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Environ. Sci. Technol. 2007, 41, 3368-3374

Functional Associations between Two Estrogen Receptors, Environmental Estrogens, and Sexual Disruption in the Roach (Rutilus rutilus) YOSHINAO KATSU,† ANKE LANGE,‡ HIROSHI URUSHITANI,† RIE ICHIKAWA,† GREGORY C. PAULL,‡ LAURA L. CAHILL,‡ SUSAN JOBLING,§ C H A R L E S R . T Y L E R , * ,‡ A N D TAISEN IGUCHI† Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, 444-8787, Japan, University of Exeter, School of Biosciences, Prince of Wales Road, Exeter, Devon, EX4 4PS, United Kingdom, and Brunel University, Department of Biological Sciences, Uxbridge, Middlesex UB8 3PH, United Kingdom

Wild male roach (Rutilus rutilus) living in U.K. rivers contaminated with estrogenic effluents from wastewater treatment works show feminized responses and have a reduced reproductive capability, but the chemical causation of sexual disruption in the roach has not been established. Feminized responses were induced in male roach exposed to environmentally relevant concentrations of the pharmaceutical estrogen 17R-ethinylestradiol, EE2 (up to 4 ng/ L), during early life (from fertilization to 84 days posthatch, dph), and these effects were signaled by altered patterns of expression of two cloned roach estrogen receptor (ER) subtypes, ERR and ERβ, in the brain and gonad/ liver. Transactivation assays were developed for both roach ER subtypes and the estrogenic potencies of steroidal estrogens differed markedly at the different ER subtypes. EE2 was by far the most potent chemical, and estrone (E1, the most prevalent environmental steroid in wastewater discharges) was equipotent with estradiol (E2) in activating the ERs. Comparison of the EC50 values for the compounds tested showed that ERβ was 3-21-fold more sensitive to natural steroidal estrogens and 54-fold more sensitive to EE2 as compared to ERR. These findings add substantial support to the hypothesis that steroidal estrogens play a significant role in the induction of intersex in roach populations in U.K. rivers and that the molecular approach described could be usefully applied to understand interspecies sensitivity to xenoestrogens.

Introduction The occurrence of intersex in some wild fish has been associated with the proximity of these fish to point source * Corresponding author phone: +44-1392-264450; +44-1392-263700; e-mail: [email protected]. † Okazaki Institute for Integrative Bioscience. ‡ University of Exeter. § Brunel University. 3368

