Aquatic Toxicology 156 (2014) 191–200
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Sensitization of vitellogenin gene expression by low doses of octylphenol is mediated by estrogen receptor autoregulation in the Bombina orientalis (Boulenger) male liver Chan Jin Park, Myung Chan Gye ∗ Department of Life Science and Institute for Natural Sciences, Hanyang University, Seoul 133-791, South Korea
a r t i c l e
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Article history: Received 8 February 2014 Received in revised form 26 August 2014 Accepted 27 August 2014 Available online 6 September 2014 Keywords: Octylphenol Xenoestrogen Vitellogenin Estrogen receptors Bombina orientalis
a b s t r a c t This study aimed to elucidate the mechanisms by which alkylphenols disrupt endocrine function in wild amphibians in Korea. To this end, the effects of 4-tert-octylphenol (OP), 17ˇ-estradiol (E2 ), and estrogen receptor (ER) agonists on the expression profiles of vitellogenin (VTG) and ERs were examined in livers obtained from male Bombina orientalis toads. A single injection of E2 (10 g/kg; 0.03 mol/kg) induced transcription of VTG mRNA at 2 days post injection; however, injection of either the ER␣-selective agonist propyl-(1H)-pyrazole-1,3,5-triyl-trisphenol (PPT, 50 g/kg; 0.12 mol/kg) or the ER-selective agonist 2,3-bis-(4-hydroxyphenyl)-propionitrile (DPN, 50 g/kg; 0.20 mol/kg) did not affect the expression of VTG. This finding suggests that both ER␣ and ER are required to induce transcription of VTG in the male B. orientalis liver. Interestingly, E2 , PPT, and DPN induced transcription of ER␣, which was also reflected on the protein level; however, these alkylphenols did not affect ER transcription. Similarly, VTG transcription was induced by a single injection of 1–100 mg/kg (0.04–484.66 mol/kg) OP, while 0.1 mg/kg (0.48 mol/kg) OP had no effect on VTG transcription. This result suggests that the lowest observable effect concentration (LOEC) of OP for induction of VTG transcription in the male liver is 1 mg/kg (4.84 mol/kg). Furthermore, treatment with E2 (10 g/kg; 0.03 mol/kg) or OP (1 mg/kg; 4.84 mol/kg) significantly upregulated ER␣ transcription, and a 10 mg/kg (48.46 mol/kg) dose of OP significantly upregulated ER transcription. The ER antagonist ICI 182,780 decreased the basal levels of ER␣ and ER mRNA, and also prevented E2 -mediated and OP-mediated induction of VTG, ER␣, and ER transcription. A second injection of 0.1 mg/kg (0.48 mol/kg) OP after a two-day interval significantly upregulated the transcription of VTG and ER␣, but not of ER. These results suggest that sensitization of VTG transcription by repeated exposure to OP is mediated by the induction of ER␣. Different combinations of alkylphenols that are ubiquitous in the freshwater system in Korea could potentially exert a synergistic effect on endocrine disruption. Thus, chronic exposure to alkylphenols, even at their NOECs, could still disrupt endocrine function in B. orientalis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Endocrine disruption is believed to be a contributing factor to the global decline of amphibians (Renner, 2002). Natural and synthetic estrogens enter water systems from anthropogenic sources via sewage treatment effluent (Desbrow et al., 1998), discharge from the industrial manufacturing of pharmaceuticals (Heberer, 2002), and agricultural runoff, which often contains animal manure that is used as fertilizer (Hanselman et al., 2003). The observation of various abnormalities, such as the feminization of wild animals, implies that water contamination with estrogenic
∗ Corresponding author. Tel.: +82 222200958; fax: +82 222989646. E-mail address: [email protected] (M.C. Gye). http://dx.doi.org/10.1016/j.aquatox.2014.08.013 0166-445X/© 2014 Elsevier B.V. All rights reserved.
compounds is an undeniable concern (Vos et al., 2000; Hutchinson et al., 2000). In amphibians, xenoestrogens induce the expression of estrogen-responsive genes, which have been associated with adverse effects on amphibian development (Kloas et al., 1999; Lutz and Kloas, 1999; Mosconi et al., 2002; Mackenzie et al., 2003; Ahn et al., 2011). Alkylphenolic compounds, which are used in a variety of human commercial products, are known to be estrogenic in vertebrates (Ying et al., 2002; Waring and Harris, 2005). Among these alkylphenols, 4-tert-octylphenol (OP) is one of the two main degradation products of alkylphenol polyethoxylates. These compounds are nonionic surfactants that are often used in household and industrial detergents (Nimrod and Benson, 1996); moreover, OP accounts for about 15% of all commercial alkylphenols in the USA and Canada (Bennett and Metcalfe, 1998). Although nonylphenol (NP), another degradation product of alkylphenol
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polyethoxylates, is the predominant contaminant in most water systems, the estrogenic potency of OP has been shown to be higher than that of NP (White et al., 1994). In Korean rivers, OP has been detected at concentrations ranging from 2.24 to 16.78 ng/L (10.86–81.33 nM) in surface water, and at 0.5 ng/L (2.42 nM) in the effluent from sewage treatment plants (Duong et al., 2010; Ra et al., 2011). Thus, OP in the aquatic environment may exert toxic effects on amphibians during their embryonic, larval, and adult life cycle stages. Due to its structural similarity to estrogen, OP can induce the transcription of estrogen-responsive genes and thus exert adverse effects on the embryonic development, sexual behavior, and sexual differentiation of amphibians (Nishimura et al., 1997; Kloas et al., 1999; Lutz and Kloas, 1999; Mayer et al., 2003; Huang et al., 2005; Porter et al., 2011). Vitellogenin (VTG) is an egg yolk precursor that is synthesized in the livers of female oviparous and ovoviviparous vertebrates (Wahli et al., 1981; Wallace, 1985). In amphibians, several natural estrogens and xenoestrogens have been shown to upregulate VTG transcription and protein production, both in male livers and in hepatocyte cultures (Wangh and Knowland, 1975; Carnevali et al., 1995; Bögi et al., 2003; Rotchell and Ostrander, 2003; Gye and Kim, 2005; Kang et al., 2006). Therefore, VTG induction in males has been used as a biomarker for measuring the estrogenicity of certain chemicals and environmental media (Sumpter and Jobling, 1995). The two estrogen receptors, ER␣ and ER, mediate the estrogenic effects of natural estrogens and xenoestrogens in both humans and wild animals (Kuiper et al., 1998; Rotchell and Ostrander, 2003; Melzer et al., 2011). In Xenopus, OP binds directly to ERs and activates the VTG promoter (Huang et al., 2005). Moreover, estrogens and xenoestrogens have also been reported to affect the expression of VTG, ER␣, and ER in fishes (Soverchia et al., 2005; Nagler et al., 2010). However, in amphibians, the transcriptional responses of ER␣ and ER to natural estrogens and xenoestrogens have not yet been investigated. An assessment of the ecological effects of endocrine disruptors on freshwater fishes and frogs in Korea revealed that the concentration of OP in the muscle tissue of adult bullfrogs (Rana catesbeiana) was 10 times higher than that in fishes (NIER, 2001). Moreover, the plasma VTG levels of male bullfrogs were similar to those of females in the surveyed areas (NIER, 2003). In the present study, we examined the effects of OP on ER␣, ER, and VTG expression. As a model organism, we selected the male fire-bellied toad, Bombina orientalis, which is a common native Korean anuran. Importantly, B. orientalis can be induced via gonadotropin to ovulate and spawn eggs at three-month intervals in the laboratory, thus permitting developmental toxicity assessment on embryos year-round (Park et al., 2010). Moreover, the sequence of B. orientalis ER␣ is available (GenBank accession no. FJ387577.1), and a homologue of VTG A2 has been identified (Lee and Gye, 2004). Furthermore, a protocol for the quantitative analysis of hepatic VTG mRNA has been established (Gye and Kim, 2005; Kang et al., 2006). In an effort to understand the mechanisms of endocrine disruption by estrogenic alkylphenols in a native Korean toad species, we examined the effects of OP on the expression of VTG, ER␣, and ER in livers from adult male B. orientalis. We also tested the hypothesis that autoregulation of ER␣ and ER in the male liver by OP may mediate VTG induction after chronic exposure to low doses of xenoestrogens.
