Lack of association of hepatic estrogen receptor-alpha expression with histopathological and biochemical findings in chronic hepatitis C

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Pathology–ResearchandPractice209 (2013) 727–730

ContentslistsavailableatScienceDirect

Pathology

–

Research

and

Practice

jo u r n al ho me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / p r p

Original

article

Lack

of

association

of

hepatic

estrogen

receptor-alpha

expression

with

histopathological

and

biochemical

ﬁndings

in

chronic

hepatitis

C

Gulbanu

Erkan

a,∗

_,

_Guldal

_Yilmaz

b

_,

_Mustafa

_Cengiz

c

_,

_Ceyla

_Konca

_Degertekin

d

_,

Gulen

Akyol

b

_,

_Seren

_Ozenirler

c

a_Ufuk_University_Hospital,_Department_of_{Gastroenterology,}_Faculty_of_Medicine,₀₆₅₂₀_Balgat,_Ankara,_Turkey

b_Gazi_University_Hospital,_Department_of_Pathology,_Faculty_of_Medicine,₀₆₅₀₀_Besevler,_Ankara,_Turkey

c_Gazi_University_Hospital,_Department_of_{Gastroenterology,}_Faculty_of_Medicine,₀₆₅₀₀_Besevler,_Ankara,_Turkey

d_Gazi_University_Hospital,_Department_of_{Endocrinology,}_Faculty_of_Medicine,₀₆₅₀₀_Besevler,_Ankara,_Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received26March2013

Receivedinrevisedform8July2013

Accepted7August2013

Keywords:

Estrogenreceptor-alpha

ChronichepatitisC

a

b

s

t

r

a

c

t

Estrogensexertaprotectiveeffectagainsthepaticsteatosisandfibrosis.Lossofestrogenreceptor-alpha (ER-␣)intheliverisassociatedwithhepaticsteatosisandinflammationinanimalmodels.We con-ductedastudyinordertoinvestigatethepresenceandextentofER-␣expressioninHCVinfection, anditsrelationshipwithhistologicalandbiochemicalfindings.Ninetybiopsy-provenchronichepatitisC (CHC)patientswereenrolledinthestudy.Liverbiopsyspecimenswereimmunohistochemicallystained forER-␣expression.NuclearER-␣expressionpercentagewascalculated.ER-␣waspositivein69ofthe patients(76%).ER-␣positiveandnegativegroupswerenotsignificantlydifferentintermsofage,gender, necroinflammatoryactivity,fibrosis,steatosis,serumlevelsofAST,ALT,ALP,GGT,andbilirubin.ER-␣ expressionpercentagewasnotcorrelatedwithfibrosis,steatosis,necroinflammatoryactivityand bio-chemicalfindings.Althoughestrogensareknowntobeprotectiveagainstfibrosisandsteatosisinanimal models,wedidnotfindanysignificantcorrelationbetweenER-␣expressionandhistopathologicaland biochemicalfindingsinCHCpatients.Thesefindingsshouldbeverifiedinfurtherlargescalestudies.

Introduction

HepatitisCvirus(HCV)isoneoftheleadingcausesofchronic

liverdiseaseworldwide [9].Thelong-termoutcomesofchronic

hepatitisC (CHC)infectionhavea widespectrum,varyingfrom

minimalchangestochronichepatitis,extensiveﬁbrosis,and

even-tuallycirrhosiswithorwithouthepatocellularcarcinoma(HCC)

[5].

The clinical course of CHC varies signiﬁcantly according to

gender: male sex is an independent predictor of more severe

liverﬁbrosis[12].Nevertheless,this differencediminishesafter

menopause;in fact,theprogression ofﬁbrosisisaccelerated in

postmenopausal women, which can be prevented by hormone

replacement therapy (HRT) [2,3]. Hepatic steatosis and insulin

resistancearealsohallmarksofchronicHCVinfection,andthey

arebothassociatedwiththeprogressionofhepaticﬁbrosis[6,15].

Datafromrecentstudiesindicatethatestrogensand/or

estro-genreceptors have an impact ontheliver steatosis [1,11] and

ﬁbrosis[16,18].Estrogensmediatetheireffectsbybindingtotheir

∗ Correspondingauthorat:UfukUniversitesiTıpFakultesiMevlanaBulvarıNo.

