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Induction of Vitellogenesis in 17A-Ethinylestradiol-Exposed Rainbow Trout (Oncorhynchus Mykiss): a Method Comparison

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Induction of Vitellogenesis in 17A-Ethinylestradiol-Exposed Rainbow Trout (Oncorhynchus Mykiss): a Method Comparison Comparative Biochemistry and Physiology Part C 132 (2002) 483–492 Induction of vitellogenesis in 17a-ethinylestradiol-exposed rainbow trout (Oncorhynchus mykiss): a method comparison Tim Verslycke*, Gert F. Vandenbergh, Bram Versonnen, Katrien Arijs, Colin R. Janssen Laboratory of Environmental Toxicology and Aquatic Ecology, Ghent University, J. Plateaustraat 22, B-9000 Ghent, Belgium Received 22 February 2002; received in revised form 27 June 2002; accepted 2 July 2002 Abstract ( ) Juvenile rainbow trout, Oncorhynchus mykiss, were exposed to the synthetic estrogen 17a-ethinylestradiol EE2 ( ) ( ) through injection 1, 10, 25 and 50 mgEE2yg fishyweek and via water exposure 1, 10 and 100 ng EE2yl . After seven (injection and water exposure) and 14 days (only for water exposure), blood and plasma vitellogenin concentrations were quantified using indirect endpoints, i.e. plasma alkaline-labile phosphorus (ALP), plasma protein and plasma calcium. In addition, the relative gonad (GSI) and liver weight (HSI) were recorded. Actual plasma vitellogenin concentrations were measured with an enzyme immunoassay. Only fish injected with 50 mgEE2yg fish had a significantly higher gonad weight. No concentration-dependent changes in the HSI were detected in fish exposed via the water, but a significant dose-dependent increase of the HSI was observed in fish injected with EE2 . Exposure of rainbow trout to EE2 had a significant effect on all tested plasma parameters. Plasma protein, phosphoprotein and calcium concentrations were significantly higher after two weeks exposure to 100 ng EE22yl. Fish injected with 10, 25 and 50 mgEEyg fish exhibited increased plasma protein concentrations after 1 week. Compared to the controls, plasma ALP and calcium levels were significantly higher in all injected fish. A significant and positive correlation was observed between all three plasma parameters and between these indirect parameters and the actual plasma vitellogenin concentrations. These findings indicate that both the plasma ALP and the plasma calcium assay have a similar sensitivity as that of available ( ) antibody-based assays EIA , at least in EE2 exposure studies, and thus these assays can provide a rapid, simple and cost-effective alternative to available immunoassays. ᮊ 2002 Elsevier Science Inc. All rights reserved. Keywords: Rainbow trout; Oncorhynchus mykiss; Vitellogenin; Ethinylestradiol; ALP; Calcium; EIA; Method comparison; Endocrine disruption 1. Introduction Vtg is present in very low concentrations in the plasma of immature or male organisms. However, ( ) Vitellogenin Vtg is an estrogen induced yolk- estrogenic compounds can also act on hepatic precursor lipophosphoprotein which is present in estrogen receptors to induce synthesis of vitello- the blood of oviparous vertebrates and inverte- genin (Pelissero et al., 1993). Vtg production by ( brates during vitellogenesis Bergink and Wallace, males and juveniles can therefore be considered as ) 1974 . During vitellogenesis the liver of females a biomarker of exposure to environmental estro- is stimulated to produce Vtg, which in turn, is gens (Heppell et al., 1995; Kime et al., 1999). incorporated into the yolk of developing oocytes. A number of different techniques has been used *Corresponding author. Tel.: q32-9-264-37-07; fax: q32- to indirectly detect blood Vtg levels. Frequently 9-264-37-66. used methods involve the biochemical determina- E-mail address: [email protected] (T. Verslycke). tion of phosphoprotein (as alkali-labile protein 1532-0456/02/$ - see front matter ᮊ 2002 Elsevier Science Inc. All rights reserved. PII: S1532-0456Ž 02. 00111-4 484 T. Verslycke et al. / Comparative Biochemistry and Physiology Part C 132 (2002) 483–492 phosphorus, ALP), total protein or calcium (Yaron compared and suggestions are made for future use et al., 1977; Whitehead et al., 1978; Korsgaard of these methods as fast and cost-effective alter- and Petersen, 1979; Scott et al., 1980; Nath and natives to the available immunoassays. Sundararaj, 1981; Craik and Harvey, 1984; Tinsley, 1985; Copeland et al., 1986; Parker and McKeown, 2. Methods 1987; Kramer et al., 1998; Christensen et al., ) 1999 . Other methods are based on immuno- 2.1. Fish agglutination (Le Bail and Breton, 1981), densi- ( tometry following electrophoresis Van Bohemen Eighty-four Juvenile rainbow trout, with an et al., 1981; Allner et al., 1999), radial immunod- ( ) average mass of 15 g, were obtained from an iffusion Hara, 1978 and radioimmunoassay aquaculturist (De Keijzerberg, The Netherlands). (Idler et al., 1979; Campbell and Idler, 1980; So ) A 10 day acclimation period