histological effect of nicotine on adrenal zona fasciculata and the effect of grape seed extract with or without withdrawal of nicotine

Histological effect of nicotine on adrenal zona fascicu-withdrawalprocess[5].Intheadrenalmedulla,nicotine bindstoitsreceptors,leadingtoincreasedheartrate,blood pressure,respiratoryrate,a

Please cite this article in press as: Khalaf HA, et al Histological effect of nicotine on adrenal zona

fascicu-ContentslistsavailableatScienceDirect

j ou rn a l h o m e pa g e :w w w e l se v i e r c o m / l o c a t e / j m a u

Original Article

Histological effect of nicotine on adrenal zona fasciculata and

the effect of grape seed extract with or without withdrawal

of nicotine

Hanaa Attia Khalafa,∗, Fatma M Ghoneima, Eetmad A Arafata,

Q5

El-Hassanen M Mahmoudb

Keywords:

nicotine

Cigarettesmokingisharmfultothehealthofbothsmokersandnonsmokers.Itisamajor causeofdeath.Thisstudyaimedtoinvestigatethestructuralchangesinthezonafasciculata

ofalbinoratscausedbynicotineandtheprotectiveeffectofgrapeseedswithorwithout thestoppageofnicotineadministration.Thirty-fiveadultmaleratswereusedandequally dividedintofivegroups:negativeandpositivecontrolgroups(GroupsIandII), nicotine-treatedgroup(GroupIII),nicotine-andgrapeseedextract-treatedgroup(GroupIV),and nicotinewithdrawalandgrapeseedextract-treatedgroup(GroupV).Adrenalglandswere dissectedandpreparedforhistologicalstudies.Themajorityofzonafasciculatacellsof GroupIIIshowedstrikingchangesintermsofswellingofthecellswithmarkedcytoplasmic vacuolation,manypyknoticnuclei,andincreasedimmunoexpressiontocaspase3 anti-bodies.Byelectronmicroscopy,amarkedincreaseinlipiddepositionwithitsappearance

inthecapillarybetweenzonafasciculatacellswasnoticed.Heterochromaticnucleiand dilatedsmoothendoplasmicreticulumwerenoted.Degeneratedmitochondriaandsome mitochondriathathadcavitationwithaprogressivelossoftheircristaewereseen.The zonafasciculatacellsofGroupIVwerepartiallyimproved,whileinGroupV,thosecells showedcompleteimprovement.Wecanconcludethatnicotinecausesseverehistological

Q6

changesinzonafasciculatacells.Grapeseedextractcanpartiallyamelioratethesechanges, andcompleterecoveryisachievedwithgrapeseedextractafterthestoppageofnicotine administration

©2016SaudiSocietyofMicroscopes.PublishedbyElsevierLtd.Thisisanopenaccess

articleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Introduction

Many thousands of components are present in a

cigarette.Nicotineisoneofthefewliquidalkaloidsthatare

commonlyabsorbedbythebodythroughcigarette smok-ing(acigarettecontainsapproximately2mgofabsorbed nicotine)[1,2]andtobaccoingestion(nicotinerepresents roughly0.6–3.0%ofthedryweightoftobacco)[3].Nico- Q7

tineexistsasapowerfulparasympathomimeticalkaloidin thenightshadegroupofplants andis alsofoundin the leavesofNicotiana rustica[4].Variousstudiesindicated thatnicotinecausesacriticalriseinserumcortisol,then cortisollevelsfallwithtime aswellasduringtheearly

http://dx.doi.org/10.1016/j.jmau.2016.11.001

( http://creativecommons.org/licenses/by-nc-nd/4.0/ ).

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Please cite this article in press as: Khalaf HA, et al Histological effect of nicotine on adrenal zona

fascicu-withdrawalprocess[5].Intheadrenalmedulla,nicotine

bindstoitsreceptors,leadingtoincreasedheartrate,blood

pressure,respiratoryrate,andbloodglucoselevelscaused

byincreasedadrenalineandnoradrenalinesecretion[6]

Inlesserdoses,nicotineactsasastimulant,whileitcanbe

riskyatahighdose(>50mg).Thisstimulatingeffectmakes

nicotinehighlyaddictive.Thisaddictivenessofnicotineis

themainreasonforthepersistentusageoftobacco

prod-ucts,whichinturnresultsinmostofthetobacco-related

diseases[7]

