Detection of Thyroid System–Disrupting Chemicals Using in Vitro and in
http://www.100md.com
《毒物学科学杂志》
Department of Biology, Faculty of Science, Shizuoka University, Shizuoka 422-8529, Japan
BioResource Center, RIKEN Tsukuba Institute, Tsukuba 305-0074, Japan
ABSTRACT
We developed a thyroid hormone (TH) inducible primary screening assay for the identification and assessment of man-made chemicals that interfere with the TH-signalling pathway within target cells. The assay was developed in a Xenopus laevis cell line that was transduced with a self-inactivating (SIN) lentivirus vector (LV) containing a luciferase gene. The luciferase activation in this cell line was TH-specific: 3,3',5-L-triiodothyronine (T3) > 3,3'5-L-triiodothyroacetic acid (Triac) > 3,3',5-D-triiodothyronine (D-T3), > L-thyroxine (T4) > 3,3',5'-L-triiodothyronine (rT3). The application of the ligand-dependent luciferase assay for screening for thyroid system-disrupting chemicals revealed that three phthalates (dicyclohexyl phthalate, n-butylbenzyl phthalate, and di-n-butyl phthalate), two herbicides (ioxynil and pentachlorophenol) and a miticide (dicofol) had 3,3',5-L-triiodothyronine- T3- antagonist activity at concentrations ranging from 10–6 to 10–5 M. These chemicals also inhibited the expression of the endogenous primary T3-response TH nuclear receptor (TR) gene. The inhibitory characteristics of these chemicals were similar for both assays performed, although the assay for T3-dependent activation of TR gene was more sensitive than the luciferase assay. These results indicate that the luciferase assay was a rapid method with a small intra-assay variation for the primary screening of thyroid system-disrupting chemicals. Of the six chemicals, only n-butylbenzyl phthalate and pentachlorophenol exhibited T3-antagonist activity in an in vivo metamorphosis-based assay. It should be noted that chemicals elicited thyroid system-disrupting activity in the luciferase assay did not always interfere with the thyroid system in vivo.
Key Words: endocrine toxicology – endocrine; thyroid, gene expression/regulation – receptor; nuclear hormone, endocrine toxicology – endocrine disruptors.
INTRODUCTION
Over the past several decades amphibian populations worldwide have tended to decrease, the number of malformed frogs has increased, and some species at certain localities have been extirpated (Houlahan et al., 2000; Ouellet et al., 1997). Possible causes for these trends include natural population fluctuation, fungal pathogens, increased ultraviolet radiation, climate change, acid precipitation, loss of suitable habitats by human activity, and/or natural and man-made environmental contaminants (Baustein and Wake, 1995; Burkhart et al., 2000; Wake, 1991). It is not clear what impact each of these possible causes has had upon amphibian populations; however, it is likely that environmental contaminants have had the greatest impact as amphibians are generally considered more sensitive to aquatic contaminants due to the permeability of their skin.
Xenopus laevis is a water-living amphibian (all of its life stages occur in water) that is used widely as a laboratory animal. Its development and the expression of its genes are well characterized. For these reasons, X. laevis has been approved as an experimental model for evaluating the effects of endocrine-disrupting chemicals (EDCs) in amphibians by the Organization for Economic Cooperation and Development. A critical process in the amphibian life cycle is metamorphosis that is obligatorily controlled by thyroid hormones (THs). This process is also influenced by several environmental factors, such as the density of the population, habitat dessication, temperature, and food availability (Denver, 1997).
THs are essential for normal brain development and behavior in higher vertebrates (Howdeshell, 2002; Zoeller et al., 2002) and postembryonic development in lower vertebrates (Dickhoff et al., 1990). However, there have been relatively few reports concerning the molecular mechanisms by which EDCs disrupt the thyroid system despite reports of structural lesions in thyroid follicular cells and irregular levels of THs in experimental animals and wildlife (Brouwer et al., 1998; Colborn, 2002) exposed to EDCs. These results suggest that there may be thyroid system–disrupting chemicals in the environment not yet identified. Our laboratory has been interested in studying the effect of EDCs, especially those that interfere with the amphibian thyroid system in vitro including TH-binding to the major plasma TH-carrier protein, transthyretin (Ishihara et al., 2003; Kudo and Yamauchi, 2005; Yamauchi et al., 2000, 2002, 2003), cellular TH-uptake (Shimada and Yamauchi, 2004) and TH-binding to TH nuclear receptor (TR) (Ishihara et al., 2003; Kudo and Yamauchi, 2005; Shimada and Yamauchi, 2004; Yamauchi et al., 2002, 2003); and in vivo including X. laevis metamorphosis (Kudo and Yamauchi, 2005). Assay methods mechanistically distinct from ours have also been used by others to investigate thyroid system–disrupting chemicals (Crump et al., 2002; DeVito et al., 1999; Veldhoen and Helbing, 2001). Together, these studies revealed that EDCs target differentially several processes within the thyroid system in a chemical-dependent manner. Bioassay methods capable of identifying thyroid system–disrupting chemicals and their effects, as a total sum of each effect on individual processes, within the thyroid system are essential for the primary screening of thyroid system–disrupting chemicals. Furthermore, although there are advantages and limitations in each bioassay, as suggested by Zacharewski (1998), important criteria for primary screening bioassays may be high throughput with low cost.
In the present study, we established a permanent X. laevis recombinant cell line as a bioassay for screening thyroid system-disrupting chemicals using a self-inactivation (SIN) lentivirus vector (LV) containing a TH response element (TRE)-SV40 promoter-luciferase reporter gene. We chose nine suspect EDCs, most of which are known to affect at least one of the following processes: 3,3',5-L-triiodothyronine- (T3-) binding to transthyretins and TRs, and cellular T3-uptake in amphibian systems (Ishihara et al., 2003; Shimada and Yamauchi, 2004; Yamauchi et al., 2002); and investigated their effects on the ligand-dependent luciferase assay, T3-dependent activation of the TR gene using the same cell line and T3-induced tadpole metamorphosis in vivo. Given the advantages of the luciferase assay, high sensitivity, rapidity and high reproducibility, its use as a high throughput primary screening system would provide large-scale assessment of environmental chemicals as thyroid system disruptors.
MATERIALS AND METHODS
Reagents.
T3, (>98% purity), L-thyroxine (T4; >98%), 3,3'5-L-triiodothyroacetic acid (Triac; 95%), 3,3',5-D-triiodothyronine (D-T3; >98%), 3,3',5'-L-triiodothyronine (rT3; 95%), 17-estradiol (E2; >98%), and pentachlorophenol (>99% purity) were obtained from Sigma (St. Louis, MI). Bisphenol A (>98% purity), dicyclohexyl phthalate (>99% purity), dicofol (Kelthane, 2,2,2-trichloro-1,1-bis(4-chlorophenyl)ethanol, analytical standard, >95% purity), and ioxynil (3,5-diiodo-4-hydroxybenzonitril, analytical standard, >99% purity) were purchased from Wako Pure Chemical Industries (Osaka, Japan) and diethyl phthalate (>99% purity) and di(2-ethylhexyl) phthalate (>99% purity) were from Kanto Chemicals (Tokyo, Japan). Di-n-butyl phthalate (>98% purity) and n-butylbenzyl phthalate (>97% purity) were obtained from Nacalai Tesque (Kyoto, Japan). 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, except for E2 which was dissolved in ethanol to a concentration of 1 mM, were dissolved in dimethylsulfoxide to a concentration of 10 mM. These chemicals were diluted with an appropriate buffer to give less than 0.5% (v/v) solvent. A control assay without the test chemicals was performed in the presence of the solvent alone and at less than 0.5% (v/v). The solvent did not affect the luciferase assay, the T3-dependent activation of TR gene, cell viability nor T3-induced tadpole metamorphosis.
Luciferase assay in a permanent Xenopus laevis recombinant cell line.
A human immunodeficiency virus-1-based LV pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G) was prepared by transient transfection of the following plasmids: packaging construct, pMDLg/pRRE; VSV-G- and Rev- expressing construct, pCMV-VSV-G-RSV-Rev; and SIN LV construct, pCS-TRE-Luc-CG, as described elsewhere (Sugiyama et al., in press). This LV contains the firefly luciferase gene under the control of the SV40 promoter and the X. laevis TREs. The structures of the TREs and pCS-TRE-Luc-CG are illustrated in Figure 1. Subconfluent monolayers of X. laevis XL58 cells were transduced with the viral vector at a multiplicity of infection of 10, and incubated in 70% Leibovitz's L-15 medium containing 10% fetal bovine serum (FBS) for 15 h at 25°C with air. A cell clone, XL58-TRE-Luc, with luciferase highly expressed in a T3-dependent manner was isolated (Sugiyama et al., in press).
XL58-TRE-Luc cells were cultured in FBS-free 70% Leibovitz's L-15 medium with or without 2 nM T3 in the presence or absence (control) of each EDC for 24 h, following incubation in 70% Leibovitz's L-15 medium containing 10% resin-stripped FBS (Samuels et al., 1979) for 15 h. Their cell lysate was assayed for firefly luciferase activity using the PicaGene Luminescence kit (Nippon Gene, Tokyo, Japan). Cell viability was examined using the Cell Count Reagent SF kit (Nacalai Tesque), according to the manufacturer's directions, based on the method described by Ishiyama et al. (1997). Protein concentration was measured by the Bradford dye-binding method using bovine -globulin as the standard (Bradford, 1976).
Metamorphosis assay.
Xenopus laevis tadpoles were purchased from Akita Xenopus Co. (Ibaraki, Japan). Tadpoles were staged according to Nieuwkoop and Faber (1975). The metamorphosis assay was performed as described previously (Kudo and Yamauchi, 2005). In brief, five tadpoles in NF stages 51–52 were transferred into a 1 L glass beaker containing 0.5 L of FETAX buffer (625 mg/L NaCl, 96 mg/L NaHCO3, 30 mg/L KCl, 15 mg/L CaCl2, 60 mg/L CaSO4.2H2O and 75 mg/L MgSO4.7H2O, pH 7.7) (Dumont et al., 1983). The FETAX buffer contained dimethylsulfoxide, T3 in dimethylsulfoxide or T3 and each chemical in dimethylsulfoxide. Tadpoles were cultured in the FETAX buffer at 22–24°C. FETAX buffer containing the chemical applications was renewed every other day. Final dimethylsulfoxide concentrations were less than 0.02% in the chemical exposed and control groups. After five days, all living tadpoles were frozen in liquid nitrogen and then stored at –80°C until RNA preparation.
