Vitellogenin-induced pathology in male summer flounder ( Paralichthys dentatus)

Aquatic Toxicology 51 (2001) 431–441

Vitellogenin-induced pathology in male summer flounder(Paralichthys dentatus)

Leroy C. Folmar a,*, George R. Gardner b, Martin P. Schreibman c,Lucia Magliulo-Cepriano c,d, Lesley J. Mills b, Gerald Zaroogian b,

Ruth Gutjahr-Gobell b, Ramona Haebler b, Doranne B. Horowitz b,Nancy D. Denslow e

a US En6ironmental Protection Agency, 1 Sabine Island Dri6e, Gulf Breeze, FL 32561, USAb US En6ironmental Protection Agency, 27 Tarzwell Dri6e, Narragansett, RI 02882, USA

c Aquatic Research and En6ironmental Assessment Center, Biology Department, Brooklyn College, CUNY, Brooklyn,NY 11210, USA

d Biology Department, State Uni6ersity of New York, Farmingdale, NY 11735, USAe Department of Biochemistry and Molecular Biology, Uni6ersity of Florida, Gaines6ille, FL 32610, USA

Received 28 January 2000; received in revised form 31 March 2000; accepted 18 April 2000

Abstract

Male summer flounder (Paralichthys dentatus) were given two injections (initially and 2 weeks later) of 17b-estra-diol (E2) totaling 0.2 (2×0.1), 2.0 (2×1.0) or 20.0 (2×10.0) mg E2/kg body weight. Blood and tissue samples werecollected 4, 6 and 8 weeks after the initial injection in the (2×0.1) mg/kg treatment, 4, 6, 8, and 15 weeks after thefirst injection in the (2×1.0) mg/kg treatment and at 4 weeks only in the (2×10.0) mg/kg treatment. Five of the 12fish injected twice with 10.0 mg/kg were moribund before the first sampling period. Circulating levels of vitellogenin(VTG) in the blood of all E2-injected fish from all treatments were comparable with those concentrations found inthe blood of wild male carp (Cyprinus carpio) and walleye (Stezostedion 6itreum) previously collected near a sewagetreatment plant (0.1–10.0 mg VTG/ml plasma). Excessive hyalin material accumulated in the livers, kidneys and testesof the treated fish. A portion of that material was identified as VTG by immunohistochemistry. The accumulation ofVTG, and possibly other estrogen-inducible proteins, resulted in hepatocyte hypertrophy, disruption of spermatogen-esis, and obstruction or rupture of renal glomeruli. Published by Elsevier Science B.V.

Keywords: Fish; Pathology; Vitellogenin; Estradiol

www.elsevier.com/locate/aquatox

1. Introduction

During the past decade, there has been a signifi-cant effort to determine the effects of xenobioticchemicals on endocrine regulation of reproduc-tion, growth and development in wildlife species. A

* Corresponding author. Tel.: +1-850-9349207; fax: +1-850-9349201.

E-mail address: [email protected] (L.C. Folmar).

0166-445X/01/$ - see front matter Published by Elsevier Science B.V.

PII: S 0166 -445X(00 )00121 -1

L.C. Folmar et al. / Aquatic Toxicology 51 (2001) 431–441432

central focus of that research has been to identifyenvironmental estrogens and determine their ef-fects on reproduction of fish. In that research, themeasurement of the egg yolk protein precursorprotein, vitellogenin (VTG), in male fish has be-come a popular biomarker of exposure to estro-genic chemicals in the aquatic environment(Purdom et al., 1994; Folmar et al., 1996; Harrieset al., 1996, 1997). Male fish possess the gene forVTG, but normally do not synthesize measurablequantities of this protein. However, when malefish are treated (by injection or aquatic exposure)with natural or xeno-estrogens, they are capableof producing circulating levels of VTG equal to orgreater than amounts found in female fish duringgonadal recrudescence (Maitre et al., 1985;LeGuellec et al., 1988). In addition to laboratoryexposures, numerous field investigations have re-ported vitellogenin (VTG) production in wild orcaged male fish exposed to domestic sewage orindustrial effluent in Norway (Knudsen et al.,1997), the US (Folmar et al., 1996, 2000b; Or-lando et al., 1999) and the UK (Purdom et al.,1994; Sumpter and Jobling, 1995; Harries et al.,1996, 1997; Lye et al., 1997).

