In Vitro and in Vivo Analysis of the Thyroid Disrupting Activities of Phenolic and Phenol Compounds in Xenopus laevis
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《毒物学科学杂志》
Department of Biology, Faculty of Science, Shizuoka University, Shizuoka 422-8529, Japan
ABSTRACT
We investigated the effects of phenolic and phenol compounds on 3,3',5-L-125I-triiodothyronine (125I-T3) binding to purified Xenopus laevis transthyretin (xTTR) and to the ligand-binding domain of X. laevis thyroid hormone receptor (xTR LBD), on T3-induced metamorphosis in X. laevis tadpoles and on the induction of T3-dependent reporter gene in a X. laevis cell line. Of the halogenated phenolic and phenol compounds tested, 3,3',5-trichlorobisphenol A and 2,4,6-triiodophenol, respectively, were the most potent competitors of 125I-T3 binding to both xTTR and xTR LBD. Most of the halogenated compounds had stronger interactions with xTTR than with xTR LBD. Generally, chlorinated derivatives with a greater degree of chlorination were more efficient competitors of T3 binding to xTTR and xTR LBD. Structures with a halogen in either ortho position or in both ortho positions, with respect to the hydroxy group, were more efficient competitors. 3,3',5-Trichlorobisphenol A and 2,4,6-triiodophenol acted as T3 antagonists in the X. laevis tadpole metamorphosis assay. Interestingly, o-t-butylphenol and 2-isopropylphenol, for which xTTR and xTR LBD had weak or no significant affinity, showed T3 antagonist activity in the metamorphosis assay. T3 antagonist activities of all these chemicals except for o-t-butylphenol were verified by T3-dependent reporter gene assay. Our results suggest that some phenolic and phenol compounds target the process of T3 binding to xTTR and xTR and/or an unknown process, and that they interfere with the intracellular T3 signaling pathway.
Key Words: thyroid hormone; transthyretin; thyroid hormone receptor; halogenated phenolic compounds; thyroid disrupting chemicals; metamorphosis; Xenopus laevis.
INTRODUCTION
An increasing number of chemicals released in the environment, although not apparently toxic, may interfere with critical endocrine systems and developmental processes in a wide range of vertebrates (Bevan et al., 2003; Sonnenschein and Soto, 1998). These endocrine-disrupting chemicals (EDCs) include pharmaceuticals, pesticides, herbicides, and industrial chemicals (Brucker-Davis, 1998; Cheek et al., 1998; Danzo, 1997; Sonnenschein and Soto, 1998). The majority of research concerning EDCs has focused on steroidgenic and antisteroidgenic effects and steroidgenesis in wildlife, experimental animals, and humans. Relatively less research has focused on their effects on the thyroid system, despite reports of abnormally structured thyroid glands and irregular levels of thyroid hormones (THs) in experimental animals (Brouwer et al., 1998; Collins and Capen, 1980; Collins et al., 1977) and wildlife (Moccia et al., 1986; Rolland, 2000; Verreault et al., 2004) exposed to EDCs.
THs are important hormones of brain development, intelligence, and behavior in higher vertebrates (Porterfield, 1994; Zoeller et al., 2002) and postembryonic development in lower vertebrates (Dickhoff et al., 1990). However, there have been few reports concerning the molecular mechanisms of thyroid system disruption by EDCs. Of the EDCs, a number of organohalogen compounds are known to interfere with the thyroid system (Brucker-Davis, 1998). Possible sites that the organohalogens target in the thyroid system include the thyroid gland (Collins et al., 1977), the plasma TH binding protein, transthyretin (TTR) (Brouwer et al., 1998), and TH metabolism (Byrne et al., 1987). The disruption of the thyroid system by EDCs is characterized by the fact that there are few chemicals that competitively interact with 3,3',5-L-triiodothyronine (T3) binding to TH receptor (TR) (Cheek et al., 1999b; Ishihara et al., 2003b). This is in contrast to the steroid system (Matthews et al., 2000), suggesting that thyroid system processes other than T3 binding to TR are targeted by EDCs, although this assumption is mainly based on in vitro studies (Cheek et al., 1999b; Gauger et al., 2004; Ishihara et al., 2003a,b; Yamauchi et al., 2003). Recently, chlorinated derivatives of bisphenol A were detected at the concentrations of 10–8 M in wastewater from paper recycling plants in Japan (Fukazawa et al., 2001). Of the chlorinated derivatives, trichlorobisphenol A and tetrachlorobisphenol A, structurally resemble T3 and L-thyroxine (T4), respectively. Therefore, it is likely that they interfere with the thyroid system.
To determine which thyroid system process is targeted by EDCs in vitro, the experimental animal model Xenopus laevis may provide the most insight. As X. laevis tadpoles are water-living animals (all of its life stages occur in water) and have thin and permeable body skin, they may be particularly sensitive to a number of EDCs present in water. X. laevis is widely used as a laboratory animal and its development and gene expression are well characterized. For these reasons, X. laevis has been approved as an experimental model for evaluating the effects of EDCs in amphibians by the Organization for Economic Cooperation and Development (OECD). As amphibian metamorphosis is obligatorily controlled by THs, amphibian tadpoles are a good model animal for understanding the molecular mechanism by which EDCs disrupt the thyroid system.
In the present study, we investigated the effects of phenolic and phenol compounds, including chlorinated and iodinated compounds, on the X. laevis thyroid system in vitro—competitive interactions of the chemicals with 125I-T3 binding to X. laevis TTR (xTTR) and the ligand-binding domain of X.laevis TR (xTR LBD)—and in vivo—amphibian metamorphosis and T3-dependent gene reporter assays. The potent competitors detected in the in vitro study exhibited a T3-antagonist activity in the in vivo assay; however, some chemicals with almost no interaction with TR in the in vitro study also exhibited a T3-antagonist activity in the in vivo study.
MATERIALS AND METHODS
Reagents. 125I-T3 (122 MBq/μg; carrier free) was purchased from NEN Life Science Products (Boston, MA). Unlabeled T3 (>98% purity) and T4 (>98% purity) were obtained from Sigma (St. Louis, MI). Bisphenol A (>98% purity), 2,4,6-triiodophenol (>98% purity) and o-t-butylphenol (>95% purity) were purchased from Wako Pure Chemical Industries (Osaka, Japan) and 4-nonylphenol (>97% purity) was from Kanto Chemicals (Tokyo, Japan). 2-Isopropylphenol (>98% purity) was obtained from Lancaster (Morecambe, England). AG 1-X8 resin was from BioRad (Hercules, CA). The chlorinated derivatives (>98% purity) of bisphenol A and nonylphenol were synthesized as described previously (Fukazawa et al., 2001) and kindly provided by Dr. Y. Terao. All other chemicals used in this study were either chromatography grade or the highest grade available and were purchased from Wako Pure Chemical Industries or Nacalai Tesque (Kyoto, Japan).
All chemicals tested as EDCs were dissolved in dimethylsulfoxide to a concentration of 10 mM. These chemicals were diluted with an appropriate buffer to give less than 0.4% (v/v) solvent. A control assay without the test chemicals was performed in the presence of the solvent alone and at less than 0.4% (v/v). The solvent did not affect the competitive 125I-T3 binding assays, the metamorphosis assay nor the gene reporter assay described below.
Preparation of recombinant xTTR and xTR LBD. Recombinant xTTR and glutathione-S-transferease (GST) fused xTR LBD were expressed in Escherichia coli BL21 and purified from the bacterial extracts by affinity column chromatography, human retinol-binding protein coupled to Sepharose 4B (Larsson et al., 1985), and glutathione coupled to Sepharose 4B (Amersham Pharmacia Biotech), respectively, as described previously (Yamauchi et al., 2002). They were stored in 10% glycerol at –85°C for later use. Protein concentration was determined by the dye-binding method using bovine -globulin as the standard (Bradford, 1976).