Real-time polymerase chain reaction.
Total RNA was extracted from XL58-TRE-Luc cells using the acid guanidinium thiocyanate-phenol-chloroform extraction procedure (Chomczynski and Sacchi, 1987) and from the frozen tadpoles using the LiCl-urea procedure (Auffray and Rougeon, 1980). To confirm its integrity, RNA (5 μg per lane) was electrophoresed in a 1% agarose gel containing 2.6 M formaldehyde, and 28S and 18S rRNA were visualized by ethidium bromide staining. The quantity of specific RNA species in each sample was 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 had been treated with reverse transcriptase (TaqMan Reverse Transcription Reagents, Applied Biosystems), as previously described (Kudo and Yamauchi, 2005). To standardize each experiment, the amount of TR transcript in each sample was divided by the amount of glyceraldehyde-phosphate dehydrogenase (GAPDH) transcript in the same sample. The primer sequences used were as follows: X. laevis TR transcript (accession number: X17385) sense 5'-CAAGCACCAAGAACGAAAACC-3' (nucleotide numbers 530–555) and antisense 5'-TTGGAAGGTCTGCTCATTCTTCTA-3' (600–580); X. laevis TR 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).
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
Ligand-Specific Luciferase Assay in XL58-TRE-Luc Cells
Figure 2 shows the specificity of the ligand-dependent luciferase assay in this recombinant cell line. Luciferase activity in XL58-TRE-Luc cells was induced 3.1- and 3.3-fold in the presence of 2 and 20 nM T3, respectively. Triac was less potent than T3 at the low concentration but was as potent as T3 at the high concentration. T4 and D-T3 were moderately potent while rT3 was less potent than the above chemicals. E2 elicited no statistically significant effect on the induction of the luciferase activity in our cell culture system. These results indicate that this luciferase gene specifically responds to THs.
Effects of EDCs on the T3-Dependent Luciferase Assay Using XL58-TRE-Luc Cells
To determine which phthalates affect the T3-dependent luciferase activity, XL58-TRE-Luc cells were incubated with or without 2 nM T3, in the presence or absence of various concentrations of each phthalate. In the absence of EDCs and presence of T3, the luciferase activity increased by 3.0-fold. Figure 3 shows dose-effects of five phthalates on the T3-dependent luciferase activity. Of the five phthalates, dicyclohexyl phthalate was the most potent antagonist with a 50% inhibitory concentration (IC50) of 11 ± 3 μM (n = 3). The IC50 rank order of antagonist activity was dicyclohexyl phthalate > n-butylbenzyl phthalate (40 ± 6 μM, n = 3) = di-n-butyl phthalate (39 ± 1 μM, n = 3) > di(2-ethylhexyl) phthalate (>50 μM, n = 3) > diethyl phthalate (>50 μM, n = 3). The effect of ioxynil, pentachlorophenol, dicofol, and bisophenol A on the T3-dependent luciferase assay was also investigated (Fig. 4). Of these chemicals, ioxynil was the most potent antagonist with an IC50 of 0.9 ± 0.3 μM (n = 3) with significant potency even at 0.2 μM. The antagonist activities of pentachlorophenol and dicofol were weaker with less than 50% inhibition at their maximum concentrations, 0.8 and 10 μM, respectively. Bisphenol A failed to elicit statistically significant responses at the concentrations tested (0.001–10 μM). Activation of luciferase activity was not detected when the chemicals were applied in the absence of T3. All of these chemicals did not affect cell viability at the highest concentrations tested under our experimental conditions (data not shown).
Effects of EDCs on the T3-Dependent Activation of TR Gene in XL58-TRE-Luc Cells
To investigate whether EDCs that disrupted the cellular T3-signaling pathway in the T3-dependent luciferase assay also affected other T3-response genes, the transcripts of endogenous primary T3-response TR genes were analyzed by real-time PCR. XL58-TRE-Luc cells were grown with or without 2 nM T3 in the presence or absence of various concentrations of each EDC. The TR transcript was not induced by T3 in XL58-TRE-luc cells under our experimental conditions. In the absence of EDCs and presence of T3, the amount of TR transcript increased by 13.3 ± 0.5 (n = 5) fold. This T3-dependent activation was significantly inhibited by all chemicals tested except for bisphenol A at the indicated or higher concentrations (Fig. 5). The inhibition percentages obtained from independent repeated experiments were 30 ± 7% diethyl phthalate (50 μM, n = 3), 29 ± 5% di(2-ethylhexyl) phthalate (50 μM, n = 3), 32 ± 5% di-n-butyl phthalate (50 μM, n = 3), 45 ± 4% n-butylbenzyl phthalate (50 μM, n = 3), 42 ± 6% dicyclohexyl phthalate (20 μM, n = 3), 45 ± 4% ioxynil (0.1 μM, n = 3), 63 ± 4% pentachlorophenol (0.08 μM, n = 3), and 32 ± 5% dicofol (10 μM, n = 3). n-Buthylbenzyl phthalate, even at the concentration of 4 μM, were still potent, with more than 50% inhibition. Activation of the TR gene was not detected when the chemicals were applied in the absence of T3.
Effects of EDCs on the T3-Dependent Activation of TR Gene in Xenopus laevis Tadpoles
To examine whether the potent chemicals selected from the above in vitro assays affect the thyroid system in vivo, X. laevis tadpoles in NF stages 51–52 tadpoles were immersed in FETAX buffer alone or in FETAX buffer containing 2 nM T3 with or without each chemical, and cultured for five days at 22–24°C. Total RNA was then extracted from tadpoles in each group and the amount of TR transcript was quantified by real-time PCR (Fig. 6). At the end of the experiments, positive control (T3-treated), test (T3 and chemical-treated), and negative control (T3-untreated) tadpoles were in NF stages 54, 53–54, and 51–52, respectively. No tadpoles died during the culture period. The amount of TR transcript increased 12.4 ± 0.7 times (data not shown) in T3-treated tadpoles, which was less than half of the increase reported in the previous in vivo study (Kudo and Yamauchi, 2005). Co-treatment of T3 with n-butylbenzyl phthalate (4 μM) or pentachlorophenol (0.1 μM) significantly inhibited the T3-induced increase in the amount of TR transcript. The inhibition percentages obtained from independent repeated experiments were 48 ± 3% n-butylbenzyl phthalate (n = 3) and 62 ± 6% pentachlorophenol (n = 3). No significant effects were detected for di-n-butyl phthalate (4 μM), ioxynil (0.1 μM), dicofol (0.1 μM), and bisphenol A (5 μM). When tadpoles were treated with di-n-butyl phthalate or dicofol, at concentrations several times higher, some of the test tadpoles died due to toxicity. Therefore, it was not possible to compare their thyroid system-disrupting activity in tadpoles with that in cultured cells at similar concentrations. These results indicated that of the six chemicals showing T3-antagonist activity in the in vitro assays only n-butylbenzyl phthalate and pentachlorophenol exhibited T3-antagonist activity in vivo at micromolar and sub-micromolar concentrations, respectively.
DISCUSSION
We have used a recombinant X. laevis cell line containing a T3-dependent reporter gene, XL58-TRE-Luc, to detect the thyroid system-disrupting activity of several suspect EDCs. This luciferase assay was highly sensitive to T3 with an ED50 of 10–11 M, in the absence of FBS (Sugiyama et al., in press) and was specific to THs: T3 > Triac > D-T3 > T4 > rT3 (Fig. 2). This specificity was in agreement with the binding specificity of amphibian and mammalian TRs (Mukku et al., 1983; Thomas et al., 1992). In the present study, the ligand-dependent luciferase assay revealed the TH-antagonist activities of at least three phthalates, dicyclohexyl phthalate, n-butylbenzyl phthalate, and di-n-butyl phthalate, and three agricultural chemicals, ioxynil, pentachlorophenol, and dicofol. The T3-dependent activation of the TR gene confirmed the T3-antagonist activities of these chemicals in the same cells, and indicated that di(2-ethylhexyl) phthalate and diethyl phthalate were also potent. Of the above chemicals, only n-butylbenzyl phthalate and pentachlorophenol elicited T3-antagonist activity in tadpoles.
Of the six chemicals found to have T3-antagonist activity using the luciferase assay, pentachlorophenol and ioxynil were previously identified as the most potent inhibitors of T3-binding to X. laevis transthyretin (IC50 values of 180–260 nM); however, none of them, at 1 μM, were potent inhibitors of T3-binding to the X. laevis TR gene (Yamauchi et al., 2002). Di(2-ethyhexyl) phthalate and pentachlorophenol are known to decrease the plasma thyroxine level and the plasma T3 and thyroxine levels, respectively, in rat (Brucker-Davis, 1998). These effects are probably due to alterations in thyroidal activity (Hinton et al., 1986) or the control of the hypothalamus/pituitary/thyroid axis (Jekat et al., 1994). These findings suggest that these chemicals target TH system processes, extracellular TH-transport system, TH-synthesis, and/or control system of TH-synthesis, before THs access the peripheral target cells. Together with our previous findings, the present study opened the possibility that some of the six chemicals interfere with a process or several processes other than T3-binding to TR in the T3-signaling pathway within peripheral target cells. This possibility has been proposed for polychlorinated biphenyls (PCBs) or hydroxylated PCBs. These compounds affected TH-dependent extension of Purkinje dendrite in mouse cerebellar culture at 10–12–10–11 M (Kimura-Kuroda et al., 2005) and suppressed T3-induced transcriptional activation of TR at 10–10 M (Iwasaki et al., 2002), although they slightly competed with T3-binding to TR at the concentrations of more than 10–6 M (Cheek et al., 1999b; Gauger et al., 2004). Miyazaki et al. (2004) found that these compounds induced partial dissociation of TR/retinoid X receptor heterodimer complex from the TRE. It remains to be clarified how the three phthalates and the three agricultural chemicals affected T3-dependent transactivation in our culture system.
