Effects of Natural and Synthetic Estrogens and Various Environmental Contaminants on Vitellogenesis in Fish Primary Hepatocytes: Comparison of Bream (Abramis brama) and Carp (Cyprinus carpio)

T. Rouhani Rankouhi*,1, J. T. Sanderson*, I. van Holsteijn*, C. van Leeuwen*, A. D. Vethaak{dagger} and M. van den Berg*

* Institute for Risk Assessment Sciences (IRAS), Utrecht University, P.O. Box 80176, 3508 TD, Utrecht, The Netherlands, and {dagger} Institute for Coastal and Marine Management (RIKZ), P.O. Box 20907, 2500 EX The Hague, The Netherlands

Received March 20, 2004; accepted May 12, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Interaction of environmental estrogens with the estrogen receptor (ER) has been shown in various fish species. Our objective was to compare the sensitivity of bream (Abramis brama) to (xeno-)estrogens with that of the carp (Cyprinus carpio), by measuring the effects of 17ß-estradiol (E2), estrone (E1), ethynylestradiol (EE2), bisphenol A (BPA), nonylphenol (NP), methoxychlor (MXCL), and halogenated aromatic hydrocarbons (HAHs) such as polychlorinated biphenyls (PCB126, PCB118), 2,3,7,8-tetrachlorodibenzo-dioxin (TCDD), and 2,3,4,7,8-pentachlorodibenzofuran (PCDF) on vitellogenesis in primary hepatocytes. Comparing the EC50 values in bream hepatocytes: EE2 (0.1–0.2 µM) < E1 (0.6–0.2 µM) < E2 (1.9 µM) with those of carp hepatocytes EE2 (0.03–0.06 µM) < E2 (0.3 µM) {approx} E1 (0.2–0.3 µM) we found differences in sensitivity and ranking of the estrogenic potency of E2 and E1, indicating interspecies differences. Exposure to BPA, NP, MXCL, and HAHs did not or only weakly induce vitellogenesis. Bream hepatocytes coexposed to E2 and TCDD, PCB126 or PCDF showed a concentration-dependent inhibition of E2-induced vitellogenesis. IC50 (concentration of a compound that elicits 50% inhibition of E2-induced vitellogenesis) values determined in bream were: TCDD (0.02–0.09 nM) < PCB126 (0.35–0.1 nM) < PCDF (2.0–0.1) and in carp were: TCDD (0.01 nM) < PCB126 (0.4 nM). PCB118 showed no (anti-)estrogenic response. IC50 values and benchmark-concentration for TCDD and PCB126 in bream and carp hepatocytes were in the same range, indicating similar sensitivity to these compounds. Due to their anti-estrogenic capacity with benchmark-concentrations in the pM range TCDD, PCDF, and PCB126 may form a potential hazard for the reproductive success of fish species by inhibition of vitellogenesis.

Key Words: bream; carp; hepatocytes; vitellogenin; (anti-)estrogenicity; dioxins.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The freshwater bream (Abramis brama), a member of the Cyprinid family, has been used for several biomonitoring studies of, for example, Dutch and German waters with regard to the presence of endocrine disrupting chemicals (Hecker et al., 2002Go). One advantage of using the bream in biomonitoring studies is its common presence in large fresh water systems. Moreover, it migrates only over short distances, and because of its ecological niche it is exposed to water-soluble pollutants as well as sediment-bound chemicals (Jedamski-Grymlas et al., 1995Go; Marth et al., 1997Go). Although the bream has been chosen as an indicator species in several studies there is only little data available on the relative sensitivity of the bream to (anti-)estrogenic compounds compared to other fish species.

Vitellogenin (vtg) has been used as a biomarker for exposure to (anti-)estrogenic compounds in several in vivo and in vitro studies with fishes (Rodgers-Gray et al., 2000Go; Shilling and Williams, 2000Go; Smeets et al., 1999aGo,bGo; Sumpter and Jobling, 1995Go; Tolar et al., 2001Go; Vethaak et al., 2002Go). Vtg, a precursor of the yolk proteins phosvitin and lipovitelline, is synthesized in the liver of female oviparous vertebrates. Vitellogenesis is induced by estrogen-dependent activation of gene expression. Although the vtg gene is present in male and juvenile oviparous vertebrates, the endogenous estrogen levels are normally too low to induce vitellogenesis (Callard and Ho, 1987Go).