125I-T3 binding assays using TTR (TTR assay) and the TR LBD (TR assay). xTTR (300 ng/tube) was incubated with 0.1 nM 125I-T3 in 250 μl of 20 mM Tris-HCl, pH 7.5, 93 mM NaCl, and 1 mM CaCl2 in the presence or absence of 5 μM unlabeled T3 for 1.0 h at 4°C. GST-xTR LBD fusion protein (54 ng/tube) was incubated with 0.1 nM 125I-T3 in 250 μl of 10 mM Tris-HCl, pH 7.5, 1.5 mM EDTA, 1 mM dithiothreitol and 10% (v/v) glycerol in the presence or absence of 1 μM unlabeled T3 for 1.5 h at 4°C. Competitive 125I-T3 binding was performed with solvent only or increasing concentrations of the unlabeled test chemical, as described previously (Yamauchi et al., 2000). For the TTR assay, protein-bound 125I-T3 was separated from free 125I-T3 by the polyethylene glycol method (Yamauchi et al., 1993). For the TR assay, the Dowex method (Lennon, 1992; Lennon et al., 1980) separated bound 125I-T3 from free 125I-T3. Radioactivity was measured in a gamma counter (Auto Well Gamma System ARC-2000, Aloka, Japan). The amount of 125I-T3 bound nonspecifically was derived from the radioactivity of samples incubated with excess unlabeled T3. The nonspecific binding value was subtracted from the amount of total bound 125I-T3 to give the value of specifically bound 125I-T3.
Metamorphosis assay. X. laevis tadpoles were purchased from Akita Xenopus Co. (Ibaraki, Japan). Tadpoles were staged according to Nieuwkoop and Faber; NF (1975). They were maintained under natural lighting conditions in a 20-l glass aquarium containing dechlorinated tap water and fed dried food commercially available for the fish Medaka (Kyorin Co., Himeji, Japan) once a week. Before starting experiments, test animals were acclimatized to laboratory conditions (25–26°C) for 24 h. During the acclimatization and exposure periods, tadpoles were not fed. Five tadpoles in NF stages 52–53 were transferred into a 1-l glass beaker containing 0.5 l of FETAX buffer (625 mg NaCl, 96 mg NaHCO3, 30 mg KCl, 15 mg CaCl2, 60 mg CaSO4.2H2O, and 75 mg MgSO4.7H2O per l distilled water, pH 7.7) (Dumont et al., 1983). The FETAX buffer contained dimethylsulfoxide, T3 in dimethylsulfoxide, or T3 and each chemical in dimethylsulfoxide. Chemical applications were renewed every other day by changing the above FETAX buffer. Final dimethylsulfoxide concentrations were less than 0.02% in the chemical-exposed and control groups. Fresh NANOpure ultrapure water (Barnstead International, Dubuque, IA) was used for preparing the FETAX buffer. During the experiments, tadpoles were anesthetized in 0.02% 3-aminobenzoic acid ethyl ester (Sigma) and photographed under a stereomicroscope (type SZ-PT, Olympus, Japan) to measure body length, tail length, interocular distance, forelimb length, and hindlimb length. After 5 or 7 days, all living tadpoles were frozen in liquid nitrogen and then stored at –80°C until RNA preparation. Each experiment was repeated at least three times using tadpoles derived from different sets of adults.
Real-time polymerase chain reaction. Total RNA was extracted from the frozen tadpoles using the LiCl-urea procedure (Auffray and Rougeon, 1980). RNA (5–10 μg per lane) was electrophoresed in a 1% agarose gel containing 2.6 M formaldehyde. After visualizing 28S and 18S rRNAs by ethidium bromide staining to check the integrity of the RNA samples and equal loading, amounts of specific RNA species were estimated by real-time polymerase chain reaction (PCR) using SYBR Green Master Mix and ABI Prism 7000 (Applied Biosystems, Foster City, CA) after the RNA samples were treated with reverse transcriptase (TaqMan Reverese Transcription Reagents, Applied Biosystems). Each PCR was run in duplicate to control for PCR variation. The thermocycler program included a step of denaturation at 95°C (10 min), and 40 cycles of 95°C (15 sec), 60°C (1 min), and 50°C (2 min). The endpoint used in real-time PCR quantification, Ct, was defined as the PCR cycle number that crosses an arbitrarily placed signal threshold and is a function of the amount of target DNA present in the starting material. Quantification was determined by applying the 2–Ct formula and calculating the average of the two values obtained for each sample. Eligibility of this formula was verified using a mixture of X. laevis cDNAs containing the xTR cDNA at three different concentrations (1:50:250). To standardize each experiment, the amount of xTR transcript was divided by the amount of glyceraldehyde dehydrogenase (GAPDH) RNA in the same samples. Primer sequences used were as follows: xTR transcript (accession number: M35356 and M35357) sense 5'-CAAGCACCAAGAACGAAAACC-3' (nucleotide numbers 15–35) and antisense 5'-TTGGAAGGTCTGCTCATTCTTCTA-3' (39–16), and X. laevis GAPDH transcript (accession number: V41753) sense 5'-CTCATGACAACAGTCCATGCTTTC-3' (558–581) and antisense 5'-CTCTGCCATCTCTCCACAGCTT-3' (639–618).
Luciferease assay. Sense and antisense oligonucleotides containing the thyroid hormone response elements (TREs), TH/bZIP TRE1 + TRE2 (–99 to –63) (Furlow and Brown, 1999), were annealed and introduced into the unique BglII/SacI site in the pGL2 promoter vector (Promega, Madison, WI) making the reporter plasmid pGL2-TRE. xTR cDNA (kindly provided by Dr. D. D. Brown) was cloned into the EcoRI site of pcDNA3 (Invitrogen, Carlsbad, CA) making the xTR expression plasmid pcDNA3-xTR. Plasmids for transfection were purified using QIAGEN (Chatsworth, CA) miniprep kits. For transient transfection assays, X. laevis XL58 cells (0.6 x 105 cells) were seeded in 24-well culture plates (Nunc, Roskilde, Denmark) and cultured in 70% Leibovitz's L-15 medium (Sigma) containing 10% resin-stripped fetal bovine serum (Samuels et al., 1979) for 15 h at 25°C with air. The next day, the cells were transfected with 500 ng pGL2-TRE, 50 ng pcDNA3-xTR, and 20 ng pRL-CMV vector (Promega) with 4 μl of the lipofection reagent DOSPER (Roche, Mannheim, Germany). After 6 h, the cells were replenished with 70% Leibovitz's L-15 medium containing 10% resin-stripped fetal bovine serum and further cultured with 2 nM T3, without T3, or with 2 nM T3 and each test chemical at defined concentrations for 24 h at 25°C. The culture media were not changed during this treatment. The cells from each well were harvested and assayed for firefly Photinus pyralis and sea pansy Renilla reniformis luciferase activities, derived from pGL2-TER and pRL-CMV, respectively, by using Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's directions. Transfection efficiency was normalized using a constant amount of the sea pansy luciferase activity.
Statistical analysis. The data are presented as mean ± SEM. Differences between groups were analyzed with either Student's t-test or Cochran-Cox test to evaluate the significance of the differences; p < 0.05 was considered statistically significant.
RESULTS
Effects of Halogenated Phenolic and Phenol Compounds on 125I-T3 binding to xTTR
To determine which halogenated phenolic and phenol compounds were strong competitors, 125I-T3 binding to xTTR was examined at 4°C in the presence of various concentrations of the phenolic compounds with two phenolic rings: bisphenol A and its chlorinated derivatives (Fig. 1A), and of the phenol compounds: nonylphenol and its chlorinated derivatives, 2,4,6-triiodophenol, 2-isopropylphenol, and o-t-butylphenol (Fig. 1B). Of bisphenol A and its chlorinated derivatives, 3,3',5-trichlorobisphenol A was the most powerful competitor, with a 50% inhibitory concentration (IC50) of 13 ± 3 nM. The rank order binding affinity was T3 (IC50 = 5.6 ± 1.3 nM) > 3,3',5-trichlorobisphenol A (13 ± 3 nM) > 3,3',5,5'-tetrachlorobisphenol A (32 ± 2 nM) > 3,3'-dichlorobisphenol A (54 ± 10 nM) > 3,5-dichlorobisphenol A (240 ± 50 nM) > 3-chlorobisphenol A (320 ± 10 nM) > bisphenol A (2100 ± 200 nM). The relative affinity of xTTR for 3,3',5-trichlorobisphenol A was 160 times greater than that for bisphenol A but only two times less than that for T3. Of the phenol compounds tested, 2,4,6-triiodophenol was the most potent competitor, with an IC50 of 54 ± 21 nM, and the rank order binding affinity within this group was T3 > 2,4,6-triiodophenol > 2,6-dichloro-4-nonylphenol (240 ± 20 nM) > 2-chloro-4-nonylphenol (5700 ± 400 nM) > 4-nonylphenol (5800 ± 600 nM) > o-t-butylphenol (11000 ± 1000 nM) > 2-isopropylphenol (15000 ± 2000 nM). The relative affinity of xTTR for 2,4,6-triiodophenol was ten times less than that for T3. The relative affinity of xTTR for 2,6-dichloro-4-nonylphenol was 24 times greater than that for 4-nonylphenol but still 43 times less than that for T3.