Using the luciferase assay, the significant effective doses of the three phthalates and the three agricultural chemicals were 10–7–10–5 M (Figs. 2 and 3). Most of these EDCs have been shown to interfere with the estrogen-signaling pathway by competitive binding to estrogen receptors (Jobling et al., 1995; Matthews et al., 2000; Nakai et al., 1999; Zacharewski et al., 1998), the MCF-7 proliferation assay (Harris et al., 1997; Okubo et al., 2003; Soto et al., 1995), yeast and mammalian two-hybrid systems (Nishihara et al., 2000; Zacharewski et al., 1998), and estrogen-dependent reporter gene assays (Harris et al., 1997; Jobling et al., 1995; Zacharewski et al., 1998), with effective concentrations of 10–6–10–3 M. Pentachlorophenol (l0–6 M) exhibited antagonist activity in a yeast progesterone-dependent reporter gene assay (Tran et al., 1996) whereas dicofol (10–6–10–5 M) exerted estrogenic activity in the yeast estrogen-dependent reporter gene assay and the MCF-7 proliferation assay (Vinggaard et al., 1999). The effective concentrations of these EDCs for our luciferase assay and most of the above assays for steroid system disruption were within a similar range. However, our cell culture system specifically responded to THs but not to E2 (Fig. 1). It is likely that these EDCs interfere with several hormonal signalling pathways simultaneously within a cell, and/or that they target a cellular site that affects several hormonal signalling pathways. The rank orders of their steroidgenic or anti-steroidgenic activities showed occasional discrepancies, which could be due to differences in the cellular environment of the cell lines and/or the species used (Matthews et al., 2000; Zacharewski et al., 1998) or differences in the assay methods and/or in the assay conditions used (Harris et al., 1997; Okubo et al., 2003; Zacharewski et al., 1998). These rank orders did not compare with that of their TH-antagonist activities as determined by our luciferase assay. Therefore, it is not possible to conclude whether the chemicals potent in the luciferase assay act on a single cellular site that affects several hormonal signalling pathways.
The inhibitory effect of the EDCs on the T3-dependent activation of the TR gene (Fig. 5) was similar to that determined using the luciferase assay. The fold induction of TR gene expression by T3 and inhibition of its T3-dependent induction by the EDCs indicated that the assay for T3-dependent activation of the TR gene was more sensitive to T3 and the EDCs used than the luciferase assay. Of the five phthalates, dicyclohexyl phthalate (20 μM) had the most potent T3-antagonist activity in the luciferase assay whereas n-butylbenzyl phthalate (4 μM) was the most potent inhibitor of the T3-dependent activation of the TR gene. This discrepancy may be, in part, due to the difference in the structure of the TREs located in the two genes.
N-Butylbenzyl phthalate (4 μM) and pentachlorophenol (0.08–0.1 μM), exhibited T3-antagonist activity in both the in vitro and in vivo assays in X. laevis. Phthalates including n-butylbenzyl phthalate are widespread in food and the environment due to their use as plasticizers in consumer products, food packaging materials and biomedical devices. n-Butylbenzyl phthalate is one of the most abundant man-made chemicals and is used in the production of vinyl floor tiles, adhesives, and synthetics leather. Phthalates can leach out of these materials into water, soil, or food over time, after use. Pentachlorophenol had been used commonly as a fungicidal substance for wood protection and leather impregnation and as a herbicide. It is also present in the gases of municipal waste incinerators. Due to its highly persistent nature, pentachlorophenol is still one of the dominant phenolic compounds in blood (Sandau et al., 2000). However, little is known about their absorption, bioaccumulation, and metabolism, and their chronic effects on humans or wildlife if administered at low concentrations over long time periods. Considering the prevalence of phenolic compounds in the environment, more research should be focused on their thyroid system-disrupting activities as well as steroid system-disrupting activities, and their cellular target sites, i.e., metabolic enzymes that activate or inactivate the chemicals and endogenous hormones, hormone receptors and/or nuclear co-regulators.
Recently, it was reported that bisphenol A (10–5–10–4 M) inhibited T3-induced metamorphosis (Iwamuro et al., 2003). In a mammalian two-hybrid system, bisphenol A (10–5 M) recruited the nuclear corepressor N-CoR to the TR then elicited T3-antagonist activity (Moriyama et al., 2002). However, in the present in vitro and in vivo assays, bisphenol A (10 μM and 5 μM, respectively) exhibited no T3-antagonist activity in X. laevis. Although we can not fully explain this discrepancy, higher concentrations of bisphenol A than we used would be necessary to elicit significant T3-antagonist activity under our experimental conditions.
The luciferase assay using XL58-TRE-Luc cells has several advantages. This cell line can produce enough endogenous TR in response to exogenously added T3. It is easy to grow and work with, and the data derived from the assay had a small intra-assay variation when compared with the transient transcription assay (Sugiyama et al., in press). Although there have been a number of reports on the effects of environmental chemicals on amphibian metamorphosis (Cheek et al., 1999a; Crump et al., 2002; Larson et al., 1998; Veldhoen and Helbing, 2001), most of these assays were not suitable for the primary screening of potential thyroid system-disrupting chemicals because of the time required and the high costs involved. Our luciferase assay would provide high throughput primary screening for chemicals with thyroid system-disrupting activity in the water environment where amphibians habitat. However, it should be noted that the luciferase assay would not provide the potential intracellular sites for thyroid disruption by EDCs, and that the in vitro assays, including the assay for T3-dependent activation of TR gene, and the transthyretin and TR binding assays, can not reproduce the pharmacokinetic and pharmacodynamic interactions and sensitive developmental windows that may occur in vivo. We actually found that different concentrations of EDCs were required to elicit a toxic effect in cultured cells and metamorphosing tadpoles. Of the chemicals that were T3-antagonistic in the luciferase assay and T3-dependent TR gene activation in cultured cells, only n-butylbenzyl phthalate and pentachlorophenol elicited T3-antagonist activity in tadpoles reflecting in part differences in the action of EDCs in the in vitro and in vivo assays, as described above. Despite these limitations, the results from the in vitro assays may indicate potential mechanisms of EDC action. Furthermore, our study indicates the importance of the combination of several assays to assess the thyroid system-disrupting activity of EDCs. The transgene by the LV system can be introduced into organisms (Miyoshi et al., 1998; Zufferey et al., 1998) as well as cell lines (Gatlin et al., 2001; Katayama et al., 2004). Therefore, the same DNA construct can be introduced into eggs, embryos, or some tissues of tadpoles. This application of the transgene would allow, to some extent, the connection of data obtained from the various in vitro assays with data obtained from in vivo metamorphosis assays.
ACKNOWLEDGMENTS
We are grateful to Dr. D. D. Brown, Carnegie Institute of Washington, MD, for kindly providing the X. laevis TR cDNA. We would like to thank Ms. J. Monk for a thorough and critical reading of the manuscript. This work was supported by Grant-in Aid for Scientific Research (B) (No. 13559001, No. 16244120) from Japan Society for the Promotion of Science, and by grants from The Sumitomo Foundation and from Toukai Foundation for Technology. The authors declare they have no competing financial interests.
REFERENCES
Auffray, C., and Rougeon, F. (1980). Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur. J. Biochem. 107, 303–314.
Baustein, A. R., and Wake, D. B. (1995). The puzzle of declining amphibian populations. Sci. Am. 272, 52–57.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
Brouwer, A., Morse, D. C., Lans, M. C., Schuur, A. G., Murk, A. J., Klasson-Wehler, E., and Visser, T. J. (1998). Interactions of persistent environmental organohalogens with the thyroid hormone system: Mechanisms and possible consequences for animal and human health. Toxicol. Ind. Health 14, 59–84.
Brucker-Davis, F. (1998). Effects of environmental synthetic chemicals on thyroid function. Thyroid 8, 827–856.
Burkhart, J. G., Ankley, G., Bell, H., Carpenter, H., Fort, D., Gardiner, D., Gardner, H., Hale, R., Helgen, J. C., Jepson, P., et al. (2000). Strategies for assessing the implications of malformed frogs for environmental health. Environ. Health Perspect. 108, 83–90.
Cheek, A. O., Ide, C. F., Bollinger, J. E., Rider, C. V., and McLachlan, J. A. (1999a). Alternation of leopard frog (Rana pipiens) metamorphosis by the herbicide acetochlor. Arch. Environ. Contam. Toxicol. 37, 70–77.
Cheek, A. O., Kow, K., Chen, J., and McLachlan, J. A. (1999b). Potential mechanisms of thyroid disruption in humans: Interaction of organochlorine compounds with thyroid receptor, transthyretin, and thyroid-binding globulin. Environ. Health Perspect. 107, 273–278.
Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.
Colborn, T. (2002). Clues from wildlife to create an assay for thyroid system disruption. Environ. Health Perspect. 110(Suppl. 3), 363–367.
Crump, D., Werry, K., Veldhoen, N., Van Aggelen, G., and Helbing, C. C. (2002). Exposure to the herbicide acetochlor alters thyroid hormone-dependent gene expression and metamorphosis in Xenopus laevis. Environ. Health Perspect. 110, 1199–1205.
Denver, R. J. (1997). Environmental stress as a developmental cue: Corticotropin-releasing hormone is a proximate mediator of adaptive phenotypic plasticity in amphibian metamorphosis. Horm. Behav. 31, 169–179.
DeVito, M., Biegel, L., Brouwer, A., Brown, S., Brucker-Davis, F., Cheek, A. O., Christensen, R., Colborn, T., Cooke, P., Crissman, J., et al. (1999). Screening methods for thyroid hormone disruptors. Environ. Health Perspect. 107, 407–415.
Dickhoff, W. W., Brown, C. L., Sullivan, C. V., and Bern, H. A. (1990). Fish and amphibian models for developmental endocrinology. J. Exp. Zool. 4(Suppl.), 90–97.
Dumont, J., Schultz, T. W., Buchanan, M., and Kao, G. (1983). Frog Embryo Teratogenesis Assay-Xenopus (FETAX)- A Short-term Assay Applicable to Complex Environmental Mixtures. In Short-term Bioassays in the Analysis of Complex Environmental Mixtures III (M. D. Waters, S. S. Sandhu, J. Lewtas, L. Claxton, N. Chernoff, and S. Nesnow, Eds.), pp.393–405. Plenum, New York.
Furlow, J. D., and Brown, D. D. (1999). In vitro and in vivo analysis of the regulation of a transcription factor gene by thyroid hormone during Xenopus laevis metamorphosis. Mol. Endocrinol. 13, 2076–2089.
Gatlin, J., Unett, D. J., Lerner, M. R., and Garcia, J. V. (2001). Efficient, long-term transgene expression in Xenopus laevis dermal melanophores. Pigment Cell Res. 14, 275–282.