Earlier studies have shown that exposure ofguppies (Poecilia reticulata) to b-hexachlorocyclo-hexane (HCH) (Wester et al., 1985) and feedingrainbow trout (Oncorhynchus mykiss) large quan-tities of estradiol (Herman and Kincaid, 1988)resulted in the accumulation of a hyalin materialin the livers and kidneys, which caused significantpathology in those tissues. Both sets of authorssuggested that the hyalin material was VTG.Here, we injected male summer flounder(Paralichthys dentatus) with estradiol-17b, mea-sured circulating plasma VTG concentrations,conducted pathological evaluations of the kid-neys, livers and testes, and using immunohisto-chemical techniques demonstrated substantialaccumulations of VTG in the kidneys and livers.

2. Methods and materials

2.1. Husbandry and E2 treatments

Two-year-old, laboratory-raised, sexually im-

mature, male summer flounder (P. dentatus)maintained in flow-through Narragansett Bay wa-ter were used in this study. Prior to treatment, fishwere randomly selected, weighed and measured,tagged with an ink gun and distributed to ran-domly arranged 20-gallon aquaria (four fish/aquarium). Aquaria were aerated and receivedfiltered Narragansett Bay water at a flow rate of 1l/min. Water temperature was maintained be-tween 15 and 17°C, salinity at 3092 parts perthousand and photoperiod on a constant 16-hlight, 8-h dark cycle. The fish were acclimated tothe aquaria for 7 days prior to treatment. A dietof striped bass pellets (Marclark Foods, St An-drews, New Brunswick, Canada) was fed, ad libi-tum, throughout the experiment.

This experiment, organized as serial exposuresbetween August 1997 and March 1998, consistedof the following two injection treatments (initialand 2 weeks later): coconut oil only, with bloodand tissue samples collected 4, 6, and 8 weeksafter the initial injection; estradiol (E2) at 0.1 mgE2 (2×0.1)/kg body weight (bw), with tissue andblood samples collected at 4, 6, and 8 weeks afterthe initial injection; estradiol at 1.0 mg E2 (2×1.0)/kg (bw), with tissue and blood samples col-lected at 4, 6, 8, 15 weeks after the initialinjection; and estradiol at 10.0 mg E2 (2×10.0)/kg (bw), with tissue and blood samples collectedat 4 weeks after the initial injection. Sixteen fishwere injected in each of the treatment and co-conut oil control groups. Uninjected flounderwere used to establish normal histological condi-tions. The 17b-estradiol (CASc 50-28-2), ob-tained from Steraloids Inc. (Wilton, NH) wasdissolved in acetone, then suspended in coconutoil (100% refined) from Spectrum Naturals, Inc.(Petaluma, CA) prior to injection. Each group offish received two injections of either 0.1, 1.0, or10.0 mg E2/kg body weight, one at zero time andthe second 2 weeks later. Stock concentrationswere adjusted so that the solvent concentrationwas maintained at 12% of the injected volume,which was 1.0 ml of the coconut oil suspensionper kg of body weight. Likewise, the coconut oilcontrol suspensions contained 12% acetone. Fish

L.C. Folmar et al. / Aquatic Toxicology 51 (2001) 431–441 433

were injected in the dorsal sinus just anterior tothe dorsal fin.