preceded the exposure et al., 1985; Sumpter, 1985 . Recently, different period. During acclimation, the fish were kept in immunoassays (EIA) have been developed for ( 200-l glass tanks with carbon-filtered tap water measuring Vtg in various teleost species Specker and a biofilter. One-third of the culture water was and Sullivan, 1994; Bon et al., 1997; Brion et al., renewed manually every 2 days. 2000). Although these assays are sensitive, their use is mostly limited to one species due to the a complexity of the Vtg molecule. Moreover, these 2.2. Exposure to 17 -ethinylestradiol assays are expensive and limited in their use due to the lack of species-specific antibodies. In the first experiment, 48 fish were exposed to ( In the present study, blood Vtg levels were 1, 10 and 100 ng EE2yl and a solvent control 0.1 ) measured in rainbow trout Oncorhynchus mykiss ml EtOHyl water for 1 weeks. After 1 week (Teleostei: Salmonidae) exposed to 17a-ethinyles- exposure, 24 fish were sampled and the remaining ( ) fish were collected and analyzed at the end of the tradiol EE2 ; water-borne and injected by different exposure period. The exposure medium was direct and indirect methods. EE2 is the main active component of contraceptive pills and is also more renewed every 2 days. EE2 concentrations were resistant to breakdown than natural estrogens (Sole´ checked with liquid chromatography coupled with ( ) et al., 2000). This synthetic compound has at least multiple mass spectrometry LC-MSn and were been partially held responsible for the estrogenic within 10% of the nominal concentrations. activity of many effluents (Desbrow et al., 1998; For the second experiment, fish were injected Routledge et al., 1998; Larsson et al., 1999). with 1, 10, 25 and 50 mgEE2yg fishyweek in half ( The rainbow trout is one of the most widely doses twice a week e.g. 0.5 mgEE2yg fish twice ) used fish species in ecotoxicology and its biology a week for the 1 mg EE2 dose . Solvent controls ( ) is well suited for laboratory studies and for in situ received the vehicle 100 ml of peanut oil only, monitoring for the presence of environmental whereas controls were not injected. In both exper- estrogens (Harries et al., 1996; Jobling et al., iments, fish were exposed in a temperature-con- ( ) 1996; Thorpe et al., 2000). Furthermore, radioim- trolled room 20 8C , with an average water munoassays (Sumpter, 1985) and enzyme immuno temperature of 18 8C. assays (Bon et al., 1997) for Vtg detection in this species are available. 2.3. Collection of plasma, liver and gonad This study evaluates the potential use of indirect indicators of plasma Vtg levels. The total protein, On day 7 and day 14 (for the water exposure alkali-labile phosphoprotein and calcium concen- only), blood samples were collected from the trations in the plasma of rainbow trout exposed to caudal artery using heparinized syringes and trans- EE2 were determined. To validate these indirect ferred into heparinized ice-cooled vials containing parameters, actual plasma Vtg concentrations were aprotinin (Sigma–Aldrich, Belgium) at a final also determined in a selected number of samples concentration of 10 mlyml. Plasma collected after with a commercially available rainbow trout vitel- centrifugation (1700=g, 10 min, 4 8C), was logenin EIA (Biosense, Norway). In addition, the shock-frozen in liquid nitrogen and kept at y80 gonadosomatic (GSI) and hepatosomatic (HSI) 8C. Liver and gonads were dissected, weighed and index were measured. The different techniques are immediately frozen in liquid nitrogen and kept at T. Verslycke et al. / Comparative Biochemistry and Physiology Part C 132 (2002) 483–492 485 y80 8C for further analysis. The GSI and HSI or time-dependent effects were observed on the were calculated as follows: GSI. Exposure of rainbow trout to EE had a signif- GSI (%) 2 icant effect on all four tested plasma parameters s(total gonad weightytotal body weight) (protein, ALP, Ca and Vtg). The plasma protein =100 concentration was significantly higher after 2 ( )s( ) ( ) HSI % total liver weightytotal body weight weeks exposure to 100 ng EE2yl P-0.01 , but =100 was not significantly different from the solvent control at 1 and 10 ng EE2yl. A significant time- 2.4. Vitellogenin analysis dependent induction of the plasma protein concen- tration was noted at the highest exposure The plasma total protein concentration was concentration (P-0.05; Fig. 1a). Plasma phospho- determined according to Bradford (1976) with protein concentrations were significantly induced ( ) bovine serum albumin Sigma–Aldrich, Belgium by EE22 at 100 ng EE yl after 1 week exposure as protein standard. Alkaline-labile phosphorus, as (P-0.001). Plasma ALP concentrations were sig- ortho-phosphate released from vitellogenin, was nificantly higher after two weeks in the 1 and 10 extracted from 30 ml of plasma according to ng EE2yl treatments compared to those observed
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