2. Materials and methods 2.1. Animals Adult B. orientalis were bred and reared in the Hanyang University Aquarium. The toads were maintained on a diurnal 14 h
light and 10 h dark cycle at 20–22 ◦ C, and were fed crickets and mealworms three times a week. Adult toads with a mean body weight (BW) of 8.0 ± 0.5 g were used for all experiments. All experiments conducted on amphibians followed the procedures outlined in the “Guidelines for the Use of Live Amphibians and Reptiles in Field and Laboratory Research” publication (ASIH, 2004) (http://www.asih.org/files/hacc-final.pdf). As a control for ER␣ and ER in Western blots, mouse uterus tissue was obtained from an adult female mouse that had been euthanized after asphyxiation in CO2 . All mouse procedures were approved by the Institutional Animal Care and Use Committee of Hanyang University (HY-IACUC11-044). 2.2. Chemical exposure The 17-estradiol (E2) (MW = 272.38; Sigma Aldrich, St. Louis, MO), 4-tert-octylphenol (OP; MW = 206.33; Aldrich Chemical Co., Milwaukee, WI), ICI 182,780 (ICI; MW = 606.77; Sigma Aldrich) as an ERs antagonist, and ER subtype specific agonists, propyl pyrazole triol (PPT; MW = 386.44; Abcam, Cambridge, MA) estrogenic compounds were used for experiments involving ER␣ and diarylpropionitrile (DPN; MW = 239.27; Abcam) was used for experiments involving ER. Selected adult male toads received an intraperitoneal injection of OP (0.01, 0.1, 1, 10, or 100 mg/kg BW; 0.04, 0.48, 4.84, 48.46, or 484.66 mol/kg BW) or E2 (10 g/kg; 0.03 mol/kg) dissolved in sesame oil. These doses were found to effectively induce VTG expression in B. orientalis in initial optimization experiments. For some experiments, toads were given 50 g/kg (0.12 mol/kg) PPT and 50 g/kg (0.20 mol/kg) DPN. The doses of PPT and DPN were determined according to previous studies (Frasor et al., 2003; Neese et al., 2010). ICI was given at 1 mg/kg (1.64 mol/kg). This dose was based on the binding affinity of ICI to ERs, which is 100-fold lower than that of E2 (Preisler-Mashek et al., 2002). ICI was given either alone or in combination with E2 , OP, PPT, or DPN. As a vehicle control (VC), males were given sesame oil only. The total injection volume was 100 L per animal. To test the effects of repeated exposure to OP on gene expression in the male liver, two OP injections were given with a two-day interval. The concentration of OP used (0.1 mg/kg; 0.48 mol/kg) was below the lowest observed effect concentration (LOEC). Toads were returned to the aquaria and sampled 48 h after the second injection. Five male toads were used in each experimental group. 2.3. Degenerate PCR and cloning of ERˇ cDNA Female toads were euthanized by inhalation of ether to minimize pain, and their livers were removed. Total liver RNA was isolated using TRI reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer’s instructions. The concentrations of all RNA preparations were determined prior to storage at −85 ◦ C until use. For cDNA synthesis, RNA (1 g) was reverse transcribed for 60 min at 42 ◦ C in a 20 L reaction with 50 units of MuLV reverse transcriptase and 2.5 M oligo d(T)16 primer, according to the manufacturer’s standard protocol (Applied Biosystems, Foster City, CA). Degenerate PCR was performed with degenerate primers for the ER cDNA sequence, which was based on multiple anuran ER mRNA sequences (Xenopus laevis, NM 001130954.1; Xenopus tropicalis, NM 001040012.1; Rana rugosa, FJ828859.1; Bufo rangeri, AB524915.1) (Table 1). PCR products were ligated into the pGEM-T Easy vector (Promega, Madison, WI), and the resultant constructs were transformed into DH5␣ competent cells. More than 10 positive colonies were selected by blue/white screening. DNA sequencing was performed using M13 primers.
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Table 1 Primers for RT-PCR analysis. Primer ER (DGP) VTG ER␣ ER -actin a
F R F R F R F R F R
Primer sequence
Product size (bp)
Reference
5 -TTC AAG AGR AGY ATY CAA GGR C-3 5 -VTG YTG MAG TTT WAR CTC TCK VA-3 5 -CAC ATG CTG ATC CAT CTG TCC TGA-3 5 -TGG CGA CCA CAC AAT CCA CA-3 5 -CAG AGC CGC CCA TCG TCT AC-3 5 -CCT GGC ACT CTC TTT GCC CA-3 5 -GGC GGC ATT CAG ACG ATC AG-3 5 -CAT TTG GCG GTT CTG CTT CG-3 5 -GAG AGG TAT CCT GAC CCT GAA GTA-3 5 -ATA ACC TTC ATA GAT GGG CAC AGT-3
669 160
NM 001130954.1a ; NM 001040012.1a ; FJ828859.1a ; AB524915.1a Lee and Gye (2004), Gye and Kim (2005)
132
FJ387577.1a
144 325
Gye and Kim (2005)
GenBank accession number; DGP: degenerate primers.
2.4. RT-PCR analysis RT-PCR was performed according to the method outlined by Gye and Kim (2005). Briefly, PCR reactions contained 0.5 L liver cDNA, 1.25 units of Ex TaqTM polymerase (Takara, Japan), 1× Ex PCRTM Buffer II (Mg2+ -free), 2.5 mM MgCl2 , 0.4 mM dNTPs, and 0.4 M of each primer in a total volume of 25 L. -actin mRNA was amplified as a reference gene (Table 1). Real-time PCR was carried out using the same primers and iQ SYBR Green Supermix reagent (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. PCR was performed in a MyiQ i Cycler (Bio-Rad) using the following thermocycling conditions: 1 cycle of 3 min at 95 ◦ C followed by 40 cycles of 95 ◦ C for 30 s, the appropriate annealing temperature for 30 s, and 72 ◦ C for 30 s. VTG, ER␣, and ER mRNA levels are expressed as arbitrary units relative to -actin mRNA, and relative quantification was performed using the comparative CT method according to the manufacturer’s protocols (Bio-Rad).
2.5. SDS-PAGE and Western blot analysis Toad livers and mouse uteri were homogenized on ice in 10 volumes of extraction buffer [20 mM Tris–HCl (pH 7.5), 1% Triton X-100] containing a protease inhibitor cocktail (Roche, Mannheim, Germany). Homogenates were then sonicated and spun by centrifugation at 12,000 × g for 10 min at 4 ◦ C. The protein concentrations of the resultant supernatants were quantified using a BCA protein assay kit (Bio-Rad). Protein samples were mixed with 5× sample buffer containing 0.15 M dithiothreitol (DTT), boiled for 5 min, and then cooled to room temperature. After clarification to remove debris, the proteins in the supernatants were resolved on 10% SDS-polyacrylamide gels. As positive controls for ER␣ and ER, mouse uterus homogenates were also included. Following electrophoretic transfer to a nitrocellulose membrane (Amersham Bioscience, Buckinghamshire, UK), the membrane was blocked overnight at 4 ◦ C in Tris-buffered saline (1× TBS, pH 7.4) containing 7% skim milk. After washing for 5 min with TBS/0.1% Tween 20 (TBST), the membrane was incubated with rabbit polyclonal antibodies against either ER␣ (catalog no. ac37438, Abcam) or ER (catalog no. sc8974, Santa Cruz Biotechnology, Santa Cruz, CA). Primary antibodies were diluted in 5% skim milk in TBST (1:1000) and incubated for 2 h at room temperature. As a loading control, membranes were also probed with rabbit anti -tubulin antibodies (catalog no. sc9104, Santa Cruz Biotechnology). As a negative control, a 1:1000 dilution of rabbit IgG (catalog no. ab53041, Abcam, Cambridge, UK) in TBST was used. After three 10 min washes in TBST, the membrane was incubated with peroxidase-conjugated goat anti-rabbit IgG antibodies (catalog no. 81-6120, Zymed, San Francisco, CA) diluted in 5% skim milk in TBST (1:1000) for 1 h. Next, the membrane was washed with TBST for 10 min and then with TBS for 10 min. Immunoreactive bands were detected using an enhanced chemiluminescence (ECL) kit (Amersham Bioscience).
The amount of protein in each lane was quantified by analyzing the corresponding band intensity using 1D scan EX software (Scanalytics Inc., Fairfax, VA). 2.6. Immunohistochemistry and image analysis Paraffin sections (5 m thick) of 4% paraformaldehyde-fixed liver tissue were prepared. After deparaffination, endogenous peroxidase activity was blocked with 3% H2 O2 for 15 min. For antibody labeling, sections were incubated for 30 min with a blocking solution [1.5% normal goat serum in phosphate-buffered saline (PBS)]. The slide was then incubated in either a 1:1000 dilution of anti-ER␣ primary antibodies (rabbit polyclonal, catalog no. ac37438, Abcam) or a 1:500 dilution of anti-ER primary antibodies (rabbit polyclonal, catalog no. sc8974, Santa Cruz Biotechnology). Antibodies were diluted in blocking solution, and incubations were performed for 2 h at room temperature. As a negative control, slides were incubated in the same concentration of normal rabbit IgG (catalog no. ab27478, Abcam) under the same conditions. After two washes in PBS, the slide was incubated with peroxidase-labeled goat antirabbit IgG antibodies diluted 1:200 in blocking solution (catalog no. 81-6120, Zymed). Immunoreactive regions were visualized by reaction with 3,3-diaminobenzidine and H2 O2 as a chromogen. Slides were then counterstained with modified Harris hematoxylin (HHS16, Sigma Aldrich, St. Louis, MO). Permanently mounted slides were observed and images were captured using a microscope (IX71, Olympus, Japan) equipped with a digital imaging system (DP71, Olympus). Quantitative imaging of ER␣ and ER staining was conducted in four different sections using a commercially available image analysis program (IMT iSolution Lite, version 7.8, iMTechnology, Vancouver, British Columbia, Canada), according to the method described by Gye et al. (2011). 2.7. Statistical analysis All VTG mRNA levels were log-transformed before comparisons between groups. Statistical significance for real-time PCR and imaging data was analyzed using one-way analysis of variance (ANOVA) followed by a Tukey post hoc test. Statistical significance was defined as a p value < 0.05. 3. Results 3.1. B. orientalis ERˇ cDNA sequence A 558-bp (186 amino acid) ER sequence was identified by sequencing the appropriate amplicon (Fig. S1). The defined partial amino acid sequence of ER contained a DNA-binding domain and a ligand-binding domain. The deduced amino acid sequence of the corresponding B. orientalis ER ORF was 82% identical with the anuran X. laevis sequence (GenBank accession no. NP 001124426)
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Fig. 1. Effects of E2 , ICI, PPT, and DPN on the mRNA expression of VTG and ERs in the B. orientalis male liver. (A) Changes in the mRNA expression levels of VTG, ER␣, and ER resulting from injection of E2 (10 g/kg; 0.03 mol/kg), PPT (50 g/kg; 0.12 mol/kg), or DPN (50 g/kg; 0.20 mol/kg). (B) Changes in the mRNA expression levels of VTG, ER␣, and ER resulting from injection of ICI alone (1 mg/kg; 1.64 mol/kg), ICI + PPT (50 g/kg; 0.12 mol/kg), or ICI + DPN (50 g/kg; 0.20 mol/kg). VC, vehicle control; E2, 17-estradiol; ICI, ICI 182,780; PPT, propyl pyrazole triol; DPN, diarylpropionitrile. (a)–(c) were significantly different from each other (p < 0.05) as determined by ANOVA. Error bars = SDs (n = 5).
and 73% identical with the human sequence (GenBank accession no. CAA67555) (Fig. S2).
