeffectofformulatedFPNonoxidant/antioxidant

statusandtheliverandkidneyfunctionbiomarkersinmale

rats.Therefore,thisstudyaimedtoevaluatetheadverse

effectsofsub-chronicexposuretoformulatedFPNon

oxi-dant/antioxidantstatusandliverandkidneybiomarkersof

malerats.

2. Materialsandmethods

2.1. Animalsandmanagement

Malealbinoratsweighing105±5gwereprocuredfrom

theAnimalBreedingHouseoftheNationalResearchCentre

(NRC),Dokki,Giza,Egypt.Ratswerehousedin

polypropyl-enecages(sixratsineach),withfreeaccessstandardpellet

diet,wateradlibitum,understandardizedhousing

condi-tions(12hlight/darkcycle,temperature(23_±2◦C)anda

minimumrelativehumidityof48%inthelaboratory.The

ratswereacclimatizedfor1weekbeforethestartofthe

experiment.Alltheratswerekeptaccordingtothe

guide-linesandwelfareregardinganimalprotectionapprovedby

NRCLocalEthicalReviewCommitteeandwasconducted

inaccordancewiththe“GuidefortheCareandUseof

Lab-oratoryAnimals”.

2.2. Chemicalsandreagents

Fipronil (Insecto SC 5%) is a product of BASF

Com-pany and manufactured by, Sinochem Group Ningbo

TechnicalCo.,Ltd.,China.Theassaykitsusedfor

biochem-ical measurementsof aspartate aminotransferases (AST,

EC2.6.1.1.), alanineaminotransferases (ALT, EC2.6.1.2),

alkalinephosphatase(ALP,EC3.1.3.1),lactate

dehydroge-nase (LDH,EC1.1.1.27), superoxidedismutase(SOD, EC

1.15.1.1), catalase(CAT, EC1.11.1.6),glutathione

perox-idase (GPx, EC 1.11.1.9), glutathione-s-transferase (GST,

EC2.5.1.13),glutathionereduced(GSH),lipidperoxidation

(LPO),albumin,uricacidandcreatininewerepurchased

fromBiodiagnosticCompany,Dokki,Giza,Egypt.Allother

chemicalswereofreagentgradesandwereobtainedfrom

reputedcompanies.

2.3. Experimentaldesign

Rats were randomly divided into four experimental

groups,sixrats.GroupI,receivedwaterandservedasa

con-trol.Theremainingthreegroups(II,IIIandIV)receivedFPN

indrinkingwateratconcentrations0.1,1.0and10mg/L

for45consecutivedays.TheconcentrationsofFPNwere

calculateddependingonthepercentageofactive

ingredi-entsofcommercialformulationofFPN.Concentrationsof

FPNwerefreshlypreparedandbodyweightswere

moni-toredweeklyduringtheexperimentalperiod.Allratswere

observedfor signsoftoxicity andmortalitydailyfor 45

days.

Theconcentrationsusedinthisstudyrepresent2.0,0.2

and0.02mg/kgb.wt.ofFPN,basedonaveragewater

con-sumptions and body weights of treated rats.The lower

concentrationofFPNrepresentsthedoseofnoobservable

adverseeffectlevel(NOAEL)ofhuman[22]with

descen-ding concentrationlevelsby 10-foldinterval, i.e.,1 and

10mg/L.

2.4. Bloodandtissuesamples

Attheendoftheexperimentalperiod,ratswerefasted

overnightandbloodsampleswerecollectedbypuncturing

theretero-orbitalvenousplexusoftheanimalswithaﬁne

sterilizedglasscapillary,thenratsweresacriﬁcedby

cer-vicaldislocation.Bloodsampleswerelefttoclotinclean

drytubesandcentrifugedat3000rpm(600xg)for10min

at4◦CusingHeraeusLabofuge400R(KendroLaboratory

ProductsGmbH,Germany)toobtainthesera.Serum

sam-pleswerestoredat−20◦_C_for_further_biochemical_analysis,

suchasAST,ALT,ALPandLDH.