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wastewater effluent discharges (reviewed in ref 1). Intersex phenotypes include individuals with feminized reproductive ducts and/or developing oocytes within their testes (2). These disruptions in sexual development are associated with abnormal concentrations of sex steroid hormones and elevated blood concentrations of the estrogen-dependent protein, vitellogenin VTG (3). Gondal duct disruption and VTG induction in roach have been induced through controlled exposures to effluents from wastewater treatment works (WwTW) (4, 5). There are several chemicals in WwTW effluents for which there is a plausible link with the feminized responses seen, and they include steroid estrogens (arising from human and animal waste; principally, the natural steroid estrogens 17β-estradiol (E2), estrone (E1), and the contraceptive pill hormone 17R-ethinylestradiol (EE2)) and some industrial chemicals, such as alkylphenols, 4-nonylphenol (NP), and 4-tert-octylphenol (OP). Feminization in fish can be induced by these compounds (e.g., refs 6-8), but the evidence of a causal link for the effects seen in wild fish and exposure to a specific chemical(s) has not been established. Steroid estrogens have been shown to be exquisitely potent in vivo but are often present in the aquatic environment at lower concentrations than might be expected to cause effects in fish. The most prevalent steroid estrogen in the environment is E1, while the most potent (but least prevalent) is generally thought to be EE2. Alkylphenols are generally present at higher concentrations in the aquatic environment than steroid estrogens but are generally much less potent as estrogens. There is a need to develop our understanding of the roles and mechanisms of action of the various steroid estrogens, and other estrogenic chemicals present in the aquatic environment, in the feminization process. Biological effects of estrogens are principally mediated through specific nuclear receptor proteins, the estrogen receptors (ERs), which function as ligand-activated transcription factors. There are at least two subtypes of ERs (ERR and ERβ) mediating the diverse functions of estrogens, and these have been cloned in a variety of vertebrate species (see ref 9). ERs exhibit broad tissue expression, consistent with the diverse roles of estrogens. In fish, tissue expression of ERR and ERβ has been shown to differ between species, but generally, it appears to be concentrated in the gonad and liver (10, 11) where estrogens have pivotal roles in gonadal sex differentiation and development (12), and in the hepatic production of the egg yolk precursor, VTG. The finding of a second form of ERβ subtype in fish, designated as ERγ or ERβ2 (13), has complicated further our understanding of estrogens and their roles and effect pathways, but ERγ is predominantly expressed in somatic tissues only (e.g., ref 14). To further our understanding of the chemical causation and ER-mediated pathway of sexual disruption in wild fish, the cDNAs encoding for ERR and ERβ were first cloned, sequenced, and characterized in the roach (Rutilus rutilus) (a wild fish species in which intersex is prevalent in rivers in Europe). Roach were then exposed to a series of environmentally relevant concentrations of the steroid estrogen EE2 during early life (until 84 days post-hatch (dph)), and the effects were determined on the dynamics of expression of the two ER subtypes and on gonadal differentiation and development. Finally, transactivational reporter gene assays for roach (r)ERR and rERβ were developed to screen environmental estrogens contained in WwTW effluents (including EE2, E2 E1, estriol (E3), NP, and OP) to investigate their relative potencies in disrupting ER-mediated signaling pathways in the roach. 10.1021/es062797l CCC: $37.00

 2007 American Chemical Society Published on Web 04/03/2007

Materials and Methods Fish Source, Culture, and Husbandry. For production of the embryos for the EE2 exposure experiment, pre-spawning sexually mature roach were brought into the aquarium facility where they were artificially induced to spawn, as described by Jobling et al. (15). The resulting embryos were maintained under flow-through conditions at 18 ( 1 °C with a fixed photoperiodic regime of 16 h:8 h light/dark. Embryos hatched 7-10 days post-fertilization and the resulting fry were fed three times a day with a commercial cyprinid dry food until 250 dph supplemented with freshly hatched Artemia sp. nauplii. Biological Sampling for Molecular Characterization of rERs. Sexually mature roach, including post-spawning fish in one group and sexually maturing fish in another (720 dph), were sacrificed with a lethal dose of anesthetic (ethyl-paminobenzoate) as approved by the U.K. Home Office (Animals (Scientific Procedures) Act 1986). Gonads, brain, liver, and muscle tissue were collected, immediately frozen in liquid nitrogen, and stored at -80 °C until RNA isolation. Molecular Cloning and Characterization of rERs. Extraction of DNase-treated total RNA from the ovary of an adult roach and cDNA synthesis were conducted as described previously (16). This cDNA was used as a template for amplification, using degenerate oligonucleotides (Table S1, Supporting Information) designed in conserved regions of the DNA-binding C-domain and the ligand-binding Edomain of ERs. The fragments obtained were subcloned and sequenced, and the 5′- and 3′-ends of the ER cDNAs were amplified as described by Katsu et al. (16). All sequences were searched for similarity using the Blast tools (National Center of Biotechnology Information (17)), and multiple sequence alignments were performed using ClustalX (18). On the basis of these alignments, a phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replicates. Tissue Distribution of ER Expression. Tissue expression of ERs was analyzed by RT-PCR in gonads, brain, liver, and muscle of the post-spawning male and female roach and in brain, liver, and gonads of sexually maturing fish. Total RNA was extracted and reverse transcribed as described previously and, in the case of the tissue derived from post-spawning fish, subjected to RT-PCR using gene-specific primers. PCR conditions were as described previously (16). Expression of the housekeeping gene β-actin was used to control for differences in loading and cDNA synthesis efficiency. At completion of the PCR, fragments were separated by agarose gel electrophoresis. Real-time PCR (Q-PCR) was carried out to determine the tissue localization of ERs in a quantitative manner in the sexually maturing roach. Using gene-specific primers, Q-PCR was carried out as described by Katsu et al. (9). Gene expression levels were normalized using the expression levels of the ribosomal protein L8 mRNA (rpl8). Sequences of all primers used are available in Table S1 (Supporting Information). Effects of EE2 Exposure during Early Life on ER Expression and Gonadal Development. Newly fertilized roach eggs were divided between eight tanks and exposed under flowthrough conditions immediately to EE2 (Sigma-Aldrich, Gillingham, U.K.) for a period of 84 days. Fish were exposed to EE2 in duplicate tanks to a series of three concentrations of environmental concentrations of EE2 (nominal 0.1, 1.0, and 10 ng/L). Duplicate control water tanks were run under the same conditions, without the addition of EE2. Water samples were taken regularly from each tank to measure the concentrations of EE2. Samples were spiked with 5% (v/v) methanol and extracted onto preconditioned solid-phase Sep-Pack C18 cartridges (Waters Ltd., Elstree, Hertsfordshire,

