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Elevation of serum 17-β-estradiol in channel catfish following injection of 17-β-estradiol, ethynyl estradiol, estrone, estriol and estradiol-17-β-glucuronide
Environmental Toxicology and Pharmacology 9 (2001) 169– 172
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Elevation of serum 17-b-estradiol in channel catfish following injection of 17-b-estradiol, ethynyl estradiol, estrone, estriol and estradiol-17-b-glucuronide Fred Tilton 1, William H. Benson 2, Daniel Schlenk * Department of Pharmacology and Research Institute of Pharmaceutical Sciences, School of Pharmacy, The Uni6ersity of Mississippi, Uni6ersity, MS 38677, USA Received 27 October 2000; received in revised form 5 January 2001; accepted 19 January 2001
Abstract 17-b-Estradiol is naturally converted in numerous organisms to various derivatives/metabolites, which may be excreted from the organism into its immediate external environment. There is a paucity of data regarding the biological effects of these derivatives/metabolites on aquatic organisms. Male channel catfish (200– 500 g, N = 5, 12 – 18 months) were injected with 1 mg/kg 17-b-estradiol (E2), ethynyl estradiol (EE2), estrone, estriol or E2-17-b-glucuronide with subsequent measurements of vitellogenin (Vtg) and serum E2 concentrations 7 days post injection. EE2 and E2 gave the largest magnitude of Vtg response followed by estrone and estriol. Exposure to EE2, estrone, and E2-17-b-glucuronide all induced significant increases in serum E2 concentrations. This study indicates that metabolites of E2 are also estrogenic and may potentially disrupt estrogen feedback loops within aquatic organisms. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Channel catfish; 17-b-estradiol; Estrone; Ethynylestradiol; Estriol
1. Introduction Several studies have measured natural and synthetic estrogens together in municipal wastewaters (Harries et al. 1997; Desbrow et al. 1998; Lye et al. 1999). The major compounds measured to date have been the natural estrogen, 17-b-estradiol (E2) (up to 50 ng/l), and its metabolite, estrone (Ternes et al. 1999; Larson et al. 1999; Desbrow et al. 1998). The synthetic estrogen, ethynyl estradiol (EE2), has also been measured up to 10 ng/l (Desbrow et al. 1998). EE2 is a component of the prescribed human contraceptive pill and post* Corresponding author. Present address: Department of Environmental Sciences, University of California, Riverside, CA 92521, USA. Tel.: + 1-662-9157330; fax: +1-662-9155148. E-mail address: [email protected] (D. Schlenk). 1 Present address: Department of Biological Sciences, University of Idaho, Moscow, ID 83844. 2 Present address: U.S. EPA, National Health and Environmental Effects Research Laboratory, Gulf Ecology Division, 1 Sabine Island Drive, Gulf Breeze, FL 32561-5299.
menopausal hormonal supplements and has a greater binding affinity to the human and channel catfish estrogen receptor (ER) (Nimrod and Benson, 1997). The major E2 metabolite, estrone has lower ER binding affinity relative to E2 (Nimrod and Benson, 1997; Arcand-Hoy and Benson, 1998). In wastewater effluent, estrone was measured at concentrations up to 80 ng/l (Ternes et al. 1999). In fish, the effects of natural and synthetic estrogens have been well established in terms of ER-mediated events; particularly expression of the phospholipoprotein, vitellogenin (Vtg) in the liver, which is often measured in the blood serum of fish as a measurement of exposure to estrogenically active chemicals (Folmer et al. 1996; Harries et al. 1997). Little work has been done to measure the effects of these estrogens on other physiological processes in fish. The aim of this study was designed to investigate what effect other estrogenic compounds (primarily Phase I and Phase II metabolites of E2) have on the levels of the endogenous hormone, E2, in male channel catfish.