Liverandkidneywereexcisedimmediatelyafter

sacri-ﬁced,cleanedinsalineandweighed.Smallpiecesofeach

liverandkidneywerecutandkeptin10%naturalformalin

forhistopathologicalstudy.Theotherportionsofliverand

kidneywerehomogenizedin10%(w/v)icecold100mM

phosphatebuffer(pH7.4)andcentrifugedat10,000rpm

(3)

obtainedandusedforoxidativestressbiomarkersstudies

(SOD,CAT,GPx,GSH,LPO)andtotalprotein.

2.5. Serumbiochemicalparameters

2.5.1. Liverandkidneyfunctionbiomarkers

SerumASTandALTweredeterminedaccordingtothe

methods ofReitman and Frankel[23],ALP accordingto

Youngetal.[24],LDHasanindicatorofnecroticcelldeath

wasdeterminedaccordingtoVassault[25],albumin,uric

acidandcreatinineaccordingtoWestgard andPoquette

[26],Tietzetal.[27]andTietz[28],respectively.Allserum

biomarkerswereperformedaccordingtothedetailsgiven

inBiodiagnostickit’sinstructions(BiodiagnosticCompany,

Dokki,Giza,Egypt).

2.5.2. Oxidativestressbiomarkersinliverandkidney

DeterminationofSOD,CAT,GPx,GST,GSHandlipid

per-oxidation(LPO)wereperformedaccordingtothedetails

giveninBiodiagnostickit’sinstructionsandtheprincipals

belowofdifferentmethodsaregivenforeachconcerned

biochemicalparameter.

SOD was determined according to the method of

Nishikimi et al. [29]. The method based on the ability

ofSODenzymetoinhibitthephenazine

methosulphate-mediatedreductionofnitrobluetetrazoliumdye.Brieﬂy,

0.05ml sample was mixed with 1.0ml buffer (pH 8.5),

0.1mlnitrobluetetrazolium(NBT)and0.1mlNADH.The

reactionwasinitiatedbyadding0.01mlphenazine

metho-sulphate(PMs),andthenabsorbancewasreadat560nm

for5min.SODactivitywasexpressedasunits/mgprotein.

CAT was determined according to the method of

Abei [30]. The method is based on the decomposition

of H2O2 by catalase. The sample containing catalase

is incubated in the presence of a known

concen-tration of H2O2. After incubation for exactly 1min,

the reaction is quenched with sodium azide. The

amount of H2O2 remaining in the reaction mixture is

then determined by the oxidative coupling reaction of

4-aminophenazone (4-aminoantipyrene, AAP) and

3,5-dichloro-2-hydroxybenzenesulfonic acid (DHBS) in the

presenceofH2O2andcatalyzedbyhorseradishperoxidase

(HRP).Theresultingquinoneiminedye

(N-(4-antipyrl)-3-chloro-5-sulfonate-p-benzoquinonemonoimine) is

mea-suredat510nm.CATactivitywasexpressedas␮mol/mg

protein.

GSTwasdeterminedaccordingtothemanufacturer’s

instructionsreferredtoHabigetal.[31].Themethodwas

basedontheconjugationof1-chloro-2,4-dinitrobenzene

(CDNB)withreducedglutathione(GSH)inareaction

cat-alyzed byGST. GST activitywasexpressedas␮mol/mg

protein.

GPxwasdeterminedspectrophotometricallyaccording

tothemethodofPagliaandValentine[32].The

estima-tionof GPxactivitywasbasedontheoxidation ofGSH

andNADPHusingglutathionereductase(GR)and

measur-ingthedecreaseinabsorbanceat340nmandexpressedin

units/mgprotein.

GSHlevelwasassessedspectrophotometrically

accord-ingtothemethodofBeutleretal.[33].Themethodwas

based on the reduction of 5,5-dithiobis(2-nitrobenzoic

acid)(DTNB)withglutathionetoproduceayellow

com-pound.Thereduced chromogendirectlyproportional to

GSHconcentrationanditsabsorbancecanbemeasuredat

405nm.GSHcontentwasexpressedin␮mol/mgprotein.