U.K.) following the manufacturer’s protocol. The extract was subsequently eluted from the column with 100% methanol and stored at 4 °C until required. A radioimmunoassay was used to verify EE2 concentrations in water using a procedure identical to that used for radioimmunoassay reported for other steroids by the CEFAS laboratory (19, 20). A polyclonal antiserum to EE2 was raised by injection of ethynylestradiol 6-carboxymethyloxime/bovine serum albumin (Steraloids Inc. Ltd., London, U.K.) into rabbits. Tritiated EE2 was purchased from Perkin-Elmer Life Sciences Inc. (Boston, MA). The antiserum had a negligible ( testis) and rERR in the liver (females > males). Effects of EE2 Exposure during Early Life on the Expression of rERR and rERβ and Associated Impacts on Sexual Development. The mean measured exposure concentrations of EE2 were below detection limit (nominal 0.1), 3370

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0.3 ( 0.1 (1.0) and 4 ( 0.3 (10) ng/L of EE2, during the 84 dph exposure. The basal concentration of VTG in control fish was 14.4 ( 0.6 ng/mL, and exposure to EE2 during early life resulted in a 12-fold induction of VTG at 4 ng of EE2/L, with no effects at the lower concentrations (Figure 2A). Histological analyses of the gonads found no observable difference in the gonadal status of fish exposed to the lowest concentration of EE2 as compared with control fish. Both groups (n ) 20 each) contained approximately 20% discernible females and fewer discernible males, and the remainder was undifferentiated. For fish exposed to 0.3 ng/L of EE2, 37% were discernible females (n ) 20), but there were no discernible males. In contrast, 95% of the fish exposed to 4 ng of EE2/L had a female-like gonadal morphology, characterized by the presence of an ovarian cavity (Figure 2B). This group of fish would have included feminized male gonad phenotypes. Expression of rERβ was higher than rERR in both the head and the body of all fish, with the exception of fish exposed to 4 ng of EE2/L (Figure 2C,D). There was a concentrationrelated induction of rERR in the body of fish exposed to EE2. In the head (brain), EE2 similarly induced an elevated rERR mRNA expression, but the effect was less marked. rERβ expression in the body was significantly elevated after exposure to 0.3 and 4 ng of EE2/L. In heads, expression of rERβ was elevated above controls at the lower two concentrations but not at 4 ng of EE2/L. Transcriptional Activities of rERs in Response to Steroidal Estrogens and Alkylphenols. Functional responses of the two rERs to environmental estrogens, including EE2, in the reporter gene assay showed that the cloned rERs encoded functional proteins. A higher luciferase activity was found for rERR as compared to rERβ, which could be due to