86-88,06520Balgat,Ankara,Turkey.Tel.:+903122044172;fax:+903122044055.

E-mailaddress:gcanbaloglu@gmail.com(G.Erkan).

speciﬁcreceptorswhichbelongtothesteroidhormonereceptor

familyofthenuclearreceptorsuperfamily.Therearetwodistinct

formsofestrogenreceptors,knownas␣and␤,whichareencoded

byseparategenes[10].Whileithaspreviouslybeenreportedthat

estrogensmaybeprotectiveagainstﬁbrosisinCHC,theeffectsof

thepresenceandextentofhepaticestrogenreceptor(ER)

expres-siononsteatosisandﬁbrosishavenotbeeninvestigatedsofar.

Inthisstudy,weaimedtodeterminewhetherthereisa

relation-shipbetweenthepresenceandextentofhepaticERexpressionand

liverfunctiontests,histopathologicalﬁndings(steatosis,ﬁbrosis,

necroinﬂammatoryactivity)inpatientswithCHC.

Materialsandmethods

Thestudypopulationconsistedof90patientswithCHCwho

werereferredtotheGastroenterologyDepartment.None ofthe

patientshad receivedanyanti-viraltreatmentbefore.A written

informedconsentwasobtainedfromeachpatient,andthestudy

protocolwasapprovedbythelocalethicscommittee.Thestudy

wasconductedaccordingtothedeclarationofHelsinki.Patients

wereincludedinthestudyiftheyhadpersistentlyelevatedserum

transaminaselevelsforatleast6monthsandliverbiopsy

spec-imenscompatiblewithCHC;andiftheywerepositive forboth

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728 G.Erkanetal./Pathology–ResearchandPractice209 (2013) 727–730

anti-HCVantibodyandHCV-RNA.Patientswereexcludedfromthe

studyiftheyhadlivercirrhosis,anycontraindicationtoliverbiopsy,

positiveserologicalmarkersforhepatitisBvirus,seropositivityfor

anti-HIVantibody,chronicalcoholconsumptioninexcessof20g

perday,anysignofautoimmuneormetabolicliverdisease,severe

cardiacand/orrenaldisease,andiftheywereontreatmentwith

interferons, immunosuppressive agents,or any medicationthat

causessteatosis.

Percutaneousliverbiopsywasperformedusing adisposable

Menghinitypeneedle(Hepaﬁx1.6mm;BraunMelsungenAG,

Mel-sungen, Germany). The biopsy specimens of liver tissues were

stainedwithhematoxylin–eosinandMassontrichromestain,and

allbiopsyspecimenswereanalyzedbytwoexperienced

patholo-gistsblindedtotheclinicaldataofpatients,usingtheIshakscoring

system[8].Thedegreeofsteatosiswasdeterminedbycalculating

thepercentageofinﬂuencedhepatocytes.Thedegreeofsteatosis

wasgradedasfollows:grade0:noneto5%ofhepatocytes

con-tainmacrovesicularfat droplets;grade 1:6–33%ofhepatocytes

containmacrovesicularfatdroplets;grade2:33–66%ofthe

hepa-tocytescontainmacrovesicularfatdroplets,andgrade3:>66%of

thehepatocytescontainmacrovesicularfatdroplets.

Liverbiopsyspecimenswerestainedimmunohistochemically

forthepresenceofER-␣expression.10%formalinsolutionwas

usedforﬁxatingtheliverbiopsyspecimens.Cross-sectionswere

obtainedfromtheparafﬁnblocksinordertoassesstheestrogen

receptorexpression.Anautomatedsystemandstreptavidin-biotin

tripleindirectimmunoperoxidasemethodswereusedfor

immuno-histochemicalstaining.TheantibodyemployedforER-␣wasIgG

typeandofmonoclonalcharacter(ER␣Antibody(1D5):sc-56833,

Santa Cruz Biotechnology, CA, USA). ER-positive breast cancer

tissuewasusedasapositivecontrol.NuclearER-␣expression

per-centagewascalculatedinallER-positivecases.Onehundredcells

werecountedinrandomlyselectedtenhighpowermicroscopic

ﬁelds(OlympusBX41)tocalculatetheER-␣percentage,andthe

sumofER-_␣-positivecellswasnoted;thentheresultwasdivided

byten.