As test compounds for this study we have chosen a variety of chemicals with a broad range of known (anti-)estrogenic potencies. The natural estrogens estradiol (E2) and estrone (E1), as well as the synthetic estrogen ethynylestradiol (EE2), which is used in contraceptives, have been shown to act as highly potent estrogens in several fish studies. (Allner et al., 1999Go; Folmar et al., 2000Go; Legler et al., 2002Go). E2, E1, and EE2 have been have been found in the lower ng/l range in effluents of sewage treatment plants (Desbrow et al., 1998Go, Ternes et al., 1999Go). In contrast to these compounds the estrogenic potencies of the environmental contaminants nonylphenol (NP), bisphenol A (BPA), and methoxychlor (MXCL) are many orders of magnitude lower that that of E2. In an in vivo study with transgenic zebrafish reporter gene assay, measuring the luciferase activity, the EC50 value for NP was more than 100 times higher compared to that of E2 (Legler et al., 2002Go). In a previous study with carp hepatocytes we found that maximum vitellogenin induction by 100 µM BPA and 50 µM MXCL was about 25–33% lower compared to that by 1 µM E2 (Rouhani Rankouhi et al., 2002Go). BPA is used in the manufacture of epoxy and polycarbonate resins and has been found in the lower µg/l range in surface water (Staples et al., 2000Go). NP is a major metabolite of alkyl phenol ethoxylates (APEs), which are used as nonionic and anionic surfactants and in rubbers and plastics. APE concentrations up to 76 µg/l were recorded in U.K. rivers (Blackburn et al., 1999Go). MXCL is a still commonly used organochlorine pesticide. According to WHO guideline values, MXCL concentration in drinking water should not exceed 20 µg/l (Younes and Galal-Gorchev, 2000Go).

We also included in our study various halogenated aromatic hydrocarbons (HAHs) that have dioxin like properties, are persistent in the environment, and bioaccumulate in the food chain. Exposure to these HAHs, for example by dietary intake, has been associated with a combination of toxic responses including carcinogenicity and adverse effects on reproduction (van den Berg et al., 1998Go). In vivo and in vitro studies in both mammals and fish have described the anti-estrogenic potency of 2,3,7,8-tetrachlorodibenzo-dioxin (TCDD), polychlorinated biphenyl (PCB126), and 2,3,4,7,8-pentachlorodibenzofuran (PCDF), based, e.g., on their ability to reduce vitellogenesis in carp hepatocytes derived from a female carp (Smeets et al., 1999bGo) or inhibition of several E2-induced responses in rodents (Safe, 1995Go). PCB118 had no measurable effect on E2-induced vtg production in female carp hepatocytes (Smeets et al., 1999bGo), which is in line with the toxic equivalency factors (TEF) for mono-ortho PCBs recommended by the WHO (PCB 118:<0.000005 versus TCDD: 1) (van den Berg et al., 1998Go).

The objective of this study was to investigate the ability of a variety of natural and synthetic chemicals with estrogenic or anti-estrogenic potency, hence expected, to interfere with vtg synthesis in bream hepatocytes. In addition, we compared the sensitivity of bream hepatocytes to (anti-)estrogenic compounds to those of the common carp (Cyprinus carpio) by measuring the effects of E2, E1, EE2, TCDD, and PCB126 on in vitro vitellogenesis under the same experimental conditions.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Animals. Male and female bream (Abramis brama) were sampled from the river Linge (around Beesd) and from the river Merwede (between Hardinxsveld and Giessendam). They were kept in an aquarium with copper-free tap water, at a temperature of 20.5 ± 0.5°C, on a 12-h day-night cycle (18 W Philips TLD 840/41 and TLD 820/21). Animals were fed with rain worms and tubifex twice a day. Rain worms and tubifex were obtained from the wholesale company for animal necessities Landman Hoevelaken in Hoevelaken (The Netherlands).

Genetically uniform, adult male carp (Cyprinus carpio) were obtained from the hatchery of the fish culture and fisheries research group at Wageningen Agriculture University. They were kept in an aquarium with copper free tap water, at a temperature of 24 ± 0.5°C, on a 12-h day-night cycle and fed with carp fish pellets (Trouvit, C-4.5 Carpe F, France) twice a day. Fish from both species were allowed to adjust to their housing conditions for at least one month prior to the start of the experiments.

In vivo induction of vtg. A female bream weighing 293 g was anesthetized in MS222 (0.3 g/l) (Sigma, St. Louis, MO) and NaHCO3 (4 g/l) and injected with 0.2 ml 17ß-estradiol (E2) (30 mg) dissolved in 900 µl ethanol and commercially available olive oil (1.5:8.5) into the dorsal muscle. The treatment was repeated three times with a one-week interval. A week after the third injection the animal was anesthetized in MS222 and NaHCO3. Blood was obtained by heart puncture and collected in heparinized vials (6 ml/tube). One hundred-fifty µl aprotinin (Roche, Mannheim, Germany) (0.1 mg/ml 0.9 % NaCl) was added to each vial. Blood was centrifuged 10 min at 2500 x g. Plasma was stored at –70°C for further analysis. The blood of a male, untreated fish weighing 183 g was collected and centrifuged as described above.

Identification of plasma vtg. The presence and the molecular weight (MW) of vtg in bream plasma were determined by electrophoresis in 7.5% polyacrylamide gels, using a silver-staining technique as described earlier and by Western blot (Rouhani Rankouhi et al., submitted). Briefly, plasma samples were dissolved in sample buffer and the presence of vtg was determined by comparing protein-bands of male plasma and E2-treated female plasma. MW of vtg was determined by comparing the vtg protein-band to the protein-bands of a high MW-range marker (14300–220000, code RPN 756, Amersham Pharmacia Biotech, England) on the silver stained gels. The Western blots were performed using a 1:5000 dilution of a monoclonal mouse anti-carp vtg-antibody (ND-2D3, Biosense Laboratories, Bergen, Norway) and a 1:7500 dilution of an alkaline phosphatase conjugated anti-mouse IgG-antibody (Sigma, St. Louis, MO). Female E2-treated plasma. was used as vtg containing sample (positive control) and male serum was used as negative control.