3.2. Effects of estrogen and selective ER-agonists on the expression profiles of VTG and ERs in the male liver In adult males, a single injection of E2 (10 g/kg; 0.03 mol/kg) upregulated the transcription of hepatic VTG by 2 days post injection; however, this effect was not observed with either PPT (50 g/kg; 0.12 mol/kg) or DPN (50 g/kg; 0.20 mol/kg). In contrast, E2 , PPT, and DPN significantly increased the levels of ER␣ mRNA, but not ER mRNA (Fig. 1A). The ERs antagonist ICI (1 mg/kg; 1.64 mol/kg) did not affect VTG transcription in the male liver; however, ICI significantly downregulated transcription of ER␣ and ER. Adult males treated with a combination of either ICI + PPT or ICI + DPN did not exhibit any significant differences in VTG or ER␣ transcription compared with VC-treated adult males. However, adult males treated with a combination of ICI + DPN exhibited significantly reduced transcription of ER compared with VC-treated adult males (Fig. 1B). Immunohistochemical analysis revealed ER␣ immunoreactivity in the nuclei and cytoplasm of hepatocytes from the E2 , PPT, and DPN treatment groups; in contrast, only basal ER␣ immunoreactivity was found in hepatocytes from the vehicle control group. Basal ER immunoreactivity was observed in hepatocytes from the E2 , PPT, DPN, and vehicle control groups (Fig. 2A). Densitometric analysis of ER␣ immunoreactivity revealed that hepatocytes from the E2 , PPT, and DPN groups exhibited significantly increased ER␣ immunoreactivity compared with hepatocytes from the vehicle control group; however, this trend was not observed with ER (Fig. 2B). Western blot analysis revealed that mouse and B. orientalis ER␣ and ER were detected by antibodies against ER␣ and ER, respectively; all bands migrated according to the predicted molecular weights (Fig. 3A). Moreover, ER␣, ER, and -tubulin were detected by antibodies against ER␣, ER, and tubulin, respectively, in the E2 , PPT, and DPN groups (Fig. 3B). Densitometric analysis revealed increased levels of ER␣ in the E2 , PPT,
and DPN groups compared with the vehicle control group; however, no differences in the levels of ER were observed between the groups (Fig. 3C). 3.3. Effects of OP on VTG and ERs transcription in the male liver The dosing of OP at 100 mg/kg (484.66 mol/kg) did not result in either death or narcosis at 2 days post injection, indicating that the OP dose used in this study did not evoke systemic toxic effects in B. orientalis. Time course experiments revealed that transcription of VTG was markedly increased by 1 day after a single injection of OP (100 mg/kg; 484.66 mol/kg) and reached maximum levels in the male liver at 2 days post injection. At 3 days post injection, the level of VTG mRNA began to decrease (Fig. S3). In contrast, a single injection of OP at doses ranging from 0.01 to 0.1 mg/kg (0.04–0.48 mol/kg) did not induce VTG transcription. However, a single injection of OP at doses ranging from 1 to 100 mg/kg (4.84–484.66 mol/kg) significantly increased hepatic VTG transcription in a dose-dependent manner. While ER␣ transcription was increased by a single injection of OP at 1 mg/kg (4.84 mol/kg), ER transcription was not affected. At doses of 10 or 100 mg/kg (48.46 and 484.66 mol/kg), OP significantly increased transcription of both ER␣ and ER (Fig. 4). 3.4. Effects of ER antagonists on estrogen-induced and OP-induced (at the LOEC) changes in VTG and ER transcription in the male liver The combination of E2 + ICI and the combination of OP + ICI significantly decreased the levels of VTG mRNA compared with E2 -treated animals (10 g/kg; 0.03 mol/kg) and OP-treated animals, respectively [LOEC for VTG mRNA (VTG LOEC), 1 mg/kg; 4.84 mol/kg]. However, VTG transcription in E2 -treated or OPtreated male livers was not completely reduced to basal levels by ICI. Moreover, E2 -induced ER␣ and E2 -induced ER transcription were both significantly downregulated by ICI, and the OP-induced
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Fig. 2. Changes in ER␣ and ER immunoreactivity in the B. orientalis male liver induced by E2 , PPT, or DPN. (A) Localization of ER␣ and ER antigens in the male liver. The regions designated by the dotted boxes are magnified in the insets. Arrows, ER␣-positive or ER-positive hepatocyte nuclei. Scale bar = 30 m. (B) Changes in ER␣ and ER immunoreactivity in the nuclei of male liver hepatocytes resulting from the injection of E2 (10 g/kg; 0.03 mol/kg), PPT (50 g/kg; 0.12 mol/kg), or DPN (50 g/kg; 0.20 mol/kg). VC, vehicle control; E2 , 17-estradiol; PPT, propyl pyrazole triol; DPN, diarylpropionitrile. Error bars = SDs (n = 4). (a)–(c) were significantly different from each other (p < 0.05) according to ANOVA.
increase in ER␣ transcription was also significantly decreased by ICI. In the OP + ICI-treated group, ER transcription was not significantly different from that in the OP-treated group; however, ER transcription was significantly lower than in the VC group (Fig. 5).
3.5. Repeated injection of low concentrations of OP induces transcription of VTG and ERs in the male liver A single dose of OP at the no effective concentration (NOEC) for VTG mRNA induction (0.1 mg/kg; 0.48 mol/kg) was followed by
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Fig. 3. Effects of E2 , PPT, and DPN on the protein levels of ER␣ and ER in the B. orientalis male liver. (A) Western blot analysis of the protein levels of ER␣ and ER in the mouse uterus (MU), B. orientalis oviduct (Ov), and liver (Li). Both ER␣ (66 kDa) and ER (56 kDa) migrated according to their predicted molecular weights in all samples. (B) Detection of ER␣ and ER in male liver samples from the VC, E2 (10 g/kg; 0.03 mol/kg), PPT (50 g/kg; 0.12 mol/kg), and DPN (50 g/kg; 0.20 mol/kg) groups. -tubulin (55 kDa) was used as a loading control. No immunoreactive bands were detected in the 55–66 kDa range when membranes were probed with rabbit normal IgG. (C) Densitometric analysis of the protein levels of ER␣ and ER in the livers of male toads injected with either E2 (10 g/kg; 0.03 mol/kg), PPT (50 g/kg; 0.12 mol/kg), or DPN (50 g/kg; 0.20 mol/kg). VC, vehicle control; E2 , 17-estradiol; PPT, propyl pyrazole triol; DPN, diarylpropionitrile.
Fig. 4. Effects of OP on VTG and ER transcription in the B. orientalis male liver. Levels of hepatic VTG, ER␣, and ER mRNA at 48 h after a single injection of OP (0, 0.01, 0.1, 1, 10, or 100 mg/kg; 0, 0.04, 0.48, 4.84, 48.46, or 484.66 mol/kg). VC, vehicle control. (a)–(c) were significantly different from each other (p < 0.05) as determined by ANOVA. Error bars = SDs (n = 5).
a second injection of the same OP dose after a 2-day interval. This treatment significantly upregulated VTG and ER␣ transcription, but not ER transcription, in the male liver. The mRNA levels of VTG and ER␣ in the group that received two injections of OP at 0.1 mg/kg (0.48 mol/kg) were higher than those in the group that received only one injection of OP at 1 mg/kg (4.84 mol/kg) (Fig. 6). 4. Discussion 4.1. OP mimics estrogen-mediated induction of VTG transcription in the male liver In the present study, OP was demonstrated to mimic the effect of estrogen on the B. orientalis male liver by using the level of VTG mRNA as a biomarker. Both E2 and OP induced VTG transcription in the adult male liver 2 days after injection; this effect was abrogated by ICI. The magnitude of action of ICI was different between the E2 + ICI and OP + ICI groups, suggesting that OP-mediated induction of VTG transcription can be attributed to OP–ER interactions, similar to the E2 –ER interactions. Although ER␣ and VTG transcription were significantly induced by E2 , ER transcription was not. Of particular note, VTG transcription was not induced by PPT or DPN alone. This finding suggests that VTG transcription in the male liver cannot be induced by activated ER␣ or ER alone; however, ER␣ transcription can be induced by either activated ER␣ or ER. The ER antagonist ICI significantly decreased the mRNA levels
of both ER␣ and ER. This finding suggests that basal transcription of ER␣ and ER is regulated by endogenous estrogen in the male liver. Furthermore, the hepatic mRNA levels of ER␣ in toads treated with either ICI + PPT or ICI + DPN were not different from the levels in VC-treated toads. This finding suggests that ICI prevents PPT-mediated and DPN-mediated upregulation of ER␣ transcription in the male liver. Since ER transcription was not induced by either PPT or DPN, and since no difference in ER transcription was observed between the ICI and ICI + DPN groups, (xeno)estrogens are probably poor inducers of ER transcription. In this study, a 1 mg/kg (4.84 mol/kg) dose of OP significantly increased the level of VTG mRNA in the male liver. Similar to E2 -mediated induction of VTG transcription, both ER␣ and ER appear to be required for OPmediated induction of VTG transcription in the male liver. Although the binding affinities of OP for ER␣ and ER have not yet been determined in amphibians, the binding affinity of OP for ER in humans and fish (channel catfish, Ictalurus punctatus) was lower than that for ER␣ (Routledge et al., 2000; Gale et al., 2004). This finding suggests that ER␣ is the predominant player in OP-mediated induction of VTG transcription in the B. orientalis male liver. In X. laevis, functional interactions between ER␣ and the transcriptional activator Sp1 have been shown to regulate estrogen-dependent VTG transcription (Batistuzzo de Medeiros et al., 1997). Therefore, we cannot exclude the possibility that OP-mediated induction of VTG transcription might also involve interactions between transcriptional activators and ERs in B. orientalis.
