Fish zona radiata (eggshell) protein: A sensitive biomarker for environmental estrogens

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Augustine Arukwe,1 Frank R.Knudsen,2 and Anders Goks0yr1

'Laboratoryof MarineMolecular Biology, DepartmentofMolecularBiology,University of Bergen,Bergen, Norway;2Department of Biology, University of Oslo, Oslo, Norway

Onecategoryof endocrine disruptingxeno- biotics that has recently raised considerable concern recently consists of chemicals with the abilityto act aseitherestrogen agonists orantagonists. By disrupting reproduction and developmental processes, xenobiotic estrogensinthe environment poseaninsidi- ous riskto bothwildlife andhumans (1-4).

Due tothelipophiic andpersistent natureof mostxenobioticestrogensand theirdegrada- tion products, many of these compounds bioaccumulateandbiomagnify. Surprisingly, awiderangeof man-made chemicalsreleased eitherdeliberatelyorunintentionallyintothe aquatic environment are estrogenic. These include some organochlorine pesticides, polychlorinated biphenyls (PCBs), surfactants, plasticizers, and somenatural chemicals such asphytoestrogens and mycoestro- gens (5).Nonylphenol (NP) is abreakdown product ofalkylphenol polyethoxylates and iswidely used in manydetergent formula- tionsfor domestic andindustrialuse. NPhas beenusedas amodelxenoestrogeninseveral studies ofendocrinedisruption(6-8).

Oneofthemostimportant responses to estrogen is the induction of protein tran- scription and translation (9). Particularly well known among these responses is the estrogenicinduction ofvitellogenin (Vtg) in femalesof lower viviparous vertebrates (9).

Vtgisthecomplexphospholipoglycoprotein synthesized bythe liverinresponsetoestro- gen stimulation. Itis secreted bythe liver and transported in the blood to the ovary, whereit issequestered andcleavedintothe yolkproteins lipovitellin and phosvitin, whichare stored in the yolk and serve as foodreservefor thedevelopingembryo (10).

More recently, it was discovered that the fish vitelline envelope orzonaradiata protein (Zrp), composed ofasmall number of

proteins, is synthesized in theteleost liver inresponse to anestrogensignal

(11,12).

Teleostean Zrp forms theeggshell that providesprotection againstmechanicaldis- turbances for the developingembryo during thefirst fragile period but it is shed whenhatching is induced by developmental and environmental signals (13,14).

Furthermore, the eggshell playsan important role during fertilization by preventing polyspermybecause sperm entrytothefish egg is restricted to a predetermined site (micropyle) at the animal pole (15). After fertilization, the eggshell undergoes a calci- um-dependent hardening process that induces a 10-foldincreasein themechani- cal strength of the eggshell (16). The eggs ofplacental mammals aresurrounded bya much thinner, transparent extracellular envelope that liesimmediately outside the plasma membrane of the egg. This envelope iscalledthe zonapellucidaand is com- posedofarelatively small number ofglyco- proteins termedZPI, ZP2, andZP3, with amolecular massof around 200, 120, and 83kDa,respectively(17,18).Thezonapel- lucidaplaysasignificant roleduring fertil- izationandinthepreventionofpolyspermy (19), as does the teleostean zonaradiata proteins,thus showingdearfunctionalsim- ilarities. Verylittle Vtg or Zrp,if any, can bedetected in male and injuvenile fish, presumably because of low estrogen concentrations, butit isknownthatthesepro- teins aresynthesized by theliver cells (in vivoand in vitro) of male andjuvenilefish treatedwith 17,B-estradiol (12,20,21).

The aim of this studyis to compara- tively evaluate theresponse ofvitellogenin andzonaradiataproteinsasenvironmental estrogen biomarkers in viviparous species using immunoassaymethods.