1382-6689/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 2 - 6 6 8 9 ( 0 1 ) 0 0 0 6 2 - X
170
F. Tilton et al. / En6ironmental Toxicology and Pharmacology 9 (2001) 169–172
2. Materials and methods Male channel catfish (200 – 500 g, N= 5, 12–18 months) were injected intraperitoneally (i.p.) with either 1 mg/kg of E2, EE2, estrone, estriol, or estradiol 17-bglucuronide and held for 7 days under flow through conditions. After 7 days, the fish were euthanized by cervical dislocation and blood was collected from the caudal vein (ventral on the tail) with an unheparinized needle and syringe. A protease inhibitor (10 ml of 5 mM phenylmethylsulfonylfluoride (PMSF Sigma c P7626)) was added to every ml of collected blood. Blood was allowed to clot on ice followed by centrifugation to separate serum (10 000×g for 10 min). Serum samples were stored at −80°C until analysis. Serum Vtg levels were determined by use of an indirect competitive enzyme linked immunosorbant assay (ELISA) with monoclonal antibodies specific for channel catfish Vtg provided by Dr Charles D. Rice (Clemson University). Briefly, 50 ml of purified Vtg (2 mg/ml) was added to each well of a 96-well plate and was incubated overnight at 4°C. The following day, the plates were washed with a 17 mM borate buffered saline buffer (BBS), 0.5 ml/l Tween 20, pH 9.6 and protein blocked with BBS and bovine serum albumin (BSA-10 mg/ml) for 1 h. Standards of purified Vtg and plasma samples were diluted appropriately in BBS-BSA and serially diluted in duplicate on the 96-well plates. After addition of the monoclonal antibody raised against channel catfish Vtg, the plates were incubated for 3 h followed by a wash with BBS-tween (Goodwin et al., 1992). An anti-mouse IgG (Fc specific) secondary antibody was diluted 1:20 K in BBS-BSA and was added to each well incubated for 4 h at room temperature. Alkaline phosphatase tablets were used to visualize each well. All samples were compared with the corresponding standard curve using WinSelect™ software and an automated plate reader developed by Tecan Corp. Serum E2 concentrations were determined by utilizing a direct competitive enzyme immunoassay (EIA). Briefly, an anti-E2 antibody, provided by Dr Munro (UC-Davis) was allowed to bind directly to a 96-well plate overnight followed by incubation of sample or standard with a predetermined dilution of E2 conjugated with horse radish peroxidase (HRP) for 2 h on the following day. After a wash, a HRP substrate was used to visualize the proportion of E2-HRP bound to the antibody by incubating the substrate in each well for 10–40 min (or optimal OD of 0.8) and read utilizing the Tecan system at 405 nm. The anti-E2 antibody was assessed for cross reactivity with the compounds used in this study, as well as many other hormones (Munro et al., 1988).
Fig. 1. Serum vitellogenin concentrations in adult male channel catfish 7 days post i.p. injection with 1 mg/kg of a synthetic and several natural estrogens. Differences between mean determined utilizing a one way analysis of variance (ANOVA) (PB 0.05).
3. Results The EIA was compared with commercially available kits (Caymen Inc.). There were no differences in data collected using either assay. The most cross-reactivity occurred with estrone (3.3%) while EE2, estrone and E2-glucuronide had no significant cross reactivity (B 1%). As expected the exposure to E2 and EE2 resulted in the largest Vtg responses (Fig. 1). The two major metabolites of E2, estrone and estriol were found to be 100 and 150X less potent at inducing a Vtg response compared with E2. Vtg was not detected in fish receiving E2-glucuronide. Serum E2 concentrations were found to be significantly elevated from control levels in E2, EE2, estrone and E2-glucuronide treated fish, 7 days post injection (Fig. 2).
Fig. 2. Serum 17-b-estradiol concentrations in adult male channel catfish 7 days post i.p. injection with 1 mg/kg of a synthetic and several natural estrogens. Differences between mean determined utilizing a one way ANOVA (P B0.05).