Gauger, K. J., Kato, Y., Haraguchi, K., Lehmler, H. J., Robertson, L. W., Bansal, R., and Zoeller, R. T. (2004). Polychlorinated biphenyls (PCBs) exert thyroid hormone-like effects in the fetal rat brain but do not bind to thyroid hormone receptors. Environ. Health Perspect. 112, 516–523.
Harris, C. A., Henttu, P., Parker, M. G., and Sumpter, J. P. (1997). The estrogenic activity of phthalate esters in vitro. Environ. Health Perspect. 105, 802–811.
Hinton, R. H., Mitchell, F. E., Mann, A., Chescoe, D., Price, S. C., Nunn, A., Grasso, P., and Bridges, J. W. (1986). Effects of phthalic acid esters on the liver and thyroid. Environ. Health Perspect. 70, 195–210.
Howdeshell, K. L. (2002). A model of the development of the brain as a construct of the thyroid system. Environ. Health Pespect. 110, 337–348.
Houlahan, J. E., Findlay, C. S., Schmidt, B. R., Meyer, A. H., and Kuzmin, S. L. (2000). Quantitative evidence for global amphibian population declines. Nature 404, 752–755.
Ishihara, A., Sawatsubashi, S., and Yamauchi K. (2003). Endocrine disrupting chemicals: interference of thyroid hormone binding to transthyretins and to thyroid hormone receptors. Mol. Cell. Endocrinol. 199, 105–117.
Ishiyama, M., Miyazono, Y., Sasamoto, K., Ohkura, Y., and Ueno, K. (1997). A highly water-soluble disulfonated terazolium salt as a chromogenic indicator for NADH as well as cell viability. Talanta 44, 1299–1305.
Iwamuro, S., Sakakibara, M., Terao, M., Ozaki, A., Kurobe, C., Shigeura, T., Kato, M., and Kikuyama, S. (2003). Teratogenic and anti-metamorphic effects of bisphenol A on embryonic and larveal Xenopus laevis. Gen. Comp. Endocrinol. 133, 189–198.
Iwasaki, T., Miyazaki, W., Takeshita, A., Kuroda, Y., and Koibuchi, N. (2002). Polychlorinated biphenyls suppress thyroid hormone-induced transactivation. Biochem Biophys Res Commun. 299, 384–388.
Jekat, F. W., Meisel, M. L., Eckard, R., and Winterhoff, H. (1994). Effects of pentachlorophenol (PCP) on the pituitary and thyroidal hormone regulation in the rat. Toxicol. Lett. 71, 9–25.
Jobling, S., Reynolds, T., White, R., Parker, M. G., and Sumpter, J. P. (1995). A variety of environmentally persistent chemicals, including some phthalate plasticizers, are weakly estrogenic. Environ. Health Perspect. 103, 582–587.
Katayama, K., Wada, K., Miyoshi, H., Ohashi, K., Tachibana, M., Furuki, R., Muzuguchi, H., Hayakawa, T., Nakajima, A., Kadowaki, T., et al. (2004). RNA interfering approach for clarifying the PPAR pathway using lentiviral vector expressing short hairpin RNA. FEBS Lett. 560, 178–182.
Kimura-Kuroda, J., Nagawa, I., and Kuroda, Y. (2005). Hydroxylated metabolites of polychlorinated biphenyls inhibit thyroid-hormone-dependent extension of cerebellar Purkinje cell dendrites. Brain Res. Dev. Brain Res. 154, 259–263.
Kudo, Y., and Yamauchi, K. (2005). In vitro and In vivo analysis of the thyroid disrupting activities of phenolic and phenol compounds in Xenopus laevis. Toxicol. Sci. 84, 29–37.
Larson, D. L., McDonald, S., Fivizzani, A. J., Newton, W. E., and Hamilton, S. J. (1998). Effects of the herbicide atrazine on Ambystoma tigrinum metamprppgosis: Duration, larval growth, and hormone response. Physiol. Zool. 71, 671–679.
Matthews, J., Celius, T., Halgren, R., and Zacharewski, T. (2000). Differential estrogen receptor binding of estrogenic substances: A species comparison. J. Steroid Biochem. Mol. Biol. 74, 223–234.
Miyazaki, W., Iwasaki, T., Takeshita, A., Kuroda, Y., and Koibuchi, N. (2004). Polychlorinated biphenyls suppress thyroid hormone receptor-mediated transcription through a novel mechanism. J. Biol. Chem. 279, 18195–18202.
Miyoshi, H., Blmer, U., Takahashi, M., Gage, F. H., and Verma, I. M. (1998). Development of a self-inactivating lentivirus vector. J. Virol. 72, 8150–8157.
Moriyama, K., Tagami, T., Akamizu, T., Usui, T., Saijo, M., Kanamoto, N., Hataya, Y., Shimatsu, A., Kuzuya, H., and Nakao, K. (2002). Thyroid hormone action is disrupted by bisphenol A as an antagonist. J. Clin. Endocrinol. Metab. 87, 5185–5190.
Mukku, V. R., Kirkland, J. L., Hardy, M., and Stancel, G. M. (1983). Evidence for thyroid hormone receptors in uterine nuclei. Metabolism 32, 142–145.
Nakai, M., Tabira, Y., Asai, D., Yakabe, Y., Shimyozu, T., Noguchi, M., Takatsuki, M., and Shimohigashi, Y. (1999). Binding characteristics of dialkyl phthalates for the estrogen receptor. Biochem. Bipphys. Res. Commun. 254, 311–314.
Nieuwkoop, P. D., and Faber, J. (1975). Normal table of Xenopus laevis (DUADIN), 2nd ed., North-Holland Publishing, Amsterdam.
Nishihara, T., Nishikawa, J., Kanayama, T., Dakeyama, F., Saito, K., Imagawa, M., Takatori, S., Kitagawa, Y., Hori, S., and Utsumi, H. (2000). Estrogenic activities of 517 chemicals by yeast two-hybrid assay. J. Health Sci. 46, 282–298.
Okubo, T., Suzuki, T., Yokoyama, Y., Kano, K., and Kano, I. (2003). Estimation of estrogenic and anti-estrogenic activities of some phthalate diesters and monoesters by MCF-7 cell proliferation assay in vitro. Biol. Pharm. Bull. 26, 1219–1224.
Ouellet, M., Bonin, J., Rodrigue, J., DesGranges, J.-L., and Lair, S. (1997). Hindlimb deformities (ectromelia, ectrodactyly) in free-living anurans from agricultural habitats. J. Wildl. Diseases 33, 95–104.
Samuels, H. H., Stanley, F., and Casanova, J. (1979). Depletion of L-3,5,3'-triiodothyronine and L-thyroxine in euthyroid calf serum for use in cell culture studies of the action of thyroid hormone. Endocrinology 105, 80–85.
Sandau, C. D., Ayotte, P., Dewailly, E., Duffe, J., and Norstrom, R. J. (2000). Analysis of hydroxylated metabolites of PCBs (OH-PCBs) and other chlorinated phenolic compounds in whole blood from Canadian Inuit. Environ. Health Perspect. 108, 611–616.
Shimada, N., and Yamauchi, K. (2004). Characteristics of 3,5,3'-triiodothyronine (T3)-uptake system of tadpole red blood cells: effect of endocrine-disrupting chemicals on cellular T3 response. J. Endocrinol. 183, 627–637.
Soto, A. M., Sonnenschein, C., Chung, K. L., Femandez, M. F., Olea, N., and Serrano, F. O. (1995). The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants. Environ. Health Perspect. 103(Suppl. 7), 113–122.
Sugiyama, S., Miyoshi, H., and Yamauchi, K. (In press). Characteristics of a thyroid hormone responsive reporter gene transduced into a Xenopus laevis cell line using lentivirus vector. Gen. Comp. Endocrinol.
Thomas, C. R., Drake, J., and Frieden, E. (1992). Thyroid hormone receptor induction by triiodothyronine in tadpole erythrocytes in vivo and in vitro and the effect of cycloheximide and actinomycin-D. Gen. Comp. Endocrinol. 86, 42–51.
Tran, D. Q., Klotz, D. M., Ladlie, B. L., Ide, C. F., McLachlan, J. A., and Arnold, S. F. (1996). Inhibition of progesterone receptor activity in yeast by synthetic chemicals. Biochem. Biophys. Res. Commun. 229, 518–523.
Veldhoen, N., and Helbing, C. C. (2001). Detection of environmental endocrine-disruptor effects on gene expression in liver Rana catesbeiana tadpoles using a tail fin biopsy technique. Environ. Toxicol. Chem. 20, 2704–2708.
Vinggaard, A. M., Breinholt, V., and Larsen, J. C. (1999). Screening of selected pesticides for oestrogen receptor activation in vitro. Food Addit. Contam. 16, 533–542.
Wake, D. B. (1991). Declining amphibian populations. Science 253, 860.
Yamauchi, K., Prapunpoj, P., and Richardson, S. J. (2000). Effect of diethylstilbestrol on thyroid hormone binding to amphibian transthyretins. Gen. Comp. Endocrinol. 119, 329–339.
Yamauchi, K., Eguchi, R., Shimada, N., and Ishihara, A. (2002). The effects of endocrine-disrupting chemicals on thyroid hormone binding to Xenopus laevis transthyretin and thyroid hormone receptor. Clin. Chem. Lab. Med. 40, 1250–1256.
Yamauchi, K., Ishihara, A., Fukazawa H., and Terao, Y. (2003). Competitive interactions of chlorinated phenol compounds with 3,3',5-triiodothyronine binding to transthyretin: detection of possible thyroid-disrupting chemicals in environmental waste water. Toxicol. Appl. Pharmacol. 187, 110–117.
Zacharewski, T. (1998). Identification and assessment of endocrine disruptors: limitations and in vivo and in vitro assays. Environ. Health Perspect. 106, 577–582.
Zacharewski, T. R., Meek, M. D., Clemons, J. H., Wu, Z. F., Fielden, M. R., and Matthews, J. B. (1998). Examination of the in vitro and in vivo estrogenic activities of eight commercial phthalate esters. Toxicol. Sci. 46, 282–293.
Zoeller, R. T., Dowling, A. L. S., Herzig, C. T. A., Iannacone, E. A., Gauger, K. J., and Bansal, R. (2002). Thyroid hormone, brain development, and the environment. Environ. Health Perspect. 110(Suppl. 3), 355–361.