Isolation of hepatocytes. Bream and carp hepatocytes were isolated using a two-step collagenase perfusion technique, based on the description of Smeets et al. (1999a)Go. Perfusions were performed from January to May. Male bream (weighing between 250 and 600 g) and male carp (weighing between 400 and 600 g) were anesthetized in water containing MS222 (0.3 g/l) and NaHCO3 (4 g/l), gentamycin (0.5 ml/l) (50 mg/l, Life Technology, Paisley, Scotland) and fungizone (0.5 ml/l) (250 µg/ml, Life Technology, Paisley, Scotland), rinsed with ethanol and fixed to the operation table. Perfusion was performed at room temperature, with a flow rate of 4.5 ml/min. A heparinized canula was inserted into the vena hepatica and connected to the pump. Perfusion media (26°C) were saturated with 95% O2 and 5% CO2. First, the liver was cleared of blood by perfusion with 200 ml Ca2+- and Mg2+-free medium containing 5 mM EDTA (0.145 M NaCl, 5.4 mM KCl, 1.1 mM KH2PO4, 12 mM NaHCO3, 3 mM Na2HPO4, 100 mM HEPES, 50 mg/l gentamycin, 0.25 µg/ml fungizone, pH 7,5). The perfusion was continued with 150 ml Ca2+- and Mg2+-free medium containing 0.26 mg/ml collagenase D (Boehringer, Mannheim, Germany, activity 0.250 U/mg) and 2.5 mM CaCl2. At the end of the procedure the liver was removed, transferred to Ca2+- and Mg2+-free medium containing 2.5% bovine serum albumine and 2.5 mM CaCl2, minced and sieved through nylon gauze with 200 µm mesh. The minced tissue was centrifuged 3 times for 5 min at 100 x g. The remaining pellet (of cells) was resuspended in culture medium and cell number was counted. Viability of the hepatocytes was assessed by trypan blue exclusion.

Cell culture and exposure. The harvested cells were cultured in phenol red-free DMEM/F12 medium (D2906, Sigma, St. Louis, MO) supplemented with 14.3 mM NaHCO3, HEPES 20 mM, 10 µM hydrocortisone, 50 mg/l gentamycin, 1 µM insulin, 1% v/v Ultroser SF (Biosepra, France, pH 7.4). Isolated hepatocytes from one individual bream (4 x 106 hepatocytes/ml) or carp (9 x 105 hepatocytes/ml) were seeded in 96-well tissue culture plates (250 µl/well) (Greiner, Alphen a/d Rijn, The Netherlands). Cells were allowed to attach for at least 1 h.

All in vitro experiments described below (with exception of NP-experiments and anti-estrogenicity experiments with carp hepatocytes, which were performed only once), have been performed in duplicate, using isolated hepatocytes of one individual fish for each set of experiment. Cells were exposed to E2 (0.1 nM to10 µM, Sigma, St. Louis, MO), or E1 (1 nM to 10 µM, Sigma, St. Louis, MO), EE2 (1 nM to 10 µM, Sigma, St. Louis, MO), BPA (1 to 100 µM, Sigma, St. Louis, MO), MXCL (1 to 100 µM, Riedel de Haën Ag, Seelze, Germany), nonylphenol (1 nM to 100 µM, Riedel de Haën Ag, Seelze, Germany), TCDD (1 pM to 100 nM, Accustandard Inc. New Haven, CT), PCB126 (1 pM to 1 µM, Promochem, Wesel, Germany), PCB 118 (1 pM to 1 µM, Promochem, Wesel, Germany), PCDF (1 pM to 1 µM, Cambridge Isotope Laboratories, Inc., Andover, MA) by adding 0.25 µl (Biohit e-line pipette, inaccuracy 1 µl ± 2.5%) of a 1000-fold stock solution (in DMSO) of each compound, or DMSO alone, to each single well. In order to test the anti-estrogenicity of compounds cells were exposed to respectively E2 10 µM (bream) and 1 µM (carp) and to TCDD (1 pM to 100 nM), PCB 126 (1 pM to 1 µM), PCB 118 (1 pM to 1 µM), or PCDF (1 pM to 1 µM). Control cells in these experiments were exposed to 0.5 µl DMSO.

Each concentration of a compound was tested in six wells. Only NP was tested in 12 wells, as this compound was tested only with hepatocytes of a single fish. After 48 h culture medium was refreshed by replacing 200 µl medium with the same volume fresh culture medium using a multi-channel pipette. Subsequently 0.2 µl of each compound was added to each well. Hepatocytes were cultured for a total period of four days. At the end of the exposure period, medium was transferred to 96-well plates and kept at –70°C prior to vitellogenin analysis. The remaining monolayers were assessed for cell viability using an MTT assay.