Lipidperoxidationwasestimatedbymeasuring

thio-barbituric acid reactive substances (TBARS) and was

expressedintermsofmalondialdehyde(MDA)contentby

acolorimetricmethodaccordingtoSatoh[34].TheMDA

valueswereexpressedasnmolesofMDA/gprotein.

Proteinconcentrationinhomogenatewasdetermined

accordingtothemethoddescribedbyLowryetal.[35].

2.5.3. Histopathologicalstudies

Liver and kidney were removed and dehydrated in

gradedserialofalcoholandembeddedinparafﬁnwax[36].

Fivemicrometerthicksectionswerecutand stainedby

hematoxylinandeosin(H&E).Oneslidewaspreparedfor

eachorgan;eachslidecontainedtwosectionsandtenﬁeld

areaswereexaminedforhistopathologicalchanges. The

examinationwasdoneusing a light microscope

(Olym-pus BX50) witha digital camera (OlympusE-410). The

histopathological alterations in liver and kidney tissues

werescoredasfollows:normalappearance(−),mild(+),

moderate(++)andsevere(+++)[36].

2.5.4. Statisticalanalysis

Alldatawereexpressedasmean±standarderror(SE).

Datawerestatisticalanalyzedbyone-wayANOVA

analy-sisfollowedbyDuncan’stestusingSPSSversion17.0for

windowsandthedifferenceswerestatisticallysigniﬁcant

atp<0.05.

3. Results

3.1. Signsoftoxicity

Nomortalityoccurredduringtheexperimentalperiod.

Generally,signsoftoxicityincludedachangeinactivityand

abnormalgaitwereobservedinratsexposedto

concentra-tion10mg/LofFPNfromthesecondweek.

3.2. Bodyandrelativeorganweights

Dataofﬁnalbodyweightsandrelativeliverandkidney

weightsofmaleratssubjectedtodifferenttreatmentsare

showninFig.1.Aslightdecreaseinthebodyweightafter45

daysofFPNtreatmentwiththethreeconcentrations,

com-paredtocontrolgroup.Theweeklybodyweightgainwas

insigniﬁcantchangedbetweentreatments.Asobservedin

thecaseofrelativeliverandkidneyweights(Fig.1BandC),

bothorganweightswerefoundtohavesigniﬁcant

alter-ationsin rat exposed to 10mg/L ofFPN. Moreover,the

relativeliverandkidneyweightsofratexposedto1mg/L

and0.1mg/Lwerealteredslightly.

3.3. Serumbiochemicalparameters

Theactivityofserumenzymes;AST,ALT,ALPandLDH

wassigniﬁcantlyincreasedafter45daysofexposureto1

and10mg/LofFPNwhencomparedtothecontrolgroup

(4)

Table1

Theactivityofsomeenzymesintheseraofmaleratsexposedtothesub-lethalconcentrationsofﬁpronilfor45days.

Treatments AST(U/L) ALT(U/L) ALP(U/L) LDH(U/L)

I 52.98±0.76a _30.60_±_0.93a _60.24_±_0.98a _112.25_±_4.12a

II 54.76±0.53a _34.62_±_0.76b _64.81_±_1.11a _116.95_±_4.28a

III 58.50±0.54b _47.40_±_0.72c _70.01_±_1.74b _150.08_±_4.51b

IV 79.20±1.16c _63.10_±_1.23d _102.75_±_2.53c _202.70_±_6.79c

Eachvalueisameanofsixanimals±SE.Meanshavingthesamelettersarenotsigniﬁcantlydifferentfromeachother,p<0.05.I:controlgroup;II,IIIand IV:groups;animalthatreceived0.1,1.0and10.0mg/LofFPNrespectively.

theactivityofALTcomparedtountreatedone,whilethe

activitiesofAST,ALPandLDHhadnosigniﬁcantchanges.