F. Tilton et al. / En6ironmental Toxicology and Pharmacology 9 (2001) 169–172
4. Discussion EE2 and E2 significantly increased Vtg at the 1 mg/kg dose. The 7-day EC50 of E2 for vitellogenic responses was determined in earlier studies with channel catfish to be 0.6 mg/kg. In the same studies, EE2 was found to be more potent at ER binding (Nimrod and Benson, 1997). There was no measurable Vtg in the E2-glucuronide treated fish even though the serum E2 levels were similar to those of estrone treated fish (Fig. 2). This indicates the possibility of deconjugation of E2-glucuronide, in vivo. As a result of deconjugation, estradiol levels may have begun to rise in the plasma without expression of the Vtg indicating serum protein binding or distribution away from the liver. These data demonstrate that the injection of structurally similar estrogens result in an increase in the endogenous parent hormone E2 (Figs. 1 and 2). E2 metabolites also elicit effects through ER mediated events and may be acting at receptors located in the CNS or peripherally (e.g. gonads) to affect feed-back loops through the pituitary – hypothalamus axis (Redding et al., 1993; Chyb et al., 1999). Understanding the binding characteristics and physiology of multiple ER’s in channel catfish will likely shed light on these processes (Xia et al., 1999, 2000). Biotransformation and clearance was possibly compromised by EE2 resulting in elevated levels of E2 (Fig. 2). EE2 acts as an inhibitor of CYP3A27, which hydroxylates E2 in trout (Miranda et al., 1998). EE2 has also been found to inhibit CYP1A and several key Phase II biotransformation enzymes (Sole et al., 2000). Non-steroidal compounds with known direct estrogenic effects such as nonylphenol and its metabolites are also known to inhibit CYP1A, which also catalyzes the hydroxylation of E2 (Arukwe et al., 1997). Inhibition of the degradative pathways of E2 (i.e hydroxylation) may enhance the persistence of E2 leading to higher serum levels. Thus, it may be possible for compounds to elicit estrogenic activity without directly interacting at the ER. These data demonstrate that exposure to estrogen derivatives and metabolites possibly disrupt feedback and/or E2 clearance pathways in fish. Since these compounds have been identified as primary environmental estrogens in various wastewater effluents, additional studies are necessary to determine the effects of combined exposure on serum E2 and reproductive performance in fish.
Acknowledgements The investigators wish to express their appreciation to Dr Charles D. Rice (Clemson University) for providing monoclonal antibodies specific for channel catfish.
171
Support for the research activities presented was provided, in part, by the US Department of Interior, US Geological Survey, Mississippi Cooperative State Resources Program and the Research Institute of Pharmaceutical Sciences, The University of Mississippi.
References Arcand-Hoy, L.D., Benson, W.H., 1998. Fish reproduction: an ecologically relevant indicator of endocrine disruption. Environ. Toxicol. Chem. 17, 49 – 57. Arukwe, A., Forlin, L., Goksoyr, A., 1997. Xenobiotic and steroid biotransformation enzymes in Atlantic salmon (Salmo salar) liver treated with an estrogenic compound, 4-nonylphenol. Environ. Toxicol. Chem. 12, 2576– 2583. Chyb, J., Mikolajczk, T., Breton, B., 1999. Post-ovulatory secretion of pituitary gonadotropins GtH I and II in the rainbow trout (Oncorhynchus mykiss): regulation by steroids and possible role of non-steroidal gonadal factors. J. Endocrinol. 163 (1), 87–97. Desbrow, C., Routledge, E.J., Brighty, G.C., Sumpter, J.P., Waldock, M.., 1998. Identification of estrogenic chemical in STW effluent. 1. Chemical fractionation and in vitro biological screening. Environ. Sci. Technol. 32, 1549– 1558. Folmer, L.C., Denslow, N.D., Rao, V., Chow, M., Cram, D.A., Enblom, J., Marcino, J., Guillette, L.J., Jr, 1996. Vitellogenin induction and reduced serum testosterone concentrations in feral male carp (Cyprinus carpio) captured near a major metropolitan sewage treatment plant. Environ. Health Perspect. 104, 1096– 1101. Goodwin, A.B., Grizzle, J.M, Bradley, J.T., Estridge, B.H., 1992. Monoclonal antibody-based immunoassay of vitellogenin in the blood of male channel catfish (Ictalurus punctatus). Comp. Biochem. Physiol. 101B, 441– 446. Harries, J.E., Sheahan, D.A., Jobling, S., Matthiessen, P., Neall, P., Sumpter, J.P., Tyler, T., Zaman, N., 1997. Estrogenic activity in five UK rivers detected by measurement of vitellogenesis in caged male trout. Environ. Toxicol. Chem. 16, 534– 542. Larson, D.G.J., Adolfsson-Erici, M., Parkkonen, J., Peffersson, M., Berg, A.H., Olsson, P-E, Forlin, L., 1999. Ethinylestradiol-an undesired fish contraceptive? Aquat. Toxicol. 