Cell viability and quantification of vitellogenin. At the end of the exposure period (on day 5) cell viability was determined by measuring the mitochondrial dehydrogenase activity using 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO) as substrate (Denizot and Lang, 1986Go).

An indirect enzyme linked immunosorbent assay (ELISA), based on the description of Smeets et al. was used to determine the level of vtg in the culture medium of bream hepatocytes (Smeets et al., 1999aGo). Plasma of an E2-treated female bream, containing approximately 59 mg/ml vtg was used for the standard curve dilution. The 96-well EIA/RIA plates (Costar, Badhoevedorp, The Netherlands) were coated for 3 h at 37° with E2-treated female bream plasma, diluted 80,000 times in SBB-buffer (50 mM NaHCO3, 50 mg/l gentamycin, pH 9.6). Male and female E2-treated plasma as well as samples from the hepatocyte assay were diluted in TBST-BSA buffer (10 mM Tris, 0.15 M NaCl, 0.1% Tween-20, 50 mg/l gentamycin, 0.25 % BSA, pH 7.5). A monoclonal mouse anti-carp vtg-antibody (ND-2D3, Biosense Laboratories, Bergen, Norway), diluted in TBST-BSA buffer containing 18 µg/ml aprotinin (final dilution 1:30,000), was used as primary antibody. An alkaline phosphatase conjugated anti-mouse IgG-antibody (Sigma, St. Louis, MO), diluted 5000 times in TBST-BSA buffer, was used as a secondary antibody. Using 4-methylumbelliferylphosphate as substrate, optical density was measured with a fluorescence plate reader (excitation filter: 360 nm, emission filter 460 nm).

Vtg production of carp hepatocytes was also measured by an indirect ELISA. Briefly, plasma of an E2-treated female carp, containing approximately 45 mg/ml vtg was used for the standard curve dilution. EIA/RIA 96-well plates were coated for 3 h at 37° with a 75,000-fold dilution of the E2-treated female carp plasma in SBB-buffer. Plasma of an E2-treated female, and medium from the hepatocyte assay were diluted in TBST-BSA buffer. The same monoclonal mouse anti-carp vtg-antibody (ND-2D3, Biosense Laboratories, Bergen, Norway, final dilution 1:30,000) was used as primary antibody. An alkaline phosphatase conjugated anti-mouse IgG-antibody (Sigma, St. Louis, MO), diluted 10,000 times in TBST-BSA buffer, was used as a secondary antibody. Optical density was measured as described before.

The detection limit of the ELISA was defined as the vtg level that corresponded to three times the SD of the basal vtg value in the standard curve. The detection limit of the ELISA in the bream assays was about 9 ng/ml and in the carp assays 6 ng/ml.

Data analysis. The vtg level in medium samples of the bream and carp hepatocyte assay were assessed by comparing the fluorescence values of the diluted medium samples to those of the standard curve. The results of each experiment were normalized to the level of vtg induced by E2 10 µM in bream or by E2 1 µM in carp hepatocytes. They were expressed as a percentage of this level for comparison purposes.

Concentration-response curves were fitted using the sigmoidal curve fit options of GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA). The benchmark dose method has been used since about 1995 by agencies such as the EPA to estimate the NOAEL values using statistical fits of dose-response-data and therefore to overcome difficulties, caused by experimental uncertainties, to determine NOEL values from dose-response-data. Benchmark concentration (BMC10) values were derived by identifying the lower (95%) confidence level of EC10 or IC10 (concentration of a compound that elicits 10% inhibition of E2-induced vitellogenesis) values from the statistically fitted concentration-reponse curves (Paustenbach, 2002Go). Statistical calculations were performed using the computer program SPSS 10.1 (Statistical Analysis, SPSS Inc., Chicago, IL). Statistical significance (p<0.01 or p<0.05) among the treatments was calculated using a one-way analysis of variance (ANOVA) and a post hoc test (least squares difference [LSD] with Bonferroni-correction).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
In Vitro Induction of Vtg in Bream and Specificity of the Antibody
The presence and the MW of vtg in bream plasma were determined by gel-electrophoresis and silver staining as well as by Western blotting. By comparing the protein-bands of male (control) with E2-treated female plasma samples we found two major protein bands (MW 200 and 160 kDa) that were present only in the female plasma. These two protein bands were considered to be vtg-like proteins as on the Western blot the anti-vtg antibody showed a strong reactivity to these protein bands (results not shown). Comparison of male with E2-treated female bream plasma dilutions in the ELISA showed that at plasma dilutions between 6,144,000 and 48,000 times the anti-vtg-antibody reacts only in female plasma. No reaction with male plasma was observed (Fig. 1). Undiluted E2-treated female bream plasma contains approximately 59 mg/ml vitellogenin.



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FIG. 1. Plasma dilution curve of male and female E2-treated plasma of Abramis brama. Error bars represent SD of two determinations.