Effects of Halogenated Phenolic and Phenol Compounds on 125I-T3 Binding to xTR LBD
All compounds tested were weak inhibitors of 125I-T3 binding to xTR (Figs. 2A and 2B) compared with their effect on 125I-T3 binding to xTTR. Of these compounds, 3,3',5-trichlorobisphenol A was the most powerful competitor, with an IC50 of 760 ± 100 nM. The rank order binding affinity for the phenolic compounds was T3 (IC50 = 0.66 ± 0.04 nM) > T4 (3.5 ± 0.6 nM) >>> 3,3',5-trichlorobisphenol A (760 ± 100 nM) > 3,3',5,5'-tetrachlorobisphenol A (1300 ± 100 nM) > 3,3'-dichlorobisphenol A (2300 ± 100 nM) > 3,5-dichlorobisphenol A (4900 ± 400 nM) > 3-chlorobisphenol A (6900 ± 400 nM) > bisphenol A (24000 ± 3000 nM) (Fig. 2A); and for the phenol compounds 2,4,6-triiodophenol (1400 ± 200 nM) > 2,6-dichloro-4-nonylphenol (3400 ± 600 nM) > 2-chloro-4-nonylphenol (7900 ± 1000 nM) > 4-nonylphenol (16000 ± 100 nM) (Fig. 2B). The relative affinity of xTR for 3,3',5-trichlorobisphenol A was 32 times greater than that for bisphenol A but only 103 less than that for T3. xTR did not show significant affinity for o-t-butylphenol and 2-isopropylphenol. The rank order binding affinities for xTR were similar to those for xTTR.
Effects of Phenolic and Phenol Compounds on X. laevis Tadpole Metamorphosis
To examine whether the potent chemicals selected from the above in vitro assays affect the thyroid system in the in vivo assay, X. laevis tadpoles in NF stages 52–53 were immersed in FETAX buffer containing 2 nM T3 with or without each chemical and were cultured for 5–7 days at 25–26°C. At the end of the experiments, tadpoles treated with neither T3 nor each chemical were in NF stages 52–53, whereas tadpoles treated with T3 alone and with both T3 and each chemical developed to NF stages 56–57 and 55–56, respectively. However, it was difficult to evaluate quantitatively the effect of each chemical on tadpole metamorphosis from the progress of the NF stage. Therefore, as end points for assessing T3 antagonist activity of the potent chemicals on T3-induced metamorphosis, the following five morphological characteristics of X. laevis tadpoles were monitored during the culture: body length, tail length, interocular distance, forelimb length, and hindlimb length. Significant morphological differences between positive (+T3) and negative (–T3) control tadpoles were found in the interocular distance alone on the fourth day and in the other three morphological characteristics except for body length on the fifth day or later (data not shown). In Figure 3, the ordinate shows the ratio of interocular distance to body length (to compensate for the body size difference of each tadpole). A significant antagonistic effect of 3,3',5-trichlorobisphenol A on T3 was detected using this ratio on the fourth day (Fig. 3A). This ratio had a variation smaller than the ratios of the other three characteristics to body length. It was concluded that the interocular distance was the most sensitive end point available for determining the T3 antagonist activity of EDCs in X. laevis tadpoles of the four morphological end points tested.
T3-induced metamorphosis was completely inhibited by 2 μM 3,3',5-trichlorobisphenol A and partially inhibited by1.2 μM 2,4,6-triiodophenol (Figs. 3A and 3B), both of which were potent in the in vitro assays. Interestingly, 6 μM o-t-butylphenol and 10 μM 2-isopropylphenol, which were moderately active in the TTR assay (Fig. 1B) but scarcely active in the TR assay (Fig. 2B), also exerted T3 antagonist activity in the X. laevis metamorphosis assay on the fourth day (Fig. 3B).
To confirm whether the EDCs' effects on T3-induced X. laevis metamorphosis were due to their interference with the T3-signaling pathway, we examined the amount of TR transcript in whole tadpoles. The tr gene is a well-known, early primary T3-response gene in metamorphosing X. laevis tadpoles (Wang and Brown 1993). In X. laevis tadpoles treated with T3 (2 nM), the amount of TR transcript increased 30–35 times on the fifth day after treatment (Figs. 4A and 4B). The amount of TR transcript on the seventh day after treatment was the same as that on the fifth day (data not shown). Cotreatment of T3 with 3,3',5-trichlorobisphenol A (2 μM), 2,4,6-triiodophenol(1.2 μM), o-t-butylphenol (6 μM) or 2-isopropylphenol (10 μM) significantly inhibited the T3-induced increase in the amount of TR transcript at the fifth or seventh day of treatment. The inhibition percentages obtained from independent repeated experiments were 39 ± 5% 3,3',5-trichlorobisphenol A (n = 5), 44 ± 7% 2,4,6-triiodophenol (n = 3), 30 ± 8% o-t-butylphenol (n = 4), and 46 ± 10% 2-isopropylphenol (n = 5). These results indicated that the four chemicals tested interfered with the T3-signaling pathway, causing the inhibition of T3-induced metamorphosis.
Effects of Phenolic and Phenol Compounds on T3-Dependent Reporter Gene Assay in X. laevis Cell Line
Finally, we investigated the effect of EDCs on a T3-dependent reporter gene assay in the X. laevis cell line, XL58 (Fig. 5). In the absence of the compounds, T3 (2 nM) increased luciferase activity by 5–6 times. Cotreatment of T3 with 3,3',5-trichlorobisphenol A (2 μM), 2,4,6-triiodophenol (1.2 μM), and 2-isopropylphenol (10 μM) significantly inhibited the T3-induced increase in luciferase activity. Cotreatment of T3 with o-t-butylphenol (6 μM) decreased T3-dependent luciferase activity, but the decrease was not significant. The inhibition percentages obtained from independent repeated experiments were 47 ± 12% 3,3',5-trichlorobisphenol A (n = 3), 40 ± 8% 2,4,6-triiodophenol (n = 3), 56 ± 5% 2-isopropylphenol (n = 4), and 74 ± 6% o-t-butylphenol (n = 3).
DISCUSSION
The present study demonstrated that 3,3',5-trichlorobisphenol A and 2,4,6-triiodophenol exhibited T3 antagonist activity at micromolar or less concentrations in all bioassays tested in X. laevis, and that o-t-butylphenol and 2-isopropylphenol exhibited T3 antagonist activity at 6–10 μM in the in vitro TTR assay and the in vivo metamorphosis assays, but not in the in vitro TR assay; concentrations up to 31 μM were tested. As o-t-butylphenol had so weak effect on the T3-dependent reporter gene assay, its T3 antagonist activity was considered not significant in our experimental conditions. These results indicate that the combination of several assays, which assess different TH system processes, is useful when evaluating the thyroid disrupting activity of EDCs, due to different chemicals targeting different processes.