45, 91 – 97. Lye, C.M, Frid, C.L.J., Gill, M.E., Cooper, D.W, Jones, D.M., 1999. Estrogenic alkylphenols in fish tissues, sediments and waters from the UK Tyne and Tees estuaries. Environ. Sci. Technol. 33, 1009– 1014. Miranda, C.L., Henderson, M.C., Buhler, D.R., 1998. Evaluation of chemicals as inhibitors of trout cytochrome P450. Toxicol. Appl. Pharm. 148, 237– 244. Munro, C.J., Lasley, B.L., 1988. Polypeptide and Steroid Hormone Detection. Liss A.R. Inc, pp. 289– 329. Nimrod, A.C., Benson, W.H., 1997. Xenobiotic interaction with and alteration of channel catfish estrogen receptor. Toxicol. Appl. Pharmacol. 147, 381– 390. Redding, M.J., Patino, R., 1993. In: Evans, D.H. (Ed.), The Physiology of Fishes; Marine Science Series. CRC, Boca Raton, pp. 503– 534. Sole, M., Porte, C., Barcelo, D., 2000. Vitellogenin induction and other biochemical responses in carp (Cyprinus carpio) after experimental injection with 17 alpha-ethynylestradiol. Arch. Environ. Contam. Toxicol. 38 (4), 494– 500. Ternes, T.A., Stumpf, M., Mueller, J., Haberer, K., Wilken, R.D., Servos, M., 1999. Behavior and occurrence of estrogens in municipal sewage treatment plants -I. Investigations in Germany, Canada and Brazil. Sci. Total Environ. 225, 81 – 90.
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F. Tilton et al. / En6ironmental Toxicology and Pharmacology 9 (2001) 169–172
Xia, Z., Gale, W.L., Chang, X., Langenau, D., Patino, R., Maule, A.G., Densmore, L.D., 2000. Phylogenetic sequence analysis, recombinant expression, and the distribution of a channel catfish estrogen receptor beta. Gen. Comp. Endocrinol. 118 (1), 139– 149.
.
Xia, Z., Patino, R., Gale, W.L., Maule, A.G., Densmore, L.D., 1999. Cloning, in vitro expression, and novel phylogenetic classification of a channel catfish estrogen receptor. Gen. Comp. Endocrinol. 113 (3), 360– 368.
www.elsevier.com/locate/etap
Elevation of serum 17-b-estradiol in channel catfish following injection of 17-b-estradiol, ethynyl estradiol, estrone, estriol and estradiol-17-b-glucuronide Fred Tilton 1, William H. Benson 2, Daniel Schlenk * Department of Pharmacology and Research Institute of Pharmaceutical Sciences, School of Pharmacy, The Uni6ersity of Mississippi, Uni6ersity, MS 38677, USA Received 27 October 2000; received in revised form 5 January 2001; accepted 19 January 2001
Abstract 17-b-Estradiol is naturally converted in numerous organisms to various derivatives/metabolites, which may be excreted from the organism into its immediate external environment. There is a paucity of data regarding the biological effects of these derivatives/metabolites on aquatic organisms. Male channel catfish (200– 500 g, N = 5, 12 – 18 months) were injected with 1 mg/kg 17-b-estradiol (E2), ethynyl estradiol (EE2), estrone, estriol or E2-17-b-glucuronide with subsequent measurements of vitellogenin (Vtg) and serum E2 concentrations 7 days post injection. EE2 and E2 gave the largest magnitude of Vtg response followed by estrone and estriol. Exposure to EE2, estrone, and E2-17-b-glucuronide all induced significant increases in serum E2 concentrations. This study indicates that metabolites of E2 are also estrogenic and may potentially disrupt estrogen feedback loops within aquatic organisms. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Channel catfish; 17-b-estradiol; Estrone; Ethynylestradiol; Estriol
1. Introduction Several studies have measured natural and synthetic estrogens together in municipal wastewaters (Harries et al. 1997; Desbrow et al. 1998; Lye et al. 1999). The major compounds measured to date have been the natural estrogen, 17-b-estradiol (E2) (up to 50 ng/l), and its metabolite, estrone (Ternes et al. 1999; Larson et al. 1999; Desbrow et al. 1998). The synthetic estrogen, ethynyl estradiol (EE2), has also been measured up to 10 ng/l (Desbrow et al. 1998). EE2 is a component of the prescribed human contraceptive pill and post* Corresponding author. Present address: Department of Environmental Sciences, University of California, Riverside, CA 92521, USA. Tel.: + 1-662-9157330; fax: +1-662-9155148. E-mail address: [email protected] (D. Schlenk). 1 Present address: Department of Biological Sciences, University of Idaho, Moscow, ID 83844. 2 Present address: U.S. EPA, National Health and Environmental Effects Research Laboratory, Gulf Ecology Division, 1 Sabine Island Drive, Gulf Breeze, FL 32561-5299.