Nicotineisassociatedwithcardiovasculardisease,

con-genitalanomalies,andpoisoning[8].Moreover,nicotine

hasbeenfoundtodistracttheantioxidantdefense

mech-anismsinrats[9,10].Oxidationisachemicalreactionthat

cancreatefreeradicals(suchassuperoxide,nitricoxide,

and hydroxylions) all ofwhich have anunpaired

elec-tron.Duringnormalmetabolism,freeradicalsarecreated,

andtheirlevelsareenhanced duringcontactwith

envi-ronmentalpollutantssuchascigarettesmoke[11].Fatsin

thecellmembranearesusceptibletodamagebythesefree

radicals.Theseelectronscanstartnewreactions,anditis

Q8

knownasreactiveoxygenspecies.Inturn,oxidationcauses

deathofordamagetothecell.Antioxidantssuppressthese

effects throughthe removalof free radicals and

inhibi-tionofotheroxidativereactions[12].Thereactiveoxygen

species are offset naturally by antioxidant defense

fac-tors,suchassuperoxidedismutase,whichalreadyexistin

ourbodyunderphysiologicalconditions.Duringoxidative

stress,oxidationoutstripsthenaturalantioxidantfactors

Q9

Consequently,itcausesdestructiveprocessesthatcanlead

tocelldeath[13]

Grape (Vitis vinifera) is one of the extensively

con-sumedfruitsintheworld.Grapehasmanyactivegradients,

including flavonoids, polyphenols, anthocyanins,

proan-thocyanidins,andprocyanidins[14].Itwashypothesized

thatproanthocyanidinextractactsasafreeradical

scav-engerandanantioxidant[15].Further,grapemodifiesa

lotof biologicalreactions, andit hasanti-inflammatory,

anticarcinogenic,andantiagingeffects;therefore,itis

con-sideredacytoprotectiveagent[16]

Thehazardous effect of nicotine regarding the

mor-phologicalchangesintheadrenalcortexisstillnotwell

established.Accordingly,theaimofthepresentstudyis

toinvestigatethestructural changescaused bynicotine

administrationtotheadrenalzonafasciculata(ZF)cellsof

albinorats,aswellasthepossibleprotectiveeffectofgrape

seeds,withorwithoutthestoppageofnicotine

administra-tion,onthesechanges

Materials and methods

Chemicals

NicotinewaspurchasedfromSigmaChemicalCo.(St

Louis,MO,USA)intheformofpowder{nicotinehydrogen

tartratesalt [(–)-1-methyl-2-(3-pyridyl) pyrrolidine

(+)-bitartratesalt]}.Nicotinewasdissolvedindistilledwater

Grape seed extract (GSE) was purchased from Arab

Gelatin Pharmaceutical Products Company, Alexandria,

Egypt.Itwasdissolvedinsaline

Investigationalprotocol TheprotocolofthisstudywasacceptedbytheEthical CommitteeoftheFacultyofMedicine,Mansoura

The presentstudyincluded 35adultmale rats(each Q11

weighing about180–200g) Priortothe study,the ani-malswerekeptinaquietandnonstressfulenvironment for 1 week.Animalswerefed adlibitumandpermitted freeaccesstowaterthroughouttheinvestigationaltime Ratsweredividedequallyintofivegroups(7ratsineach group):.GroupI(negativecontrolgroup):Animalswere given saline via intragastrictube and injected subcuta-neouslywithdistilledwaterfor1month.GroupII(positive control group): Animals received GSE daily (at a dose

200mg/kgbodyweight/d),dissolvedinsaline,viaanintra- Q12

gastrictubefor1month[17].GroupIII(nicotine-treated group):Animalsweregivennicotinepowderliquefiedwith distilledwaterandinjectedsubcutaneouslyat2.5mg/kg/d [18] for 1 month Group IV (nicotine- and GSE-treated group):Animalswere simultaneouslygiven GSE(atthe samedoseasthatofGroupII)andnicotine(atadosesimilar

tothatofGroupIII)for1month.GroupV(nicotine with-drawalandGSE-treatedgroup):Eachanimalreceivedthe samedoseofnicotineandGSEasthoseofGroupIVfor1 month;theywerestudied2monthsafterthestoppageof nicotineadministrationtoassessitswithdrawaleffect Q13