Zufferey, R., Dull, T., Mandel, R. J., Bukovsky, A., Quiroz, D., Naldini, L., and Trono, D. (1998). Self-inactvating lentivirus vector for safe and efficient in vivo gene delivery. J. Virol. 72, 9873–9880.(Shin-ichiro Sugiyama, Naoyuki Shimada, H)
BioResource Center, RIKEN Tsukuba Institute, Tsukuba 305-0074, Japan
ABSTRACT
We developed a thyroid hormone (TH) inducible primary screening assay for the identification and assessment of man-made chemicals that interfere with the TH-signalling pathway within target cells. The assay was developed in a Xenopus laevis cell line that was transduced with a self-inactivating (SIN) lentivirus vector (LV) containing a luciferase gene. The luciferase activation in this cell line was TH-specific: 3,3',5-L-triiodothyronine (T3) > 3,3'5-L-triiodothyroacetic acid (Triac) > 3,3',5-D-triiodothyronine (D-T3), > L-thyroxine (T4) > 3,3',5'-L-triiodothyronine (rT3). The application of the ligand-dependent luciferase assay for screening for thyroid system-disrupting chemicals revealed that three phthalates (dicyclohexyl phthalate, n-butylbenzyl phthalate, and di-n-butyl phthalate), two herbicides (ioxynil and pentachlorophenol) and a miticide (dicofol) had 3,3',5-L-triiodothyronine- T3- antagonist activity at concentrations ranging from 10–6 to 10–5 M. These chemicals also inhibited the expression of the endogenous primary T3-response TH nuclear receptor (TR) gene. The inhibitory characteristics of these chemicals were similar for both assays performed, although the assay for T3-dependent activation of TR gene was more sensitive than the luciferase assay. These results indicate that the luciferase assay was a rapid method with a small intra-assay variation for the primary screening of thyroid system-disrupting chemicals. Of the six chemicals, only n-butylbenzyl phthalate and pentachlorophenol exhibited T3-antagonist activity in an in vivo metamorphosis-based assay. It should be noted that chemicals elicited thyroid system-disrupting activity in the luciferase assay did not always interfere with the thyroid system in vivo.
Key Words: endocrine toxicology – endocrine; thyroid, gene expression/regulation – receptor; nuclear hormone, endocrine toxicology – endocrine disruptors.
INTRODUCTION
Over the past several decades amphibian populations worldwide have tended to decrease, the number of malformed frogs has increased, and some species at certain localities have been extirpated (Houlahan et al., 2000; Ouellet et al., 1997). Possible causes for these trends include natural population fluctuation, fungal pathogens, increased ultraviolet radiation, climate change, acid precipitation, loss of suitable habitats by human activity, and/or natural and man-made environmental contaminants (Baustein and Wake, 1995; Burkhart et al., 2000; Wake, 1991). It is not clear what impact each of these possible causes has had upon amphibian populations; however, it is likely that environmental contaminants have had the greatest impact as amphibians are generally considered more sensitive to aquatic contaminants due to the permeability of their skin.
Xenopus laevis is a water-living amphibian (all of its life stages occur in water) that is used widely as a laboratory animal. Its development and the expression of its genes are well characterized. For these reasons, X. laevis has been approved as an experimental model for evaluating the effects of endocrine-disrupting chemicals (EDCs) in amphibians by the Organization for Economic Cooperation and Development. A critical process in the amphibian life cycle is metamorphosis that is obligatorily controlled by thyroid hormones (THs). This process is also influenced by several environmental factors, such as the density of the population, habitat dessication, temperature, and food availability (Denver, 1997).
THs are essential for normal brain development and behavior in higher vertebrates (Howdeshell, 2002; Zoeller et al., 2002) and postembryonic development in lower vertebrates (Dickhoff et al., 1990). However, there have been relatively few reports concerning the molecular mechanisms by which EDCs disrupt the thyroid system despite reports of structural lesions in thyroid follicular cells and irregular levels of THs in experimental animals and wildlife (Brouwer et al., 1998; Colborn, 2002) exposed to EDCs. These results suggest that there may be thyroid system–disrupting chemicals in the environment not yet identified. Our laboratory has been interested in studying the effect of EDCs, especially those that interfere with the amphibian thyroid system in vitro including TH-binding to the major plasma TH-carrier protein, transthyretin (Ishihara et al., 2003; Kudo and Yamauchi, 2005; Yamauchi et al., 2000, 2002, 2003), cellular TH-uptake (Shimada and Yamauchi, 2004) and TH-binding to TH nuclear receptor (TR) (Ishihara et al., 2003; Kudo and Yamauchi, 2005; Shimada and Yamauchi, 2004; Yamauchi et al., 2002, 2003); and in vivo including X. laevis metamorphosis (Kudo and Yamauchi, 2005). Assay methods mechanistically distinct from ours have also been used by others to investigate thyroid system–disrupting chemicals (Crump et al., 2002; DeVito et al., 1999; Veldhoen and Helbing, 2001). Together, these studies revealed that EDCs target differentially several processes within the thyroid system in a chemical-dependent manner. Bioassay methods capable of identifying thyroid system–disrupting chemicals and their effects, as a total sum of each effect on individual processes, within the thyroid system are essential for the primary screening of thyroid system–disrupting chemicals. Furthermore, although there are advantages and limitations in each bioassay, as suggested by Zacharewski (1998), important criteria for primary screening bioassays may be high throughput with low cost.
In the present study, we established a permanent X. laevis recombinant cell line as a bioassay for screening thyroid system-disrupting chemicals using a self-inactivation (SIN) lentivirus vector (LV) containing a TH response element (TRE)-SV40 promoter-luciferase reporter gene. We chose nine suspect EDCs, most of which are known to affect at least one of the following processes: 3,3',5-L-triiodothyronine- (T3-) binding to transthyretins and TRs, and cellular T3-uptake in amphibian systems (Ishihara et al., 2003; Shimada and Yamauchi, 2004; Yamauchi et al., 2002); and investigated their effects on the ligand-dependent luciferase assay, T3-dependent activation of the TR gene using the same cell line and T3-induced tadpole metamorphosis in vivo. Given the advantages of the luciferase assay, high sensitivity, rapidity and high reproducibility, its use as a high throughput primary screening system would provide large-scale assessment of environmental chemicals as thyroid system disruptors.
MATERIALS AND METHODS
Reagents.
T3, (>98% purity), L-thyroxine (T4; >98%), 3,3'5-L-triiodothyroacetic acid (Triac; 95%), 3,3',5-D-triiodothyronine (D-T3; >98%), 3,3',5'-L-triiodothyronine (rT3; 95%), 17-estradiol (E2; >98%), and pentachlorophenol (>99% purity) were obtained from Sigma (St. Louis, MI). Bisphenol A (>98% purity), dicyclohexyl phthalate (>99% purity), dicofol (Kelthane, 2,2,2-trichloro-1,1-bis(4-chlorophenyl)ethanol, analytical standard, >95% purity), and ioxynil (3,5-diiodo-4-hydroxybenzonitril, analytical standard, >99% purity) were purchased from Wako Pure Chemical Industries (Osaka, Japan) and diethyl phthalate (>99% purity) and di(2-ethylhexyl) phthalate (>99% purity) were from Kanto Chemicals (Tokyo, Japan). Di-n-butyl phthalate (>98% purity) and n-butylbenzyl phthalate (>97% purity) were obtained from Nacalai Tesque (Kyoto, Japan). 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, except for E2 which was dissolved in ethanol to a concentration of 1 mM, were dissolved in dimethylsulfoxide to a concentration of 10 mM. These chemicals were diluted with an appropriate buffer to give less than 0.5% (v/v) solvent. A control assay without the test chemicals was performed in the presence of the solvent alone and at less than 0.5% (v/v). The solvent did not affect the luciferase assay, the T3-dependent activation of TR gene, cell viability nor T3-induced tadpole metamorphosis.
Luciferase assay in a permanent Xenopus laevis recombinant cell line.
A human immunodeficiency virus-1-based LV pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G) was prepared by transient transfection of the following plasmids: packaging construct, pMDLg/pRRE; VSV-G- and Rev- expressing construct, pCMV-VSV-G-RSV-Rev; and SIN LV construct, pCS-TRE-Luc-CG, as described elsewhere (Sugiyama et al., in press). This LV contains the firefly luciferase gene under the control of the SV40 promoter and the X. laevis TREs. The structures of the TREs and pCS-TRE-Luc-CG are illustrated in Figure 1. Subconfluent monolayers of X. laevis XL58 cells were transduced with the viral vector at a multiplicity of infection of 10, and incubated in 70% Leibovitz's L-15 medium containing 10% fetal bovine serum (FBS) for 15 h at 25°C with air. A cell clone, XL58-TRE-Luc, with luciferase highly expressed in a T3-dependent manner was isolated (Sugiyama et al., in press).
XL58-TRE-Luc cells were cultured in FBS-free 70% Leibovitz's L-15 medium with or without 2 nM T3 in the presence or absence (control) of each EDC for 24 h, following incubation in 70% Leibovitz's L-15 medium containing 10% resin-stripped FBS (Samuels et al., 1979) for 15 h. Their cell lysate was assayed for firefly luciferase activity using the PicaGene Luminescence kit (Nippon Gene, Tokyo, Japan). Cell viability was examined using the Cell Count Reagent SF kit (Nacalai Tesque), according to the manufacturer's directions, based on the method described by Ishiyama et al. (1997). Protein concentration was measured by the Bradford dye-binding method using bovine -globulin as the standard (Bradford, 1976).
Metamorphosis assay.
Xenopus laevis tadpoles were purchased from Akita Xenopus Co. (Ibaraki, Japan). Tadpoles were staged according to Nieuwkoop and Faber (1975). The metamorphosis assay was performed as described previously (Kudo and Yamauchi, 2005). In brief, five tadpoles in NF stages 51–52 were transferred into a 1 L glass beaker containing 0.5 L of FETAX buffer (625 mg/L NaCl, 96 mg/L NaHCO3, 30 mg/L KCl, 15 mg/L CaCl2, 60 mg/L CaSO4.2H2O and 75 mg/L MgSO4.7H2O, pH 7.7) (Dumont et al., 1983). The FETAX buffer contained dimethylsulfoxide, T3 in dimethylsulfoxide or T3 and each chemical in dimethylsulfoxide. Tadpoles were cultured in the FETAX buffer at 22–24°C. FETAX buffer containing the chemical applications was renewed every other day. Final dimethylsulfoxide concentrations were less than 0.02% in the chemical exposed and control groups. After five days, all living tadpoles were frozen in liquid nitrogen and then stored at –80°C until RNA preparation.