 
Cell Viability
Cell viability was determined by comparing mitochondrial MTT reduction activity of hepatocytes exposed to medium containing DMSO (controls) or medium containing the chemicals dissolved in DMSO. Exposure of bream hepatocytes to 10 µM E2, E1, EE2, or NP resulted in increased MTT activity up to 200%, compared to the controls (100% = 0.48 [±0.03] absorption units) (Figs. 2, 3, and 4), whereas exposure to MXCL (0–100 µM) and PCB118 (0–1 µM) had little or no influence on MTT activity (Figs. 5 and 6). BPA at concentrations of ≥100 µM, PCB126 of ≥10 nM, TCDD and PCDF at concentrations of ≥1 nM, resulted in a reduced MTT activity ranging between 40 and 70%, compared to the controls (Figs. 5, 6, and 7). Exposure of carp hepatocytes to ≥100 nM E2 or E1, EE2 ≥1 µM, PCB126 ≥1 nM or TCDD 10 pM and higher resulted in a reduced MTT-activity ranging between 60 and 80%, compared to the controls (100% = 0.51 [±0.09] absorption units) (Figs. 2, 3, 6, and 7). Higher MTT-values indicate stimulation of MTT activity by a compound and have to our knowledge no adverse affect on viability of cells. The observed reduced MTT activity in bream and carp hepatocytes, indicates lower viability of cells compared to the controls. Therefore caution should be taken with some vtg-measurements at higher concentrations as these may not only be related to (anti-) estrogenic properties of these compounds.



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FIG. 2. Effects of E2 treatment on MTT activity and vitellogenesis in Abramis brama and Cyprinus carpio hepatocytes. Error bars represent SD of six determinations. **Values significantly different from their controls: p < 0.01.

 


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FIG. 3. Effects of E1 and EE2 treatment on MTT activity and vitellogenesis in Abramis brama and Cyprinus carpio hepatocytes. Error bars represent SD of six determinations. **Values significantly different from their controls: p < 0.01. *Values significantly different from their controls: p < 0.05.

 


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FIG. 4. Effects of NP treatment on MTT activity and vitellogenesis in Abramis brama hepatocytes. Error bars represent SD of 12 determinations. **Values significantly different from their controls: p < 0.01. *Values significantly different from their controls: p < 0.05.

 


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FIG. 5. Effects of BPA or MXCL treatment on MTT activity and vitellogenesis in Abramis brama hepatocytes. Error bars represent SD of six determinations. **Values significantly different from their controls: p < 0.01.

 


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FIG. 6. Effects of PCB 126 or PCB118 on MTT activity and effects of PCB 126 or PCB118 and E2 treatment on vitellogenesis in Abramis brama and Cyprinus carpio hepatocytes. Error bars represent SD of six determinations. **Values significantly different from their controls: p < 0.01.

 


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FIG. 7. Effects of TCDD or PCDF on MTT activity and effects of TCDD or PCDF and E2 treatment on vitellogenesis in Abramis brama and Cyprinus carpio hepatocytes. Error bars represent SD of six determinations. **Values significantly different from their controls: p < 0.01.

 
Vtg Induction
We observed relative large differences in inter-individual responses of the hepatocytes to estrogens, especially with bream hepatocytes. These differences were probably caused by seasonal differences (perfusions were performed from January to May) as well as differences in age of the individual fish (indicated by differences in weight). Differences in response of especially of the bream may also have been caused by induction of receptor tolerance or priming due to prior exposure to xenobiotica. It has been shown that exposure of salamonid and cyprinid fish to estrogenic effluents induced vitellogenesis after three and seven days (Jobling et al., 2003Go). We tried to minimize the effects of eventually prior exposure by keeping the fish for at least 1 month in an aquarium with tap water prior to the experiments. Fish may also have been exposed to estrogens via dietary intake while being kept in aquariums. As in all experiments with both fish species (Tables 1, 2, and 3) constantly very low vtg levels (2–7 ng/ml) could be detected in our control hepatocytes it is not very likely that dietary intake of estrogens did influence our results. Additionally, unlike the genetically uniform carp strain, we had to use wild bream in this study. Therefore the higher genetic variance between the individual bream, may have essentially contributed to the interindividual variance. Due to these interindividual differences in response, we normalized the results of each experiment to the highest observed vitellogenin level (efficacy) induced by E2, which occurred at 10 µM in bream hepatocytes and at 1 µM in carp hepatocytes. In vitro vtg synthesis was subsequently expressed as a percentage of this level for comparison purposes. Moreover we decided to present BMC10 as well as EC50, relative potency (RPEC50: EC50 E2/EC50 compound), IC50, and the relative inhibitory potency (RPIC50: IC50 TCDD/IC50 compound) derived from each experiment and not the average values of different sets of experiments.