2.2. Vitellogenin

Blood was drawn from the caudal vessel usinga heparinized (1000 U/ml sodium solution rinse)tuberculin syringe with a 22 gauge needle.Drawn blood was transferred to 1.5-mlpolypropylene microfuge tubes containing 25 mlheparin and 25 ml of the protease inhibitoraprotinin (5–10 TIU/ml). The samples were cen-trifuged at 2000×g for 3 min, the plasma aspi-rated and transferred to a second microfugetube containing 4 ml aprotinin/100 ml plasma,and frozen at −80°C until analyzed. The pres-ence of VTG in the plasma was first verified bysodium dodecyl sulfate-polyacrylamide gel elec-trophoresis (SDS-PAGE) and then quantified byan enzyme-linked immunosorbent assay (ELISA)(Folmar et al., 1996) using a monoclonal anti-body (3G2-4G11 — University of Florida)raised against VTG from the striped bass (Mo-rone saxatilis). Statistically significant differencesin plasma VTG concentrations between the testgroups were determined by ANOVA (Sokal andRohlf, 1981).

2.3. Pathology

After the fish were exsanguinated, livers, kid-neys and testes were dissected and fixed in eitherDietrich’s solution or 10% neutral buffered for-malin. Representative cross- and longitudinalsections, 3–5 mm in thickness, were trimmedfrom the preserved tissue, processed in a Shan-don Hypercenter 2 and embedded in paraffinblocks. Sections, 5–6 mm in thickness, weremounted on glass slides, stained with Gill’s he-matoxylin and counter-stained with alcoholiceosin or stained with Periodic Acid Schiff’s(PAS) for glycogen, neutral muco-substancesand polysaccharides. The PAS staining was per-formed with and without diastase treatments tofurther characterize the clear eosinophilic mate-rial observed in the hepatocytes. Cover slips

were added and the slides examined by light mi-croscopy.

2.4. Immunohistochemistry

Liver, kidney and gonad tissues from animalsrepresenting each of the treatment groups weresubsectioned from the original paraffin blocksand re-embedded in paraffin. Liver and gonadtissues were double embedded into a singleblock. Tissues were sectioned at 6 mm, and threesections from each block were arranged onFisher Plus (+ ) charged slides for immunohis-tochemical treatment.

The sections were deparaffinized in xylene,then rehydrated in descending alcohol washes.Following rehydration, the slides were washedtwice in Tris buffered saline (TBS), pH 7.4, for5 min at room temperature. The tissue was thenincubated in blocking solution (normal goatserum, NGS, provided with the Vectastain Elitekit) for 30 min at room temperature. Next, theslides were incubated at 4°C for 48 h with theprimary antibody (monoclonal antibody 3G2-4G11, University of Florida), diluted 1:1000 inTBS. The following morning the slides werewashed twice with TBS for 5 min at room tem-perature. The slides were then incubated in bi-otinylated anti-mouse antisera for 30 min,followed by two 5-min washes with TBS, all atroom temperature. The samples were next incu-bated with Vectastain Elite Avidin BiotinylatedComplex for 1 h, followed with two 5-minwashes with TBS all at room temperature. Thesamples were then incubated with the chroma-gen (diaminobenzidine) solution for 10 min. Af-ter staining, the samples were dehydrated inascending alcohol washes, and cleared in xylol.Finally, a coverslip with permount was added,and the slides were evaluated qualitatively forthe presence of VTG in the tissues.

Validation procedures included replacement ofthe primary antibody (anti-VTG) with normalserum, and the elimination of sequential steps inthe immunohistochemical procedure. These con-trol procedures resulted in the elimination of allVTG immunoreactivity.