SerumHCV-RNAlevelsweredeterminedbyreverse

transcrip-tase– PCRusinga commercialkit(MagAttract VirusMiniM48

Kit,RealTimeTM_HCV_{Ampliﬁcation}_Reagent_Kit,_Abbott)_and

Anti-HCVantibodiesweredeterminedbyELISA(chemiluminescence)

(ADVIA®_CentaurTM_HCV_assay,_Bayer_Healthcare_LLC,_Diagnostics

Division,Tarrytown,NY,USA).

Statisticalanalysis

Statisticalanalyseswere performedusingthe SPSSsoftware

version17.Thevariableswereinvestigatedusinganalytical

meth-odssuchasKolmogorov–Smirnov/Shapiro–Wilk’stestandvisual

(histograms,probabilityplots)todeterminewhethertheywere

Table1

Histopathologicalandbiochemicalﬁndingswithrelationtogender.

Female(n=58) Male(n=32) p Age 54.1±12.8 59.3±9.4 0.045 Necroinﬂammatoryactivity 6.5±2.0 6.4±1.9 0.949 Fibrosisscore 1.9±1.1 2.3±1.4 0.255 Steatosispercentage 13.5±19.1 12.7±17.2 0.915 ERpercentage 14.4±19.1 17.6±23.1 0.673 AST(IU/l) 42.5±25.9 77.1±150.7 0.241 ALT(IU/l) 52.2±42.2 100.8±135.4 0.042 ALP(IU/l) 88.3±28.8 100.6±46.4 0.284 GGT(IU/l) 48±57.5 101.3±223.5 0.004 Totalbilirubin(mg/dl) 0.7±0.4 0.8±0.4 0.428 Directbilirubin(mg/dl) 0.3±0.2 0.4±0.3 0.404 ERpositivity,n(%) 45(77.6%) 24(75%) 0.986 Diabetesmellitus,n(%) 9(15.5%) 8(25%) 0.413 Resultsexpressedasmean±SD.

Abbreviations:ER,estrogenreceptor;SD,standarddeviation.

normallydistributed.Descriptiveanalyseswerepresentedusing

mediansfor thenon-normallydistributed andordinalvariables.

AstheERpositivity,ERpercentage, necroinﬂammatoryactivity,

ﬁbrosis,steatosismeasurements,notnormallydistributed

mea-surementandtheordinalvariablesamongthemenopausalstatusof

femalesandmalepatientswereanalyzedbyKruskal–Wallistests.

TheMann–WhitneyUtestwasperformedtotestthesigniﬁcance

ofpairwisedifferencesusingBonferronicorrectiontoadjustfor

multiplecomparisons.Whileinvestigatingtheassociationbetween

non-normallydistributedvariablesandordinalvariables,the

cor-relationcoefﬁcientsandtheirsigniﬁcancewerecalculatedusing

Spearmantest.Anoverall5%type-1errorlevelwasusedtoinfer

statisticalsigniﬁcance.

Results

Ninety patients with CHC, 58 of whom were female (64%),

were enrolled in the study. Biochemical and histopathological

ﬁndings according to gender are presented in Table 1. Serum

levelsof ALT (100.8±135.4 vs. 52.2±42.2IU/ml, p=0.042) and

GGT(101.3±223.5vs.48±57.5IU/ml,p=0.004)weresigniﬁcantly

higher in male patients when compared to those in women.

Nevertheless,necroinﬂammatoryactivity,ﬁbrosis,steatosis,

ER-␣positivityandexpressionpercentagedidnotdiffersigniﬁcantly

withregardtogender.

Overall,ER-␣waspositivein69ofthepatients(76%)(Fig.1).

ER-␣-positiveandnegativegroupswerenotsigniﬁcantlydifferentin

termsofage,gender,necroinﬂammatoryactivity,ﬁbrosis,steatosis,

serumlevelsofAST,ALT,ALP,GGT,andbilirubin(Table2).