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Fig. 5. Effects of ICI on E2 -induced and OP-induced upregulation of VTG and ER transcription in the B. orientalis male liver. Hepatic VTG and ER mRNA expression levels were examined after injection of ICI (1 mg/kg; 1.64 mol/kg), in combination with either E2 (10 g/kg; 0.03 mol/kg) or OP (1 mg/kg; 4.84 mol/kg), into adult male B. orientalis toads. VC, vehicle control. (a)–(d) were significantly different from each other (p < 0.05) according to ANOVA. Error bars = SDs (n = 5).
Fig. 6. Effects of repeated OP injections on VTG and ER expression in the B. orientalis adult male liver. mRNA expression levels of VTG, ER␣, and ER following a single injection of OP at 0.1 mg/kg (0.48 mol/kg), a double (with a two-day interval) injection of OP at 0.1 mg/kg (0.48 mol/kg), or a single injection of OP at 1 mg/kg (4.84 mol/kg). VC, vehicle control. (a)–(c) were significantly different from each other (p < 0.05) according to ANOVA. Error bars = SDs (n = 5).
4.2. Autoregulation of ER mRNA by estrogen and OP in the male liver In animals, ER mRNA and protein production is regulated by estrogen, most commonly through auto-induction (Bagamasbad and Denver, 2011). In fishes, expression of ER␣ is induced by (xeno)estrogens. In rainbow trout (Oncorhynchus mykiss), functional estrogen-responsive elements (EREs) have been located in a 0.2-kb transcription start site upstream of the ER␣ gene (Petit et al., 1999). Moreover, E2 and nonylphenol (NP) have been shown to induce VTG transcription in hepatocytes at 1 and 10 M, respectively. In hatched larvae, ER transcription has been shown to be induced by 0.1 M E2 and 21 M NP following exposure during the embryonic stage (Madigou et al., 2001). Moreover, E2 , NP, bisphenol A (BPA), and OP have all also been shown to drive ER␣ transcription in the liver (Min et al., 2003; Andreassen et al., 2005; Osachoff et al., 2013). In male medaka (Oryzias latipes), E2 (100 ng/L; 0.37 nM), NP (0.5 mg/L; 2.27 M), and BPA (8 mg/L; 35.04 M) have all been shown to upregulate hepatic VTG and ER␣ transcription, but not ER transcription (Yamaguchi et al., 2005, 2008). In largemouth bass (Micropterus salmoides), E2 (0.5 mg/kg; 1.83 mol/kg) has been shown to upregulate ER␣ and VTG transcription, but not ER transcription, in the male liver (Sabo-Attwood et al., 2004). However, in goldfish (Carassius auratus), both ER and VTG transcription have been shown to be significantly upregulated by 0.1 M E2 or NP (Soverchia et al., 2005). These results indicate that the sensitivities of ER␣ and ER to estrogen-mediated transcriptional activation may differ among various fish species. In Xenopus, the ERE is located in the protein coding region of the ER␣ gene (Lee et al., 1995). In the Japanese giant salamander (Andrias japonicus), transcription of ER␣ has been shown to be upregulated by E2 in CHO cells cotransfected with a plasmid containing an ERE (Katsu et al., 2006). In amphibians, E2 , NP, and BPA have all been shown to upregulate
hepatic ER␣ transcription (Westley and Knowland, 1979; Barton and Shapiro, 1988; Levy et al., 2004; Lutz et al., 2005). Similarly, we found that the levels of hepatic ER␣ mRNA and protein were both significantly increased by E2 (10 g/kg; 0.03 mol/kg), PPT (50 g/kg; 0.12 mol/kg), and DPN (50 g/kg; 0.20 mol/kg) at 2 days post injection in male B. orientalis. Although the EREs of the B. orientalis ER genes have not yet been characterized, it is highly likely that B. orientalis ER␣ is autoregulated via transactivation of an ERE by activated ERs. However, unlike ER␣, transcription of ER was not induced by estrogen or ER agonists, at least when used at the same concentrations that induced ER␣ transcription. This finding suggests that ER␣ and ER exhibit different sensitivities to estrogen-mediated transcriptional activation in the B. orientalis male liver. In support of this idea, CHO cells transfected with the ER␣ or ER protein coding regions (including functional EREs) exhibit higher estrogen-mediated transactivation of the ER␣ genes compared with the ER genes of anurans (X. tropicalis and B. rangeri) (Katsu et al., 2010). In contrast, ER transactivation was more readily induced by estrogen in caudata (Hynobius tokyoensis and Ambystoma mexicanum) and urodela (Cynops pyrrhogaster) (Katsu et al., 2010), indicating that the sensitivities of ER␣ and ER to estrogen-mediated transcriptional activation are different among various amphibian species. In the B. orientalis male liver, the LOECs for induction of ER␣ and ER with a single dose of OP were 1 mg/kg (4.84 mol/kg) and 10 mg/kg (48.46 mol/kg), respectively. This finding indicates that ER␣ is 10-fold more sensitive to OP-mediated gene transcription compared with ER. Although both ER␣ and ER are required for the induction of VTG transcription in the B. orientalis male liver, induction of ER␣ by E2 and OP might also be important for the induction of VTG transcription. Together, OP–ERs may autoregulate ERs, which in turn transactivate transcription of ER-dependent target genes, including VTG, in the B. orientalis male liver (Fig. 7).
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Fig. 7. Proposed mechanism for OP-mediated sensitization of VTG expression involving ER autoregulation in the B. orientalis male liver. Transcription of VTG and ER␣ was induced by either a single injection or repeated injections of either E2 or OP. Moreover, transcription of ER was induced by a high dose of OP (10 mg/kg; 48.46 mol/kg). Either an ER␣-specific agonist (PPT) or an ER-specific agonist (DPN) induced transcription of ER␣, but not of VTG or ER. Thus, either activation of ER␣ or ER is sufficient to upregulate transcription of ER␣, but not of VTG or ER, in B. orientalis. Although test doses of either E2 or OP did not induce ER expression, a high dose of OP did induce ER expression. Thus, upregulation of ER␣ by OP, even at a low dose, can sensitize VTG expression in the B. orientalis male liver.
4.3. Sensitization of VTG mRNA expression by low levels of OP and estrogen In X. laevis, the binding affinity of OP for ER is 15 times lower than that of E2 ; furthermore, the ability of OP to upregulate VTG expression is 200 times lower than that of E2 (Huang et al., 2005). In X. laevis hepatocytes, the binding affinity of OP for ERs has been shown to be lower than that of NP and BPA (Lutz and Kloas, 1999; Kloas et al., 1999). Similarly, in male crucian carp (C. carassius), the VTG LOEC of OP (20 g/L; 0.10 M) has been shown to be lower than that of the VTG LOEC of BPA (100 g/L; 0.48 M) (Zhang et al., 2010). We found that repeated injection of OP at its NOEC (0.1 mg/kg; 0.48 mol/kg) sensitized transcription of both VTG and ER␣, which resulted in a marked increase in VTG and ER␣ transcription compared with toads given a single dose of OP at a higher concentration (1 mg/kg; 4.84 mol/kg). Considering the observations that the levels of hepatic ER␣ mRNA and protein were significantly increased by E2 , PPT, and DPN at 2 days post injection, and that the levels of ER were not altered by the same treatments, sensitization of VTG gene transcription by repeated dosing of OP at the NOEC might be predominantly mediated by ER␣. In flounder (Platichthys flesus), OP has been shown to accumulate in the liver; moreover, the tissue concentration of OP was positively correlated with the plasma VTG concentration (Madsen et al., 2002). This finding suggests that chronic exposure to low concentrations of OP could cause bioaccumulation and thereby disrupt endocrine function in male amphibians by sensitizing ER signaling pathways. 4.4. Environmental concerns In Korea, many toad species including B. orientalis breed in rice fields, puddles, and riverside pools in freshwater systems (AmphibiaWeb, http://amphibiaweb.org). Therefore, they are vulnerable to exposure to a variety of EDCs throughout their life cycles. In the B. orientalis male liver, the VTG NOEC for OP was 0.1 mg/kg
(0.48 mol/kg). Exposure to OP in combination with other xenoestrogens, such as BPA and NP, is most likely unavoidable for toads in contaminated habitats. Thus, OP may elicit endocrine disruption in B. orientalis, even when it is only present at the NOEC, due to the presence of other xenoestrogens in the aquatic media. In one approach for defining predicted no-effect concentrations (PNECs), the constant application factors usually range between 10 and 1000, depending on pragmatic assumptions relating to single substances versus real world mixtures, acute–chronic ratios, and interspecies differences. These factors are then applied to the available toxicity data, including the LOEC (EC, 2003). According to this guideline, an acceptable exposure value for OP without inducing overt endocrine disruption in adult B. orientalis toads is 0.1–10 g/kg/day (0.48–48.46 nmol/kg/day; 1/10–1/1000 of the LOEC from this study). In the basins of the Korean Yeongsan and Seomjin Rivers, the concentrations of OP, NP, and BPA in surface water have been found to range from 2.24 to 16.78 ng/L (10.86–81.33 nM), 114.63 to 336.14 ng/L (0.52–1.53 M), and 7.54 to 335.56 ng/L (0.03–1.47 M), respectively (Ra et al., 2011). Moreover, sediment samples from the Taehwa River, Yeocheon River, and Ulsan Bay area have been found to contain levels of OP ranging from