Aftereachexperiment,animalsweresacrificed.Partial fixationofspecimenswasdonebyintracardiacperfusion using 2.5% phosphate bufferedglutaraldehyde (PH 7.4) Adrenalglandsweredissected,weighted,andpreparedfor histological, immunohistochemical, and electron micro-scopicstudies

Histologicalstudy TherightadrenalglandswerecutandfixedinBouin’s solution.Then, dehydration ofthespecimens in alcohol wasdone,followedbyclearinginxyleneandembeddingin paraffin.Usingarotarymicrotome,sectionsof5␮m thick-ness wereobtained, whichwereplaced onclean slides Next, slides were stained with hematoxylin and eosin accordingtothemethodofBancroftandLayton[19]

Immunohistochemicalstudy Sections were placed on positive slides and then immunostained by the avidin–biotin technique [20] Deparaffinizationandrehydrationoftheslideswere per-formed,followedbyrinsingintapwater.Forblockingof endogenous peroxidaseactivity,theslides were embed-dedin0.01%H2O2;thentheantigenicsitewasunmasked

by putting sections in 0.01M citrate buffer (pH 6) for

30minutes.Afterthatboilingwasdoneinamicrowavefor

6minutes.Toomitnonspecificbackground,theslideswere Q14

incubatedfor20minutesindilutednormalrabbitserum, followed by incubation in primary antibody (caspase 3/CPP32rabbitpolyclonalantibody)at1/50–1/00dilution for2hours.Subsequently,theslideswereincubatedusing Q15

theavidin–biotin complexsubstrate for 60minutes and then in peroxidase substrate solutionfor 6–10minutes

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Please cite this article in press as: Khalaf HA, et al Histological effect of nicotine on adrenal zona

fascicu-Table 1

< 0.001 ** 0.222 *** 0.560 ****

* Group I versus Group II.

** Group I versus Group III; significant

*** Group I versus Group IV.

**** Group I versus Group V

Lastly, counterstaining by hematoxylin was performed

Forthenegativecontrolslide,theused1ryantibodywas

exchangedbyphosphatebuffersaline.Tonsilwas

consid-eredapositivecontrol[21]

Thepolyclonalrabbitcaspase3/CPP32antibody

[Diag-nosticBiosystems, Pleasanton,CA,USA(format:purified

immunoglobulinfractionofrabbitantiserumagainst

cas-pase3;EmergoEurope,theHague,TheNetherlands)]was

usedforstainingthecytoplasmicantigens

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Electronmicroscopicstudy

Smallpiecesoftheleftadrenalcortex wereusedfor

transmissionelectronmicroscopyandfixedfor2hoursin

2.5%glutaraldehydebufferedwith0.1McacodylateatpH

7.2;thenthespecimenswerewashedbythisbuffer.Then

postfixationattheroomtemperaturewasperformedby

1%osmiumtetroxidebufferedwithphosphatefor2hours,

followedbyalcoholdehydration.Specimenswere

embed-dedinepoxyresinmixture,afterimmersioninpropylene

oxide.Semi-thinsectionsofabout1␮mthicknesswerecut,

stainedby1%toluidineblue,andthen examinedwitha

lightmicroscope.Next,ultrathinsections(80–90nm)were

obtainedusinganLKBultratome,andstainedbyuranyl

acetateandleadcitrate[22].Theultrastructuralanalysis

wasdoneusingatransmissionelectronmicroscope(Joel

TEMCS100)intheElectronMicroscopicUnit,Facultyof

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Science,El-Shatby,AlexandriaUniversity,Egypt

Morphometricstudy

Byimageanalyses,theareapercentofthe

immunore-action of the studied group was evaluated using five

immune-stained slidesfor everygroup.Theslides were

photographed with an Olympus digital camera

(E24-10 mega pixel; Olympus, China) fitted on an Olympus

Q18

microscopethrough a 0.5× photoadaptor, using a 40× objective lens The resulted images were evaluated by

anIntelCore13computerusingVideoTestMorphology software (Video Test, Saint Petersburg, Russia) using a Q19

specificbuilt-inroutineforcalculatingtheareapercentof immunoreaction[23]

Biochemicalstudy Bloodwascollectedfromtheleftventricleoftheratsto measurethelevelofmalondialdehyde(MDA)[24]