FIGURE 2. Effects of 17r-ethinylestradiol (EE2) exposure during early life on the expression of rERr and rERβ and associated impacts on sexual development. (A) Vitellogenin concentrations in whole-body homogenates of roach exposed for 84 dph, results are shown as the mean ( SEM, and the numbers in parentheses indicate the number of samples analyzed. (B) Transverse section of roach gonad exposed to 4 ng/L of EE2 up to 84 dph. pgc: primordial germ cell; oc: ovarian-like cavity; L: liver; and pw: peritoneal wall; arrows indicate the points of attachment to pw. Bar 50 µm. Relative expression of rERr (C) and rERβ (D) in bodies and heads of juvenile roach (84 dph) exposed to different concentrations of EE2. Gene expression results (n ) 19-20) are presented as box plots where the boundary of the box closest to zero indicates the 25th percentile, a line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Whiskers above and below the box indicate the 90th and 10th percentiles, and black dots represent the fifth and 95th percentiles. Different letters above boxes indicate significant differences (p < 0.01). n.d.: nondetectable. a differential degradation rate of receptor proteins or a differential stability of the receptor-DNA or receptor-ligand complexes. Concentration-dependent luciferase induction occurred for E1, E2, E3, and EE2 in both transcriptional assays (Figure 3). In the rERR assay, EE2 was the most potent (significant at 10-10 M), and activating concentrations for other steroidal estrogens were 10-9 M for E2 and E1 and 10-8

M for E3. In rERR, both OP and NP induced luciferase activity at 10-6 M. Similarly for rERβ, EE2 was the most potent of the chemicals tested and induced transcriptional activity at 10-11 M. Significant increases in transcriptional activity in rERβ for the other steroidal estrogens occurred at concentrations of 10-10 M for E1 and E2 and 10-8 M for E3. NP and OP did not induce significant transactivational activity with rERβ. VOL. 41, NO. 9, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Dose-dependent effects of 17β-estradiol (E2), estrone (E1), estriol (E3), 17r-ethinylestradiol (EE2), nonylphenol (NP), and 4-tert-octylphenol (OP) on the transcriptional activation of rERr (A) and rERβ (B). Luciferase activities are presented relative to the luciferase activity of the solvent control. Asterisk: p < 0.01 vs DMSO controls (Dunnett’s test). Testosterone and progesterone were unable to induce expression of the reporter gene mediated by either of the rERs (data not shown). The maximal luciferase activity was achieved with E1 and E2 at concentrations of 10-7 M for rERR and 10-9 M for rERβ. EC50 values were calculated from the concentrationrelated responses for the four steroidal estrogens and used for determining their relative potency (the ratio between the EC50 of E2 and the EC50 of the tested compound; Table 1). For rERR, the relative potency of EE2 was similar to E2 (112%) but lower for E1 (34.8%) and E3 (4.8%). For rERβ, the relative potencies were 665.3% for EE2, 82.4% for E1, and 1.7% for E3.

Discussion The relative importance of the presence of the various steroid (and other) estrogens in the aquatic environment is de-