menopausal hormonal supplements and has a greater binding affinity to the human and channel catfish estrogen receptor (ER) (Nimrod and Benson, 1997). The major E2 metabolite, estrone has lower ER binding affinity relative to E2 (Nimrod and Benson, 1997; Arcand-Hoy and Benson, 1998). In wastewater effluent, estrone was measured at concentrations up to 80 ng/l (Ternes et al. 1999). In fish, the effects of natural and synthetic estrogens have been well established in terms of ER-mediated events; particularly expression of the phospholipoprotein, vitellogenin (Vtg) in the liver, which is often measured in the blood serum of fish as a measurement of exposure to estrogenically active chemicals (Folmer et al. 1996; Harries et al. 1997). Little work has been done to measure the effects of these estrogens on other physiological processes in fish. The aim of this study was designed to investigate what effect other estrogenic compounds (primarily Phase I and Phase II metabolites of E2) have on the levels of the endogenous hormone, E2, in male channel catfish.
1382-6689/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 2 - 6 6 8 9 ( 0 1 ) 0 0 0 6 2 - X
170
F. Tilton et al. / En6ironmental Toxicology and Pharmacology 9 (2001) 169–172
2. Materials and methods Male channel catfish (200 – 500 g, N= 5, 12–18 months) were injected intraperitoneally (i.p.) with either 1 mg/kg of E2, EE2, estrone, estriol, or estradiol 17-bglucuronide and held for 7 days under flow through conditions. After 7 days, the fish were euthanized by cervical dislocation and blood was collected from the caudal vein (ventral on the tail) with an unheparinized needle and syringe. A protease inhibitor (10 ml of 5 mM phenylmethylsulfonylfluoride (PMSF Sigma c P7626)) was added to every ml of collected blood. Blood was allowed to clot on ice followed by centrifugation to separate serum (10 000×g for 10 min). Serum samples were stored at −80°C until analysis. Serum Vtg levels were determined by use of an indirect competitive enzyme linked immunosorbant assay (ELISA) with monoclonal antibodies specific for channel catfish Vtg provided by Dr Charles D. Rice (Clemson University). Briefly, 50 ml of purified Vtg (2 mg/ml) was added to each well of a 96-well plate and was incubated overnight at 4°C. The following day, the plates were washed with a 17 mM borate buffered saline buffer (BBS), 0.5 ml/l Tween 20, pH 9.6 and protein blocked with BBS and bovine serum albumin (BSA-10 mg/ml) for 1 h. Standards of purified Vtg and plasma samples were diluted appropriately in BBS-BSA and serially diluted in duplicate on the 96-well plates. After addition of the monoclonal antibody raised against channel catfish Vtg, the plates were incubated for 3 h followed by a wash with BBS-tween (Goodwin et al., 1992). An anti-mouse IgG (Fc specific) secondary antibody was diluted 1:20 K in BBS-BSA and was added to each well incubated for 4 h at room temperature. Alkaline phosphatase tablets were used to visualize each well. All samples were compared with the corresponding standard curve using WinSelect™ software and an automated plate reader developed by Tecan Corp. Serum E2 concentrations were determined by utilizing a direct competitive enzyme immunoassay (EIA). Briefly, an anti-E2 antibody, provided by Dr Munro (UC-Davis) was allowed to bind directly to a 96-well plate overnight followed by incubation of sample or standard with a predetermined dilution of E2 conjugated with horse radish peroxidase (HRP) for 2 h on the following day. After a wash, a HRP substrate was used to visualize the proportion of E2-HRP bound to the antibody by incubating the substrate in each well for 10–40 min (or optimal OD of 0.8) and read utilizing the Tecan system at 405 nm. The anti-E2 antibody was assessed for cross reactivity with the compounds used in this study, as well as many other hormones (Munro et al., 1988).