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TABLE 1 Summary of the Results of Two Sets of Experiments with Abramis brama Hepatocytes Exposed to E2, E1, EE2, BPA, NP, TCDD, and PCDF

 

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TABLE 2 Summary of the Results of Experiments with Abramis brama and Cyprinus carpio Hepatocytes, Coexposed to E2 and Several Halogenated Aromatic Hydrocarbons (HAH)

 

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TABLE 3 Summary of the Results of Two Sets of Experiments with Cyprinus carpio Hepatocytes Exposed to E2, E1, EE2

 
Vtg Induction in Bream Hepatocytes
Fish hepatocytes treated with the endogenous estrogens E2 and E1 and the synthetic estrogen EE2 showed significant, concentration-dependent induction of vtg. In bream hepatocytes maximum vtg concentrations were measured at exposure levels of 10 µM for E2 and E1 and 1 µM for EE2 (Figs. 2 and 3). Vitellogenesis was induced by exposure to 10 µM E2 or E1 and to 1 µM EE2 up to 100%, 135–174%, and 135–162%, respectively (Table 1). Statistical analysis showed that there is no significant difference between these maximum vtg values. We found for E2 an EC50 at 1.9 µM. The following order for EC50 values for Abramis brama was determined: EE2 (0.1–0.2 µM) < E1 (0.6–0.2 µM) < E2 (1.9 µM). Based on the EC50 values the following order was determined for the RPEC50: E2 (1) < E1 (3.2–9.5) < EE2 (9.5–19). BMC10 values for vtg induction were between 77–181 nM for E2, 19–131 nM for E1 and 15–35 nM for EE2 (Table l).

Exposure to NP, BPA, and MXCL resulted in weak or no induction of vitellogenesis (Figs. 4 and 5; Table 1). Exposure to NP 100 nM and 1 µM, BPA 10–50 µM induced vitellogenesis up to 0.3 and 0.9%, respectively. BMC10 values were for NP 0.2 nM and for BPA 134 nM. It was noticeable that we found, that exposure to TCDD 0.1–1 nM and PCDF 1 nM caused a slight, but statistically significant vtg induction up to 5 and 0.3% respectively (Table 1). As full concentration-response curves were not obtained we did not calculate EC50 values or RPEC50 for these compounds.

Antagonism of E2-induced vtg synthesis was determined by co-exposing the hepatocytes to E2 and HAHs. All tested HAHs, except for PCB118, reduced E2-induced vtg synthesis (Figs. 6 and 7). For TCDD we found an IC50 value between 0.09–0.02 nM. The following order for IC50 values was determined: TCDD (0.09–0.02 nM) < PCB126 (0.35–0.1 nM) < PCDF (2.0–0.1). Based on the IC50 values the following order was determined RPIC50 values of anti-estrogens: PCB126 (0.06–0.9) {approx} PCDF (0.05–0.2) < TCDD (1). BMC10 values for inhibitory effects on vtg induction were between 5–4 pM for TCDD, 5–41 pM for PCB126 and between 65–282 pM for PCDF (Table 2).

Vtg Induction in Carp Hepatocytes
Carp hepatocytes exposed to E2, E1, or EE2 showed significant concentration-dependent induction of vtg. Maximum induction for all these estrogens was achieved at exposure levels of 1 µM (Figs. 2 and 3). Exposure to 1 µM E2, E1, or EE2 induced vtg synthesis up to 100%, 52–90%, and 55–108% respectively of E2 (100%) (Table 3). Statistically significant differences between these maximum vtg values (p < 0.01), which we believe to be an artifact, were found only in one experiment between E2 induction up to 100%, E1 up to 52%, and EE2 up to 55%. For E2 we found an EC50 value at 0.3 µM. The following order for EC50 values was determined: EE2 (0.03–0.06 µM) < E2 (0.3 µM) {approx} E1 (0.2–0.3 µM). Based on the EC50 values the following order was determined for the relative potency: E2 (1) {approx} E1 (1–1.5) < EE2 (5–10). BMC10 values for vtg induction were between 33–67 nM for E2, 41 nM for E1, and between 3–9 nM for EE2. The results of the experiments with carp hepatocytes exposed to E2, E1, or EE2 are summarized in Table 3.

When carp hepatocytes were co-exposed to E2 and TCDD or PCB126 inhibitory effects on vtg induction were observed (Figs. 6 and 7). The IC50 values for TCDD and PCB126 were 0.01 nM and 0.4 nM respectively. Based on the IC50 (concentration of a compound that elicits 50% inhibition of E2-induced vitellogenesis) values the following order was determined for the relative inhibitory potency: PCB126 (0.03) < TCDD (1). We found for exposure to TCDD and PCB 126 BMC10 values for inhibitory effects on vtg induction of 1 and 47 pM respectively (Table 2).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
In Vivo Induction of Vtg in Bream, Specificity of Antibody
After staining male and female plasma proteins, two major protein-bands in the range of MW 200 and 160 kDa were found exclusively in plasma of female, E2-treated bream, indicating vitellogenesis was successfully induced by E2 treatment. The 160 kDa protein-band was quantitatively the most dominant one. These data are consistent with data, on multiple MW subunits of goldfish vtg (De Vlaming et al., 1980Go). In another study with bream only one vtg subunit (154 kDa) was found in the plasma (Allner et al., 1999Go). Staining methods with different sensitivities (Coomassie blue versus silver staining in our experiment) may explain the additional vtg subunit we found. As in the ELISA assay the vtg-antibody only cross-reacted with E2-treated female plasma, we considered the antibody to be specific against bream vtg and suitable for semiquantitative vtg measurement.