Materials and Methods

Chemicals. 4-nonylphenol (85% ofp-iso- mers) was purchased from Fluka Chemika- Biochemika (Buchs, Switzerland). The impurities in 4-nonylphenol consist mainly of phenol (8-13%), tripropylene(-1%),and 2,4-dinonylphenol (-1%).Aprotinin,

17P-

estradiol (E2), Ponceau S, o-phenylenedi- aminedihydrochloride (OPD),

N,N,NA,N'-

tetramethylethylenediamine (TEMED),and 4-chloro-1-naphtholwere purchasedfrom Sigma Chemical Co. (St. Louis, MO).

Equipmentand other chemicals [goat anti- rabbit-horseradish peroxidase (GAR-HRP)]

forWestern blottingand ELISAwerepur- chased from Bio-RadLaboratories(Hercules, CA). MaxiSorp microtiter plateswere pur- chasedfromNunc(Roskilde, Denmark).All otherchemicalswereof thehighestcommer- cially available grade.

Experiment

1. Inexperiment 1,juvenile Atlantic salmon, Salmosalar,approximately 1+ years old and with a body weightof 75-100 g, were purchased from

S&vareid

smolt producerA/S in Fusacounty (near Bergen,Norway). The fish were ahomoge- nous group belonging to the Norwegian salmon strain (NLA-stamme). They were maintained at the Industrial Laboratory (ILAB) at the High Technology Center in Bergen (HIB) under natural photoperiod (Bergen, 600N) and continouslyrunning sea water withsalinity of35% (ppt) at a constant temperature of 10°C. At thestart ofthis experiment, 36 individuals from the group were randomly selected and trans- ferredto aseparate tank.Theywerefurther divided into six subgroups ofsix fish each and acclimatized to seawaterwith salinity of34% (ppt) at 10°C ± 0.4°C for 1 week

AddresscorrespondencetoA.Arukwe, Laboratory ofMarineMolecularBiology,UniversityofBergen, HIB, N-5020Bergen, Norway.

Wethankthe NorwegianResearchCouncil (NFR, Program forEcotoxicology) and the European Environmental Research Organisation (EERO EnvironmentalToxicology-Network) for financial support. We alsothank Kjersti Milde for rabbit immunization and RongChunjung for thezona radiataprotein antiserum.Theexcellent technical assistance/support ofM.L.Wiborg,E.Mona, andJ.

Stenersen attheBiology Departmentof Oslo Universityand0. SundbergatEssoSlagentangen are greatlyappreciated. Wealso wanttothank BerntWaltherforcomments onthemanuscript.

Received13September1996;accepted5November 1996.

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Articles-Environmentalestrogenbiomarkers

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Figure1.Western blotanalysis of vitellogenin (Vtg)andzonaradiataproteins (Zrp)in plasmaof control (C), 4-nonylphenol (NP; single ip injectionat1, 5, 25,and 125mg/kg bodyweight),and17,-estradiol (E2);5 mg/kg body weighttreatedjuvenilesalmon.Highrangeprestainedmolecularweightstandards(Std)were runin the first lane. (A) Vtg(180 kDa)and(B) Zrp (zrp-a,60kDa;zrp-3,55 kDa;zrp-y= 50kDa)were

probedwithhomologousprimary polyclonalrabbit anti-salmonVtgandZrp antibodies, respectively.Goat anti-rabbit horseradish peroxidase (GAR-HRP)wasusedas asecondaryantibodyinbothcases.Plasma from control and NP treated(50nI)andE2treated(10nl)salmonwasappliedperwell(eachlanerepre- sentsplasmafromanindividualfish).

before the start of the experiment. The

groups were given single intraperitoneal (ip) injections ofNP [1, 5, 25, and 125 mg/kg body weight (bw)],

170-estradiol

(5 mg/kg bw; positive control), and vehicle (negative control), respectively. The nonylphenol doses were chosen tospan a

widerangeof doses thatareenvironmental- lyrelevant. All the subgroupswerekeptin

separate 150-liter tanks. NP wasdissolved in 1:1 ratio of acetone/Echantillon Alkamuls EL-620 (vehicle), Rhone- Poulenc (Paris, France). Theanimalswere

starved during the 14-day experimental period. Fishwereanesthetized with benzo- caine(50mgin1liter^seawater)and blood

was collected from the caudal vessel in heparinized precooled syringes and imme- diately centrifuged (5,000 ^rpm for5min).