The rank order of binding affinities in the in vitro TTR and TR assays gave similar results, although the TTR assay was more sensitive to the chemicals tested than the TR assay. Both xTTR and xTR had higher affinities for the chlorinated derivatives of bisphenol A and of nonylphenol than for their parent molecules. The relative potency of the chlorinated derivatives was generally dependent upon the degree of chlorination. xTTR and xTR have a binding preference for the two groups of chemicals. The first group contains chlorinated phenolic compounds that have two phenolic rings with chlorines in either ortho position or in both ortho positions with respect to the hydroxy group, such as 3,3',5-trichlorobisphenol A and 3,3',5,5'-tetrachlorobisphenol A. The second group contains chlorinated or brominated phenols that have a single phenol ring with halogens in both ortho positions with respect to the hydroxy group, such as 2,6-dichloro-4-nonylphenol and 2,4,6-triiodophenol. The competitive binding characteristics of these chemicals were similar to previous studies, which analyzed the interactions of the same chlorinated phenolic and phenol compounds with TTRs and TRs from different species (Yamauchi et al., 2003) and different halogenated phenolic and phenol compounds with human TTR (Meerts et al., 2000; van den Berg, 1990) and rat TR (Kitamura et al., 2002).
The binding preference of xTTR and xTR for the chlorinated phenolic compounds, belonging to the first group, may reflect their TH binding properties and the chemical's structural resemblance to THs. Mammalian TTRs have 4–8 times higher affinity for T4 than for T3 (Chang et al., 1999; Robbins, 1996) while xTTR has 102 times higher affinity for T3 than for T4 (Yamauchi et al., 2002). Interestingly, hTTR has 18 times higher affinity for 3,3',5,5'-tetrabromobisphenol A than for 3,3',5-tribromobisphenol A (Meerts et al., 2000), although xTTR has 2.5 times higher affinity for 3,3',5-trichlorobisphenol A than for 3,3',5,5'-tetrabromobisphenol A. Therefore, it may be concluded that TRs from lower and higher vertebrates and TTRs from lower vertebrates preferentially bind the phenolic compounds with a T3-like structure, while TTRs from higher vertebrates preferentially bind the phenolic compounds with a T4-like structure. This raises the possibility that some EDCs differently affect TTR-mediated plasma TH homeostasis in tadpoles and mammals.
The binding mode of pentabromophenol and tribromophenol provides an example of how the halogenated phenols, belonging to the second group, bind to xTTR. Pentabromophenol and tribromophenol bind to human TTR exclusively in the "reversed mode" with their hydroxyl group oriented toward the mouth of the binding pocket, while T4, which structurally resembles the chemicals belonging to the first group, binds in the "normal mode" with its hydroxyl group oriented toward the center of the binding pocket (Ghosh et al., 2000). Therefore, it is likely that compounds belonging to the first group with a double-ring structure and compounds belonging to the second group with a single-ring structure bind to xTTR in the normal and reverse mode, respectively. These structure–activity relationships may provide clues for deducing the thyroid-disrupting activity of other phenolic and phenol compounds.
Our in vitro and in vivo studies strongly suggest that halogenated phenolic and phenol compounds, such as 3,3'5-trichlorobisphenol A and 2,4,6-triiodophenol at micromolar concentrations, can directly affect T3 binding to xTTR and xTR and exert their T3 antagonist activity in vivo (Table 1), and that o-t-butylphenol and 2-isopropylphenol cause the inhibition of T3-dependent pathways by a molecular mechanism distinct from that for the above halogenated compounds. This is because o-t-butylphenol and 2-isopropylphenol did not compete with 125I-T3 binding to xTR even at 31 μM, but exhibited TH antagonist activity at 6–10 μM in the metamorphosis assay (Table 1). Therefore, further investigation into which cellular process other than the competitive interaction with T3 binding to xTR is targeted by o-t-butylphenol and 2-isopropylphenol will be necessary.
During the in vivo experiments, the concentrations of T3 and each EDC that remained in the FETAX buffer, where tadpoles were reared, were quantified, by adding small amount of 125I-T3 into the buffer at the start of the experiments followed by counting its radioactivity in a gamma counter and directly by high-performance liquid chromatography, respectively. The concentrations of T3, o-t-butylphenol, and 2-isoprophylphenol in the FETAX buffer maintained more than 2/3 of the initial concentrations at 48 h after starting the experiments or exchanging a new buffer, whereas those of 3,3',5-trichlorobisphenol A and 2,4,6-triiodophenol decreased quickly with a half-life of 4–8 h, although the concentrations of all chemicals maintained almost the initial levels when the FETAX buffer containing the chemicals was kept in the absence of the tadpoles for 48 h (data not shown). It is likely that the thyroid-disrupting activity of 3,3',5-trichlorobisphenol A and 2,4,6-triiodophenol was underestimated for our in vivo studies. Exchange of the breeding buffer for the fresh one at least on every several hours or usage of several times volume of the breeding buffer would be necessary to keep the initial concentrations of the chemicals in the breeding buffer. The precise mechanism by which the concentrations of 3,3',5-trichlorobisphenol A and 2,4,6-triiodophenol decreased quickly is under investigation.
Recently, it was reported that bisphenol A interfered with the thyroid system by recruiting the nuclear corepressor N-CoR to human TR (Moriyama et al., 2002), and that hydroxylated polychlorinated biphenyls (OH-PCBs) caused the partial dissociation of TR/retinoidxreceptor heterodimer complex from TRE (Miyazaki et al., 2004). The effective concentrations, 0.1–1.0 μM for bisphenol A and 0.1 pM for OH-PCBs, were 2–8 orders of magnitude lower than the IC50 values of human TR, 200 μM for bisphenol A (Moriyama et al., 2002) and 10–100 μM for OH-PCBs (Cheek et al., 1999b), indicating that these compounds affect TR activation without displacing T3, which is in agreement with a recent report (Gauger et al., 2004). This situation was similar to the results for o-t-butylphenol and 2-isopropylphenol obtained in our studies. Furthermore, chlorinated derivatives of bisphenol A have a molecular structure that resembles those of bisphenol A and OH-PCBs. Therefore, it is possible that phenolic and phenol compounds exert T3 antagonist activity by a mechanism similar to that of bisphenol A or hydroxylated PCBs. Other molecular mechanisms by which the environmental chemicals interfere with the intracellular thyroid signaling pathway have been proposed and include TH metabolizing enzymes (Brouwer et al., 1998; Cheek et al., 1999a; Crump et al., 2002), a crosstalk between TH and other hormonal pathways (Crump et al., 2002), and other nuclear regulatory proteins (Crump et al., 2002). Further studies will be necessary to address the precise molecular mechanisms by which phenols and phenolic compounds affect the thyroid signaling pathway.
Bisphenol A is an essential component of epoxy resin. Nonylphenol is an industrial additive used in a wide variety of detergents and plastics. Simple phenols, such as butylphenols and isopropylphenols, have been used in the manufacture of surface-active agents, plasticizers, and phenolic resins, and in many industrial products including oil additives, oil demulsifiers, and antioxidants. A dozen simple phenols are widely distributed in the water environment, such as river water, lake water, groundwater, and seawater (JEA, 1999). Bisphenol A is easily chlorinated by sodium hypochlorite (Fukazawa et al., 2002), which is used as a bleaching agent in paper recycling plants and as a disinfection agent in sewage treatment plants. In effluents from paper manufacturing plants in Japan, the maximum concentration for the chlorinated derivatives was 7.6 nM and for bisphenol A was 1.6 μM (Fukazawa et al., 2001). The concentration of 2,4,6-triiodophenol in the environment has been unknown, although iodinated phenols can be easily generated from phenol in iodide water in the presence of chlorine under the experimental conditions (Patnaik and Khoury, 2003). Considering the IC50 values of xTTR for bisphenol A and of 3,3',5-trichlorobisphenol A, 2.1 μM and 13 nM, respectively, and the effective concentration of bisphenol A required to recruit the nuclear corepressor N-CoR to human TR as reported by Moriyama et al. (2002), 0.1–1.0 μM, their interference with the X. laevis thyroid system could be possible at the concentrations reported in specific environments, although we cannot elucidate how strong an impact these chemicals have on TTR-mediated TH homeostasis from our study.
ACKNOWLEDGMENTS
We are grateful to Dr. Y. Terao, University of Shizuoka, Japan, for kindly providing the chlorinated derivatives of bisphenol A and of nonylphenol, and to Dr. D. D. Brown, Carnegie Institute of Washington, MD, for kindly providing the xTR cDNA.
This work was supported by Grant-in Aid for Scientific Research on Priority Area (A) (No. 13027236, No. 14042223) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grant-in Aid for Scientific Research (B) (No. 13559001, No. 16244120) from Japan Society for the Promotion of Science.