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Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox
Sensitization of vitellogenin gene expression by low doses of octylphenol is mediated by estrogen receptor autoregulation in the Bombina orientalis (Boulenger) male liver Chan Jin Park, Myung Chan Gye ∗ Department of Life Science and Institute for Natural Sciences, Hanyang University, Seoul 133-791, South Korea
a r t i c l e
i n f o
Article history: Received 8 February 2014 Received in revised form 26 August 2014 Accepted 27 August 2014 Available online 6 September 2014 Keywords: Octylphenol Xenoestrogen Vitellogenin Estrogen receptors Bombina orientalis
a b s t r a c t This study aimed to elucidate the mechanisms by which alkylphenols disrupt endocrine function in wild amphibians in Korea. To this end, the effects of 4-tert-octylphenol (OP), 17ˇ-estradiol (E2 ), and estrogen receptor (ER) agonists on the expression profiles of vitellogenin (VTG) and ERs were examined in livers obtained from male Bombina orientalis toads. A single injection of E2 (10 g/kg; 0.03 mol/kg) induced transcription of VTG mRNA at 2 days post injection; however, injection of either the ER␣-selective agonist propyl-(1H)-pyrazole-1,3,5-triyl-trisphenol (PPT, 50 g/kg; 0.12 mol/kg) or the ER-selective agonist 2,3-bis-(4-hydroxyphenyl)-propionitrile (DPN, 50 g/kg; 0.20 mol/kg) did not affect the expression of VTG. This finding suggests that both ER␣ and ER are required to induce transcription of VTG in the male B. orientalis liver. Interestingly, E2 , PPT, and DPN induced transcription of ER␣, which was also reflected on the protein level; however, these alkylphenols did not affect ER transcription. Similarly, VTG transcription was induced by a single injection of 1–100 mg/kg (0.04–484.66 mol/kg) OP, while 0.1 mg/kg (0.48 mol/kg) OP had no effect on VTG transcription. This result suggests that the lowest observable effect concentration (LOEC) of OP for induction of VTG transcription in the male liver is 1 mg/kg (4.84 mol/kg). Furthermore, treatment with E2 (10 g/kg; 0.03 mol/kg) or OP (1 mg/kg; 4.84 mol/kg) significantly upregulated ER␣ transcription, and a 10 mg/kg (48.46 mol/kg) dose of OP significantly upregulated ER transcription. The ER antagonist ICI 182,780 decreased the basal levels of ER␣ and ER mRNA, and also prevented E2 -mediated and OP-mediated induction of VTG, ER␣, and ER transcription. A second injection of 0.1 mg/kg (0.48 mol/kg) OP after a two-day interval significantly upregulated the transcription of VTG and ER␣, but not of ER. These results suggest that sensitization of VTG transcription by repeated exposure to OP is mediated by the induction of ER␣. Different combinations of alkylphenols that are ubiquitous in the freshwater system in Korea could potentially exert a synergistic effect on endocrine disruption. Thus, chronic exposure to alkylphenols, even at their NOECs, could still disrupt endocrine function in B. orientalis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Endocrine disruption is believed to be a contributing factor to the global decline of amphibians (Renner, 2002). Natural and synthetic estrogens enter water systems from anthropogenic sources via sewage treatment effluent (Desbrow et al., 1998), discharge from the industrial manufacturing of pharmaceuticals (Heberer, 2002), and agricultural runoff, which often contains animal manure that is used as fertilizer (Hanselman et al., 2003). The observation of various abnormalities, such as the feminization of wild animals, implies that water contamination with estrogenic
∗ Corresponding author. Tel.: +82 222200958; fax: +82 222989646. E-mail address: [email protected] (M.C. Gye). http://dx.doi.org/10.1016/j.aquatox.2014.08.013 0166-445X/© 2014 Elsevier B.V. All rights reserved.
compounds is an undeniable concern (Vos et al., 2000; Hutchinson et al., 2000). In amphibians, xenoestrogens induce the expression of estrogen-responsive genes, which have been associated with adverse effects on amphibian development (Kloas et al., 1999; Lutz and Kloas, 1999; Mosconi et al., 2002; Mackenzie et al., 2003; Ahn et al., 2011). Alkylphenolic compounds, which are used in a variety of human commercial products, are known to be estrogenic in vertebrates (Ying et al., 2002; Waring and Harris, 2005). Among these alkylphenols, 4-tert-octylphenol (OP) is one of the two main degradation products of alkylphenol polyethoxylates. These compounds are nonionic surfactants that are often used in household and industrial detergents (Nimrod and Benson, 1996); moreover, OP accounts for about 15% of all commercial alkylphenols in the USA and Canada (Bennett and Metcalfe, 1998). Although nonylphenol (NP), another degradation product of alkylphenol
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polyethoxylates, is the predominant contaminant in most water systems, the estrogenic potency of OP has been shown to be higher than that of NP (White et al., 1994). In Korean rivers, OP has been detected at concentrations ranging from 2.24 to 16.78 ng/L (10.86–81.33 nM) in surface water, and at 0.5 ng/L (2.42 nM) in the effluent from sewage treatment plants (Duong et al., 2010; Ra et al., 2011). Thus, OP in the aquatic environment may exert toxic effects on amphibians during their embryonic, larval, and adult life cycle stages. Due to its structural similarity to estrogen, OP can induce the transcription of estrogen-responsive genes and thus exert adverse effects on the embryonic development, sexual behavior, and sexual differentiation of amphibians (Nishimura et al., 1997; Kloas et al., 1999; Lutz and Kloas, 1999; Mayer et al., 2003; Huang et al., 2005; Porter et al., 2011). Vitellogenin (VTG) is an egg yolk precursor that is synthesized in the livers of female oviparous and ovoviviparous vertebrates (Wahli et al., 1981; Wallace, 1985). In amphibians, several natural estrogens and xenoestrogens have been shown to upregulate VTG transcription and protein production, both in male livers and in hepatocyte cultures (Wangh and Knowland, 1975; Carnevali et al., 1995; Bögi et al., 2003; Rotchell and Ostrander, 2003; Gye and Kim, 2005; Kang et al., 2006). Therefore, VTG induction in males has been used as a biomarker for measuring the estrogenicity of certain chemicals and environmental media (Sumpter and Jobling, 1995). The two estrogen receptors, ER␣ and ER, mediate the estrogenic effects of natural estrogens and xenoestrogens in both humans and wild animals (Kuiper et al., 1998; Rotchell and Ostrander, 2003; Melzer et al., 2011). In Xenopus, OP binds directly to ERs and activates the VTG promoter (Huang et al., 2005). Moreover, estrogens and xenoestrogens have also been reported to affect the expression of VTG, ER␣, and ER in fishes (Soverchia et al., 2005; Nagler et al., 2010). However, in amphibians, the transcriptional responses of ER␣ and ER to natural estrogens and xenoestrogens have not yet been investigated. An assessment of the ecological effects of endocrine disruptors on freshwater fishes and frogs in Korea revealed that the concentration of OP in the muscle tissue of adult bullfrogs (Rana catesbeiana) was 10 times higher than that in fishes (NIER, 2001). Moreover, the plasma VTG levels of male bullfrogs were similar to those of females in the surveyed areas (NIER, 2003). In the present study, we examined the effects of OP on ER␣, ER, and VTG expression. As a model organism, we selected the male fire-bellied toad, Bombina orientalis, which is a common native Korean anuran. Importantly, B. orientalis can be induced via gonadotropin to ovulate and spawn eggs at three-month intervals in the laboratory, thus permitting developmental toxicity assessment on embryos year-round (Park et al., 2010). Moreover, the sequence of B. orientalis ER␣ is available (GenBank accession no. FJ387577.1), and a homologue of VTG A2 has been identified (Lee and Gye, 2004). Furthermore, a protocol for the quantitative analysis of hepatic VTG mRNA has been established (Gye and Kim, 2005; Kang et al., 2006). In an effort to understand the mechanisms of endocrine disruption by estrogenic alkylphenols in a native Korean toad species, we examined the effects of OP on the expression of VTG, ER␣, and ER in livers from adult male B. orientalis. We also tested the hypothesis that autoregulation of ER␣ and ER in the male liver by OP may mediate VTG induction after chronic exposure to low doses of xenoestrogens.