Real-time polymerase chain reaction.
Total RNA was extracted from XL58-TRE-Luc cells using the acid guanidinium thiocyanate-phenol-chloroform extraction procedure (Chomczynski and Sacchi, 1987) and from the frozen tadpoles using the LiCl-urea procedure (Auffray and Rougeon, 1980). To confirm its integrity, RNA (5 μg per lane) was electrophoresed in a 1% agarose gel containing 2.6 M formaldehyde, and 28S and 18S rRNA were visualized by ethidium bromide staining. The quantity of specific RNA species in each sample was 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 had been treated with reverse transcriptase (TaqMan Reverse Transcription Reagents, Applied Biosystems), as previously described (Kudo and Yamauchi, 2005). To standardize each experiment, the amount of TR transcript in each sample was divided by the amount of glyceraldehyde-phosphate dehydrogenase (GAPDH) transcript in the same sample. The primer sequences used were as follows: X. laevis TR transcript (accession number: X17385) sense 5'-CAAGCACCAAGAACGAAAACC-3' (nucleotide numbers 530–555) and antisense 5'-TTGGAAGGTCTGCTCATTCTTCTA-3' (600–580); X. laevis TR 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).
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
Ligand-Specific Luciferase Assay in XL58-TRE-Luc Cells
Figure 2 shows the specificity of the ligand-dependent luciferase assay in this recombinant cell line. Luciferase activity in XL58-TRE-Luc cells was induced 3.1- and 3.3-fold in the presence of 2 and 20 nM T3, respectively. Triac was less potent than T3 at the low concentration but was as potent as T3 at the high concentration. T4 and D-T3 were moderately potent while rT3 was less potent than the above chemicals. E2 elicited no statistically significant effect on the induction of the luciferase activity in our cell culture system. These results indicate that this luciferase gene specifically responds to THs.
Effects of EDCs on the T3-Dependent Luciferase Assay Using XL58-TRE-Luc Cells
To determine which phthalates affect the T3-dependent luciferase activity, XL58-TRE-Luc cells were incubated with or without 2 nM T3, in the presence or absence of various concentrations of each phthalate. In the absence of EDCs and presence of T3, the luciferase activity increased by 3.0-fold. Figure 3 shows dose-effects of five phthalates on the T3-dependent luciferase activity. Of the five phthalates, dicyclohexyl phthalate was the most potent antagonist with a 50% inhibitory concentration (IC50) of 11 ± 3 μM (n = 3). The IC50 rank order of antagonist activity was dicyclohexyl phthalate > n-butylbenzyl phthalate (40 ± 6 μM, n = 3) = di-n-butyl phthalate (39 ± 1 μM, n = 3) > di(2-ethylhexyl) phthalate (>50 μM, n = 3) > diethyl phthalate (>50 μM, n = 3). The effect of ioxynil, pentachlorophenol, dicofol, and bisophenol A on the T3-dependent luciferase assay was also investigated (Fig. 4). Of these chemicals, ioxynil was the most potent antagonist with an IC50 of 0.9 ± 0.3 μM (n = 3) with significant potency even at 0.2 μM. The antagonist activities of pentachlorophenol and dicofol were weaker with less than 50% inhibition at their maximum concentrations, 0.8 and 10 μM, respectively. Bisphenol A failed to elicit statistically significant responses at the concentrations tested (0.001–10 μM). Activation of luciferase activity was not detected when the chemicals were applied in the absence of T3. All of these chemicals did not affect cell viability at the highest concentrations tested under our experimental conditions (data not shown).
Effects of EDCs on the T3-Dependent Activation of TR Gene in XL58-TRE-Luc Cells
To investigate whether EDCs that disrupted the cellular T3-signaling pathway in the T3-dependent luciferase assay also affected other T3-response genes, the transcripts of endogenous primary T3-response TR genes were analyzed by real-time PCR. XL58-TRE-Luc cells were grown with or without 2 nM T3 in the presence or absence of various concentrations of each EDC. The TR transcript was not induced by T3 in XL58-TRE-luc cells under our experimental conditions. In the absence of EDCs and presence of T3, the amount of TR transcript increased by 13.3 ± 0.5 (n = 5) fold. This T3-dependent activation was significantly inhibited by all chemicals tested except for bisphenol A at the indicated or higher concentrations (Fig. 5). The inhibition percentages obtained from independent repeated experiments were 30 ± 7% diethyl phthalate (50 μM, n = 3), 29 ± 5% di(2-ethylhexyl) phthalate (50 μM, n = 3), 32 ± 5% di-n-butyl phthalate (50 μM, n = 3), 45 ± 4% n-butylbenzyl phthalate (50 μM, n = 3), 42 ± 6% dicyclohexyl phthalate (20 μM, n = 3), 45 ± 4% ioxynil (0.1 μM, n = 3), 63 ± 4% pentachlorophenol (0.08 μM, n = 3), and 32 ± 5% dicofol (10 μM, n = 3). n-Buthylbenzyl phthalate, even at the concentration of 4 μM, were still potent, with more than 50% inhibition. Activation of the TR gene was not detected when the chemicals were applied in the absence of T3.
Effects of EDCs on the T3-Dependent Activation of TR Gene in Xenopus laevis Tadpoles
To examine whether the potent chemicals selected from the above in vitro assays affect the thyroid system in vivo, X. laevis tadpoles in NF stages 51–52 tadpoles were immersed in FETAX buffer alone or in FETAX buffer containing 2 nM T3 with or without each chemical, and cultured for five days at 22–24°C. Total RNA was then extracted from tadpoles in each group and the amount of TR transcript was quantified by real-time PCR (Fig. 6). At the end of the experiments, positive control (T3-treated), test (T3 and chemical-treated), and negative control (T3-untreated) tadpoles were in NF stages 54, 53–54, and 51–52, respectively. No tadpoles died during the culture period. The amount of TR transcript increased 12.4 ± 0.7 times (data not shown) in T3-treated tadpoles, which was less than half of the increase reported in the previous in vivo study (Kudo and Yamauchi, 2005). Co-treatment of T3 with n-butylbenzyl phthalate (4 μM) or pentachlorophenol (0.1 μM) significantly inhibited the T3-induced increase in the amount of TR transcript. The inhibition percentages obtained from independent repeated experiments were 48 ± 3% n-butylbenzyl phthalate (n = 3) and 62 ± 6% pentachlorophenol (n = 3). No significant effects were detected for di-n-butyl phthalate (4 μM), ioxynil (0.1 μM), dicofol (0.1 μM), and bisphenol A (5 μM). When tadpoles were treated with di-n-butyl phthalate or dicofol, at concentrations several times higher, some of the test tadpoles died due to toxicity. Therefore, it was not possible to compare their thyroid system-disrupting activity in tadpoles with that in cultured cells at similar concentrations. These results indicated that of the six chemicals showing T3-antagonist activity in the in vitro assays only n-butylbenzyl phthalate and pentachlorophenol exhibited T3-antagonist activity in vivo at micromolar and sub-micromolar concentrations, respectively.
DISCUSSION
We have used a recombinant X. laevis cell line containing a T3-dependent reporter gene, XL58-TRE-Luc, to detect the thyroid system-disrupting activity of several suspect EDCs. This luciferase assay was highly sensitive to T3 with an ED50 of 10–11 M, in the absence of FBS (Sugiyama et al., in press) and was specific to THs: T3 > Triac > D-T3 > T4 > rT3 (Fig. 2). This specificity was in agreement with the binding specificity of amphibian and mammalian TRs (Mukku et al., 1983; Thomas et al., 1992). In the present study, the ligand-dependent luciferase assay revealed the TH-antagonist activities of at least three phthalates, dicyclohexyl phthalate, n-butylbenzyl phthalate, and di-n-butyl phthalate, and three agricultural chemicals, ioxynil, pentachlorophenol, and dicofol. The T3-dependent activation of the TR gene confirmed the T3-antagonist activities of these chemicals in the same cells, and indicated that di(2-ethylhexyl) phthalate and diethyl phthalate were also potent. Of the above chemicals, only n-butylbenzyl phthalate and pentachlorophenol elicited T3-antagonist activity in tadpoles.
Of the six chemicals found to have T3-antagonist activity using the luciferase assay, pentachlorophenol and ioxynil were previously identified as the most potent inhibitors of T3-binding to X. laevis transthyretin (IC50 values of 180–260 nM); however, none of them, at 1 μM, were potent inhibitors of T3-binding to the X. laevis TR gene (Yamauchi et al., 2002). Di(2-ethyhexyl) phthalate and pentachlorophenol are known to decrease the plasma thyroxine level and the plasma T3 and thyroxine levels, respectively, in rat (Brucker-Davis, 1998). These effects are probably due to alterations in thyroidal activity (Hinton et al., 1986) or the control of the hypothalamus/pituitary/thyroid axis (Jekat et al., 1994). These findings suggest that these chemicals target TH system processes, extracellular TH-transport system, TH-synthesis, and/or control system of TH-synthesis, before THs access the peripheral target cells. Together with our previous findings, the present study opened the possibility that some of the six chemicals interfere with a process or several processes other than T3-binding to TR in the T3-signaling pathway within peripheral target cells. This possibility has been proposed for polychlorinated biphenyls (PCBs) or hydroxylated PCBs. These compounds affected TH-dependent extension of Purkinje dendrite in mouse cerebellar culture at 10–12–10–11 M (Kimura-Kuroda et al., 2005) and suppressed T3-induced transcriptional activation of TR at 10–10 M (Iwasaki et al., 2002), although they slightly competed with T3-binding to TR at the concentrations of more than 10–6 M (Cheek et al., 1999b; Gauger et al., 2004). Miyazaki et al. (2004) found that these compounds induced partial dissociation of TR/retinoid X receptor heterodimer complex from the TRE. It remains to be clarified how the three phthalates and the three agricultural chemicals affected T3-dependent transactivation in our culture system.