AsshowninTable2thecomparativeanalysisoftotal

protein,uricacidandcreatinineingroupIVwas

signiﬁ-cantlyincreased.Ontheotherhand,signiﬁcantincreasein

totalproteinandcreatininewererecordedinratsexposed

0 50 100 150 200 250 300 I II III IV Bo dy w eigh t ( g) Groups a _a _a a

A

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 I II III IV Re la ve li ve r w ei g ht ( %) Group a a ab b

B

0 0.2 0.4 0.6 0.8 1 1.2 I II III IV Rela v e kidn ey weig ht (% ) Group a a a b

C

Fig.1.Bodyweight(A)andrelativeweightsofliver(B)andkidney(C)of maleratsexposed01,1.0and10.0mg/Lofﬁpronil(groupsII,IIIandIV, respectively)for45daysindrinkingwaterandcontrolgroup(I).Values representedaremeans±SE.Meanshavingthesamelettersarenot signif-icantlydifferentfromeachother,p<0.05.Relativeorganweight=[organ weight/bodyweight]×100.

to1mg/LofFPN(groupIII).TheresultsrevealedthatFPN

changedserumbiomarkersinaconcentrationdependent

manner.

3.4. Oxidativestressbiomarkers

AsshowninTable3andFig.2,oxidativestress

biomark-ers;SOD,CAT,GST,GPx,GSHandLPOweredeterminedin

liverandkidneytissuesofmaleratsexposedto0.1,1and

10mg/LofFPNfor45days.Ratsexposedto1mg/LofFPN

(groupIII)showedsigniﬁcantchangesintheactivityofGPx,

GSHandLPOlevelinlivertissueandSOD,GST,GPx,GSH

activitiesandLPOlevelinkidneytissue.IngroupIV,rats

exposedto10mg/LofFPN,signiﬁcantchangesinall

oxida-tivestressbiomarkers(SOD,CAT,GST,GPx,GSHandLPO)

wererecordedinbothorgans.Thepresentresultsrevealed

thatFPNcausedstatisticallysigniﬁcantchangesin

oxida-tivestressbiomarkersintheliverandkidneyhomogenates

inaconcentrationdependentmanner.

3.5. Histopathologicalobservations

Figs.3and4andTable4showhistopathological

alter-ationsandscoreintheliverandkidneyofmaleratsexposed

to0.1,1and10mg/LofFPNfor45dayscomparedto

con-trol. Liver sectionsin thecontrolrats(group I) showed

thenormalhistopathologicalstructureofthehepatic

lob-ule(Fig.3a).Severehistopathologicalalterations,including

degeneration,inﬁltration,inﬂammatorycells, cell

prolif-erationandfocalhepatichemorrhagewerenotedinthe

liverof maleratsexposedto10mg/LofFPN (groupIV)

(Fig.3b).Ratsexposedto1mg/LofFPN(groupIII)showed

degenerationof hepatocytesand portalinﬁltrationwith

inﬂammatory cells (Fig.3c). Mild alterations, including,

congestion,vacuolizationandcysticdilation ofbileduct

werenotedintheliverofmaleratsexposedto0.1mg/Lof

FPN(groupII)(Fig.3d).

Lightmicroscopicofkidneysectionsinthecontrolrats

(groupI)showedthenormalhistopathologicalstructureof

renalparenchyma(Fig.4a).Severehistopathological

alter-ations, includingnecrosis,inﬂammatory cellinﬁltration,

atrophyofglomerulartuft,vacuolation,focalhemorrhage

(Fig.4b)werenotedinthekidneyofmaleratsexposed

to10mg/Lof FPN(groupIV).Rats exposedto1mg/Lof

FPN (group III) showed vacuolation of epithelial lining

renal tubulesand focal hemorrhage (Fig.4c). Kidney of

ratsexposedto0.1mg/LofFPN(groupII)appearedas

nor-malcontrolwithmildvacuolationofepithelialliningrenal

(5)

Table2

Effectofﬁpronilonalbumin,totalprotein,uricacidandcreatininelevelsinseraofmalerats.