2. Materials and methods 2.1. Animals Adult B. orientalis were bred and reared in the Hanyang University Aquarium. The toads were maintained on a diurnal 14 h
light and 10 h dark cycle at 20–22 ◦ C, and were fed crickets and mealworms three times a week. Adult toads with a mean body weight (BW) of 8.0 ± 0.5 g were used for all experiments. All experiments conducted on amphibians followed the procedures outlined in the “Guidelines for the Use of Live Amphibians and Reptiles in Field and Laboratory Research” publication (ASIH, 2004) (http://www.asih.org/files/hacc-final.pdf). As a control for ER␣ and ER in Western blots, mouse uterus tissue was obtained from an adult female mouse that had been euthanized after asphyxiation in CO2 . All mouse procedures were approved by the Institutional Animal Care and Use Committee of Hanyang University (HY-IACUC11-044). 2.2. Chemical exposure The 17-estradiol (E2) (MW = 272.38; Sigma Aldrich, St. Louis, MO), 4-tert-octylphenol (OP; MW = 206.33; Aldrich Chemical Co., Milwaukee, WI), ICI 182,780 (ICI; MW = 606.77; Sigma Aldrich) as an ERs antagonist, and ER subtype specific agonists, propyl pyrazole triol (PPT; MW = 386.44; Abcam, Cambridge, MA) estrogenic compounds were used for experiments involving ER␣ and diarylpropionitrile (DPN; MW = 239.27; Abcam) was used for experiments involving ER. Selected adult male toads received an intraperitoneal injection of OP (0.01, 0.1, 1, 10, or 100 mg/kg BW; 0.04, 0.48, 4.84, 48.46, or 484.66 mol/kg BW) or E2 (10 g/kg; 0.03 mol/kg) dissolved in sesame oil. These doses were found to effectively induce VTG expression in B. orientalis in initial optimization experiments. For some experiments, toads were given 50 g/kg (0.12 mol/kg) PPT and 50 g/kg (0.20 mol/kg) DPN. The doses of PPT and DPN were determined according to previous studies (Frasor et al., 2003; Neese et al., 2010). ICI was given at 1 mg/kg (1.64 mol/kg). This dose was based on the binding affinity of ICI to ERs, which is 100-fold lower than that of E2 (Preisler-Mashek et al., 2002). ICI was given either alone or in combination with E2 , OP, PPT, or DPN. As a vehicle control (VC), males were given sesame oil only. The total injection volume was 100 L per animal. To test the effects of repeated exposure to OP on gene expression in the male liver, two OP injections were given with a two-day interval. The concentration of OP used (0.1 mg/kg; 0.48 mol/kg) was below the lowest observed effect concentration (LOEC). Toads were returned to the aquaria and sampled 48 h after the second injection. Five male toads were used in each experimental group. 2.3. Degenerate PCR and cloning of ERˇ cDNA Female toads were euthanized by inhalation of ether to minimize pain, and their livers were removed. Total liver RNA was isolated using TRI reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer’s instructions. The concentrations of all RNA preparations were determined prior to storage at −85 ◦ C until use. For cDNA synthesis, RNA (1 g) was reverse transcribed for 60 min at 42 ◦ C in a 20 L reaction with 50 units of MuLV reverse transcriptase and 2.5 M oligo d(T)16 primer, according to the manufacturer’s standard protocol (Applied Biosystems, Foster City, CA). Degenerate PCR was performed with degenerate primers for the ER cDNA sequence, which was based on multiple anuran ER mRNA sequences (Xenopus laevis, NM 001130954.1; Xenopus tropicalis, NM 001040012.1; Rana rugosa, FJ828859.1; Bufo rangeri, AB524915.1) (Table 1). PCR products were ligated into the pGEM-T Easy vector (Promega, Madison, WI), and the resultant constructs were transformed into DH5␣ competent cells. More than 10 positive colonies were selected by blue/white screening. DNA sequencing was performed using M13 primers.
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Table 1 Primers for RT-PCR analysis. Primer ER (DGP) VTG ER␣ ER -actin a
F R F R F R F R F R
Primer sequence
Product size (bp)
Reference
5 -TTC AAG AGR AGY ATY CAA GGR C-3 5 -VTG YTG MAG TTT WAR CTC TCK VA-3 5 -CAC ATG CTG ATC CAT CTG TCC TGA-3 5 -TGG CGA CCA CAC AAT CCA CA-3 5 -CAG AGC CGC CCA TCG TCT AC-3 5 -CCT GGC ACT CTC TTT GCC CA-3 5 -GGC GGC ATT CAG ACG ATC AG-3 5 -CAT TTG GCG GTT CTG CTT CG-3 5 -GAG AGG TAT CCT GAC CCT GAA GTA-3 5 -ATA ACC TTC ATA GAT GGG CAC AGT-3
669 160
NM 001130954.1a ; NM 001040012.1a ; FJ828859.1a ; AB524915.1a Lee and Gye (2004), Gye and Kim (2005)
132
FJ387577.1a
144 325
Gye and Kim (2005)
GenBank accession number; DGP: degenerate primers.
2.4. RT-PCR analysis RT-PCR was performed according to the method outlined by Gye and Kim (2005). Briefly, PCR reactions contained 0.5 L liver cDNA, 1.25 units of Ex TaqTM polymerase (Takara, Japan), 1× Ex PCRTM Buffer II (Mg2+ -free), 2.5 mM MgCl2 , 0.4 mM dNTPs, and 0.4 M of each primer in a total volume of 25 L. -actin mRNA was amplified as a reference gene (Table 1). Real-time PCR was carried out using the same primers and iQ SYBR Green Supermix reagent (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. PCR was performed in a MyiQ i Cycler (Bio-Rad) using the following thermocycling conditions: 1 cycle of 3 min at 95 ◦ C followed by 40 cycles of 95 ◦ C for 30 s, the appropriate annealing temperature for 30 s, and 72 ◦ C for 30 s. VTG, ER␣, and ER mRNA levels are expressed as arbitrary units relative to -actin mRNA, and relative quantification was performed using the comparative CT method according to the manufacturer’s protocols (Bio-Rad).
2.5. SDS-PAGE and Western blot analysis Toad livers and mouse uteri were homogenized on ice in 10 volumes of extraction buffer [20 mM Tris–HCl (pH 7.5), 1% Triton X-100] containing a protease inhibitor cocktail (Roche, Mannheim, Germany). Homogenates were then sonicated and spun by centrifugation at 12,000 × g for 10 min at 4 ◦ C. The protein concentrations of the resultant supernatants were quantified using a BCA protein assay kit (Bio-Rad). Protein samples were mixed with 5× sample buffer containing 0.15 M dithiothreitol (DTT), boiled for 5 min, and then cooled to room temperature. After clarification to remove debris, the proteins in the supernatants were resolved on 10% SDS-polyacrylamide gels. As positive controls for ER␣ and ER, mouse uterus homogenates were also included. Following electrophoretic transfer to a nitrocellulose membrane (Amersham Bioscience, Buckinghamshire, UK), the membrane was blocked overnight at 4 ◦ C in Tris-buffered saline (1× TBS, pH 7.4) containing 7% skim milk. After washing for 5 min with TBS/0.1% Tween 20 (TBST), the membrane was incubated with rabbit polyclonal antibodies against either ER␣ (catalog no. ac37438, Abcam) or ER (catalog no. sc8974, Santa Cruz Biotechnology, Santa Cruz, CA). Primary antibodies were diluted in 5% skim milk in TBST (1:1000) and incubated for 2 h at room temperature. As a loading control, membranes were also probed with rabbit anti -tubulin antibodies (catalog no. sc9104, Santa Cruz Biotechnology). As a negative control, a 1:1000 dilution of rabbit IgG (catalog no. ab53041, Abcam, Cambridge, UK) in TBST was used. After three 10 min washes in TBST, the membrane was incubated with peroxidase-conjugated goat anti-rabbit IgG antibodies (catalog no. 81-6120, Zymed, San Francisco, CA) diluted in 5% skim milk in TBST (1:1000) for 1 h. Next, the membrane was washed with TBST for 10 min and then with TBS for 10 min. Immunoreactive bands were detected using an enhanced chemiluminescence (ECL) kit (Amersham Bioscience).
The amount of protein in each lane was quantified by analyzing the corresponding band intensity using 1D scan EX software (Scanalytics Inc., Fairfax, VA). 2.6. Immunohistochemistry and image analysis Paraffin sections (5 m thick) of 4% paraformaldehyde-fixed liver tissue were prepared. After deparaffination, endogenous peroxidase activity was blocked with 3% H2 O2 for 15 min. For antibody labeling, sections were incubated for 30 min with a blocking solution [1.5% normal goat serum in phosphate-buffered saline (PBS)]. The slide was then incubated in either a 1:1000 dilution of anti-ER␣ primary antibodies (rabbit polyclonal, catalog no. ac37438, Abcam) or a 1:500 dilution of anti-ER primary antibodies (rabbit polyclonal, catalog no. sc8974, Santa Cruz Biotechnology). Antibodies were diluted in blocking solution, and incubations were performed for 2 h at room temperature. As a negative control, slides were incubated in the same concentration of normal rabbit IgG (catalog no. ab27478, Abcam) under the same conditions. After two washes in PBS, the slide was incubated with peroxidase-labeled goat antirabbit IgG antibodies diluted 1:200 in blocking solution (catalog no. 81-6120, Zymed). Immunoreactive regions were visualized by reaction with 3,3-diaminobenzidine and H2 O2 as a chromogen. Slides were then counterstained with modified Harris hematoxylin (HHS16, Sigma Aldrich, St. Louis, MO). Permanently mounted slides were observed and images were captured using a microscope (IX71, Olympus, Japan) equipped with a digital imaging system (DP71, Olympus). Quantitative imaging of ER␣ and ER staining was conducted in four different sections using a commercially available image analysis program (IMT iSolution Lite, version 7.8, iMTechnology, Vancouver, British Columbia, Canada), according to the method described by Gye et al. (2011). 2.7. Statistical analysis All VTG mRNA levels were log-transformed before comparisons between groups. Statistical significance for real-time PCR and imaging data was analyzed using one-way analysis of variance (ANOVA) followed by a Tukey post hoc test. Statistical significance was defined as a p value < 0.05. 3. Results 3.1. B. orientalis ERˇ cDNA sequence A 558-bp (186 amino acid) ER sequence was identified by sequencing the appropriate amplicon (Fig. S1). The defined partial amino acid sequence of ER contained a DNA-binding domain and a ligand-binding domain. The deduced amino acid sequence of the corresponding B. orientalis ER ORF was 82% identical with the anuran X. laevis sequence (GenBank accession no. NP 001124426)
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Fig. 1. Effects of E2 , ICI, PPT, and DPN on the mRNA expression of VTG and ERs in the B. orientalis male liver. (A) Changes in the mRNA expression levels of VTG, ER␣, and ER resulting from injection of E2 (10 g/kg; 0.03 mol/kg), PPT (50 g/kg; 0.12 mol/kg), or DPN (50 g/kg; 0.20 mol/kg). (B) Changes in the mRNA expression levels of VTG, ER␣, and ER resulting from injection of ICI alone (1 mg/kg; 1.64 mol/kg), ICI + PPT (50 g/kg; 0.12 mol/kg), or ICI + DPN (50 g/kg; 0.20 mol/kg). VC, vehicle control; E2, 17-estradiol; ICI, ICI 182,780; PPT, propyl pyrazole triol; DPN, diarylpropionitrile. (a)–(c) were significantly different from each other (p < 0.05) as determined by ANOVA. Error bars = SDs (n = 5).
and 73% identical with the human sequence (GenBank accession no. CAA67555) (Fig. S2).