Fig. 1. Serum vitellogenin concentrations in adult male channel catfish 7 days post i.p. injection with 1 mg/kg of a synthetic and several natural estrogens. Differences between mean determined utilizing a one way analysis of variance (ANOVA) (PB 0.05).
3. Results The EIA was compared with commercially available kits (Caymen Inc.). There were no differences in data collected using either assay. The most cross-reactivity occurred with estrone (3.3%) while EE2, estrone and E2-glucuronide had no significant cross reactivity (B 1%). As expected the exposure to E2 and EE2 resulted in the largest Vtg responses (Fig. 1). The two major metabolites of E2, estrone and estriol were found to be 100 and 150X less potent at inducing a Vtg response compared with E2. Vtg was not detected in fish receiving E2-glucuronide. Serum E2 concentrations were found to be significantly elevated from control levels in E2, EE2, estrone and E2-glucuronide treated fish, 7 days post injection (Fig. 2).
Fig. 2. Serum 17-b-estradiol concentrations in adult male channel catfish 7 days post i.p. injection with 1 mg/kg of a synthetic and several natural estrogens. Differences between mean determined utilizing a one way ANOVA (P B0.05).
F. Tilton et al. / En6ironmental Toxicology and Pharmacology 9 (2001) 169–172
4. Discussion EE2 and E2 significantly increased Vtg at the 1 mg/kg dose. The 7-day EC50 of E2 for vitellogenic responses was determined in earlier studies with channel catfish to be 0.6 mg/kg. In the same studies, EE2 was found to be more potent at ER binding (Nimrod and Benson, 1997). There was no measurable Vtg in the E2-glucuronide treated fish even though the serum E2 levels were similar to those of estrone treated fish (Fig. 2). This indicates the possibility of deconjugation of E2-glucuronide, in vivo. As a result of deconjugation, estradiol levels may have begun to rise in the plasma without expression of the Vtg indicating serum protein binding or distribution away from the liver. These data demonstrate that the injection of structurally similar estrogens result in an increase in the endogenous parent hormone E2 (Figs. 1 and 2). E2 metabolites also elicit effects through ER mediated events and may be acting at receptors located in the CNS or peripherally (e.g. gonads) to affect feed-back loops through the pituitary – hypothalamus axis (Redding et al., 1993; Chyb et al., 1999). Understanding the binding characteristics and physiology of multiple ER’s in channel catfish will likely shed light on these processes (Xia et al., 1999, 2000). Biotransformation and clearance was possibly compromised by EE2 resulting in elevated levels of E2 (Fig. 2). EE2 acts as an inhibitor of CYP3A27, which hydroxylates E2 in trout (Miranda et al., 1998). EE2 has also been found to inhibit CYP1A and several key Phase II biotransformation enzymes (Sole et al., 2000). Non-steroidal compounds with known direct estrogenic effects such as nonylphenol and its metabolites are also known to inhibit CYP1A, which also catalyzes the hydroxylation of E2 (Arukwe et al., 1997). Inhibition of the degradative pathways of E2 (i.e hydroxylation) may enhance the persistence of E2 leading to higher serum levels. Thus, it may be possible for compounds to elicit estrogenic activity without directly interacting at the ER. These data demonstrate that exposure to estrogen derivatives and metabolites possibly disrupt feedback and/or E2 clearance pathways in fish. Since these compounds have been identified as primary environmental estrogens in various wastewater effluents, additional studies are necessary to determine the effects of combined exposure on serum E2 and reproductive performance in fish.
Acknowledgements The investigators wish to express their appreciation to Dr Charles D. Rice (Clemson University) for providing monoclonal antibodies specific for channel catfish.
171
Support for the research activities presented was provided, in part, by the US Department of Interior, US Geological Survey, Mississippi Cooperative State Resources Program and the Research Institute of Pharmaceutical Sciences, The University of Mississippi.