Treatments Totalprotein(g/dl) Albumin(g/dl) Uricacid(mg/dl) Creatinine(mg/dl)

I 6.79±0.21a _4.79_±_0.09a _6.31_±_0.28a _0.95_±_0.02a

II 7.21±0.19ab _4.78_±_0.06a _6.58_±_0.22ab _0.99_±_0.03a

III 7.52±0.16b _4.74_±_0.10a _7.02_±_0.31ab _1.18_±_0.04b

IV 8.51±0.32c _4.53_±_0.11a _7.25_±_0.16b _1.37_±_0.03c

Eachvalueisameanofsixanimals±SE.Meanshavingthesamelettersarenotsigniﬁcantlydifferentfromeachother,p<0.05.I:controlgroup;II,IIIand IV:groups;animalthatreceived0.1,1.0and10.0mg/LofFPNrespectively.

Table3

Effectofﬁpronilonoxidativestressbiomarkersinlivertissuesofmalerats. Treatments Oxidativestressbiomarker

SOD(U/mg protein) CAT(␮mol/mg protein) GST(␮mol/mg protein) GPx(U/mg protein) GSH(␮mol/mg protein) LPO(nmol/g protein) I 6.67±0.11b _13.85_±_0.25b _0.43_±_0.017b _7.78_±_0.34c _0.078_±_0.002c _74.25_±_2.44a II 6.82±0.14b _13.56_±_0.42b _0.42_±_0.007b _5.87_±_0.31b _0.076_±_0.002c _78.17_±_3.23ab III 6.86±0.23b _12.98_±_0.28b _0.41_±_0.004b _5.23_±_0.39b _0.071_±_0.0013b _87.62_±_2.56b IV 5.47±0.07a _7.91_±_0.26a _0.33_±_0.012a _3.52_±_0.27a _0.048_±_0.002a _111.76_±_4.21c

Eachvalueisameanofsixanimals±SE.Meanshavingthesamelettersarenotsigniﬁcantlydifferentfromeachother,p<0.05.I:controlgroup;II,IIIand IV:groups;animalthatreceived0.1,1.0and10.0mg/LofFPN,respectively.

Table4

Theseverityofthereactioninliverandkidneytissueofdifferentgroupsaccordingtothehistopathologicalalterations.

Histopathologicalalterations Treatments

I II III IV

Liver

Ballooningdegenerationofhepatocytes – + +++ +++

Congestionofcentralvein – + – –

Portalinﬁltrationwithinﬂammatorycells – + ++ +++

Ovalcellsproliferation – – – ++

Focalhepatichemorrhage – – – ++

Cysticdilatationofbileduct – – – –

Kidneys

Focalnecrosisofrenaltubulesassociatedwithinﬂammatorycellsinﬁltration. – – – +++

Vacuolationofepithelialliningrenaltubules. – + ++ +++

Focalrenalhemorrhage – – ++ ++

+++Severe;++moderate;+mild;–nil.I:controlgroup;II,IIIandIV:groupsreceivedFPNatconcentrationsof0.1,1.0and10.0mg/L. 4. Discussion

NomortalitywasobservedinratsexposedtotheFPN

at differentconcentrationsfor 45 days.While the

clini-calsignsrelatedtoFPN-treatment(group IV)includeda

changeinactivityandabnormalgaitwereobservedfrom

thesecondweek.ThepotentialforFPNtoinducespeciﬁc

neurotoxicitywasreportedbyotherstudiesinmice[37],

rats[38]andhuman[16].