pendent on their ability to disrupt the normal pattern of estrogen receptor-mediated signaling in the body of exposed organisms. We demonstrated that ERR and ERβ signaling pathways operate in both male and female roach and that exposure to EE2 during early life altered ER subtype expression and induced gonadal feminization of males. Transactivational assays demonstrated that the steroid estrogens and alkylphenolic xenoestrogens (to a lesser extent) activated both these ER pathways. Of particular interest was the finding that the relative potencies of the various estrogens differed through the different ER subtypes. Tissue-specific effects of estrogens in roach are likely related to the differential tissue distribution and regulation of the different ER receptor subtypes. The detection of both rER mRNAs in brain, gonad, and liver in post-spawning fish is consistent with the broad range of functions of estrogens (10, 23-25). The higher level of rERR expression in the female liver is consistent with a dominant role of ERR in the hepatic induction of VTG (26, 27), but the functional significance of expression of rERβ in hepatic tissue of both sexes is not known. In mammals, estrogens are known to play multi-faceted roles in ovarian function, all of which are mediated via the ERs (28, 29). Increasingly, however, it is recognized that estrogens also play a role in the fertility in males (30, 31). Within the ovary and testis of maturing roach, expression levels of rERR did not differ between the sexes, whereas rERβ was significantly more highly expressed in the ovary as compared with the testis. These findings contrast with results obtained for sexually mature fathead minnows, where ERR was shown to be predominant in the ovary and ERβ predominant in the testis (14). These data indicate species differences in the expression of ER subtypes in gonads and emphasize the importance of staging the fish precisely when reporting and assessing functional relationships for the expression of specific genes (here, ERs in the gonad). rERR was expressed at a higher level as compared with rERβ in the brain for both sexes, and there were no genderspecific differences in expression levels of rERR and rERβ, consistent with findings in other fish species (32, 33). Distinct distributions of the ER subtypes have been demonstrated in fish (13), suggesting some distinct roles for the different ERs, but any functional division for ERR and ERβ in the brain has not been determined. Exposure of roach during early life to an environmentally relevant concentration of EE2 (4 ng/L) disrupted the normal dynamics of sexual development, inducing VTG and a concentration-related feminization of the gonadal ducts in male fish, comparable with the effects seen in studies in zebrafish and fathead minnow for similar exposure regimes (6, 34). Concentration-related up-regulation of rERR and rERβ expression in both the body and the head was associated with the altered sexual phenotype in the roach. The highest induction was for rERR (in the body), and this is consistent with other studies in fish where ERR has been shown to be

TABLE 1. Gene Transactivational Activities of Chemicals Mediated by rERr and rERβa rERβb

rERr

17β-estradiol Estrone Estriol 17R-ethinylestradiol a

EC50 (M)

RPb

4.957 × 10-10 (3.763 × 10-10 to 6.53 × 10-10) 1.424 × 10-9 (31.067 × 10-9 to 1.901 × 10-9) 1.036 × 10-8 (9.014 × 10-9 to 1.191 × 10-8) 4.427 × 10-10 (3.086 × 10-10 to 6.352 × 10-10)

100

(%)

34.81 4.79 111.97

Numbers in brackets represent 95% confidence intervals for the EC50 values.

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b

EC50 (M) 5.49 × 10-11 (3.66 × 10-11 to 8.236 × 10-11) 6.667 × 10-11 (4.538 × 10-11 to 9.794 × 10-11) 3.279 × 10-9 (2.636 × 10-9 to 4.08 × 10-9) 8.252 × 10-12 (5.753 × 10-12 to 1.1184 × 10-11) RP: relative potency.