Statisticalanalysis Weights of adrenal, morphometric, and biochemical datawereevaluatedusingtheStudentttest,and calcu-latedasmeanvalue±standard deviation.A probability valueofp<0.05wasconsideredsignificantandp<0.01 highlysignificant[25]

Result

Statisticaldata

In Table 1, the adrenal weightof groupIII was sig-nificantly increased compared with that of the control groups.Inaddition,theadrenalweightofGroupIVwas increasedcomparedwiththatofthecontrolgroups,but thisincreasewasstatisticallynonsignificant.However,the adrenalweightofGroupVwassimilartothatofthecontrol groups

TheserumlevelofMDAwassignificantlyincreasedin GroupIIIcomparedwiththatinGroupI.Moreover,a non-significantincreaseoftheserumlevelofMDAwasdetected

inGroupIVcomparedwiththatofGroupI,buttheMDA levelofGroupVwassimilartothatofGroupI(Table2)

Table 2

< 0.001 ** 0.073 *** 0.978 ****

* Group I versus Group II.

** Group I versus Group III.

*** Group I versus Group IV; significant.

**** Group I versus Group V.

MDA = malondialdehyde.

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fascicu-Theareapercentofimmunoexpressionofcaspase3was

significantly increasedin Group IIIand nonsignificantly

increasedinGroupIVcomparedwiththecontrolgroups

ThisvalueinGroupVwassimilartothatinGroupI(Table3)

Histologicalresult

Lightmicroscopicexamination

Examinationofthehematoxylinandeosin-stained

sec-tionsofthecontrolgroupsrevealedthenormalhistological

architectureoftheZF.TheZFwasthemiddleandthe

broad-Q20

estzoneoftheadrenalcortex.Itwasplacedbetweenthe

zonaglomerulosaandthezonareticularis(Figure1A).Cells

oftheZFwerearrangedinparallelcordsseparatedbyblood

sinusoids,andthecordswereofoneortwocellsinwidth Thecells werepolyhedralwithcentral,rounded vesicu-larnuclei.Thecytoplasmwasfaintlystainedacidophilic andvacuolated.Binucleatedcellscouldbeseen(Figure1B) Immunostainedsectionsshowedweakpositiveand min-imal cytoplasmicreactions tocaspase 3 of theZF cells (Figure2A)

Inthenicotine-treatedgroup(GroupIII),therewasa disorganizationoftheZFarrangement.Themajorityofthe

ZFcellsshowedstrikingchangesintheformofswelling

ofthecells withmarkedcytoplasmicvacuolation.Many nucleiappearedpyknotic,anddilatationofbloodsinusoids were observed (Figures 1 and 1D) In the immunos-tained sections, increased immunoexpression of the ZF

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cells arranged in parallel cords disjoined by blood sinusoids (zigzag arrows) The cords are one or two cells in width The cells have central, rounded vesicular nuclei (arrows) and vacuolated, faintly stained acidophilic cytoplasm Binucleated cells (crossed arrow) are seen (C,D) Some swollen cells (curved arrows) with cytoplasmic vacuolation (arrow heads) are noticed in the ZF of Group III (nicotine-treated group) Many pyknotic nuclei (tailed arrows) are seen Note the appearance of dilated blood sinusoids (zigzag arrows) (E) Most of the ZF cells of Group IV (nicotine- and GSE-treated group) are normal The cells are polyhedral and arranged in parallel cords disjoined by blood sinusoids (zigzag arrow) The cells have central rounded vesicular nuclei (arrows) and vacuolated, faintly stained acidophilic cytoplasm Note the appearance of a few pyknotic nuclei (tailed arrows) and cytoplasmic vacuolation (arrow heads) (F) ZF cells of group V (nicotine withdrawal and GSE-treated group) showing a normal histological structure of the ZF The cells are polyhedral and arranged in parallel cords disjoined by blood sinusoids (zigzag arrow) The cells have central, rounded vesicular nuclei (arrows) and faintly stained acidophilic vacuolated cytoplasm Binucleated cells (crossed arrow) can be seen.

C = capsule; GSE = grape seed extract; H&E = hematoxylin and eosin; ZF = zona fasciculata; ZG = zona glomerulosa; ZR = zona reticularis.

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fascicu-Table 3

< 0.001 ** 0.233 *** 0.555 ****

* Group I versus Group II.