Inthepresentstudy,exposureofrattotheFPNresulted

inaslightlyreducedbodyweightandsigniﬁcantelevation

inrelativeliverandkidneyweightsespeciallyathigh

con-centration(groupIV).Theslightdecreaseinbodyweight

maybeduetotheoxidativestressand neurotoxicityof

FPN,whiletheincreaseinrelativeliverandkidneyweights

couldbeattributedtotherelationshipbetweentheliver

weightincreaseandvarioustoxicologicaleffectsortothe

reduction in body weight gain of experimentalanimals

[17–19,39].FPNtreatmentcausedanincreaseinliverand

thyroidglandweightofrats[40].

The results of the present study indicate that

sub-chronicexposureto1and10mg/LofFPN(groupsIIIand

IV)causeliverand kidneydamage intreated rats

com-paredtocontrolasshownbyincreasesinserummarker

enzymesAST,ALT,ALPandLDHalongwithincreasesin

creatinine,uricacidandtotalproteinlevelsina

concen-trationdependentmanner.Liverenzymesinserum e.g.,

AST,ALT, ALP and LDH are mainly usedin the

evalua-tionofhepaticdamage.Transaminases(ASTandALT)play

animportantroleinaminoacidscatabolismand

biosyn-thesis.Theyareresponsiblefordetoxiﬁcationprocesses,

metabolismandbiosynthesisofenergeticmacromolecules

fordifferentessentialfunctions[41,42]andusedas

spe-ciﬁcindicatorsforliverdamage[43].Theincreaseinthese

enzymesmaybeduetoliverdysfunctionanddisturbance

inthebiosynthesisoftheseenzymeswithalterationinthe

permeability ofthelivermembrane takesplace [44].In

thecurrentstudy,theelevationinLDHactivityinserum

ofrat exposed toFPN maybedue tothehepatocelluar

necrosisandleakageof theenzymeintotheblood[45].

OraladministrationofFPNatdose0.5mgkg−1day−1for21

daysinducedsigniﬁcantincreasesinplasmaLDH,AST,acid

phosphatase, gamma-glutamyl transferase, total plasma

(6)

0 1 2 3 4 5 6 I II III IV S OD (U/mg pr ot e in ) Group c c b a SOD 0 2 4 6 8 10 12 I II III IV CAT (μmol/ mg pr otein) Group b b _b a CAT 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 I II III IV GST (μmol/ mg protein) Group GST c b b a 0 1 2 3 4 5 6 I II III IV GP x ( U/ mg protein ) Group c b b a GPx 0 0.01 0.02 0.03 0.04 0.05 0.06 I II III IV GSH ( μm o l/ mg p ro tein) Group d c b a GSH 0 20 40 60 80 100 120 I II III IV LPO (n m ol/g pr ot e in ) Group a ab b c LPO

Fig.2.Oxidativestressbiomarkers;SOD,CAT,GPx,GST,GSH,LPOinkidneytissueofmaleratsexposedto01,1.0and10mg/Lofﬁpronil(groupsII,IIIand IV,respectively)for45daysindrinkingwaterandcontrolgroup(I).valuesrepresentedaremeans±SE.Meanshavingthesamelettersarenotsigniﬁcantly differentfromeachother,p<0.05.

resultsaresupportedbyotherstudiesconductedonothers

insecticides[17,18,39,45,47,48].

Serumuricacidandcreatinineconcentrationswere

sig-niﬁcantly increased in FPN-treated rats(group IV).The

increasedinuricacidmaybeduetodegradationofpurines

and pyrimidines or increase uric acid concentrationby

eitheroverproductionortheinabilityofexcretion[39,47]

andanelevationofcreatininelevelin theblood isthus

anindicationofimpairedkidneyfunction[49,50].In

addi-tion, the increase of total protein in FPN-treated rats

(groupsIIIand IV) maybedue to theliverand kidney

dysfunctions,partiallybecauseofthehighelevationofthe

serumenzymes[17,18,39].Otherinvestigationsshowedan

increaseofurea,creatinineandtotalproteinintheserumin

ratsexposedtodifferentpesticides[43,44,49].Ratorgans

affectedbychronicFPNexposureincludethethyroid,liver

andkidney[51].