None of the postmenopausal patients were on HRT. When

the patients were divided into three groups as male patients,

Table2

DemographichistopathologicalandbiochemicalﬁndingsrelativetoERpresence.

ERnegative(n=21) ERpositive(n=69) p Age 56.9±10.5 55.6±12.4 0.673 Necroinﬂammatoryactivity 5.9±1.6 6.6±2.1 0.115 Fibrosisscore 2.0±1.4 2.1±1.2 0.397 Steatosispercentage 13.7±20.9 13.0±17.7 0.703 AST(IU/l) 74.9±185.3 48.7±32.2 0.237 ALT(IU/l) 73.6±77.7 68.2±76.0 0.335 ALP(IU/l) 94.4±33.0 92.2±37.4 0.807 GGT(IU/l) 73.4±77.7 65.0±156.9 0.380 Totalbilurubin(mg/dl) 0.7±0.4 0.8±0.4 0.406 Directbilurubin(mg/dl) 0.4±0.3 0.3±0.2 0.738 Diabetesmellitus,n(%) 6(28.6%) 11(15.9%) 0.329 Gender(Female/male),n(%) 13/8(61.9%/38.1%) 45/24(65.2%/34.8%) 0.986

Resultsexpressedasmean±SD.

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G.Erkanetal./Pathology–ResearchandPractice209 (2013) 727–730 729

Table3

Histopathologicalﬁndingswithregardtogenderandmenopausalstatus.

Male(n=32) Premenopausal(n=20) Postmenopausal (n=38) p(acrossall groups) p(premenopausal– postmenopausal) Necroinﬂammatoryactivity 6.4±1.9 6.3±2.8 6.6±1.6 0.916 0.708 Fibrosisscore 2.3±1.4 1.7±1.2 2.0±1.1 0.292 0.265 Steatosispercentage 12.7±17.2 13.0±20.3 13.8±18.8 0.986 0.908 ERpercentage 17.6±23.1 16.7±22.7 13.2±17.1 0.851 0.655 ERPositivity,n(%) 24(75%) 17(85%) 28(73.7%) 0.602 0.509

Resultsexpressedasmean±SD.

premenopausal female patients, and postmenopausal female

patients, ER-␣ positivity, ER-␣ expression percentage,

necroin-ﬂammatoryactivity,ﬁbrosis,andsteatosiswerenotfoundtobe

signiﬁcantlydifferentamongthe3groups(Table3).Therewasno

signiﬁcantcorrelationbetweenER-␣expressionpercentage and

necroinﬂammatoryactivity,ﬁbrosis,steatosis,serumlevelsofAST,

Fig.1. Variousdegreesofestrogenreceptor-alphaexpression:noexpression(a),

weakexpression(b),andstrongexpression(c)inpatientswithchronichepatitisC.

ALT,ALP,GGT,andbilirubin(p>0.05).Steatosispercentagedidnot

signiﬁcantlycorrelatewithnecroinﬂammatoryactivity(r=0.160,

p=0.133)orﬁbrosis(r=0.086,p=0.421).

Discussion

Recentﬁndingsindicatethatestrogensand/orestrogen

recep-torsmaymodifyliversteatosis,inﬂammation,andﬁbrosis[13,16].

Chowetal.haveshownthatonlyER-␣isexpressedinthemouse

liver,andaselectiveER-␣agonistalleviateshepaticsteatosisinthe

malearomataseknock-outmice[1].Ribasetal.demonstratedthat

lossofER-␣causesdiminishedoxygenutilization,lipiddeposition,

andinﬂammationintheliver[13].Ontheotherhand,estradiol

isapotentendogenousantioxidantthatcounteractsliverﬁbrosis

inanimalmodels[16].Moreover,neutralizingantibodiesagainst

estradiolinmaleratsandovariectomyinfemaleratswerefound

tobeassociatedwithincreasedﬁbrogenesis[19].