The authors declare they have no competing financial interests.
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ABSTRACT
We investigated the effects of phenolic and phenol compounds on 3,3',5-L-125I-triiodothyronine (125I-T3) binding to purified Xenopus laevis transthyretin (xTTR) and to the ligand-binding domain of X. laevis thyroid hormone receptor (xTR LBD), on T3-induced metamorphosis in X. laevis tadpoles and on the induction of T3-dependent reporter gene in a X. laevis cell line. Of the halogenated phenolic and phenol compounds tested, 3,3',5-trichlorobisphenol A and 2,4,6-triiodophenol, respectively, were the most potent competitors of 125I-T3 binding to both xTTR and xTR LBD. Most of the halogenated compounds had stronger interactions with xTTR than with xTR LBD. Generally, chlorinated derivatives with a greater degree of chlorination were more efficient competitors of T3 binding to xTTR and xTR LBD. Structures with a halogen in either ortho position or in both ortho positions, with respect to the hydroxy group, were more efficient competitors. 3,3',5-Trichlorobisphenol A and 2,4,6-triiodophenol acted as T3 antagonists in the X. laevis tadpole metamorphosis assay. Interestingly, o-t-butylphenol and 2-isopropylphenol, for which xTTR and xTR LBD had weak or no significant affinity, showed T3 antagonist activity in the metamorphosis assay. T3 antagonist activities of all these chemicals except for o-t-butylphenol were verified by T3-dependent reporter gene assay. Our results suggest that some phenolic and phenol compounds target the process of T3 binding to xTTR and xTR and/or an unknown process, and that they interfere with the intracellular T3 signaling pathway.
Key Words: thyroid hormone; transthyretin; thyroid hormone receptor; halogenated phenolic compounds; thyroid disrupting chemicals; metamorphosis; Xenopus laevis.
INTRODUCTION
An increasing number of chemicals released in the environment, although not apparently toxic, may interfere with critical endocrine systems and developmental processes in a wide range of vertebrates (Bevan et al., 2003; Sonnenschein and Soto, 1998). These endocrine-disrupting chemicals (EDCs) include pharmaceuticals, pesticides, herbicides, and industrial chemicals (Brucker-Davis, 1998; Cheek et al., 1998; Danzo, 1997; Sonnenschein and Soto, 1998). The majority of research concerning EDCs has focused on steroidgenic and antisteroidgenic effects and steroidgenesis in wildlife, experimental animals, and humans. Relatively less research has focused on their effects on the thyroid system, despite reports of abnormally structured thyroid glands and irregular levels of thyroid hormones (THs) in experimental animals (Brouwer et al., 1998; Collins and Capen, 1980; Collins et al., 1977) and wildlife (Moccia et al., 1986; Rolland, 2000; Verreault et al., 2004) exposed to EDCs.
THs are important hormones of brain development, intelligence, and behavior in higher vertebrates (Porterfield, 1994; Zoeller et al., 2002) and postembryonic development in lower vertebrates (Dickhoff et al., 1990). However, there have been few reports concerning the molecular mechanisms of thyroid system disruption by EDCs. Of the EDCs, a number of organohalogen compounds are known to interfere with the thyroid system (Brucker-Davis, 1998). Possible sites that the organohalogens target in the thyroid system include the thyroid gland (Collins et al., 1977), the plasma TH binding protein, transthyretin (TTR) (Brouwer et al., 1998), and TH metabolism (Byrne et al., 1987). The disruption of the thyroid system by EDCs is characterized by the fact that there are few chemicals that competitively interact with 3,3',5-L-triiodothyronine (T3) binding to TH receptor (TR) (Cheek et al., 1999b; Ishihara et al., 2003b). This is in contrast to the steroid system (Matthews et al., 2000), suggesting that thyroid system processes other than T3 binding to TR are targeted by EDCs, although this assumption is mainly based on in vitro studies (Cheek et al., 1999b; Gauger et al., 2004; Ishihara et al., 2003a,b; Yamauchi et al., 2003). Recently, chlorinated derivatives of bisphenol A were detected at the concentrations of 10–8 M in wastewater from paper recycling plants in Japan (Fukazawa et al., 2001). Of the chlorinated derivatives, trichlorobisphenol A and tetrachlorobisphenol A, structurally resemble T3 and L-thyroxine (T4), respectively. Therefore, it is likely that they interfere with the thyroid system.
To determine which thyroid system process is targeted by EDCs in vitro, the experimental animal model Xenopus laevis may provide the most insight. As X. laevis tadpoles are water-living animals (all of its life stages occur in water) and have thin and permeable body skin, they may be particularly sensitive to a number of EDCs present in water. X. laevis is widely used as a laboratory animal and its development and gene expression are well characterized. For these reasons, X. laevis has been approved as an experimental model for evaluating the effects of EDCs in amphibians by the Organization for Economic Cooperation and Development (OECD). As amphibian metamorphosis is obligatorily controlled by THs, amphibian tadpoles are a good model animal for understanding the molecular mechanism by which EDCs disrupt the thyroid system.
In the present study, we investigated the effects of phenolic and phenol compounds, including chlorinated and iodinated compounds, on the X. laevis thyroid system in vitro—competitive interactions of the chemicals with 125I-T3 binding to X. laevis TTR (xTTR) and the ligand-binding domain of X.laevis TR (xTR LBD)—and in vivo—amphibian metamorphosis and T3-dependent gene reporter assays. The potent competitors detected in the in vitro study exhibited a T3-antagonist activity in the in vivo assay; however, some chemicals with almost no interaction with TR in the in vitro study also exhibited a T3-antagonist activity in the in vivo study.
MATERIALS AND METHODS
Reagents. 125I-T3 (122 MBq/μg; carrier free) was purchased from NEN Life Science Products (Boston, MA). Unlabeled T3 (>98% purity) and T4 (>98% purity) were obtained from Sigma (St. Louis, MI). Bisphenol A (>98% purity), 2,4,6-triiodophenol (>98% purity) and o-t-butylphenol (>95% purity) were purchased from Wako Pure Chemical Industries (Osaka, Japan) and 4-nonylphenol (>97% purity) was from Kanto Chemicals (Tokyo, Japan). 2-Isopropylphenol (>98% purity) was obtained from Lancaster (Morecambe, England). AG 1-X8 resin was from BioRad (Hercules, CA). The chlorinated derivatives (>98% purity) of bisphenol A and nonylphenol were synthesized as described previously (Fukazawa et al., 2001) and kindly provided by Dr. Y. Terao. All other chemicals used in this study were either chromatography grade or the highest grade available and were purchased from Wako Pure Chemical Industries or Nacalai Tesque (Kyoto, Japan).
All chemicals tested as EDCs were dissolved in dimethylsulfoxide to a concentration of 10 mM. These chemicals were diluted with an appropriate buffer to give less than 0.4% (v/v) solvent. A control assay without the test chemicals was performed in the presence of the solvent alone and at less than 0.4% (v/v). The solvent did not affect the competitive 125I-T3 binding assays, the metamorphosis assay nor the gene reporter assay described below.
Preparation of recombinant xTTR and xTR LBD. Recombinant xTTR and glutathione-S-transferease (GST) fused xTR LBD were expressed in Escherichia coli BL21 and purified from the bacterial extracts by affinity column chromatography, human retinol-binding protein coupled to Sepharose 4B (Larsson et al., 1985), and glutathione coupled to Sepharose 4B (Amersham Pharmacia Biotech), respectively, as described previously (Yamauchi et al., 2002). They were stored in 10% glycerol at –85°C for later use. Protein concentration was determined by the dye-binding method using bovine -globulin as the standard (Bradford, 1976).