References Arcand-Hoy, L.D., Benson, W.H., 1998. Fish reproduction: an ecologically relevant indicator of endocrine disruption. Environ. Toxicol. Chem. 17, 49 – 57. Arukwe, A., Forlin, L., Goksoyr, A., 1997. Xenobiotic and steroid biotransformation enzymes in Atlantic salmon (Salmo salar) liver treated with an estrogenic compound, 4-nonylphenol. Environ. Toxicol. Chem. 12, 2576– 2583. Chyb, J., Mikolajczk, T., Breton, B., 1999. Post-ovulatory secretion of pituitary gonadotropins GtH I and II in the rainbow trout (Oncorhynchus mykiss): regulation by steroids and possible role of non-steroidal gonadal factors. J. Endocrinol. 163 (1), 87–97. Desbrow, C., Routledge, E.J., Brighty, G.C., Sumpter, J.P., Waldock, M.., 1998. Identification of estrogenic chemical in STW effluent. 1. Chemical fractionation and in vitro biological screening. Environ. Sci. Technol. 32, 1549– 1558. Folmer, L.C., Denslow, N.D., Rao, V., Chow, M., Cram, D.A., Enblom, J., Marcino, J., Guillette, L.J., Jr, 1996. Vitellogenin induction and reduced serum testosterone concentrations in feral male carp (Cyprinus carpio) captured near a major metropolitan sewage treatment plant. Environ. Health Perspect. 104, 1096– 1101. Goodwin, A.B., Grizzle, J.M, Bradley, J.T., Estridge, B.H., 1992. Monoclonal antibody-based immunoassay of vitellogenin in the blood of male channel catfish (Ictalurus punctatus). Comp. Biochem. Physiol. 101B, 441– 446. Harries, J.E., Sheahan, D.A., Jobling, S., Matthiessen, P., Neall, P., Sumpter, J.P., Tyler, T., Zaman, N., 1997. Estrogenic activity in five UK rivers detected by measurement of vitellogenesis in caged male trout. Environ. Toxicol. Chem. 16, 534– 542. Larson, D.G.J., Adolfsson-Erici, M., Parkkonen, J., Peffersson, M., Berg, A.H., Olsson, P-E, Forlin, L., 1999. Ethinylestradiol-an undesired fish contraceptive? Aquat. Toxicol. 45, 91 – 97. Lye, C.M, Frid, C.L.J., Gill, M.E., Cooper, D.W, Jones, D.M., 1999. Estrogenic alkylphenols in fish tissues, sediments and waters from the UK Tyne and Tees estuaries. Environ. Sci. Technol. 33, 1009– 1014. Miranda, C.L., Henderson, M.C., Buhler, D.R., 1998. Evaluation of chemicals as inhibitors of trout cytochrome P450. Toxicol. Appl. Pharm. 148, 237– 244. Munro, C.J., Lasley, B.L., 1988. Polypeptide and Steroid Hormone Detection. Liss A.R. Inc, pp. 289– 329. Nimrod, A.C., Benson, W.H., 1997. Xenobiotic interaction with and alteration of channel catfish estrogen receptor. Toxicol. Appl. Pharmacol. 147, 381– 390. Redding, M.J., Patino, R., 1993. In: Evans, D.H. (Ed.), The Physiology of Fishes; Marine Science Series. CRC, Boca Raton, pp. 503– 534. Sole, M., Porte, C., Barcelo, D., 2000. Vitellogenin induction and other biochemical responses in carp (Cyprinus carpio) after experimental injection with 17 alpha-ethynylestradiol. Arch. Environ. Contam. Toxicol. 38 (4), 494– 500. Ternes, T.A., Stumpf, M., Mueller, J., Haberer, K., Wilken, R.D., Servos, M., 1999. Behavior and occurrence of estrogens in municipal sewage treatment plants -I. Investigations in Germany, Canada and Brazil. Sci. Total Environ. 225, 81 – 90.
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F. Tilton et al. / En6ironmental Toxicology and Pharmacology 9 (2001) 169–172
Xia, Z., Gale, W.L., Chang, X., Langenau, D., Patino, R., Maule, A.G., Densmore, L.D., 2000. Phylogenetic sequence analysis, recombinant expression, and the distribution of a channel catfish estrogen receptor beta. Gen. Comp. Endocrinol. 118 (1), 139– 149.
.
Xia, Z., Patino, R., Gale, W.L., Maule, A.G., Densmore, L.D., 1999. Cloning, in vitro expression, and novel phylogenetic classification of a channel catfish estrogen receptor. Gen. Comp. Endocrinol. 113 (3), 360– 368.