Sensitivity of Bream Hepatocytes to E2 Compared with Other Fish Species
Based on EC50 and BMC10 values our bream hepatocytes are less sensitive to E2 than our carp hepatoytes. Hepatocytes of both species are less sensitive to E2 in our assay than rainbow trout hepatocytes with an EC50 value of 1–10 nM and a lowest observed effect concentrator (LOEC) value of 0–0.1 nM (Tremblay and van der Kraak, 1998Go). In another study, carp hepatocytes had slightly lower EC50 (EC50 50–150 nM) and LOEC values (2 nM) (Smeets et al., 1999aGo) than in our study. Different culture periods (five versus four days in our assay) and different solvents (ethanol versus DMSO in our study) may explain the minor differences in EC50 and LOEC/BMC10 values in the two carp assays. However, bream hepatocytes are clearly less sensitive to E2 exposure than those of the carp or rainbow trout, which may be a reflection of interspecies differences in binding affinities of E2 for the ER.

Vtg Induction by E2, E1, and EE2
Exposure to E2, E1, and EE2 increased vtg synthesis in bream and carp hepatocytes in a concentration-dependent way. In both types of hepatocytes we found no significant difference between maximum vtg levels induced by these estrogens, indicating that these compounds are full agonists for the ER. In carp hepatocytes EE2 was more potent than E2 and E1, whereas in bream hepatocytes, both EE2 and E1 were more potent than E2. The higher potency of EE2 compared with E2 in both fish species can be explained by a higher binding affinity of EE2 for the ER. In in vitro ligand binding studies with hepatic ER of channel catfish, the affinity of EE2 (IC50: 0.43 nM) was five times higher than E2 (IC50: 2.20 nM) (Nimrod and Benson, 1996Go). Consistent with carp hepatocytes in this study, we have previously shown that E2 was more potent in inducing vtg synthesis than E1 (EC50: E2 about 100 nM; E1 about 600 nM) (Rouhani Rankouhi et al., 2002Go). Different exposure periods in the two carp assays (eight days versus four days in this study) may explain the differences in magnitude of EC50 values.

As the higher potency of E2 compared to E1 has been shown, for example, in previous studies with carp hepatocytes, it was unexpected to find in our bream hepatocytes the RP of E1 to be higher than that of E2. However, in in vivo studies with male fathead minnow, E1 induced vtg synthesis at lower concentrations than E2 did (0.118 nM versus 0.367 nM) (Panter et al., 1998Go). As we used in our carp and bream study the same experimental conditions, such as exposure regime and media-composition for both assays, we can exclude modulatory effects, such as differential binding to serum proteins, influencing the bio-availability of estrogens. Therefore differences in ranking of the estrogenic potency of E2 and E1 in bream and carp hepatocytes may rather be explained by interspecies differences in relative binding affinities of estrogens for the ER, metabolic rates, or in the passage across the cell-membrane. Different binding affinities of estrogens to the membrane-bound ER may affect genomic actions of estrogens via differences in nongenomic responses (Revelli et al., 1998Go; Watson, 1999Go).

Vtg Induction by BPA, MXCL, NP
In bream hepatocytes, exposure to the environmental contaminants BPA, NP, or MXCL had either a weak or no influence on vitellogenesis compared to E2. In a previous study with primary carp hepatocytes, vtg induction by 100 µM BPA or 50 µM MXCL was 3- and 4-fold respectively, whereas exposure to 1 µM E2 resulted in a 100-fold induction (Rouhani Rankouhi et al., 2002Go). Comparing these data to those of our bream study we may conclude that carp hepatocytes are about a three- to four-fold more sensitive to exposure to BPA and MXCL than our bream hepatocytes. The lower estrogenic activity of BPA and MXCL can be explained by lower binding affinity to the ER. The IC50 value of MXCL in in vitro binding inhibition studies with ER of channel catfish is about 6000 times higher than that of E2 (Nimrod and Benson, 1996Go).

As in our bream assay NP induced vitellogenesis marginally, we conclude that NP, like BPA, is a very weak partial agonist of the ER. NP has been shown to act as a weak estrogen in several in vivo fish studies. After ip injection of juvenile salmon with 25 mg/kg NP vtg level in plasma increased (1.5-fold) compared to the control (Arukwe et al., 2000Go). Intermittent exposure of adult male rainbow trout to 1 and 10 µg NP/l induced plasma vtg levels up to a 11-fold (Schwaiger et al., 2002Go).

(Anti-)estrogenic Effects of HAHs
As expected, we did not measure any vtg induction in bream hepatocytes exposed to most HAHs. Only TCDD and PCDF induced vitellogenesis weakly. Further research will be necessary to elucidate the biological relevance of these findings.

Bream hepatocytes co-exposed to E2 and TCDD, PCB126 or PCDF showed, in contrast to PCB118, a concentration-dependent inhibition of vitellogenesis. As IC50 and BMC10 values of TCDD and PCB126 in bream and carp hepatocytes were in the same range, we assume that they have similar sensitivities towards these compounds. In another study with female carp hepatocytes, higher IC50 values for TCDD (IC50 value of 0.1 nM) and PCB126 (IC50 values 1 nM) were found, whereas, like in our bream study PCB118 showed no anti-estrogenic effect (Smeets et al., 1999bGo). As TCDD, PCDF, and PCB126 are agonists of the aryl hydrocarbon receptor (Ah), which is associated with inhibition of several ER-mediated responses, the observed reduction of vitellogenesis is probably caused by Ah mediated down-regulation of the ER mediated vtg induction (Safe, 1995Go; Smeets et al., 1999bGo).