3. Results

3.1. Vitellogenin

Measurements of the plasma levels of VTG (inmg/ml plasma) from the various estradiol treat-ment groups are reported in Table 1. In the(2×0.1) mg E2/kg treatment, plasma sampleswere collected from four fish at 4, 6 and 8 weeksfollowing the first injection. At both the 4- and6-week sampling periods, one of the four fish wasfound to be a female and was removed from theexperiment. Plasma samples from the 8-week col-lection were thawed in a freezer malfunction, andtherefore not analyzed. Plasma concentrations ofVTG decreased from 2.1590.1 to 1.6290.9 mg/ml plasma between weeks 4 and 6 of this treat-ment. Plasma samples from four male fish werecollected at 4, 6, 8, and 15 weeks in the (2×1.0)mg/kg treatment. A steady decrease of plasmaVTG from 9.6895.7 to 0.0990.07 mg/mlplasma was observed between the 4- and 15-weeksampling periods of this treatment. In the (2×10.0) mg/kg treatment, five of 12 fish were dead ormoribund before the 4-week sampling period.Therefore, at the 4-week sampling period, plasmaand tissues were collected from all of the remain-ing fish in that treatment. There was no significantdifference between plasma VTG concentrations inthe (2×1.0) and (2×10.0) mg/kg estradiol treat-ments at the 4-week post-injection sampling pe-riod; however, both of those treatments resulted

in significantly greater (P50.05, ANOVA)plasma VTG concentrations than in the (2×0.1)mg/kg injected fish. At the 6-week post-injectionsampling period, there were no significant differ-ences in plasma VTG concentrations between the(2×1.0) and (2×0.1) mg/kg treatments.

3.2. Pathology

3.2.1. Li6er

3.2.1.1. Coconut oil-injected fish. In the H&Estained liver tissue, the nuclear membrane wasweakly basophilic, while the cytoplasm containedneither basophilic or eosinophilic material. Glyco-gen granules, identified by PAS stain, weresparsely distributed throughout the cytoplasm.The nuclei were normal and the nucleoli werebarely visible. Some vacuolation and lipid masseswere also observed. There was a complete absenceof immune response to the VTG antisera in allcontrol tissues examined.

3.2.1.2. Estradiol-treated fish. Gross inspection ofthe livers from the estradiol-treated fish showedthem to be softer than normal and friable in thosefish receiving the highest injection dosage. Thiscondition appeared to be caused by increasedlipid and abnormal accumulations of a cleareosinophilic material (hyalin) in the hepatocytes.Using H&E stain, we found a concentration-de-pendent increase in nuclear chromatin, cytoplas-

Table 1Plasma vitellogenin (mg/ml) concentrations (mean9S.D.) in male Summer flounder injected twice with 0.1, 1.0 or 10.0 mg17b-estradiol/ kg body weight at the beginning of the experiment and again 2 weeks latera

6 weeks 15 weeksTotal 17b-estradiol injected (mg/kg body wt.) 8 weeks4 weeks

2.1590.12×0.1 1.6290.9 No samplesNo samples(n=3)1 (n=3)

0.0990.079.6895.7 0.9891.114.7594.12×1.0(n=4)2a (n=4)ab (n=4)b (n=4)b

2×10.0 9.1693.3 No samples No samplesNo samples(n=7)2

a Plasma samples were collected at 4 weeks (2×0.1, 2×1.0, 2×10.0 mg/kg), 6 weeks (2×0.1, 2×1.0 mg/kg), 8 weeks (2×1.0mg/kg) and 15 weeks (2×1.0 mg/kg) after the first injection. Coconut oil-injected fish (4) were sacrificed at each of the four samplingperiods. No VTG was measured in the coconut oil or uninjected control fish at any time. In the 2×1.0 mg/kg E2 treatments,temporal groups with different letters (a,b) are significantly different (P50.05). At the 4-week sampling period, concentration groupswith different numbers (1,2) are significantly different (P50.05).