ResultsrevealedthatFPNtreatmentcausedoxidative

stressintheliverandkidneyofmalerats,whichis

evi-dentfromthegenerationoflipidperoxidation(LPO).LPO

is knowntodisturbtheintegrityofcellularmembranes

andimplicatedinthepathogenesisofvariousliverand

kid-neyinjuries[52,53].Therefore,ithasusedasbiomarkers

of pesticides induced oxidative stresses [54] and

sug-gested as one of the molecular mechanisms involved

in pesticides-induced toxicity [55]. Increased levels of

malondialdehyde (MDA), a lipid peroxidation produced

in theliverand kidneyof FPN-treatedratsmaybe due

toincreased production ofreactiveoxygen metabolites,

especiallyhydroxylradicalsandalterantioxidantdefense

system [19]. Tukhtaev et al. [20] found that prolonged

exposureto low dosesof FPN increasedLPO inliver of

pregnantratsandtheiroffspring.Availablestudiesindicate

thatinsecticidesincreasedLPOinanimals[45,52,53].

FPN-treatment increased oxidative stress by altering

theenzymeactivitiesassociatedwithantioxidantdefense

mechanismsintheliverandkidneyofmalerats.Itcaused

decreasesintheactivityofantioxidantenzymesSOD,CAT,

GPxandGSTandlevelofGSHinaconcentrationdependent

(7)

Fig.3.PhotomicrographofliversectionsstainedbyH&Eforhistopathologicalchangesshowing:control(groupI)showing(A)thenormalhistopathological structureofhepaticlobule(H)(64×).10mg/LofFPN(groupIV)showing(B)ballooningdegeneration(D)ofhepatocytes,portalinfiltrationwithinflammatory (I)cellsandovalcellproliferation(400×)and(B1)showingballooningdegeneration(D)ofhepatocytesandfocalhepatichemorrhage(H)(400×).FPN at1.0mg/L(groupIII)showing(CandC1)degenerationofhepatocytesandportalinfiltrationwithinflammatorycells(400×).FPNat0.1mg/L(groupII) showing(D)congestion(C)ofcentralvein(400×)and(D1)cytoplasmicvacuolization(V)offocalhepatocytesandcysticdilationofbileduct(400×).

togethertopreventtheeffectofROS intissuesand are

activeinthedefenseagainstoxidativecellinjurybymeans

oftheirbeingfreeradicalscavengers[56].Therefore,SOD,

CATandGPxareconsideredﬁrstdefensesthatprotectcell

macromolecules fromoxidativedamage.In thisrespect,

SOD acceleratesthedismutationof superoxideanionto

lessreactivemolecule(H2O2)whichisrapidlyconvertedto

waterandoxygenbyCATandGPx[52,57].Inthepresent

study,thedecreasedinSOD,CATandGPxactivityinthe

liverandkidneyinratsexposedtoFPNcouldbedueto

excessproductionofO2•−whichrapidlyconvertedtoH2O2

by SODand towater byCATand GPx.Previousstudies

reportedthatpesticidese.g.chlorpyrifos,triazophosleads

todecreasein SOD,CATandGPxinliverand kidneyof

rats[17,18,21,45,52,53,57].Inagreementwithourstudies,

asigniﬁcantdecreaseinSODactivitywasobservedinliver

ofpregnantratsandtheiroffspringexposedtolowdoses

ofFPN[21],alsoSOD,CATandGPxactivityweredecreased

bydiazinontreatmentinmice[58].FPNcanberesponsible

forincreasesintheproductionofreactiveoxygenspecies

(ROS)incells,whichleadtoincreasedlipidperoxidation

levelsandoxidativestress[63].Ithasbeenreportedthat

FPNwasresponsibleforoxidativestressinC.carpio,which

wasevidentthroughalterationsonantioxidantenzymes

andincreasedlipidperoxidationlevels[59].