Plasma samples collected from the cen-

trifuged blood^were stored at-200C until analyzed.

Experiment2. Experiment 2 wasper-

formedusingjuvenileAdanticsalmon(mean weight 25 g) obtained from Drammen County Fish Administration (Drammen, Norway) and keptinhigh quality running freshwater at 10°Canda12 hrlight:12 hr dark photoperiod at the Biology Department, UniversityofOslo. Four tanks receiving high quality drinking^waterfroma

municipal^watersource wereinstalledatthe

oil refinery^treatmentplant. One of these tanks receiveddrinking^waterthroughoutthe experimental period,whereas theremaining threereceived effluent from the oil refinery

treatmentworks mixed with drinkingwater to afinal concentrationof1, 10,and 50%, respectively. Twentyfishwere transported from the Biology Department of Oslo University, released into each of the four tanks,andkeptthere for 4 weeks.Theywere

fedeverysecondday throughouttheexperi- mental period.After 4weeks, the fish were

anesthetizedand blood^wascollected. Plasma samplescollected from thecentrifugedblood

werestoredat-200C untilanalyzed.

An^oil refinery^treatmentplant (ORTP) receives wastewater from the oil refining

processcontaining^oil^remnants and chemicals used inthe oilrefiningprocess.Among these chemicals arealkylphenols used as

emulsifying agents in the oil refining

process. The basis for the ORTP doses

comesfromaprevious study^atthesamesite (Knudsenet al., unpublished data) where rainbow trout (Oncorhynchus mykiss) ^were exposed^to2.5, 10, and 20% ORTP effluent for a period of3 weeks. A significant increase inplasma Vtg levels^was observed onlyinthegroup exposedto20% effluent.

There were no significantdifferences in mortality between the different groups of fish in theexperiment. The recorded 30%

mortality was probably caused by stressfrom handling duringblood samplingrather than bytoxiceffluent.

Production ofpolyclonal vitellogenin antiserum. Weisolated, purified, andpro- duced polyclonal rabbit antibodies to salmon vitellogenin. Vitellogenin synthesis was inducedinjuvenile fish by weekly ip injections of 17,-estradiol (dissolved and sonicatedinpeanutoil)at adose level of10 mg/kg bw/week. Blood samples werecol- lected 27 days afterthefirstinjection from the caudal vessel using heparinized precooled syringes. To avoidVtgproteolysis, the fish were injected with a trypsin inhibitor,aprotinin [10-20trypsininhibitor units (TIU/ml) dissolved in 0.9% NaCI], injectedinthe caudal vessel20 min before sampling. The bloodwasimmediatelycen- trifuged(5,000 rpmfor5min).Allprepara- tiveprocedureswerecarried out at4°C. Vtg was purified by selective precipitation as describedbyWileyetal. (22) andNorberg and Haux (23). Additional protection against Vtgdegradation was provided by adding 100

lil

ofaprotinin to theVtgsolu- tionbeforedissolvingitinNaCl. Polyclonal Vtgantibodywasprepared in rabbitfrom nitrocellulose-blotted Vtg fractions, essentially^as described by Diano et al. (24).

Production ofapolyclonal antibody using thismethodgaveahighlyspecificpolyclonal antiserum, thus avoiding thelongpurifica- tion ofantigenic protein and the use of Freund's adjuvant. Wehave tested several dilutions ofthis antibodyand itsability to cross-react with Vtgdegradation products;

dilutions of up to 1:11,000 were able to detect the Vtganditsdegradation products inplasma (results not shown). The polyclonal salmonZrpantibodywas agift from ChunjungRong(Bergen, Norway) andwas prepared against solubilized and purified salmonzonaradiata proteins.

Immunochemical studies. Immuno- chemical analysis ofplasma samples was performed using Western blotting and indirectELISA. For Westernblotting, proteins wereseparatedusing4%stacking and 9% separatingsodiumdodecyl sulfate poly- acrylamide gel electrophoresis (SDS- PAGE) asdescribed by Laemmli (25) and blotting wasperformed as described by Towbin et al. (26). Indirect ELISA was performed essentially as described by Goks0yr

(27.