3.2. Effects of estrogen and selective ER-agonists on the expression profiles of VTG and ERs in the male liver In adult males, a single injection of E2 (10 g/kg; 0.03 mol/kg) upregulated the transcription of hepatic VTG by 2 days post injection; however, this effect was not observed with either PPT (50 g/kg; 0.12 mol/kg) or DPN (50 g/kg; 0.20 mol/kg). In contrast, E2 , PPT, and DPN significantly increased the levels of ER␣ mRNA, but not ER mRNA (Fig. 1A). The ERs antagonist ICI (1 mg/kg; 1.64 mol/kg) did not affect VTG transcription in the male liver; however, ICI significantly downregulated transcription of ER␣ and ER. Adult males treated with a combination of either ICI + PPT or ICI + DPN did not exhibit any significant differences in VTG or ER␣ transcription compared with VC-treated adult males. However, adult males treated with a combination of ICI + DPN exhibited significantly reduced transcription of ER compared with VC-treated adult males (Fig. 1B). Immunohistochemical analysis revealed ER␣ immunoreactivity in the nuclei and cytoplasm of hepatocytes from the E2 , PPT, and DPN treatment groups; in contrast, only basal ER␣ immunoreactivity was found in hepatocytes from the vehicle control group. Basal ER immunoreactivity was observed in hepatocytes from the E2 , PPT, DPN, and vehicle control groups (Fig. 2A). Densitometric analysis of ER␣ immunoreactivity revealed that hepatocytes from the E2 , PPT, and DPN groups exhibited significantly increased ER␣ immunoreactivity compared with hepatocytes from the vehicle control group; however, this trend was not observed with ER (Fig. 2B). Western blot analysis revealed that mouse and B. orientalis ER␣ and ER were detected by antibodies against ER␣ and ER, respectively; all bands migrated according to the predicted molecular weights (Fig. 3A). Moreover, ER␣, ER, and -tubulin were detected by antibodies against ER␣, ER, and tubulin, respectively, in the E2 , PPT, and DPN groups (Fig. 3B). Densitometric analysis revealed increased levels of ER␣ in the E2 , PPT,
and DPN groups compared with the vehicle control group; however, no differences in the levels of ER were observed between the groups (Fig. 3C). 3.3. Effects of OP on VTG and ERs transcription in the male liver The dosing of OP at 100 mg/kg (484.66 mol/kg) did not result in either death or narcosis at 2 days post injection, indicating that the OP dose used in this study did not evoke systemic toxic effects in B. orientalis. Time course experiments revealed that transcription of VTG was markedly increased by 1 day after a single injection of OP (100 mg/kg; 484.66 mol/kg) and reached maximum levels in the male liver at 2 days post injection. At 3 days post injection, the level of VTG mRNA began to decrease (Fig. S3). In contrast, a single injection of OP at doses ranging from 0.01 to 0.1 mg/kg (0.04–0.48 mol/kg) did not induce VTG transcription. However, a single injection of OP at doses ranging from 1 to 100 mg/kg (4.84–484.66 mol/kg) significantly increased hepatic VTG transcription in a dose-dependent manner. While ER␣ transcription was increased by a single injection of OP at 1 mg/kg (4.84 mol/kg), ER transcription was not affected. At doses of 10 or 100 mg/kg (48.46 and 484.66 mol/kg), OP significantly increased transcription of both ER␣ and ER (Fig. 4). 3.4. Effects of ER antagonists on estrogen-induced and OP-induced (at the LOEC) changes in VTG and ER transcription in the male liver The combination of E2 + ICI and the combination of OP + ICI significantly decreased the levels of VTG mRNA compared with E2 -treated animals (10 g/kg; 0.03 mol/kg) and OP-treated animals, respectively [LOEC for VTG mRNA (VTG LOEC), 1 mg/kg; 4.84 mol/kg]. However, VTG transcription in E2 -treated or OPtreated male livers was not completely reduced to basal levels by ICI. Moreover, E2 -induced ER␣ and E2 -induced ER transcription were both significantly downregulated by ICI, and the OP-induced
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Fig. 2. Changes in ER␣ and ER immunoreactivity in the B. orientalis male liver induced by E2 , PPT, or DPN. (A) Localization of ER␣ and ER antigens in the male liver. The regions designated by the dotted boxes are magnified in the insets. Arrows, ER␣-positive or ER-positive hepatocyte nuclei. Scale bar = 30 m. (B) Changes in ER␣ and ER immunoreactivity in the nuclei of male liver hepatocytes resulting from the injection of E2 (10 g/kg; 0.03 mol/kg), PPT (50 g/kg; 0.12 mol/kg), or DPN (50 g/kg; 0.20 mol/kg). VC, vehicle control; E2 , 17-estradiol; PPT, propyl pyrazole triol; DPN, diarylpropionitrile. Error bars = SDs (n = 4). (a)–(c) were significantly different from each other (p < 0.05) according to ANOVA.
increase in ER␣ transcription was also significantly decreased by ICI. In the OP + ICI-treated group, ER transcription was not significantly different from that in the OP-treated group; however, ER transcription was significantly lower than in the VC group (Fig. 5).
3.5. Repeated injection of low concentrations of OP induces transcription of VTG and ERs in the male liver A single dose of OP at the no effective concentration (NOEC) for VTG mRNA induction (0.1 mg/kg; 0.48 mol/kg) was followed by
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Fig. 3. Effects of E2 , PPT, and DPN on the protein levels of ER␣ and ER in the B. orientalis male liver. (A) Western blot analysis of the protein levels of ER␣ and ER in the mouse uterus (MU), B. orientalis oviduct (Ov), and liver (Li). Both ER␣ (66 kDa) and ER (56 kDa) migrated according to their predicted molecular weights in all samples. (B) Detection of ER␣ and ER in male liver samples from the VC, E2 (10 g/kg; 0.03 mol/kg), PPT (50 g/kg; 0.12 mol/kg), and DPN (50 g/kg; 0.20 mol/kg) groups. -tubulin (55 kDa) was used as a loading control. No immunoreactive bands were detected in the 55–66 kDa range when membranes were probed with rabbit normal IgG. (C) Densitometric analysis of the protein levels of ER␣ and ER in the livers of male toads injected with either E2 (10 g/kg; 0.03 mol/kg), PPT (50 g/kg; 0.12 mol/kg), or DPN (50 g/kg; 0.20 mol/kg). VC, vehicle control; E2 , 17-estradiol; PPT, propyl pyrazole triol; DPN, diarylpropionitrile.
Fig. 4. Effects of OP on VTG and ER transcription in the B. orientalis male liver. Levels of hepatic VTG, ER␣, and ER mRNA at 48 h after a single injection of OP (0, 0.01, 0.1, 1, 10, or 100 mg/kg; 0, 0.04, 0.48, 4.84, 48.46, or 484.66 mol/kg). VC, vehicle control. (a)–(c) were significantly different from each other (p < 0.05) as determined by ANOVA. Error bars = SDs (n = 5).
a second injection of the same OP dose after a 2-day interval. This treatment significantly upregulated VTG and ER␣ transcription, but not ER transcription, in the male liver. The mRNA levels of VTG and ER␣ in the group that received two injections of OP at 0.1 mg/kg (0.48 mol/kg) were higher than those in the group that received only one injection of OP at 1 mg/kg (4.84 mol/kg) (Fig. 6). 4. Discussion 4.1. OP mimics estrogen-mediated induction of VTG transcription in the male liver In the present study, OP was demonstrated to mimic the effect of estrogen on the B. orientalis male liver by using the level of VTG mRNA as a biomarker. Both E2 and OP induced VTG transcription in the adult male liver 2 days after injection; this effect was abrogated by ICI. The magnitude of action of ICI was different between the E2 + ICI and OP + ICI groups, suggesting that OP-mediated induction of VTG transcription can be attributed to OP–ER interactions, similar to the E2 –ER interactions. Although ER␣ and VTG transcription were significantly induced by E2 , ER transcription was not. Of particular note, VTG transcription was not induced by PPT or DPN alone. This finding suggests that VTG transcription in the male liver cannot be induced by activated ER␣ or ER alone; however, ER␣ transcription can be induced by either activated ER␣ or ER. The ER antagonist ICI significantly decreased the mRNA levels
of both ER␣ and ER. This finding suggests that basal transcription of ER␣ and ER is regulated by endogenous estrogen in the male liver. Furthermore, the hepatic mRNA levels of ER␣ in toads treated with either ICI + PPT or ICI + DPN were not different from the levels in VC-treated toads. This finding suggests that ICI prevents PPT-mediated and DPN-mediated upregulation of ER␣ transcription in the male liver. Since ER transcription was not induced by either PPT or DPN, and since no difference in ER transcription was observed between the ICI and ICI + DPN groups, (xeno)estrogens are probably poor inducers of ER transcription. In this study, a 1 mg/kg (4.84 mol/kg) dose of OP significantly increased the level of VTG mRNA in the male liver. Similar to E2 -mediated induction of VTG transcription, both ER␣ and ER appear to be required for OPmediated induction of VTG transcription in the male liver. Although the binding affinities of OP for ER␣ and ER have not yet been determined in amphibians, the binding affinity of OP for ER in humans and fish (channel catfish, Ictalurus punctatus) was lower than that for ER␣ (Routledge et al., 2000; Gale et al., 2004). This finding suggests that ER␣ is the predominant player in OP-mediated induction of VTG transcription in the B. orientalis male liver. In X. laevis, functional interactions between ER␣ and the transcriptional activator Sp1 have been shown to regulate estrogen-dependent VTG transcription (Batistuzzo de Medeiros et al., 1997). Therefore, we cannot exclude the possibility that OP-mediated induction of VTG transcription might also involve interactions between transcriptional activators and ERs in B. orientalis.