** Group I versus Group III; significant.

*** Group I versus Group IV.

**** Group I versus Group V.

cells tocaspase3 antibodieswasprominentlyobserved

(Figure2B)

In comparison with Group III, the light microscopic

examinationofhematoxylinandeosin-stainedsectionsof

thenicotine-andGSE-treatedgroup(GroupIV)revealeda

normalarchitectureofthecortex.However,afewZFcells

wereswollen,andothershadpyknoticnucleiand

vacuo-latedcytoplasm(Figure1E).Theimmunostainedsections

oftheZFcellsofthisgroupshowedamildpositive

cyto-plasmicreactiontocaspase3antibodies(Figure1C)

In the nicotine withdrawal and GSE-treated group

(Group V), the ZF showed an almost normal

histologi-cal structure The cells were arranged in parallel cords

separatedbyblood sinusoids Thecellswerepolyhedral

with central, rounded vesicular nuclei and faintly

aci-dophilicvacuolatedcytoplasm(Figure1F).Examinationof

theimmunostainedsectionsofthisgrouprevealeda

reac-tionalmostsimilartothatofthecontrolgroupswherethere

wasaminimalpositivecytoplasmicreactiontocaspase3 antibodiesintheZF(Figure2D)

Electronmicroscopicresults Examinationof the ultrathin sectionsof the control groupsshowedthenormalultrastructuralimageoftheZF cells.Thenucleuswaseuchromaticwithprominent nucle-olus.Themitochondriaweresphericalandvariableinsize withdenselypackedvesicular cristae.Plenty ofsmooth endoplasmicreticulum(sER)tubulesanda lipiddroplet wereseen(Figure3A)

TheZFofGroupIIIshowedamarkedincreaseinlipid deposition,withtheappearanceofalipiddropletinthe capillarybetweentheZFcells(Figure3B).Somenucleihad irregularoutlineswithcondensedchromatin(Figures3B and3E)andotherswereheterochromatic(Figures3 and 3D).Manydegeneratedmitochondria(Figure3D), bizarre-shapedmitochondria(Figure3D),andsomemitochondria

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fascicu-Please cite this article in press as: Khalaf HA, et al Histological effect of nicotine on adrenal zona

fascicu-possessing cavitation with a progressive loss of their

cristae(Figures3Dand3E)wereallseenwithdilatedsER

(Figures3 and3E).Manyautophagicbodies(Figures3D

and3E)weredetected

UltrastructuralexaminationoftheZFcellsofGroupIV

showedanearlynormalstructureoftheZFcells

Euchro-matic nucleus with a prominent nucleolus and a few

lipiddropletswereseen.However,fewmitochondriawere

degeneratedandotherswerevacuolated.Moreover,some

dilatedsERweredetected(Figure3F)

Q21

AnelectronmicroscopicimageoftheZFcellsofGroup

V showed an almost normal histological structure An

euchromaticnucleus,afewlipiddroplets,andsERwere

seen.Mitochondriaweresphericalandvariableinsizewith

denselypackedvesicularcristae(Figure3G)

Q22

Discussion

Cigarettesmokingisharmfultothehealthofboth

smok-ersandnonsmokers.Itisamajorcauseofdeath,accounting

foroneinfivedeathsintheUnitedStates[26].Nicotineis

oneofthedangerouscomponentsofacigarette[27].Its

biologicaleffectsarewidespreadandextendtoallsystems

ofthebodyincludingcardiovascular,respiratory,renal,and

reproductivesystems.Inseveralstudies,nicotinehasalso

beenfoundtobecarcinogenic[6].Furthermore,nicotine

hasbeenfoundtodistracttheantioxidantdefense

mecha-nismsinrats,andincreaselipidperoxidationanddeplete

antioxidantsintissues[9,10]

The currentstudy wascarriedout toinvestigatethe

structuralchangescausedbynicotineadministrationtothe

adrenalZFcells,aswellasthepossibleeffectofgrapeseeds

withorwithoutthestoppageofnicotineadministration

In our work, the majority of the ZF cells of the

rats treated by nicotine (Group III) showed destructive

structuralandultrastructuralchanges.Themoststriking

changes detectedwere in theformof swelling of cells,

markedcytoplasmicvacuolation,anda markedincrease

inlipiddeposition.Somemitochondriaweredegenerated

andotherspossessedcavitationwithaprogressivelossof

cristae.Manypyknoticshrunkennucleiwereseen,andit

wasprovedbyasignificantincreaseintheareapercent

ofthecaspase3-stainedsections.DilatedsERand many

autophagicvacuolescontainingdegeneratedmitochondria

couldbedetected

Thisresultwasinharmonywiththoseofotherauthors;