Fig. 1. Liver section from male summer flounder (P. dentatus)receiving two injections of 10.0 mg E2/kg, sampled at 4 weeksafter the initial injection (H&E, 400× , I–I=13 mm). Sectiondisplays acute VTG-induced hepatocellular disarray, hypertro-phy and necrosis. Note nuclear chromatin, cytoplasmicbasophilia, hyalin material (arrowhead) and small lipiddroplets within hepatocytes.

hepatic sinusoidal, portal and arterial circulationwere also intensely eosinophilic. Nuclei and nucle-oli were enlarged in the hepatocytes from fish inthe (2×1.0) and (2×10.0) mg/kg estradiol treat-ments. These alterations were most apparent atthe 4-week sampling period, and while retainingconcentration dependence, showed incremental re-versal of their intensity at the 6- and 8-weeksampling periods. At the 4-week sampling period,an increase in the intensity of basophilia associ-ated with nuclear and cytoplasmic membranesand excess accumulation of hyalin occurred in allof the (2×1.0) and (2×10.0) mg/kg estradiol-in-jected male fish, but in none of the (2×0.1)mg/kg injected male fish. The hyalin material wasrandomly localized in focal areas in the (2×1.0)mg/kg injected fish, but widespread throughoutthe organ in the (2×10.0) mg/kg injected fish(Fig. 1). The hyalin material observed 4 weeksafter the first treatment in the (2×1.0 mg/kg)-in-jected fish, was absent in the fish sampled fromthat treatment group, 6 and 8 weeks after firstinjection. This pattern paralleled that observed forbasophilic material (RER) in the cytoplasm. Theestradiol injections also appeared to be lipogenic,with substantial increases in cellular lipids accom-panying increases in hyalin material.

Immunohistochemical responses to the VTGantisera observed in association with the plasmamembrane, hyalin droplets, and the intercellularspaces and non-cellular fluids of hepatic vascularsystem were substantially greater in fish from the(2×1.0) and (2×10.0) mg E2/kg treatments (Fig.2) than in animals given (2×0.1) mg/kg at the4-week post-injection sampling period. In the(2×1.0) mg/kg treatment, liver tissue from theanimals sampled at 4 weeks after the initial injec-tion demonstrated substantial amounts of im-munoreactive hyalin material, while tissue sectionsfrom the 8- and 15-week post-injection fishshowed little or no immunoreactive response.Pancreatic tissue present in the liver was notimmunoreactive in any animal.

3.2.2. KidneyWe observed no significant pathological abnor-

malities in the kidneys collected from flounderinjected with coconut oil, (2×0.1) or (2×1.0)

Fig. 2. Liver section from male summer flounder (P. dentatus)receiving two injections of 10.0 mg E2/kg, sampled at 4 weeksafter the initial injection (400× , I–I=13 mm). Slide illustratesarea of advanced injury within a field of hepatocytes stainedwith VTG antisera. Positive immunoreaction occurred in hep-atocellular membranes, intracellular hyalin (arrowheads) andfluids of the hepatic vascular system.

mic basophilia (RER) and eosinophilic material(hyalin) in the cytoplasm of hepatocytes from fishin the three treatment groups (Fig. 1). Addition-ally, red blood cells and non-cellular fluids of the


mg/kg estradiol at any of the sampling periods.However, all of the flounder from the (2×10.0)mg/kg treatment (Fig. 3) presented hyalinuria, acondition characterized by accumulation of hyalinbodies in the capillary loops of the glomeruli and

hyalin casts present as excretion products in thevarious compartments of the uriniferous tubules,collecting tubules and ducts and the urinary blad-der. In this state of glomerular damage, filtratedrained freely into Bowman’s Space and intoremaining nephron components.

All fish injected with estradiol demonstrateddose-dependent increases in VTG immunoreactivehyalin material in kidney glomeruli at the 4-weekpost-injection sampling period (Fig. 4), as well asin peritubular spaces and cells, and non-cellularcomponents of blood vessels. As with the liver,increasing intensity of the immunoreactive re-sponse was directly related to E2 dosage at 4weeks post-injection, but absent in the (2×1.0)mg/kg fish sampled at 8 and 15 weeks post-injec-tion. Hematopoietic tissue, which was abundantin all sections, did not demonstrate VTGimmunoreactivity.