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Fig. 5. Effects of ICI on E2 -induced and OP-induced upregulation of VTG and ER transcription in the B. orientalis male liver. Hepatic VTG and ER mRNA expression levels were examined after injection of ICI (1 mg/kg; 1.64 mol/kg), in combination with either E2 (10 g/kg; 0.03 mol/kg) or OP (1 mg/kg; 4.84 mol/kg), into adult male B. orientalis toads. VC, vehicle control. (a)–(d) were significantly different from each other (p < 0.05) according to ANOVA. Error bars = SDs (n = 5).
Fig. 6. Effects of repeated OP injections on VTG and ER expression in the B. orientalis adult male liver. mRNA expression levels of VTG, ER␣, and ER following a single injection of OP at 0.1 mg/kg (0.48 mol/kg), a double (with a two-day interval) injection of OP at 0.1 mg/kg (0.48 mol/kg), or a single injection of OP at 1 mg/kg (4.84 mol/kg). VC, vehicle control. (a)–(c) were significantly different from each other (p < 0.05) according to ANOVA. Error bars = SDs (n = 5).
4.2. Autoregulation of ER mRNA by estrogen and OP in the male liver In animals, ER mRNA and protein production is regulated by estrogen, most commonly through auto-induction (Bagamasbad and Denver, 2011). In fishes, expression of ER␣ is induced by (xeno)estrogens. In rainbow trout (Oncorhynchus mykiss), functional estrogen-responsive elements (EREs) have been located in a 0.2-kb transcription start site upstream of the ER␣ gene (Petit et al., 1999). Moreover, E2 and nonylphenol (NP) have been shown to induce VTG transcription in hepatocytes at 1 and 10 M, respectively. In hatched larvae, ER transcription has been shown to be induced by 0.1 M E2 and 21 M NP following exposure during the embryonic stage (Madigou et al., 2001). Moreover, E2 , NP, bisphenol A (BPA), and OP have all also been shown to drive ER␣ transcription in the liver (Min et al., 2003; Andreassen et al., 2005; Osachoff et al., 2013). In male medaka (Oryzias latipes), E2 (100 ng/L; 0.37 nM), NP (0.5 mg/L; 2.27 M), and BPA (8 mg/L; 35.04 M) have all been shown to upregulate hepatic VTG and ER␣ transcription, but not ER transcription (Yamaguchi et al., 2005, 2008). In largemouth bass (Micropterus salmoides), E2 (0.5 mg/kg; 1.83 mol/kg) has been shown to upregulate ER␣ and VTG transcription, but not ER transcription, in the male liver (Sabo-Attwood et al., 2004). However, in goldfish (Carassius auratus), both ER and VTG transcription have been shown to be significantly upregulated by 0.1 M E2 or NP (Soverchia et al., 2005). These results indicate that the sensitivities of ER␣ and ER to estrogen-mediated transcriptional activation may differ among various fish species. In Xenopus, the ERE is located in the protein coding region of the ER␣ gene (Lee et al., 1995). In the Japanese giant salamander (Andrias japonicus), transcription of ER␣ has been shown to be upregulated by E2 in CHO cells cotransfected with a plasmid containing an ERE (Katsu et al., 2006). In amphibians, E2 , NP, and BPA have all been shown to upregulate
hepatic ER␣ transcription (Westley and Knowland, 1979; Barton and Shapiro, 1988; Levy et al., 2004; Lutz et al., 2005). Similarly, we found that the levels of hepatic ER␣ mRNA and protein were both significantly increased by E2 (10 g/kg; 0.03 mol/kg), PPT (50 g/kg; 0.12 mol/kg), and DPN (50 g/kg; 0.20 mol/kg) at 2 days post injection in male B. orientalis. Although the EREs of the B. orientalis ER genes have not yet been characterized, it is highly likely that B. orientalis ER␣ is autoregulated via transactivation of an ERE by activated ERs. However, unlike ER␣, transcription of ER was not induced by estrogen or ER agonists, at least when used at the same concentrations that induced ER␣ transcription. This finding suggests that ER␣ and ER exhibit different sensitivities to estrogen-mediated transcriptional activation in the B. orientalis male liver. In support of this idea, CHO cells transfected with the ER␣ or ER protein coding regions (including functional EREs) exhibit higher estrogen-mediated transactivation of the ER␣ genes compared with the ER genes of anurans (X. tropicalis and B. rangeri) (Katsu et al., 2010). In contrast, ER transactivation was more readily induced by estrogen in caudata (Hynobius tokyoensis and Ambystoma mexicanum) and urodela (Cynops pyrrhogaster) (Katsu et al., 2010), indicating that the sensitivities of ER␣ and ER to estrogen-mediated transcriptional activation are different among various amphibian species. In the B. orientalis male liver, the LOECs for induction of ER␣ and ER with a single dose of OP were 1 mg/kg (4.84 mol/kg) and 10 mg/kg (48.46 mol/kg), respectively. This finding indicates that ER␣ is 10-fold more sensitive to OP-mediated gene transcription compared with ER. Although both ER␣ and ER are required for the induction of VTG transcription in the B. orientalis male liver, induction of ER␣ by E2 and OP might also be important for the induction of VTG transcription. Together, OP–ERs may autoregulate ERs, which in turn transactivate transcription of ER-dependent target genes, including VTG, in the B. orientalis male liver (Fig. 7).
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Fig. 7. Proposed mechanism for OP-mediated sensitization of VTG expression involving ER autoregulation in the B. orientalis male liver. Transcription of VTG and ER␣ was induced by either a single injection or repeated injections of either E2 or OP. Moreover, transcription of ER was induced by a high dose of OP (10 mg/kg; 48.46 mol/kg). Either an ER␣-specific agonist (PPT) or an ER-specific agonist (DPN) induced transcription of ER␣, but not of VTG or ER. Thus, either activation of ER␣ or ER is sufficient to upregulate transcription of ER␣, but not of VTG or ER, in B. orientalis. Although test doses of either E2 or OP did not induce ER expression, a high dose of OP did induce ER expression. Thus, upregulation of ER␣ by OP, even at a low dose, can sensitize VTG expression in the B. orientalis male liver.
4.3. Sensitization of VTG mRNA expression by low levels of OP and estrogen In X. laevis, the binding affinity of OP for ER is 15 times lower than that of E2 ; furthermore, the ability of OP to upregulate VTG expression is 200 times lower than that of E2 (Huang et al., 2005). In X. laevis hepatocytes, the binding affinity of OP for ERs has been shown to be lower than that of NP and BPA (Lutz and Kloas, 1999; Kloas et al., 1999). Similarly, in male crucian carp (C. carassius), the VTG LOEC of OP (20 g/L; 0.10 M) has been shown to be lower than that of the VTG LOEC of BPA (100 g/L; 0.48 M) (Zhang et al., 2010). We found that repeated injection of OP at its NOEC (0.1 mg/kg; 0.48 mol/kg) sensitized transcription of both VTG and ER␣, which resulted in a marked increase in VTG and ER␣ transcription compared with toads given a single dose of OP at a higher concentration (1 mg/kg; 4.84 mol/kg). Considering the observations that the levels of hepatic ER␣ mRNA and protein were significantly increased by E2 , PPT, and DPN at 2 days post injection, and that the levels of ER were not altered by the same treatments, sensitization of VTG gene transcription by repeated dosing of OP at the NOEC might be predominantly mediated by ER␣. In flounder (Platichthys flesus), OP has been shown to accumulate in the liver; moreover, the tissue concentration of OP was positively correlated with the plasma VTG concentration (Madsen et al., 2002). This finding suggests that chronic exposure to low concentrations of OP could cause bioaccumulation and thereby disrupt endocrine function in male amphibians by sensitizing ER signaling pathways. 4.4. Environmental concerns In Korea, many toad species including B. orientalis breed in rice fields, puddles, and riverside pools in freshwater systems (AmphibiaWeb, http://amphibiaweb.org). Therefore, they are vulnerable to exposure to a variety of EDCs throughout their life cycles. In the B. orientalis male liver, the VTG NOEC for OP was 0.1 mg/kg
(0.48 mol/kg). Exposure to OP in combination with other xenoestrogens, such as BPA and NP, is most likely unavoidable for toads in contaminated habitats. Thus, OP may elicit endocrine disruption in B. orientalis, even when it is only present at the NOEC, due to the presence of other xenoestrogens in the aquatic media. In one approach for defining predicted no-effect concentrations (PNECs), the constant application factors usually range between 10 and 1000, depending on pragmatic assumptions relating to single substances versus real world mixtures, acute–chronic ratios, and interspecies differences. These factors are then applied to the available toxicity data, including the LOEC (EC, 2003). According to this guideline, an acceptable exposure value for OP without inducing overt endocrine disruption in adult B. orientalis toads is 0.1–10 g/kg/day (0.48–48.46 nmol/kg/day; 1/10–1/1000 of the LOEC from this study). In the basins of the Korean Yeongsan and Seomjin Rivers, the concentrations of OP, NP, and BPA in surface water have been found to range from 2.24 to 16.78 ng/L (10.86–81.33 nM), 114.63 to 336.14 ng/L (0.52–1.53 M), and 7.54 to 335.56 ng/L (0.03–1.47 M), respectively (Ra et al., 2011). Moreover, sediment samples from the Taehwa River, Yeocheon River, and Ulsan Bay area have been found to contain levels of OP ranging from