The higher RP of TCDD to inhibit E2 induced vtg synthesis compared to PCB126 was found in hepatocytes of both fish species. These results are in line with the TEF values for dioxinlike compounds, recommended by the WHO, which are used for fish risk assessment. Fish, compared to birds and mammals, are very insensitive to mono-ortho-subsituted PCBs such as PCB118. For fish, following TEF values has been suggested: PCB118 (<0.000005) < PCB126 (0.005) < 2.3.4.7.8-PCDF (0.5) < TCDD (1) (van den Berg et al., 1998Go).

Environmental Relevance
In contrast to MXCL, BPA, and NP, activation of the ER through E2, E1, and EE2 can easily be influenced in vitro in bream. These estrogens have been detected in effluents of sewage treatment plants in several countries at levels in the lower ng/l range (Belfroid et al., 1999Go; Desbrow et al., 1998Go; Ternes et al., 1999Go) and have been shown to induce vitellogenesis in situ, in juvenile or adult male fish, indicating an estrogenic stimulation of the liver (Larsson et al., 1999Go; Purdom et al., 1994Go; Sumpter and Jobling, 1995Go). In our experiments E2, E1, and EE2 induced vitellogenesis in bream and carp hepatocytes at about 100 nM and 10 nM respectively. In in vivo studies with immature carp, EE2 was found to be even more potent in inducing vitellogenesis (LOEC value: 10 ng/l ~ 33 pM) (Purdom et al., 1994Go) than in our in vitro study (BMC10: 6–18 nM). Therefore we suggest that sewage effluents containing various estrogenic compounds in low nM ranges may interfere with the ER-system of the carp before any effects are observed in a fish such as bream. Consequently biomonitoring studies with the bream may underestimate the estrogenic potency of compounds present in the environment. It should be noted that in in vivo situations, bioactivation may increase the estrogenic potency of weak estrogens. For example MXCL induced vitellogenesis in sheepshead minnow in vivo was higher than expected based on its estrogenic potency in in vitro test systems (Folmar et al., 2002Go).

In bream hepatocytes, TCDD, PCDF, and PCB126 acted as potent anti-estrogens. Due to their persistence, residues of PCDD/F and PCB mixtures are omnipresent in the environment and may form a potential hazard for the reproductive success of fish species by inhibition of vitellogenesis. In partial life cycle studies exposure of zebrafish to the anti-estrogen tamoxifen caused inhibition of vitellogenesis and decreased egg production (Wester et al., 2003Go).


    CONCLUSIONS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Abramis brama and Cyprinus carpio are commonly occurring fish species in Europe and Asia. In this study we compared the sensitivity of the bream and the carp to (xeno-) estrogens by measuring the effects of natural estrogens and anthropogenic compounds on vtg induction in primary hepatocytes. E2, E1, and EE2 were shown to be full agonists, by activating the ER and inducing vitellogenesis. We found differences in ranking of the estrogenic potency of E2 and E1 in bream (RPEC50: E2<E1<EE2) and carp hepatocytes (RPEC50: E2 {approx} E1<EE2). Comparing EC50 values for E2, our bream hepatocytes were about 6 fold less sensitive to E2 than those of the carp. Like in carp hepatocytes exposure to BPA and MXCL had only weak or no effect on in vitro vitellogenesis in bream. Also NP and HAHs had only weak or no effect on vtg induction in bream hepatocytes. Thus in environmental situations the estrogenic, ER mediated effect of these xeno-estrogens for the bream may be less significant than those of the steroidal estrogens such as E2 and EE2. Unlike PCB118, which did not influence E2-induced vitellogenesis in bream hepatocytes, co-exposure to E2 and TCDD, PCB126 or PCDF showed a concentration-dependent inhibition of E2-induced vitellogenesis at concentrations as low as 5–282 pM. The BMC10 values for these anti-estrogenic effects were more than a 600 fold lower, compared with those for the estrogenic effects of E2 and E1. As IC50 and BMC10 values for TCDD and PCB126 in our study with bream and carp hepatocytes were in the same range, we conclude that both fish are similar in sensitivity towards these compounds. Due to their anti-estrogenic capacity at low concentrations TCDD, PCDF, and PCB126 may form a significant potential hazard for the reproductive success of fish species by inhibition of vitellogenesis.


    ACKNOWLEDGMENTS
 
This study was financially supported in part by ALTERRA (contract number: 635-35333.01), Green World Research, (Wageningen, The Netherlands), and RIKZ (contract number: 22000524), Institute for Coastal and Marine Management (The Netherlands).


    NOTES
 

1 To whom correspondence should be addressed. Fax: + 31-30-2535077. E-mail: T.Rouhani{at}iras.uu.nl.


    REFERENCES
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 ABSTRACT
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 RESULTS
 DISCUSSION
 CONCLUSIONS
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