Using the luciferase assay, the significant effective doses of the three phthalates and the three agricultural chemicals were 10–7–10–5 M (Figs. 2 and 3). Most of these EDCs have been shown to interfere with the estrogen-signaling pathway by competitive binding to estrogen receptors (Jobling et al., 1995; Matthews et al., 2000; Nakai et al., 1999; Zacharewski et al., 1998), the MCF-7 proliferation assay (Harris et al., 1997; Okubo et al., 2003; Soto et al., 1995), yeast and mammalian two-hybrid systems (Nishihara et al., 2000; Zacharewski et al., 1998), and estrogen-dependent reporter gene assays (Harris et al., 1997; Jobling et al., 1995; Zacharewski et al., 1998), with effective concentrations of 10–6–10–3 M. Pentachlorophenol (l0–6 M) exhibited antagonist activity in a yeast progesterone-dependent reporter gene assay (Tran et al., 1996) whereas dicofol (10–6–10–5 M) exerted estrogenic activity in the yeast estrogen-dependent reporter gene assay and the MCF-7 proliferation assay (Vinggaard et al., 1999). The effective concentrations of these EDCs for our luciferase assay and most of the above assays for steroid system disruption were within a similar range. However, our cell culture system specifically responded to THs but not to E2 (Fig. 1). It is likely that these EDCs interfere with several hormonal signalling pathways simultaneously within a cell, and/or that they target a cellular site that affects several hormonal signalling pathways. The rank orders of their steroidgenic or anti-steroidgenic activities showed occasional discrepancies, which could be due to differences in the cellular environment of the cell lines and/or the species used (Matthews et al., 2000; Zacharewski et al., 1998) or differences in the assay methods and/or in the assay conditions used (Harris et al., 1997; Okubo et al., 2003; Zacharewski et al., 1998). These rank orders did not compare with that of their TH-antagonist activities as determined by our luciferase assay. Therefore, it is not possible to conclude whether the chemicals potent in the luciferase assay act on a single cellular site that affects several hormonal signalling pathways.
The inhibitory effect of the EDCs on the T3-dependent activation of the TR gene (Fig. 5) was similar to that determined using the luciferase assay. The fold induction of TR gene expression by T3 and inhibition of its T3-dependent induction by the EDCs indicated that the assay for T3-dependent activation of the TR gene was more sensitive to T3 and the EDCs used than the luciferase assay. Of the five phthalates, dicyclohexyl phthalate (20 μM) had the most potent T3-antagonist activity in the luciferase assay whereas n-butylbenzyl phthalate (4 μM) was the most potent inhibitor of the T3-dependent activation of the TR gene. This discrepancy may be, in part, due to the difference in the structure of the TREs located in the two genes.
N-Butylbenzyl phthalate (4 μM) and pentachlorophenol (0.08–0.1 μM), exhibited T3-antagonist activity in both the in vitro and in vivo assays in X. laevis. Phthalates including n-butylbenzyl phthalate are widespread in food and the environment due to their use as plasticizers in consumer products, food packaging materials and biomedical devices. n-Butylbenzyl phthalate is one of the most abundant man-made chemicals and is used in the production of vinyl floor tiles, adhesives, and synthetics leather. Phthalates can leach out of these materials into water, soil, or food over time, after use. Pentachlorophenol had been used commonly as a fungicidal substance for wood protection and leather impregnation and as a herbicide. It is also present in the gases of municipal waste incinerators. Due to its highly persistent nature, pentachlorophenol is still one of the dominant phenolic compounds in blood (Sandau et al., 2000). However, little is known about their absorption, bioaccumulation, and metabolism, and their chronic effects on humans or wildlife if administered at low concentrations over long time periods. Considering the prevalence of phenolic compounds in the environment, more research should be focused on their thyroid system-disrupting activities as well as steroid system-disrupting activities, and their cellular target sites, i.e., metabolic enzymes that activate or inactivate the chemicals and endogenous hormones, hormone receptors and/or nuclear co-regulators.
Recently, it was reported that bisphenol A (10–5–10–4 M) inhibited T3-induced metamorphosis (Iwamuro et al., 2003). In a mammalian two-hybrid system, bisphenol A (10–5 M) recruited the nuclear corepressor N-CoR to the TR then elicited T3-antagonist activity (Moriyama et al., 2002). However, in the present in vitro and in vivo assays, bisphenol A (10 μM and 5 μM, respectively) exhibited no T3-antagonist activity in X. laevis. Although we can not fully explain this discrepancy, higher concentrations of bisphenol A than we used would be necessary to elicit significant T3-antagonist activity under our experimental conditions.
The luciferase assay using XL58-TRE-Luc cells has several advantages. This cell line can produce enough endogenous TR in response to exogenously added T3. It is easy to grow and work with, and the data derived from the assay had a small intra-assay variation when compared with the transient transcription assay (Sugiyama et al., in press). Although there have been a number of reports on the effects of environmental chemicals on amphibian metamorphosis (Cheek et al., 1999a; Crump et al., 2002; Larson et al., 1998; Veldhoen and Helbing, 2001), most of these assays were not suitable for the primary screening of potential thyroid system-disrupting chemicals because of the time required and the high costs involved. Our luciferase assay would provide high throughput primary screening for chemicals with thyroid system-disrupting activity in the water environment where amphibians habitat. However, it should be noted that the luciferase assay would not provide the potential intracellular sites for thyroid disruption by EDCs, and that the in vitro assays, including the assay for T3-dependent activation of TR gene, and the transthyretin and TR binding assays, can not reproduce the pharmacokinetic and pharmacodynamic interactions and sensitive developmental windows that may occur in vivo. We actually found that different concentrations of EDCs were required to elicit a toxic effect in cultured cells and metamorphosing tadpoles. Of the chemicals that were T3-antagonistic in the luciferase assay and T3-dependent TR gene activation in cultured cells, only n-butylbenzyl phthalate and pentachlorophenol elicited T3-antagonist activity in tadpoles reflecting in part differences in the action of EDCs in the in vitro and in vivo assays, as described above. Despite these limitations, the results from the in vitro assays may indicate potential mechanisms of EDC action. Furthermore, our study indicates the importance of the combination of several assays to assess the thyroid system-disrupting activity of EDCs. The transgene by the LV system can be introduced into organisms (Miyoshi et al., 1998; Zufferey et al., 1998) as well as cell lines (Gatlin et al., 2001; Katayama et al., 2004). Therefore, the same DNA construct can be introduced into eggs, embryos, or some tissues of tadpoles. This application of the transgene would allow, to some extent, the connection of data obtained from the various in vitro assays with data obtained from in vivo metamorphosis assays.
ACKNOWLEDGMENTS
We are grateful to Dr. D. D. Brown, Carnegie Institute of Washington, MD, for kindly providing the X. laevis TR cDNA. We would like to thank Ms. J. Monk for a thorough and critical reading of the manuscript. This work was supported by Grant-in Aid for Scientific Research (B) (No. 13559001, No. 16244120) from Japan Society for the Promotion of Science, and by grants from The Sumitomo Foundation and from Toukai Foundation for Technology. The authors declare they have no competing financial interests.
REFERENCES
Auffray, C., and Rougeon, F. (1980). Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur. J. Biochem. 107, 303–314.
Baustein, A. R., and Wake, D. B. (1995). The puzzle of declining amphibian populations. Sci. Am. 272, 52–57.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
Brouwer, A., Morse, D. C., Lans, M. C., Schuur, A. G., Murk, A. J., Klasson-Wehler, E., and Visser, T. J. (1998). Interactions of persistent environmental organohalogens with the thyroid hormone system: Mechanisms and possible consequences for animal and human health. Toxicol. Ind. Health 14, 59–84.
Brucker-Davis, F. (1998). Effects of environmental synthetic chemicals on thyroid function. Thyroid 8, 827–856.
Burkhart, J. G., Ankley, G., Bell, H., Carpenter, H., Fort, D., Gardiner, D., Gardner, H., Hale, R., Helgen, J. C., Jepson, P., et al. (2000). Strategies for assessing the implications of malformed frogs for environmental health. Environ. Health Perspect. 108, 83–90.
Cheek, A. O., Ide, C. F., Bollinger, J. E., Rider, C. V., and McLachlan, J. A. (1999a). Alternation of leopard frog (Rana pipiens) metamorphosis by the herbicide acetochlor. Arch. Environ. Contam. Toxicol. 37, 70–77.
Cheek, A. O., Kow, K., Chen, J., and McLachlan, J. A. (1999b). Potential mechanisms of thyroid disruption in humans: Interaction of organochlorine compounds with thyroid receptor, transthyretin, and thyroid-binding globulin. Environ. Health Perspect. 107, 273–278.
Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.
Colborn, T. (2002). Clues from wildlife to create an assay for thyroid system disruption. Environ. Health Perspect. 110(Suppl. 3), 363–367.
Crump, D., Werry, K., Veldhoen, N., Van Aggelen, G., and Helbing, C. C. (2002). Exposure to the herbicide acetochlor alters thyroid hormone-dependent gene expression and metamorphosis in Xenopus laevis. Environ. Health Perspect. 110, 1199–1205.
Denver, R. J. (1997). Environmental stress as a developmental cue: Corticotropin-releasing hormone is a proximate mediator of adaptive phenotypic plasticity in amphibian metamorphosis. Horm. Behav. 31, 169–179.
DeVito, M., Biegel, L., Brouwer, A., Brown, S., Brucker-Davis, F., Cheek, A. O., Christensen, R., Colborn, T., Cooke, P., Crissman, J., et al. (1999). Screening methods for thyroid hormone disruptors. Environ. Health Perspect. 107, 407–415.
Dickhoff, W. W., Brown, C. L., Sullivan, C. V., and Bern, H. A. (1990). Fish and amphibian models for developmental endocrinology. J. Exp. Zool. 4(Suppl.), 90–97.
Dumont, J., Schultz, T. W., Buchanan, M., and Kao, G. (1983). Frog Embryo Teratogenesis Assay-Xenopus (FETAX)- A Short-term Assay Applicable to Complex Environmental Mixtures. In Short-term Bioassays in the Analysis of Complex Environmental Mixtures III (M. D. Waters, S. S. Sandhu, J. Lewtas, L. Claxton, N. Chernoff, and S. Nesnow, Eds.), pp.393–405. Plenum, New York.
Furlow, J. D., and Brown, D. D. (1999). In vitro and in vivo analysis of the regulation of a transcription factor gene by thyroid hormone during Xenopus laevis metamorphosis. Mol. Endocrinol. 13, 2076–2089.
Gatlin, J., Unett, D. J., Lerner, M. R., and Garcia, J. V. (2001). Efficient, long-term transgene expression in Xenopus laevis dermal melanophores. Pigment Cell Res. 14, 275–282.
Gauger, K. J., Kato, Y., Haraguchi, K., Lehmler, H. J., Robertson, L. W., Bansal, R., and Zoeller, R. T. (2004). Polychlorinated biphenyls (PCBs) exert thyroid hormone-like effects in the fetal rat brain but do not bind to thyroid hormone receptors. Environ. Health Perspect. 112, 516–523.