Osman[18]reportedthataccumulationoflipiddroplets

andappearanceofcytoplasmicvacuolationintheZFcells

aftertreatmentwithnicotinemaybeduetothe

impair-ment in the synthesis of glucocorticoids As the ZF is

responsibleforsynthesisandsecretionofglucocorticoids, thedisruptedsteroidogenesishadavitaltoolinthetoxicity Q23

oftheadrenalcortex.Thismayoccurasaresultof disrup-tionofcytochromeP450enzymes;therefore,cholesterol biosynthesiswillbeinhibited.Thiswillleadto accumula-tionoflipiddropletsandcytoplasmicvacuolationoftheZF cells[28].Thisalsowasconstantwiththedataofprevious researcherswhonoticedaccumulationoflipiddropletsin thecellsoftheZFandzonareticularisaftersuppressionof steroidogenesisviadexamethasoneadministration[29]

Theadrenalglandexpressesthelevel-limitingenzyme

of steroidogenesis, acute regulatory protein (StAR), and cytochrome P450 cholesterol side chain cleavage (P450scc), which is crucial for the secretion of steroid hormones [30] StAR settles the passage of cholesterol from the outer to the inner mitochondrial membrane, whichisthefirstandrate-limiting stagein steroid syn-thesis, while P450scc splits the cholesterol side chain, convertingcholesteroltopregnenolone,thepredecessorof steroidhormones[31].Itwasfoundthatnicotinetreatment repressed StAR/P450scc expressions, thereby inhibiting cortisol secretion Furthermore, the expression of StAR stayedinhibitedmorethan15daysafterthestoppageof nicotinetreatment[32]

Anotherinvestigatorstatedthattheswellingand vac-uolationthatwasdetectedinthemitochondriaoftheZF cellspossiblyresultedfromthesuppressionofcholesterol

to pregnenolone conversion Consequently, cholesterol accumulates within the mitochondria Subsequently, it undergoessignificanthypertrophyandcavitation[28]

As the mitochondria and sER play important roles

in steroidogenesis, the lesions detected in them were sufficient toinhibitsteroid synthesis,leading tofurther accumulationofcholesterolinthemitochondria Q24

The two main apoptotic ways within a cell are the extrinsicpathway(receptorpathway)andtheintrinsicone (mitochondrial pathway) The intrinsic pathway is trig-geredbymanyintrinsicsignalsincludingoxidativestress viatheinvolvementofthemitochondria[33].Caspase3is activatedbybothextrinsicandintrinsicpathways,soitis usedasanapoptoticmarker

Many studies revealed that caspase 3 immunoreac-tionwasincreasedbyapoptosis[34].Moreover,nicotine activated specific intracellular death-related pathways, leading toincreased caspase3 immunoreaction in Ley-digcells[35].Thesefindingsarealsoincloseagreement withthatpresented byMachaalaniand colleagues[36], whoobservedthatpostnatalnicotineexposurecanleadto increasedcaspase3reactioninthehypoglossal,gracile,and dentategyrusofthebrainofmalepiglets.Increasedcaspase

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fascicu-3expressionwasdetectedinhumangingivalfibroblasts

treatedwithnicotineduetodecreasedcellintegrityand

increasedapoptosis[37]