3.2.3. TestesThere were no obvious differences in develop-

mental state or histology between uninjected fishand those injected with coconut oil or (2×0.1)mg/kg estradiol. All stages of sperm developmentwere present, tubules were filled with sperm, andthe number of cysts were decreasing. However,five of the 12 fish injected with (2×1.0) mg/kgand eight of nine fish injected with (2×10.0)mg/kg estradiol displayed inhibited testiculargrowth. Those 13 fish all presented reduced testic-ular mass or atrophy, dead germ cells, residualsperm in the efferent ducts, increased interstitialcomponents and proliferation of spermatagonia.Eosin-positive (hyalin) material was observed inthe arterial vessels, and the interstitial spaces andcapillaries of the testicular tubules from all of the(2×1.0) and (2×10.0) mg/kg estradiol injectedfish (Fig. 5). No hyalin material was observed inthe testes of the coconut oil or (2×0.1) mg/kgestradiol injected fish.

Once again, the increase in intensity of theVTG immunoresponse in testicular tissue ap-peared dose-dependent and was seen only in thefish sampled at 4 and 6 weeks after the E2 injec-tions. A significant degree of immunoreactivitywas observed in hyalin material within seminifer-ous ducts and interstitial tissues of the testicular

Fig. 3. Kidney section from male summer flounder (P. den-tatus) receiving two injections of 10.0 mg E2/kg sampled at 4weeks after the initial injection (H&E, 400× , I–I=13 mm).Slide illustrates VTG-induced glomerular injury with hyalinmaterial localized in glomerular tufts (arrowhead), Bowman’sSpace (small arrow), and renal tubule (large arrow).

Fig. 4. Kidney section from male summer flounder (P. den-tatus) receiving two injections of 10 mg E2/kg, sampled at 4weeks after the initial injection (400× , I–I=13 mm). Slideillustrates glomerular injury associated with accumulation ofVTG immunoreactive hyalin material within the glomerularcapsule (arrowhead), Bowman’s Space (small arrow), and re-nal tubules (large arrow).


Fig. 5. Testis section from male summer flounder (P. dentatus)receiving two injections of 10 mg E2/kg, sampled at 4 weeksafter the initial injection (H&E, 400× , I–I=13 mm). Noteaccumulation of hyalin material along epithelial lining ofseminiferous ducts (arrows).

4. Discussion

Although the exposure route (injection) andconcentrations (pharmacological doses) lack obvi-ous environmental relevance, plasma concentra-tions of VTG resulting from the three E2treatments (Table 1) were comparable to plasmaVTG concentrations measured in feral male carp(Cyprinus carpio) (Folmar et al., 1996) andwalleye (Stizostedion 6itreum) (Folmar et al.,2000b) collected from the Mississippi River nearthe effluent outfall of a major metropolitansewage treatment plant. Our light microscopicevaluations of the liver and kidney of E2-injectedflounder revealed accumulations of a hyalin mate-rial in the hepatocytes and in the glomeruli of thekidney, confirming previous observations made byHerman and Kincaid (1988) in rainbow trout(Salmo gairdneri ) fed an E2 supplemented diet(30 mg E2/kg feed) and by Wester et al. (1985) inguppies (Poecilia reticulata) exposed to \0.1 mg/l b-hexachlorocyclohexane (b-HCH). Herman andKincaid (1988) conjectured that the accumulatedlipoprotein was most likely VTG, while Wester etal. (1985) tentatively identified VTG by slab gelelectrophoresis. Our immunohistochemical evalu-ations have confirmed that a portion of theprotein accumulated in those tissues, as well as inthe vasculature of the testes, was VTG; however,substantial amounts of the accumulated proteindid not respond to the VTG antibody.