Despitea similarprevalence, chronicinfectionwithHCVhas

beenreportedtobemoresevereinmenthanthatinwomen.In

alargecross-sectionalstudy,malesexwasanindependent

pre-dictorofprogressiontocirrhosisinCHC,increasingtheriskover

2.5-fold[12].DiMartinoetal.evaluatedtheeffectsofpast

pregnan-cies,oralcontraceptiveuse,menopause,andHRTonliverﬁbrosis

progressioninHCV-infectedwomen.Theyfoundthatmenopause

isassociatedwithacceleratedliverﬁbrosisprogression,aneffect

whichmaybebluntedbyHRT.Pregnanciesmayhaveafavorable

impactonthelong-termprogressionofliverﬁbrosis[3].Inarecent

studybyVillaetal.,circulatingestradiollevelwasindependently

associatedwithﬁbrosis:theseverityofﬁbrosisincreasedwiththe

magnitudeofestrogendeprivation[18].Thesereportsreinforcethe

hypothesisofaprotectiveroleofestrogensintheprogressionof

ﬁbrosis.

Hepaticsteatosisisawell-establishedhistologicalhallmarkof

chronic HCV infection. Simple steatosis (SS) occurs in 40–70%,

andnonalcoholicsteatohepatitis(NASH)occursinroughly19%of

patientswithchronicHCVinfection[7,14].HepaticsteatosisinCHC

patientsisassociatedwithhepaticﬁbrosis[4,15].

Inthisstudy,weinvestigatedtheeffectofER-␣expressionon

steatosisandﬁbrosisinpatientswithCHC.WhileER-␣expression

mightbeexpectedtobeprotectiveagainststeatosis,ﬁbrosis,and

inflammation,wedidnotfindanysignificantdifferencebetween

theER-␣positiveandnegativegroupsintermsofsteatosis,

inflam-mation,fibrosis,andbiochemicalfindings.Likewise,whileithas

beenreportedintheliteraturethatHCVinfectionprogressesmore

rapidlyinmenthaninwomen,andthatﬁbrosisisacceleratedin

thepostmenopausalperiod,wedidnotﬁndanysigniﬁcant

differ-encebetweenmen,premenopausalwomen,andpostmenopausal

womenintermsofER-␣positivity,ER-␣expressionpercentage,

steatosis,necroinﬂammatoryactivity,andﬁbrosis.Moreover,

ER-␣ expression percentage was not signiﬁcantly correlated with

steatosis, necroinﬂammatory activity, ﬁbrosis, and biochemical

parameters. Steatosis percentage did not signiﬁcantly correlate

withnecroinﬂammatoryactivityorﬁbrosis,either.These

discrep-anciesbetweenourﬁndingsandpreviousreports[2–4,15,18]may

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730 G.Erkanetal./Pathology–ResearchandPractice209 (2013) 727–730

andgeneticfactorsmayalsohavecausedourﬁndingstocontradict

withpreviousreports.

ER-␣expressionmayberelatedtoserumestrogenlevels[17],

butthereisnopublisheddataontheimpactofserumestrogen

levelsonhepaticER-␣expressioninCHC.Themainlimitationof

ourstudyisthelackofdataonserumestrogenlevelsofthepatients.

WhiletheimpactofER-␣expressiononsteatosis,ﬁbrosis,and

inﬂammationhasbeenextensivelystudiedinanimalmodels,its

impactonthesameprocessesinhumansandhowiteffectscourse

ofCHC areunknown.Tothebestof ourknowledge,this isthe

ﬁrsthumanstudyinvestigatingtheimpactofER-␣expressionon

histopathologicandbiochemicalﬁndingsinchronicHCVinfection.

Furtherstudiesinalargenumberofpatientsandindifferentethnic

populationsmaybeneededtoverifytheimpactofER-␣expression

onthehistopathologicalandbiochemicalﬁndingsinCHC.

Authors’contributions

G.Erkan,S.Ozenirler,G.Yilmaz,G.Akyoldesignedthestudy;G.

Erkan,C.KoncaDegertekinandM.Cengizrecruitedthepatients,G.

YilmazandG.Akyolperformedthehistopathologicexaminations,

andG.Erkanwrotethemanuscript.

Disclosure

None.

Conﬂictofinterest

None.

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