Inthepresentstudy,therewasastatisticallysignificant

increaseintheadrenalweightandtheserumlevelofMDA

ofGroupIIIcomparedwiththatofGroupI

Enlargementoftheadrenalssuggeststhepossibilityof

cellulardamagebynicotine,leading toaccumulationof

lipidsinadrenalcells andswellingofcellorganelles(as

seenonthehistologicalsections)andhenceanincreasein

theadrenalweight

ThesedatawereconfirmedpreviouslybyIranloyeand

Bolarinwa[38].Theynoticedthattherewasanincreasein

theweightsofadrenalsafter30daysofnicotine

adminis-tration

MDAis a predictor of lipid peroxidation (used as a

markerforthedetectionoftissuedamagecausedbyfree

radicals);sotheincreasedserumlevelofMDAobservedin

thepresentworksuggeststhatnicotineinducesoxidative

stress.Thecurrentresultswereinagreementwiththedata

ofRazaliandcolleagues[39]whoannouncedthatfatand

cholesterolofthecellmembranearetargetsoffree

radi-calattack;hence,lipidperoxidationcanoccur,asevident

fromtheincreasedMDAlevel.Thesedataalsocoincidewith

thatattainedbyParlakpinarandcolleagues[40]whostated

that,innormalcircumstances,reactiveoxygenspeciesare

eradicatedbyintrinsicantioxidantenzymessuchas

super-oxidedismutase,catalase,andglutathioneperoxidase.This

is achievedby increased MDAlevels and a decrease in

superoxidedismutaseactivity

Nicotineisconsideredapotentoxidantasitproduces

freeradicalsthatreactwiththecellmembrane,leadingto

oxidativedamageandcellulardeath[41]

Moreover, it was established that nicotine induces

oxidativestressboth invitroand invivo, increasingthe

amountsoffreeradicalsandlipidperoxidationproductsin

culturedcells[42].Nicotineisfirstoxidizedintocotinine

intheliver,generatingfreeradicalsandinducingoxidative

injurytotissues[43]

Thefindings of thepresent work revealed a normal

architectureoftheZF ofGroupIV,in spiteof the

pres-ence of a few swollen cells with pyknotic nuclei and

vacuolatedcytoplasm.Degeneratedandvacuolated

mito-chondriawerealsodetected.Therewasa mild positive

cytoplasmicreactiontocaspase3antibodythatwasproved

by the morphometric analysis In addition, the adrenal

weightandserumlevelofMDAofGroupIVwere

nonsignif-icantlyincreasedcomparedwiththoseofGroupI

Fromtheseresults,itwasevidentthattheameliorative

effectofGSEispossiblyduetoitsantioxidantpotency

TheseresultsagreewiththosepublishedbyAhmedand

colleagues[44]whorevealedthatGSEprotectstheratliver

againsthepatotoxins and reducesliver MDA.They

pos-tulatedthatthis wasduetotheactionof GSEasa free

radicalscavenger.Also,Zhangetal[45]statedthatGSEis

Q25

apotentantioxidant,andpreventsarsenic-inducedrenal

fibrosisand dysfunction.Furthermore,itwasfoundthat

GSEprotectsmanyorgans(heart,kidney,andliver)from

theoxidativestressofcisplatin[17]

GSEisasourceofproanthocyanidins,aclassof

pheno-liccompoundsthatincreaseintracellularvitaminClevels,

decreasecapillarypermeability,actasfreeradicals scav-engers,andinhibitlipidperoxidation[46]

Thestructure oftheZFcells ofGroupVof ourwork wasintegraltothearchitectureofGroupI.Furthermore, therewasaminimalpositiveimmunoreactiontocaspase3 antibodysimilartothatofthecontrolgroupsanditwas confirmedbythemorphometric analysis.Moreover,the adrenalweightandserumlevelofMDAofthisgroupwere similartothoseofGroupI

SimilarresultswereobtainedbyNesseimetal[47],who statedthatthehistologicalchangesofseminiferoustubules

ofratswerepartiallyrecoveredafternicotinewithdrawal, particularlyat smalldoses (0.2mgnicotineper day) In addition,thesedatawereinagreementwiththedataof El-Meligyetal[48],whoannouncedthatstoppageof nico-tineadministrationleadstopartialimprovementofovarian anduterinedestructivechanges.Theyaddedthatcomplete recoverymayoccurifthestoppagewasaccompaniedbyan antioxidanttherapy

Conclusion

Fromthepreviousresults,wecanconcludethatnicotine causesseverehistologicalandbiochemicalchangesofthe

ZFcells.Thesechangesarebasedmainlyontheoxidative stresspotentialityofnicotine;GSEcanpartiallyameliorate these changes However,complete recovery isobtained withGSEafterthestoppageofnicotineadministration

We recommend thatit is necessarytostopsmoking duetoitshazardouseffects,andanantioxidantmustbe givenafterthestoppageofnicotineusetogaincomplete recovery

Conflicts of interest

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