Some of the unreactive protein could have beenVTG not recognized by the heterologous anti-body; however, male fish have also been shown toexpress other estrogen (or xenoestrogen)-inducibleproteins, most notably, three of the choriogenic(vitelline envelope/zona radiata) proteins (Hyllneret al., 1991, 1994; Hyllner and Haux, 1992; Op-pen-Berntsen et al., 1992a,b, 1994; Larsson et al.,1994; Arukwe et al., 1997, 1998; Murata et al.,1997; Celius and Walther, 1998a,b; Celius et al.,1999; Yadetie et al., 1999). In females, increasingE2 production during the early phase of gonadalrecrudescence stimulates the liver to synthesizethese proteins, which are transported to the ovarywhere they contribute to the formation of thevitelline envelope in the developing oocytes (Op-pen-Berntsen et al., 1992a,b). During oogenesis,

Fig. 6. Testis section from male summer flounder (P. dentatus)receiving two injections of 10 mg E2/kg, sampled at 4 weeksafter the initial injection and stained with VTG antisera(200× ). Note immunoreactive material in peritubular spaces.

tubules in animals receiving the (2×1.0) and(2×10.0) mg/kg E2 injections (Fig. 6). Fish giventhe (2×0.1) mg/kg injections showed very slightimmunohistochemical staining that was barelyabove background. No immunoreactivity wasobserved in spermatogenic cells. No im-munoreactivity was observed in the testes fromany of the fish collected at 8 or 15 weekspost-injection.


the orderly synthesis of the zona radiata proteins(ZrPs), followed by VTG, is regulated by differ-ences in E2 response thresholds (Celius andWalther, 1998a). This same sensitivity pattern wasobserved in fish treated with the xenoestrogeno,p-DDT (Celius and Walther, 1998b). Simulta-neous exposure to more than one estrogen re-sulted in an additive upregulation of ZrP synthesisin rainbow trout (Knudsen et al., 1998). Whenestrogen exposure concentrations exceed thethreshold for VTG message induction and synthe-sis, ZrP and VTG production have been shown tobe equimolar (Oppen-Berntsen et al., 1992a). Alsolike VTG, the choriogenins are ‘orphan’ proteinsin male fish and lack a depositional site (ovary) tosequester them from the general circulation.Therefore, the large concentrations of theseproteins in the general circulation overwhelm thenormal pathways for protein elimination resultingin their accumulation in the kidney and liver.

Table 1 clearly shows a significant (P50.05)decrease in plasma VTG concentrations between 4and 15 weeks in male fish receiving the (2×1.0)mg/kg estradiol treatment. Certainly, the reduc-tion in plasma VTG over time can be explainedpartially by reduced VTG synthesis associatedwith decreasing estradiol stimulation. In femalefish, decreasing concentrations of VTG in theplasma also result from incorporation of VTGinto developing oocytes; however, in males a re-duction in plasma VTG concentrations must beaccomplished by alternate pathways. Transportacross the gills and mucous accumulation couldaccount for some reduction in plasma VTG; how-ever, the most direct route for elimination of VTGfrom the blood of male fish is through the kidney.Although the plasma concentrations of VTG werethe same in fish from the (2×1.0) and (2×10.0)mg/kg estradiol treatments at the 4-week samplingperiod, there was a substantially greater accumu-lation of protein in the kidneys of the (2×10.0)mg/kg injected fish. Unfortunately, we have noplasma VTG analyses for the flounder prior to the4-week sampling period; however, based upon ourexperience with other species (Folmar et al.,2000a), the plasma levels of VTG in the (2×10.0)mg/kg treatment probably increased faster andwere greater than the (2×1.0) mg/kg injected fish

during the first 4 weeks of the experiment. Nor-mally, the molecular mass of proteins in glomeru-lar filtrate does not exceed that of albumin (66kDa) (Koger et al., 1999); therefore, the massivedeposition of protein in the tubules likely repre-sents that material which could not be resorbed,while the excess protein found in Bowman’s cap-sule resulted from glomerular rupturing or hemor-rhaging. These pathologies associated with the(2×10.0) mg/kg estradiol treatment could havecaused acute renal failure which resulted in themortality observed in the third week after the firstinjection of that test group.