Harris, C. A., Henttu, P., Parker, M. G., and Sumpter, J. P. (1997). The estrogenic activity of phthalate esters in vitro. Environ. Health Perspect. 105, 802–811.
Hinton, R. H., Mitchell, F. E., Mann, A., Chescoe, D., Price, S. C., Nunn, A., Grasso, P., and Bridges, J. W. (1986). Effects of phthalic acid esters on the liver and thyroid. Environ. Health Perspect. 70, 195–210.
Howdeshell, K. L. (2002). A model of the development of the brain as a construct of the thyroid system. Environ. Health Pespect. 110, 337–348.
Houlahan, J. E., Findlay, C. S., Schmidt, B. R., Meyer, A. H., and Kuzmin, S. L. (2000). Quantitative evidence for global amphibian population declines. Nature 404, 752–755.
Ishihara, A., Sawatsubashi, S., and Yamauchi K. (2003). Endocrine disrupting chemicals: interference of thyroid hormone binding to transthyretins and to thyroid hormone receptors. Mol. Cell. Endocrinol. 199, 105–117.
Ishiyama, M., Miyazono, Y., Sasamoto, K., Ohkura, Y., and Ueno, K. (1997). A highly water-soluble disulfonated terazolium salt as a chromogenic indicator for NADH as well as cell viability. Talanta 44, 1299–1305.
Iwamuro, S., Sakakibara, M., Terao, M., Ozaki, A., Kurobe, C., Shigeura, T., Kato, M., and Kikuyama, S. (2003). Teratogenic and anti-metamorphic effects of bisphenol A on embryonic and larveal Xenopus laevis. Gen. Comp. Endocrinol. 133, 189–198.
Iwasaki, T., Miyazaki, W., Takeshita, A., Kuroda, Y., and Koibuchi, N. (2002). Polychlorinated biphenyls suppress thyroid hormone-induced transactivation. Biochem Biophys Res Commun. 299, 384–388.
Jekat, F. W., Meisel, M. L., Eckard, R., and Winterhoff, H. (1994). Effects of pentachlorophenol (PCP) on the pituitary and thyroidal hormone regulation in the rat. Toxicol. Lett. 71, 9–25.
Jobling, S., Reynolds, T., White, R., Parker, M. G., and Sumpter, J. P. (1995). A variety of environmentally persistent chemicals, including some phthalate plasticizers, are weakly estrogenic. Environ. Health Perspect. 103, 582–587.
Katayama, K., Wada, K., Miyoshi, H., Ohashi, K., Tachibana, M., Furuki, R., Muzuguchi, H., Hayakawa, T., Nakajima, A., Kadowaki, T., et al. (2004). RNA interfering approach for clarifying the PPAR pathway using lentiviral vector expressing short hairpin RNA. FEBS Lett. 560, 178–182.
Kimura-Kuroda, J., Nagawa, I., and Kuroda, Y. (2005). Hydroxylated metabolites of polychlorinated biphenyls inhibit thyroid-hormone-dependent extension of cerebellar Purkinje cell dendrites. Brain Res. Dev. Brain Res. 154, 259–263.
Kudo, Y., and Yamauchi, K. (2005). In vitro and In vivo analysis of the thyroid disrupting activities of phenolic and phenol compounds in Xenopus laevis. Toxicol. Sci. 84, 29–37.
Larson, D. L., McDonald, S., Fivizzani, A. J., Newton, W. E., and Hamilton, S. J. (1998). Effects of the herbicide atrazine on Ambystoma tigrinum metamprppgosis: Duration, larval growth, and hormone response. Physiol. Zool. 71, 671–679.
Matthews, J., Celius, T., Halgren, R., and Zacharewski, T. (2000). Differential estrogen receptor binding of estrogenic substances: A species comparison. J. Steroid Biochem. Mol. Biol. 74, 223–234.
Miyazaki, W., Iwasaki, T., Takeshita, A., Kuroda, Y., and Koibuchi, N. (2004). Polychlorinated biphenyls suppress thyroid hormone receptor-mediated transcription through a novel mechanism. J. Biol. Chem. 279, 18195–18202.
Miyoshi, H., Blmer, U., Takahashi, M., Gage, F. H., and Verma, I. M. (1998). Development of a self-inactivating lentivirus vector. J. Virol. 72, 8150–8157.
Moriyama, K., Tagami, T., Akamizu, T., Usui, T., Saijo, M., Kanamoto, N., Hataya, Y., Shimatsu, A., Kuzuya, H., and Nakao, K. (2002). Thyroid hormone action is disrupted by bisphenol A as an antagonist. J. Clin. Endocrinol. Metab. 87, 5185–5190.
Mukku, V. R., Kirkland, J. L., Hardy, M., and Stancel, G. M. (1983). Evidence for thyroid hormone receptors in uterine nuclei. Metabolism 32, 142–145.
Nakai, M., Tabira, Y., Asai, D., Yakabe, Y., Shimyozu, T., Noguchi, M., Takatsuki, M., and Shimohigashi, Y. (1999). Binding characteristics of dialkyl phthalates for the estrogen receptor. Biochem. Bipphys. Res. Commun. 254, 311–314.
Nieuwkoop, P. D., and Faber, J. (1975). Normal table of Xenopus laevis (DUADIN), 2nd ed., North-Holland Publishing, Amsterdam.
Nishihara, T., Nishikawa, J., Kanayama, T., Dakeyama, F., Saito, K., Imagawa, M., Takatori, S., Kitagawa, Y., Hori, S., and Utsumi, H. (2000). Estrogenic activities of 517 chemicals by yeast two-hybrid assay. J. Health Sci. 46, 282–298.
Okubo, T., Suzuki, T., Yokoyama, Y., Kano, K., and Kano, I. (2003). Estimation of estrogenic and anti-estrogenic activities of some phthalate diesters and monoesters by MCF-7 cell proliferation assay in vitro. Biol. Pharm. Bull. 26, 1219–1224.
Ouellet, M., Bonin, J., Rodrigue, J., DesGranges, J.-L., and Lair, S. (1997). Hindlimb deformities (ectromelia, ectrodactyly) in free-living anurans from agricultural habitats. J. Wildl. Diseases 33, 95–104.
Samuels, H. H., Stanley, F., and Casanova, J. (1979). Depletion of L-3,5,3'-triiodothyronine and L-thyroxine in euthyroid calf serum for use in cell culture studies of the action of thyroid hormone. Endocrinology 105, 80–85.
Sandau, C. D., Ayotte, P., Dewailly, E., Duffe, J., and Norstrom, R. J. (2000). Analysis of hydroxylated metabolites of PCBs (OH-PCBs) and other chlorinated phenolic compounds in whole blood from Canadian Inuit. Environ. Health Perspect. 108, 611–616.
Shimada, N., and Yamauchi, K. (2004). Characteristics of 3,5,3'-triiodothyronine (T3)-uptake system of tadpole red blood cells: effect of endocrine-disrupting chemicals on cellular T3 response. J. Endocrinol. 183, 627–637.
Soto, A. M., Sonnenschein, C., Chung, K. L., Femandez, M. F., Olea, N., and Serrano, F. O. (1995). The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants. Environ. Health Perspect. 103(Suppl. 7), 113–122.
Sugiyama, S., Miyoshi, H., and Yamauchi, K. (In press). Characteristics of a thyroid hormone responsive reporter gene transduced into a Xenopus laevis cell line using lentivirus vector. Gen. Comp. Endocrinol.
Thomas, C. R., Drake, J., and Frieden, E. (1992). Thyroid hormone receptor induction by triiodothyronine in tadpole erythrocytes in vivo and in vitro and the effect of cycloheximide and actinomycin-D. Gen. Comp. Endocrinol. 86, 42–51.
Tran, D. Q., Klotz, D. M., Ladlie, B. L., Ide, C. F., McLachlan, J. A., and Arnold, S. F. (1996). Inhibition of progesterone receptor activity in yeast by synthetic chemicals. Biochem. Biophys. Res. Commun. 229, 518–523.
Veldhoen, N., and Helbing, C. C. (2001). Detection of environmental endocrine-disruptor effects on gene expression in liver Rana catesbeiana tadpoles using a tail fin biopsy technique. Environ. Toxicol. Chem. 20, 2704–2708.
Vinggaard, A. M., Breinholt, V., and Larsen, J. C. (1999). Screening of selected pesticides for oestrogen receptor activation in vitro. Food Addit. Contam. 16, 533–542.
Wake, D. B. (1991). Declining amphibian populations. Science 253, 860.
Yamauchi, K., Prapunpoj, P., and Richardson, S. J. (2000). Effect of diethylstilbestrol on thyroid hormone binding to amphibian transthyretins. Gen. Comp. Endocrinol. 119, 329–339.
Yamauchi, K., Eguchi, R., Shimada, N., and Ishihara, A. (2002). The effects of endocrine-disrupting chemicals on thyroid hormone binding to Xenopus laevis transthyretin and thyroid hormone receptor. Clin. Chem. Lab. Med. 40, 1250–1256.
Yamauchi, K., Ishihara, A., Fukazawa H., and Terao, Y. (2003). Competitive interactions of chlorinated phenol compounds with 3,3',5-triiodothyronine binding to transthyretin: detection of possible thyroid-disrupting chemicals in environmental waste water. Toxicol. Appl. Pharmacol. 187, 110–117.
Zacharewski, T. (1998). Identification and assessment of endocrine disruptors: limitations and in vivo and in vitro assays. Environ. Health Perspect. 106, 577–582.
Zacharewski, T. R., Meek, M. D., Clemons, J. H., Wu, Z. F., Fielden, M. R., and Matthews, J. B. (1998). Examination of the in vitro and in vivo estrogenic activities of eight commercial phthalate esters. Toxicol. Sci. 46, 282–293.
Zoeller, R. T., Dowling, A. L. S., Herzig, C. T. A., Iannacone, E. A., Gauger, K. J., and Bansal, R. (2002). Thyroid hormone, brain development, and the environment. Environ. Health Perspect. 110(Suppl. 3), 355–361.
Zufferey, R., Dull, T., Mandel, R. J., Bukovsky, A., Quiroz, D., Naldini, L., and Trono, D. (1998). Self-inactvating lentivirus vector for safe and efficient in vivo gene delivery. J. Virol. 72, 9873–9880.(Shin-ichiro Sugiyama, Naoyuki Shimada, H)