RP (%) 100 82.35 1.67 665.29

the most responsive ER gene to estrogenic stimulation (14, 26, 27). These different effects of EE2 on the expression of rERs and the vitellogenic response in exposed fish support the hypothesis that ER subtypes do not contribute equally to estrogen-dependent gene regulation, including vitellogenesis (27). It is likely that altered gonadal expression of ER contributes to the elevated levels of expression of rERs observed in the whole-body analysis of EE2 exposed roach. This is supported by the effects seen on the gonad, with a feminization of the developing testis. Other studies have shown elevated gonadal ER expression after exposure of adult medaka and male zebrafish to EE2 (35, 36). The induction of both rERs in heads of fish exposed to the two lower concentrations of EE2 might reflect a positive feedback regulation of ER expression stimulated by the exogenous estrogen. In the reported gene assays, E1, E3, and EE2 induced concentration-dependent activation of both rERs. For the natural estrogens, transactivation was significantly induced in an order of effectiveness of E2 ∼ E1 > E3 for both rER subtypes, similar to the preferences of ERs isolated from rat and Atlantic croaker (25, 37). EC50 values indicated that rERβ was more sensitive to steroidal estrogens as compared to rERR: for E3 3-fold higher, for E2 9-fold higher, for E1 21-fold higher, and for EE2 54-fold higher. Recently, Le Page et al. (38) similarly reported a higher sensitivity of zebrafish (zf)ERβ for E2 (7-fold) and EE2 (10-fold), but in contrast to the present study, zfERR was slightly more sensitive to E1 than zfERβ (relative sensitivity zfERβ/zfERR ) 0.6). The relative potency of EE2 as compared to E2 to induce rERβ (665%) is similar to the potency described for zfERβ (513%), whereas its relative potency to induce rERR is about three times lower as compared to zfERR. It has been shown that exposure of fish to alkylphenols induces feminizing responses (39, 40) and that these responses are likely to be induced through the activation of ERs. In this study, OP and NP had a weak activation of rERR only. It should be realized, however, that ERR and ERβ can recruit coactivators in the presence of estrogens and xenoestrogens, and it has been proposed that tissue-specific estrogenic and/or antiestrogenic actions of certain xenoestrogens may be associated with alterations in the tertiary structure of ERR and/or ERβ following ligand binding; changes that are sensed by cellular factors (coactivators) required for normal gene expression. It is still unclear whether xenoestrogens affect the normal behavior of ERR and/or ERβ subsequent to receptor binding (41). In conclusion, the differences in structure between rERR and rERβ and differences in their tissue expression imply distinct physiological functions in roach. In mammals, studies on the different ER subtypes have demonstrated that ERR is critical for fertility in both sexes but that ERβ is not (42, 43). Furthermore, ERβ appears to be the only ER found in germ cells, indicating a more fundamental role for ERβ in germ cell development/differentiation (44). The expression data for ER in roach indicate that both rERR and rERβ have functional roles in the normal development of the gonad and brain in both sexes, and both subtypes signal for ERmediated disruption of sexual development. The reporter gene assays constructed show that both rER subtypes can be activated by the major environmental estrogens and mediate disruptions in sexual development and function; however, the two ER subtypes appear to differ in their sensitivities and responsiveness to different environmental estrogens. The in vivo study for EE2 and ER activation assays strongly supports the hypothesis that EE2 has a major role in the evolution of intersex in wild fish, even though EE2 is normally present in effluents in the low nanogram per liter concentration range, because of its high potency as compared with E2 or E1. E1 is generally found at much higher concentrations than E2 in the aquatic environment and is commonly thought to be of

lesser importance due to its lower reported potency in some assays. The results, from the rER transactivation assays, however, suggest that E1 (with a similar potency to E2) may well be far more important in the ER-mediated disruption of sexual development in wild roach than has been believed previously.

Acknowledgments We thank Jan Shears (University of Exeter) for her technical assistance, Prof. S. Kato (Institute of Molecular and Cellular Bioscience, University of Tokyo) for kindly providing the reporter plasmid pGL3-ERE-tk-Luc, and Alan Henshaw and staff (Calverton Fish Farm, U.K. Environment Agency) for supplying the pre-spawning, sexually mature roach. We also gratefully acknowledge the contribution of Prof. Alexander Scott (CEFAS Laboratory Weymouth) for his EE2 radioimmunoassay. This work was funded by grants from the Ministry of Environment, Japan, to Y.K. and T.I., and from the U.K. Natural Environmental Research Council within the Environmental Genomics program (NER/T/S/2002/00182) to C.R.T. G.C.P. was funded by the U.K. Environment Agency to C.R.T.

Supporting Information Available Details on PCR primers (Table S1), domain structures, and sequence identities for isolated roach ERs (Figure S1) and a phylogenetic tree for the full ORF amino acid sequences of ERs (Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org.

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Received for review November 24, 2006. Revised manuscript received February 22, 2007. Accepted February 26, 2007. ES062797L