In the liver, the intense cytoplasmic basophiliareflecting proliferation of the rough endoplasmicreticulum is characteristic of protein synthesis as-sociated with vitellogenesis (Ng and Idler, 1983).With the passing of time, the intensity of thebasophilic staining decreased, accompanied by adecrease in hyalin material. During the 4-weekperiod following the first injection, estradiol up-regulated synthesis and accumulation of VTG,other estrogen-inducible proteins, and lipids re-sulting in hypertrophy of the hepatocytes. Thiscondition could have resulted from impaired ex-port (exocytosis) of newly synthesized proteinsdue to the unfavorable concentration gradientillustrated by the substantial amounts ofeosinophilic material observed in the hepatic cir-culatory system. Alternatively, some of theseproteins could have been imported from the hep-atic portal circulation in a futile attempt to me-tabolize them (Mommsen and Walsh, 1988). Ourobservations of apparent lipogenesis support thefindings of Haux and Norberg (1985) who ob-served similar responses in the livers of rainbowtrout exposed to estradiol.

Immunohistochemical detection of VTG accu-mulation in the testes was unexpected, despite aprevious report that VTG was detected in gonadalhomogenates of tilapia (Oreochromis aureus) in-jected with 4 mg/kg estradiol (Ding et al., 1989).Our observations suggest that VTG accumulatesin the testis vasculature (which would have beenincluded in the homogenates of Ding et al., 1989)through obstructive blockage, rather than in thegonadal tissue itself. This conclusion is furthersupported by the apparent lack of specific VTG


receptors in the testes of fish (Tyler and Lub-berink, 1996). Atrophy of the testis, impairedspermatogenesis and a reduced gonadosomatic in-dex (GSI) in our E2-injected flounder were similarto those reported for: E2-injected eelpout, Zoarces6i6iparous (Christiansen et al., 1997), rainbowtrout, Salmo gairdneri (Billard et al., 1981; Joblinget al., 1996) and black porgy, Acanthopagrusschlegli (Chang et al., 1995); fathead minnows,Pimephales promelas receiving a flow-through ex-posure to 100 ng E2/l for 21 days (Panter et al.,1998); and rainbow trout, Oncorhynchus mykissreceiving a 3-week aquatic exposure to 30 mg/lnonylphenol (Jobling et al., 1996). Differences inthe severity of E2-induced effects on fish testishave been related to the stage of development atthe time of the exposure (Billard et al., 1981). Thecomplex etiology of these testicular pathologies isunknown, but likely results from estradiol influ-ence on the brain–pituitary–gonadal axis of an-drogen synthesis (Trudeau et al., 1991).

5. Conclusions

Two injections of estradiol-17b (0.1, 1.0 or 10.0mg/kg) at 2-week intervals resulted in circulatingblood levels of VTG comparable with those foundin feral male fish collected near a sewage treat-ment discharge site on the Mississippi River. Con-currently, substantial accumulations of VTG andother estrogen-inducible hyalin material were ob-served in the livers, kidneys and testes of theexperimental fish. Blood levels of VTG decreasedwith time (from 4 to 15 weeks) in the (2×1.0)mg/kg treatment, accompanied by delayed, butcorresponding, reductions of VTG in the liver,kidney and testes of the treated fish. The impairedspermatogenesis observed in the E2-treated fishwas more likely the result of estradiol-inducedalterations in androgen steroidogenesis than VTGaccumulation; however, the mortality observed inthe 10.0 mg/kg estradiol treatment likely resultedfrom acute renal failure associated with excessiveVTG accumulation in the kidney. Accumulationsof estrogen responsive proteins associated withchronic exposures to low concentrations of estro-genic chemicals may not produce the dramatic

liver or kidney pathologies and possible mortali-ties observed here, but could cause mortalitythrough compromised liver and kidney functionresulting in a reduced ability to metabolize xeno-biotic chemicals or resist disease.

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