125I-T3 binding assays using TTR (TTR assay) and the TR LBD (TR assay). xTTR (300 ng/tube) was incubated with 0.1 nM 125I-T3 in 250 μl of 20 mM Tris-HCl, pH 7.5, 93 mM NaCl, and 1 mM CaCl2 in the presence or absence of 5 μM unlabeled T3 for 1.0 h at 4°C. GST-xTR LBD fusion protein (54 ng/tube) was incubated with 0.1 nM 125I-T3 in 250 μl of 10 mM Tris-HCl, pH 7.5, 1.5 mM EDTA, 1 mM dithiothreitol and 10% (v/v) glycerol in the presence or absence of 1 μM unlabeled T3 for 1.5 h at 4°C. Competitive 125I-T3 binding was performed with solvent only or increasing concentrations of the unlabeled test chemical, as described previously (Yamauchi et al., 2000). For the TTR assay, protein-bound 125I-T3 was separated from free 125I-T3 by the polyethylene glycol method (Yamauchi et al., 1993). For the TR assay, the Dowex method (Lennon, 1992; Lennon et al., 1980) separated bound 125I-T3 from free 125I-T3. Radioactivity was measured in a gamma counter (Auto Well Gamma System ARC-2000, Aloka, Japan). The amount of 125I-T3 bound nonspecifically was derived from the radioactivity of samples incubated with excess unlabeled T3. The nonspecific binding value was subtracted from the amount of total bound 125I-T3 to give the value of specifically bound 125I-T3.
Metamorphosis assay. X. laevis tadpoles were purchased from Akita Xenopus Co. (Ibaraki, Japan). Tadpoles were staged according to Nieuwkoop and Faber; NF (1975). They were maintained under natural lighting conditions in a 20-l glass aquarium containing dechlorinated tap water and fed dried food commercially available for the fish Medaka (Kyorin Co., Himeji, Japan) once a week. Before starting experiments, test animals were acclimatized to laboratory conditions (25–26°C) for 24 h. During the acclimatization and exposure periods, tadpoles were not fed. Five tadpoles in NF stages 52–53 were transferred into a 1-l glass beaker containing 0.5 l of FETAX buffer (625 mg NaCl, 96 mg NaHCO3, 30 mg KCl, 15 mg CaCl2, 60 mg CaSO4.2H2O, and 75 mg MgSO4.7H2O per l distilled water, pH 7.7) (Dumont et al., 1983). The FETAX buffer contained dimethylsulfoxide, T3 in dimethylsulfoxide, or T3 and each chemical in dimethylsulfoxide. Chemical applications were renewed every other day by changing the above FETAX buffer. Final dimethylsulfoxide concentrations were less than 0.02% in the chemical-exposed and control groups. Fresh NANOpure ultrapure water (Barnstead International, Dubuque, IA) was used for preparing the FETAX buffer. During the experiments, tadpoles were anesthetized in 0.02% 3-aminobenzoic acid ethyl ester (Sigma) and photographed under a stereomicroscope (type SZ-PT, Olympus, Japan) to measure body length, tail length, interocular distance, forelimb length, and hindlimb length. After 5 or 7 days, all living tadpoles were frozen in liquid nitrogen and then stored at –80°C until RNA preparation. Each experiment was repeated at least three times using tadpoles derived from different sets of adults.
Real-time polymerase chain reaction. Total RNA was extracted from the frozen tadpoles using the LiCl-urea procedure (Auffray and Rougeon, 1980). RNA (5–10 μg per lane) was electrophoresed in a 1% agarose gel containing 2.6 M formaldehyde. After visualizing 28S and 18S rRNAs by ethidium bromide staining to check the integrity of the RNA samples and equal loading, amounts of specific RNA species were estimated by real-time polymerase chain reaction (PCR) using SYBR Green Master Mix and ABI Prism 7000 (Applied Biosystems, Foster City, CA) after the RNA samples were treated with reverse transcriptase (TaqMan Reverese Transcription Reagents, Applied Biosystems). Each PCR was run in duplicate to control for PCR variation. The thermocycler program included a step of denaturation at 95°C (10 min), and 40 cycles of 95°C (15 sec), 60°C (1 min), and 50°C (2 min). The endpoint used in real-time PCR quantification, Ct, was defined as the PCR cycle number that crosses an arbitrarily placed signal threshold and is a function of the amount of target DNA present in the starting material. Quantification was determined by applying the 2–Ct formula and calculating the average of the two values obtained for each sample. Eligibility of this formula was verified using a mixture of X. laevis cDNAs containing the xTR cDNA at three different concentrations (1:50:250). To standardize each experiment, the amount of xTR transcript was divided by the amount of glyceraldehyde dehydrogenase (GAPDH) RNA in the same samples. Primer sequences used were as follows: xTR transcript (accession number: M35356 and M35357) sense 5'-CAAGCACCAAGAACGAAAACC-3' (nucleotide numbers 15–35) and antisense 5'-TTGGAAGGTCTGCTCATTCTTCTA-3' (39–16), and X. laevis GAPDH transcript (accession number: V41753) sense 5'-CTCATGACAACAGTCCATGCTTTC-3' (558–581) and antisense 5'-CTCTGCCATCTCTCCACAGCTT-3' (639–618).
Luciferease assay. Sense and antisense oligonucleotides containing the thyroid hormone response elements (TREs), TH/bZIP TRE1 + TRE2 (–99 to –63) (Furlow and Brown, 1999), were annealed and introduced into the unique BglII/SacI site in the pGL2 promoter vector (Promega, Madison, WI) making the reporter plasmid pGL2-TRE. xTR cDNA (kindly provided by Dr. D. D. Brown) was cloned into the EcoRI site of pcDNA3 (Invitrogen, Carlsbad, CA) making the xTR expression plasmid pcDNA3-xTR. Plasmids for transfection were purified using QIAGEN (Chatsworth, CA) miniprep kits. For transient transfection assays, X. laevis XL58 cells (0.6 x 105 cells) were seeded in 24-well culture plates (Nunc, Roskilde, Denmark) and cultured in 70% Leibovitz's L-15 medium (Sigma) containing 10% resin-stripped fetal bovine serum (Samuels et al., 1979) for 15 h at 25°C with air. The next day, the cells were transfected with 500 ng pGL2-TRE, 50 ng pcDNA3-xTR, and 20 ng pRL-CMV vector (Promega) with 4 μl of the lipofection reagent DOSPER (Roche, Mannheim, Germany). After 6 h, the cells were replenished with 70% Leibovitz's L-15 medium containing 10% resin-stripped fetal bovine serum and further cultured with 2 nM T3, without T3, or with 2 nM T3 and each test chemical at defined concentrations for 24 h at 25°C. The culture media were not changed during this treatment. The cells from each well were harvested and assayed for firefly Photinus pyralis and sea pansy Renilla reniformis luciferase activities, derived from pGL2-TER and pRL-CMV, respectively, by using Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's directions. Transfection efficiency was normalized using a constant amount of the sea pansy luciferase activity.
Statistical analysis. The data are presented as mean ± SEM. Differences between groups were analyzed with either Student's t-test or Cochran-Cox test to evaluate the significance of the differences; p < 0.05 was considered statistically significant.
RESULTS
Effects of Halogenated Phenolic and Phenol Compounds on 125I-T3 binding to xTTR
To determine which halogenated phenolic and phenol compounds were strong competitors, 125I-T3 binding to xTTR was examined at 4°C in the presence of various concentrations of the phenolic compounds with two phenolic rings: bisphenol A and its chlorinated derivatives (Fig. 1A), and of the phenol compounds: nonylphenol and its chlorinated derivatives, 2,4,6-triiodophenol, 2-isopropylphenol, and o-t-butylphenol (Fig. 1B). Of bisphenol A and its chlorinated derivatives, 3,3',5-trichlorobisphenol A was the most powerful competitor, with a 50% inhibitory concentration (IC50) of 13 ± 3 nM. The rank order binding affinity was T3 (IC50 = 5.6 ± 1.3 nM) > 3,3',5-trichlorobisphenol A (13 ± 3 nM) > 3,3',5,5'-tetrachlorobisphenol A (32 ± 2 nM) > 3,3'-dichlorobisphenol A (54 ± 10 nM) > 3,5-dichlorobisphenol A (240 ± 50 nM) > 3-chlorobisphenol A (320 ± 10 nM) > bisphenol A (2100 ± 200 nM). The relative affinity of xTTR for 3,3',5-trichlorobisphenol A was 160 times greater than that for bisphenol A but only two times less than that for T3. Of the phenol compounds tested, 2,4,6-triiodophenol was the most potent competitor, with an IC50 of 54 ± 21 nM, and the rank order binding affinity within this group was T3 > 2,4,6-triiodophenol > 2,6-dichloro-4-nonylphenol (240 ± 20 nM) > 2-chloro-4-nonylphenol (5700 ± 400 nM) > 4-nonylphenol (5800 ± 600 nM) > o-t-butylphenol (11000 ± 1000 nM) > 2-isopropylphenol (15000 ± 2000 nM). The relative affinity of xTTR for 2,4,6-triiodophenol was ten times less than that for T3. The relative affinity of xTTR for 2,6-dichloro-4-nonylphenol was 24 times greater than that for 4-nonylphenol but still 43 times less than that for T3.