Cross-reactions ofVtg and Zrpwereprobed withhomologousprimary polyclonal rabbit anti-salmon antibodies (diluted 1:2,000). Peroxidase conjugated

GAR-HRP (Bio-Rad) diluted 1:3,000 and H202/4-chloro-l-naphthol (HRP color development reagent; Bio-Rad) were used as thedetection system. Statistical analysis of ELISA absorbance was performed on

kDa SW NP1 NP5 NP25 C

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log-transformed data using Dunnett's test.

The levelof significance was set atp0.05, unlessotherwisestated,with JMPSoftware (version 3.1.6) for Statistical Visualization (SAS Institute, Cary,NC).

Forvalidationof the ELISA assay, pooled plasma samples from control and 5 mg and 125 mg NP/kgfish wereseriallydiluted in coating buffer (50 mM Na-bicarbonate buffer, pH 9.5) in eppendorftubes. The diluted sampleswereadsorbedto microtiter wellsandincubated with homologous rabbit anti-salmon vitellogenin and zonaradiata protein antibodies, respectively, asdescribed above.

Results

In plasma fromAtlantic salmon (Salmo salar)exposedtodifferent doses ofNPviaip injection, across-reacting Vtg protein (180 kDa) wasdetected onlyin plasmafrom the grouptreatedwiththehighest dose (125 mg NP/kg fish, Fig. 1A) using Westernblotting with homologouspolyclonal antibodies. At thisdose, Zrp-a (60kDa),

Zrp-0

(55kDa) and Zrp-,y(50 kDa) were alsodetected; at the lower doses, only a cross-reacting zrp protein(possiblyadoubleband)wasdetected in plasmaat the 5-position (Fig. 1 B).

Consistentwith the immunoblot analysis, an indirect ELISAofVtg andZrp levels reflected the differences in inducibility of these proteins(Fig.2).Results of the ELISA assayvalidationtests arepresentedinFigure 3. Immunoreactive Vtgand Zrpproteins weredominantinplasma from 125 mgNP treated fish (alsoZrp immunoreactive proteins in 5 mg NPtreatedfish). This assay methodrevealed theabsence ofVtgandZrp incontrolfish (alsoVtgin5 mgNPtreated fish).Aparallel decreaseinabsorbancewith the antigen (Vtg and Zrp) dilution was observed(Fig. 3).

Forvalidation ofthe laboratory experiment results,weperformed asecondstudy under semifield conditions, exposing juvenile salmonto ORTPeffluent. Again, Vtg (Fig. 4A) and Zrp-aand Zrp-,y(Fig. 4B) were detected only in plasma from the group exposed to 50% mixture (v/v) of ORTPeffluent, whereasZrp-1 reaction was detected in the groups exposed to 1 and 10% ORTP effluent (Fig. 4B). Indirect ELISAanalysis of thesesamplesagain con- firmed the immunoblot analysis (Fig. 5), althoughthe

Zrp-I

responseta1%ORTP, readily visibleon Western blots, was less apparentinELISA.

Discussion

Immunochemical analysis, usingWestern blotting andELISAwith polyclonalhomol- ogous antiseratoAtlanticsalmon Vtgand Zrp,wasusedinthisstudytomeasureplas-

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wU 1.8

1.2

0.6

0.0 LL".

Control NP1 NP5 NP25 NP125

Dose(mg/kgfish)

Figure2. Indirect ELISA analysis of vitellogenin(Vtg) and zona radiata proteins (Zrp) levels in plasma of control(C),4-nonylphenol(NP;single ipinjectionat1, 5, 25, and 125mg/kg bodyweight),and17p-estradiol(E2)treatedjuvenile salmon. Data aregivenas meanELISA absorbance values(492nm)±standard deviation(n=6pertreatmentgroup).

*p<0.001.