GSTconstituteafamilyofenzymesthatplayan

impor-tantrole inmetabolism,particularlyindetoxiﬁcationof

xenobiotic e.g. pesticides and intracellular transport of

metabolites[60].Theseenzymescatalyzetheconjugation

ofreactiveelectrophileswithglutathione(GSH),andhave

been implicated with the potential of forming reactive

intermediatesinparticularwhenGSHlevelsinthecellsare

attenuated,consequentlyresultingintoxicologicaleffects

[61,62].Inthepresentstudy,theactivityofGSTandthe

levelofGSHweresigniﬁcantlydecreasedinliverand

(8)

Fig.4. PhotomicrographofkidneysectionsstainedbyH&Eforhistopathologicalchangesshowing:control(groupI)showing(A)thenormal histopatholog-icalstructureoftheglomerular(G)andtubules(T)atthecortex(64×).10mg/LofFPN(groupIV)showing(B)focalnecrosis(N)ofrenaltubulesassociated withinﬂammatory(I)cellsinﬁltrationaswellasatrophyofglomerulartuft(400×)and(B1)vacuolation(V)ofepithelialliningrenaltubulesandfocal hemorrhage(H)(400×).FPNat1.0mg/L(groupIII)showing(C)vacuolation(V)ofepithelialliningrenaltubulesandfocalhemorrhage(H)(400×).FPNat 0.1mg/L(groupII)showing(D)vacuolation(V)ofepithelialliningrenaltubules(400×).

inantioxidantenzymeactivitiesinliverandkidneytissues

wasduetocellularinjuryanddeathofhealthycellsthatare

abletorespondtotheoxidativeinsult.Inaddition,itmay

beduetotheinsufﬁcientdetoxiﬁcationcapacityofFPNand

damagecausedbyreactiveoxygenspecies.FPNreduced

theactivityofGSTandGPxintadpolesoffrogs[60].

Sim-ilarsigniﬁcantdecreasedinGSTactivitywereobservedin

theliverofchlorpyrifostreatedrats[17].

The change in oxidative stress, liver and kidney

biomarkersinratsexposedtotheFPNcorroboratedthe

histopathologicallesionsobservedinthisstudy.The

his-tologicalobservationsofliverrevealseveredegeneration,

inﬁltration,inﬂammationandfocalhepatichemorrhage.In

kidneyseverenecrosis,inﬂammation,atrophyof

glomeru-lartuft,vacuolationandfocalhemorrhagewereobserved.

Theseobservationsindicatedmarkedchangesinthe

over-allhistoarchitectureofliverandkidneyinresponsetoFPN.

ThesechangescouldbeduetoFPNtoxiceffects

primar-ilybythegenerationofreactiveoxygenspecies causing

(9)

HistologicalobservationsontheliverandkidneyofFPN

treatedratswerecomparablewithotherstudiesconducted

onchlorpyrifos[18],malathion[63],methylparathion[64],

fenitrothion [65] and otherinsecticides[66] that reveal

insecticidesareknowntoinduceanumberof

histopatho-logical changesin liverand kidney.Theobservations in

thepreviousmentionedstudiesareincorroborationand

supportourresults.

5. Conclusion

Inviewofthedataofthepresentstudy,itcanbe

sug-gested that FPN induced liver and kidney damage that

corroboratedwiththehistopathologicallesions.FPN

expo-sureproducedmarkedelevationsinLPOandalterationsin

antioxidantbiomarkersinliverandkidneytissues.

There-fore,thechangesinliverandkidneyfunctionscouldbe

due tothegenerationofROS,which causingdamageto

membraneandallcellcomponents.

Conﬂictofinterest

Nonedeclared.

Transparencydocument

TheTransparencydocumentassociatedwiththisarticle

canbefoundintheonlineversion.

Acknowledgment

TheauthorsthankProf.Dr.KawkabAbdel-Aziz,

Pathol-ogy Department, Faculty of Veterinary Medicine, Cairo

University,Egyptfordemonstratingthehistopathological

slides.

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