Effects of Halogenated Phenolic and Phenol Compounds on 125I-T3 Binding to xTR LBD
All compounds tested were weak inhibitors of 125I-T3 binding to xTR (Figs. 2A and 2B) compared with their effect on 125I-T3 binding to xTTR. Of these compounds, 3,3',5-trichlorobisphenol A was the most powerful competitor, with an IC50 of 760 ± 100 nM. The rank order binding affinity for the phenolic compounds was T3 (IC50 = 0.66 ± 0.04 nM) > T4 (3.5 ± 0.6 nM) >>> 3,3',5-trichlorobisphenol A (760 ± 100 nM) > 3,3',5,5'-tetrachlorobisphenol A (1300 ± 100 nM) > 3,3'-dichlorobisphenol A (2300 ± 100 nM) > 3,5-dichlorobisphenol A (4900 ± 400 nM) > 3-chlorobisphenol A (6900 ± 400 nM) > bisphenol A (24000 ± 3000 nM) (Fig. 2A); and for the phenol compounds 2,4,6-triiodophenol (1400 ± 200 nM) > 2,6-dichloro-4-nonylphenol (3400 ± 600 nM) > 2-chloro-4-nonylphenol (7900 ± 1000 nM) > 4-nonylphenol (16000 ± 100 nM) (Fig. 2B). The relative affinity of xTR for 3,3',5-trichlorobisphenol A was 32 times greater than that for bisphenol A but only 103 less than that for T3. xTR did not show significant affinity for o-t-butylphenol and 2-isopropylphenol. The rank order binding affinities for xTR were similar to those for xTTR.
Effects of Phenolic and Phenol Compounds on X. laevis Tadpole Metamorphosis
To examine whether the potent chemicals selected from the above in vitro assays affect the thyroid system in the in vivo assay, X. laevis tadpoles in NF stages 52–53 were immersed in FETAX buffer containing 2 nM T3 with or without each chemical and were cultured for 5–7 days at 25–26°C. At the end of the experiments, tadpoles treated with neither T3 nor each chemical were in NF stages 52–53, whereas tadpoles treated with T3 alone and with both T3 and each chemical developed to NF stages 56–57 and 55–56, respectively. However, it was difficult to evaluate quantitatively the effect of each chemical on tadpole metamorphosis from the progress of the NF stage. Therefore, as end points for assessing T3 antagonist activity of the potent chemicals on T3-induced metamorphosis, the following five morphological characteristics of X. laevis tadpoles were monitored during the culture: body length, tail length, interocular distance, forelimb length, and hindlimb length. Significant morphological differences between positive (+T3) and negative (–T3) control tadpoles were found in the interocular distance alone on the fourth day and in the other three morphological characteristics except for body length on the fifth day or later (data not shown). In Figure 3, the ordinate shows the ratio of interocular distance to body length (to compensate for the body size difference of each tadpole). A significant antagonistic effect of 3,3',5-trichlorobisphenol A on T3 was detected using this ratio on the fourth day (Fig. 3A). This ratio had a variation smaller than the ratios of the other three characteristics to body length. It was concluded that the interocular distance was the most sensitive end point available for determining the T3 antagonist activity of EDCs in X. laevis tadpoles of the four morphological end points tested.
T3-induced metamorphosis was completely inhibited by 2 μM 3,3',5-trichlorobisphenol A and partially inhibited by1.2 μM 2,4,6-triiodophenol (Figs. 3A and 3B), both of which were potent in the in vitro assays. Interestingly, 6 μM o-t-butylphenol and 10 μM 2-isopropylphenol, which were moderately active in the TTR assay (Fig. 1B) but scarcely active in the TR assay (Fig. 2B), also exerted T3 antagonist activity in the X. laevis metamorphosis assay on the fourth day (Fig. 3B).
To confirm whether the EDCs' effects on T3-induced X. laevis metamorphosis were due to their interference with the T3-signaling pathway, we examined the amount of TR transcript in whole tadpoles. The tr gene is a well-known, early primary T3-response gene in metamorphosing X. laevis tadpoles (Wang and Brown 1993). In X. laevis tadpoles treated with T3 (2 nM), the amount of TR transcript increased 30–35 times on the fifth day after treatment (Figs. 4A and 4B). The amount of TR transcript on the seventh day after treatment was the same as that on the fifth day (data not shown). Cotreatment of T3 with 3,3',5-trichlorobisphenol A (2 μM), 2,4,6-triiodophenol(1.2 μM), o-t-butylphenol (6 μM) or 2-isopropylphenol (10 μM) significantly inhibited the T3-induced increase in the amount of TR transcript at the fifth or seventh day of treatment. The inhibition percentages obtained from independent repeated experiments were 39 ± 5% 3,3',5-trichlorobisphenol A (n = 5), 44 ± 7% 2,4,6-triiodophenol (n = 3), 30 ± 8% o-t-butylphenol (n = 4), and 46 ± 10% 2-isopropylphenol (n = 5). These results indicated that the four chemicals tested interfered with the T3-signaling pathway, causing the inhibition of T3-induced metamorphosis.
Effects of Phenolic and Phenol Compounds on T3-Dependent Reporter Gene Assay in X. laevis Cell Line
Finally, we investigated the effect of EDCs on a T3-dependent reporter gene assay in the X. laevis cell line, XL58 (Fig. 5). In the absence of the compounds, T3 (2 nM) increased luciferase activity by 5–6 times. Cotreatment of T3 with 3,3',5-trichlorobisphenol A (2 μM), 2,4,6-triiodophenol (1.2 μM), and 2-isopropylphenol (10 μM) significantly inhibited the T3-induced increase in luciferase activity. Cotreatment of T3 with o-t-butylphenol (6 μM) decreased T3-dependent luciferase activity, but the decrease was not significant. The inhibition percentages obtained from independent repeated experiments were 47 ± 12% 3,3',5-trichlorobisphenol A (n = 3), 40 ± 8% 2,4,6-triiodophenol (n = 3), 56 ± 5% 2-isopropylphenol (n = 4), and 74 ± 6% o-t-butylphenol (n = 3).
DISCUSSION
The present study demonstrated that 3,3',5-trichlorobisphenol A and 2,4,6-triiodophenol exhibited T3 antagonist activity at micromolar or less concentrations in all bioassays tested in X. laevis, and that o-t-butylphenol and 2-isopropylphenol exhibited T3 antagonist activity at 6–10 μM in the in vitro TTR assay and the in vivo metamorphosis assays, but not in the in vitro TR assay; concentrations up to 31 μM were tested. As o-t-butylphenol had so weak effect on the T3-dependent reporter gene assay, its T3 antagonist activity was considered not significant in our experimental conditions. These results indicate that the combination of several assays, which assess different TH system processes, is useful when evaluating the thyroid disrupting activity of EDCs, due to different chemicals targeting different processes.
The rank order of binding affinities in the in vitro TTR and TR assays gave similar results, although the TTR assay was more sensitive to the chemicals tested than the TR assay. Both xTTR and xTR had higher affinities for the chlorinated derivatives of bisphenol A and of nonylphenol than for their parent molecules. The relative potency of the chlorinated derivatives was generally dependent upon the degree of chlorination. xTTR and xTR have a binding preference for the two groups of chemicals. The first group contains chlorinated phenolic compounds that have two phenolic rings with chlorines in either ortho position or in both ortho positions with respect to the hydroxy group, such as 3,3',5-trichlorobisphenol A and 3,3',5,5'-tetrachlorobisphenol A. The second group contains chlorinated or brominated phenols that have a single phenol ring with halogens in both ortho positions with respect to the hydroxy group, such as 2,6-dichloro-4-nonylphenol and 2,4,6-triiodophenol. The competitive binding characteristics of these chemicals were similar to previous studies, which analyzed the interactions of the same chlorinated phenolic and phenol compounds with TTRs and TRs from different species (Yamauchi et al., 2003) and different halogenated phenolic and phenol compounds with human TTR (Meerts et al., 2000; van den Berg, 1990) and rat TR (Kitamura et al., 2002).