**p<O.0001.

1.5

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8e

=0 S .0 U)

2

1.5

1

0.5

0U.1 0

10 100 1,000 0.1 10 100 1,000

Plasma dilution(x1,000)

Figure3.Validation of ELISA assay for vitellogenin (Vtg)andzona radiataproteins(Zrp).TheELISA responses(absorbanceat492nm)areplotted against total plasma dilution(log-scale).Absorbancesare shownasabsolute values since blankswere notsubtracted.

malevels of Vtgand Zrp ofcontrol, NP,

170-estradiol,

and ORTP effluent-treated juvenile salmon. The specificity ofthese antibodies has been verified byseveral immunological techniquessuch as immuno- histochemistry, immunoblotting, and immunoprecipitation withvery high anti- genic recognition, inaddition to crossreac- tivity towards heterologousAtlantic cod (Gadus morhua) Zrp (21,28) and Vtg

(results not shown). Validation of the ELISAassayusedinthisstudy showsapar- allel decrease in absorbance with antigen dilution.However, the Vtgantiserumshows higher affinity foritsantigen than theZrp antiserum (Fig. 3). Similarresults werealso observedusingserialdilutionof the Vtg and Zrpantiserarespectively (resultsnotshown).

Our results representthefirstreporton the comparative estrogenic effects ofan

1

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Articles-Environmentalestrogenbiomarkers

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Figure4. Western blotanalysisofvitellogenin (Vtg;180kDa)andzonaradiataprotein (Zrp;zrp-a,60kDa;

zrp-J3,55kDa;zrp-y=50kDa) levels inplasma from juvenile Atlanticsalmon after4-weekexposureto1, 10,and50%mixture of oil refinerytreatmentplant (ORTP) effluent.(A)VTG and(B)Zrpwereanalyzed usinghomologousantibodiesasdescribed(see legendtoFig. 1).Plasma(50nl)wasappliedperwell.

environmentalestrogenontheinduction of Vtg and Zrp in fish orlower vertebrates.

The detection ofVtg andZrpinplasma of nonylphenol-treated juvenile salmon shows that Zrp-j is^more responsive than Zrp-ax, Zrp-y, and Vtg,^astheseproteinswereonly detectable in the groups treated with 125

mgNPand50%ORTPeffluent. Induction ofan immunoreacting protein band (or doubleband) ^atthe ,-position ofZrp^sug-

geststhat theseproteins aresynthesizedfirst during zonagenesis (hepatic synthesis of

zonaradiata proteins and transport inthe blood for deposition in the ovaries). The reproducibilityof thelaboratoryexperiment was verified byperforming asecond inde- pendent experimentinthelaboratory.Inthe second experiment, juvenile salmon from thesame group used inexperiment 1 were

exposedtoasingle ip injectionof NPat25 and 125 mg/kgfish for 1 week.Again, Vtg and the threeZrp monomerswere induced inthe group treated with 125 ^mg,whereas onlyZrp-3 wasinduced in thegrouptreated with 25 mg NP (results not shown).

Furthermore, these results have also been analyzed using monoclonal anti-salmon vitellogenin antibody producedinourlabo-

ratory(Nilsenetal., unpublished data)with similarresults.

Although these results are in accor-

dance with theestrogeniceffects of NP and otheralkylphenol polyethoxylates (APEs) reported earlier (7,8,29,30), ^we were able

todetectVtg only^at^veryhighNPconcen-

trations. In contrast,

Zrp-P

^wasdetectable

at lower NPconcentrations. In the litera-

ture, theinduction ofvitellogenin has been singledoutasthemosteffective biomarker

ofestrogenic contaminants inviviparous

vertebrates (31,32). The sensitivityof Zrp cross-reaction demonstratedin this report

points ^to the zona radiata proteins as a moresensitive biomarker for estrogenic effects ofenvironmental pollutants. The sensitivityofZrp^atlow NPdosesisproba- blyaresult of thephysiologicalfunction of theeggshell as protectorof the growing embryo. Ithas beendemonstrated that the formation of theeggshell envelope precedes the active uptake ofvitellogenin during

oocytedevelopment (12,33).

Inaddition to their implications^to the individual fish exposedto xenoestrogens,

these findings mayalso havean ecological significance. ElevatedVtgconcentrations in theplasmaof male fish exposedto sewage treatmenteffluent have earlier beenreported

as afeminization response (34). However, theecologicalconsequencesofxenoestrogen-

inducedhepatic Vtg synthesis isunknown due to the absence ofunequivocalevidence thatfish,oranyotheraquaticorganisms,are

adverselyaffectedby livingin andaccumu-

lating estrogenic chemicals. Giventhepivotal role played by endogenous estrogens in reproduction, such assexual differentiation

atthe egg/embryoand adultsexual matura-

tionstages,it is obvious thatthemostlikely

processtobe affectedisreproduction.Inthis

0.0 Control 1 ¹⁰ 50

ORTP effluent(%)

Figure 5. Immunochemical analysis using indirect ELISA ofvitellogenin (Vtg) andzonaradiataprotein (Zrp)inplasma from control juvenile Atlantic salmon

orafter 4-weekexposureto1, 10,and50%mixture of oilrefinerytreatmentplant(ORTP) effluentData

aregivenas meanELISA absorbancevalues^±standard deviation(n⁼6pertreatmentgroup).For fur- therexplanabon,seelegendtoFigure2.

*p<O.001.

**p<O.0001.

respect,smallchanges^{in zona}radiata^protein synthesis mightcause the thickness and mechanical strength of the eggshell to be altered, thus causing^aloss in itsabilityto

provideprotectionagainstmechanicaldistur- bances during the veryfragile period of

oocyte developmentand theprevention of polyspermyduringfertilization.

Although Vtg is acritical factor that provides the growingembryo with ^neces-

sarynutrients,itcould bearguedthatmore

subtle variations inVtgcontentwouldnot

be ofgreat significance to the survival of theoffspring.For the malefish,accumula- tionofhigh VtgandpossiblyZrp^concentrations might be deleterious becausehigh Vtg concentrations have been associated withkidney failure andincreasedmortality

rates (35). Furthermore, the inhibited ^tes- ticulargrowth reportedrecently by Jobling

etal. (7) would, although notyetdemon- strated, reduce fertility. However, these effects may be relatedto other nonvitel- logenic responses toenvironmental estrogens, e.g., through steroid biotransforma- tionpathways. Wehave observed that low levels of NP also affect progesterone

hydroxylase activities in salmon liver microsomes, inadditiontotheirinhibiting

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E CN

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effect on cytochrome P450 1A expression (Arukwe et al., unpublished data).

Xenoestrogen-induced changes in zona radiata protein synthesis appear to have a higher potential for ecologically adverse effects than vitellogenin induction because critical population parameters such as off- spring survival and recruitment may be more directlyaffected. However, a definite evidence for this hypothesis can only be obtainedthroughlong-termexposurestud- ies of fish to low levels of xenoestrogens.

Such studies and other xenoestrogen-associated reproductive effects are currently underwayinourlaboratory.

Another critical factor in xenoestrogen effects is the role of synergy when animals areexposedtolow dosesofseveraldifferent chemicals. Arnold et al. (36) recently demonstrated a 1,000-fold increase in the estrogenic potencyofchemicalswhencom- bined as compared to when given alone, using a simple yeast estrogen system (YES) containing the human estrogen receptor.

Such

synergistic

interactions are ofcourse

ofprofound environmental significance because most organisms are exposed to a cocktail of different environmental chemi- calsrather thantosingle compounds.

It isimpossible to determine empirical- ly the sensitivity, susceptibility, or resis- tanceofevery species toeach typeofcom- pound. Therefore, anunderstandingof the general principles and mechanisms that determine chemical and biological speci- ficityof environmental estrogens and their impairment of

reproductive

processes in aquatic organisms is very important (37).

These findings mayalso lead tothedevel- opment ofnew andsensitive biomarkers for the effects ofenvironmental estrogens inthe ecosystem.

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