The binding preference of xTTR and xTR for the chlorinated phenolic compounds, belonging to the first group, may reflect their TH binding properties and the chemical's structural resemblance to THs. Mammalian TTRs have 4–8 times higher affinity for T4 than for T3 (Chang et al., 1999; Robbins, 1996) while xTTR has 102 times higher affinity for T3 than for T4 (Yamauchi et al., 2002). Interestingly, hTTR has 18 times higher affinity for 3,3',5,5'-tetrabromobisphenol A than for 3,3',5-tribromobisphenol A (Meerts et al., 2000), although xTTR has 2.5 times higher affinity for 3,3',5-trichlorobisphenol A than for 3,3',5,5'-tetrabromobisphenol A. Therefore, it may be concluded that TRs from lower and higher vertebrates and TTRs from lower vertebrates preferentially bind the phenolic compounds with a T3-like structure, while TTRs from higher vertebrates preferentially bind the phenolic compounds with a T4-like structure. This raises the possibility that some EDCs differently affect TTR-mediated plasma TH homeostasis in tadpoles and mammals.
The binding mode of pentabromophenol and tribromophenol provides an example of how the halogenated phenols, belonging to the second group, bind to xTTR. Pentabromophenol and tribromophenol bind to human TTR exclusively in the "reversed mode" with their hydroxyl group oriented toward the mouth of the binding pocket, while T4, which structurally resembles the chemicals belonging to the first group, binds in the "normal mode" with its hydroxyl group oriented toward the center of the binding pocket (Ghosh et al., 2000). Therefore, it is likely that compounds belonging to the first group with a double-ring structure and compounds belonging to the second group with a single-ring structure bind to xTTR in the normal and reverse mode, respectively. These structure–activity relationships may provide clues for deducing the thyroid-disrupting activity of other phenolic and phenol compounds.
Our in vitro and in vivo studies strongly suggest that halogenated phenolic and phenol compounds, such as 3,3'5-trichlorobisphenol A and 2,4,6-triiodophenol at micromolar concentrations, can directly affect T3 binding to xTTR and xTR and exert their T3 antagonist activity in vivo (Table 1), and that o-t-butylphenol and 2-isopropylphenol cause the inhibition of T3-dependent pathways by a molecular mechanism distinct from that for the above halogenated compounds. This is because o-t-butylphenol and 2-isopropylphenol did not compete with 125I-T3 binding to xTR even at 31 μM, but exhibited TH antagonist activity at 6–10 μM in the metamorphosis assay (Table 1). Therefore, further investigation into which cellular process other than the competitive interaction with T3 binding to xTR is targeted by o-t-butylphenol and 2-isopropylphenol will be necessary.
During the in vivo experiments, the concentrations of T3 and each EDC that remained in the FETAX buffer, where tadpoles were reared, were quantified, by adding small amount of 125I-T3 into the buffer at the start of the experiments followed by counting its radioactivity in a gamma counter and directly by high-performance liquid chromatography, respectively. The concentrations of T3, o-t-butylphenol, and 2-isoprophylphenol in the FETAX buffer maintained more than 2/3 of the initial concentrations at 48 h after starting the experiments or exchanging a new buffer, whereas those of 3,3',5-trichlorobisphenol A and 2,4,6-triiodophenol decreased quickly with a half-life of 4–8 h, although the concentrations of all chemicals maintained almost the initial levels when the FETAX buffer containing the chemicals was kept in the absence of the tadpoles for 48 h (data not shown). It is likely that the thyroid-disrupting activity of 3,3',5-trichlorobisphenol A and 2,4,6-triiodophenol was underestimated for our in vivo studies. Exchange of the breeding buffer for the fresh one at least on every several hours or usage of several times volume of the breeding buffer would be necessary to keep the initial concentrations of the chemicals in the breeding buffer. The precise mechanism by which the concentrations of 3,3',5-trichlorobisphenol A and 2,4,6-triiodophenol decreased quickly is under investigation.
Recently, it was reported that bisphenol A interfered with the thyroid system by recruiting the nuclear corepressor N-CoR to human TR (Moriyama et al., 2002), and that hydroxylated polychlorinated biphenyls (OH-PCBs) caused the partial dissociation of TR/retinoidxreceptor heterodimer complex from TRE (Miyazaki et al., 2004). The effective concentrations, 0.1–1.0 μM for bisphenol A and 0.1 pM for OH-PCBs, were 2–8 orders of magnitude lower than the IC50 values of human TR, 200 μM for bisphenol A (Moriyama et al., 2002) and 10–100 μM for OH-PCBs (Cheek et al., 1999b), indicating that these compounds affect TR activation without displacing T3, which is in agreement with a recent report (Gauger et al., 2004). This situation was similar to the results for o-t-butylphenol and 2-isopropylphenol obtained in our studies. Furthermore, chlorinated derivatives of bisphenol A have a molecular structure that resembles those of bisphenol A and OH-PCBs. Therefore, it is possible that phenolic and phenol compounds exert T3 antagonist activity by a mechanism similar to that of bisphenol A or hydroxylated PCBs. Other molecular mechanisms by which the environmental chemicals interfere with the intracellular thyroid signaling pathway have been proposed and include TH metabolizing enzymes (Brouwer et al., 1998; Cheek et al., 1999a; Crump et al., 2002), a crosstalk between TH and other hormonal pathways (Crump et al., 2002), and other nuclear regulatory proteins (Crump et al., 2002). Further studies will be necessary to address the precise molecular mechanisms by which phenols and phenolic compounds affect the thyroid signaling pathway.
Bisphenol A is an essential component of epoxy resin. Nonylphenol is an industrial additive used in a wide variety of detergents and plastics. Simple phenols, such as butylphenols and isopropylphenols, have been used in the manufacture of surface-active agents, plasticizers, and phenolic resins, and in many industrial products including oil additives, oil demulsifiers, and antioxidants. A dozen simple phenols are widely distributed in the water environment, such as river water, lake water, groundwater, and seawater (JEA, 1999). Bisphenol A is easily chlorinated by sodium hypochlorite (Fukazawa et al., 2002), which is used as a bleaching agent in paper recycling plants and as a disinfection agent in sewage treatment plants. In effluents from paper manufacturing plants in Japan, the maximum concentration for the chlorinated derivatives was 7.6 nM and for bisphenol A was 1.6 μM (Fukazawa et al., 2001). The concentration of 2,4,6-triiodophenol in the environment has been unknown, although iodinated phenols can be easily generated from phenol in iodide water in the presence of chlorine under the experimental conditions (Patnaik and Khoury, 2003). Considering the IC50 values of xTTR for bisphenol A and of 3,3',5-trichlorobisphenol A, 2.1 μM and 13 nM, respectively, and the effective concentration of bisphenol A required to recruit the nuclear corepressor N-CoR to human TR as reported by Moriyama et al. (2002), 0.1–1.0 μM, their interference with the X. laevis thyroid system could be possible at the concentrations reported in specific environments, although we cannot elucidate how strong an impact these chemicals have on TTR-mediated TH homeostasis from our study.
ACKNOWLEDGMENTS
We are grateful to Dr. Y. Terao, University of Shizuoka, Japan, for kindly providing the chlorinated derivatives of bisphenol A and of nonylphenol, and to Dr. D. D. Brown, Carnegie Institute of Washington, MD, for kindly providing the xTR cDNA.
This work was supported by Grant-in Aid for Scientific Research on Priority Area (A) (No. 13027236, No. 14042223) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grant-in Aid for Scientific Research (B) (No. 13559001, No. 16244120) from Japan Society for the Promotion of Science.
The authors declare they have no competing financial interests.
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