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Department of Biology, Faculty of Science, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan
(Requests for offprints should be addressed to K Yamauchi; Email: sbkyama{at}ipc.shizuoka.ac.jp)
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Abstract |
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gene by T3. However, not all chemicals competed with T3 binding to the receptor at the same concentrations. Our results raise the possibility that the T3-uptake system on the plasma membrane of the tadpole RBCs could be a candidate target site for some EDCs and can modulate cellular T3 response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our recent studies have focused on the effects of endocrine-disrupting chemicals (EDCs) on the amphibian and avian thyroid systems, especially on T3 binding to the major plasma TH-binding protein, transthyretin (TTR), and to TR. These studies revealed that most EDCs tested at micromolar concentrations did not compete with T3 binding to TR but did compete with T3 binding to TTR (Yamauchi et al. 2000, 2002, 2003, Ishihara et al. 2003a, 2003b). This was in contrast to the steroid system where a number of chemicals are capable of competing with estrogen and progesterone binding to their respective receptors (Tran et al. 1996, Matthews et al. 2000). To understand the action of EDCs in the thyroid system, a research focus on processes other than the competitive binding to TR is required. Recent studies on the effects of agricultural chemicals on the thyroid system support their action on the processes other than the competitive inhibition of T3 binding to TRs. The herbicide acetochlor, for which amphibian TRs and TTRs had no significant affnity (Yamauchi et al. 2002, Ishihara et al. 2003a), altered TH-dependent gene expression and amphibian metamorphosis (Cheek et al. 1999, Veldhoen & Helbing 2001, Crump et al. 2002). Xenopus tadpoles previously exposed to methoprene did not respond to exogenously added T4 (Fort et al. 2000), although Xenopus TR and TTR showed no significant affnity for this chemical (Yamauchi et al. 2002). However, there have been few reports that focus on processes other than competitive binding of EDCs to TTR and TR in the thyroid system.
To elucidate the role of a T3-uptake system on the cellular T3 response and identify what kind of EDCs target the T3-uptake system and interfere with the thyroid system, we investigated the characteristics of T3 uptake into Rana catesbeiana tadpole red blood cells (RBCs). We chose 35 chemicals: 11 chemicals that inhibit the activity of TH transporters (Blondeau et al. 1988, McLeese & Eales 1996a) and 24 chemicals known to affect plasma TH levels in mammals (Brucker-Davis 1998), and examined their effects on [125I]T3 uptake into the tadpole RBCs. In this study, it was found that T3 was transported into tadpole RBCs by the System T-linked transporter. Several compounds, including EDCs, significantly inhibited [125I]T3 uptake in a dose- dependent manner. Furthermore, some of these inhibitors of the T3-uptake system suppressed the induction of the early primary T3-response gene tr by T3. We report here that the T3-uptake system on the plasma membrane of tadpole RBCs can modulate T3 signaling by controlling the amount of intracellular T3, and that it could be a candidate target site for some EDCs that affect the thyroid system.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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[125I]T3 (122 MBq/µg; carrier-free) was purchased from NEN Life Science Products (Boston, MA, USA). Unlabeled T3, bisphenol A, 1-aminopyrene, benzo[a]pyrene, pentachlorophenol, 2-amino[2,2,1]heptane-2-carboxylic acid (BCH), monodansylcadaverine and ouabain were obtained from Sigma (St Louis, MO, USA). Ethinylestradiol, dicyclohexyl phthalate, tributyltin chloride, benzophenone, bis(2-ethylhexyl) adipate, p-t-octylphenol, mirex (dodecachloropentacyclo [5.3.0.02,6.03,9.04,8]decane, analytical standard), malathion (diethyl(dimethoxythio-phosphorylthio)succinate, analytical standard), dicofol (Kelthane; 2,2,2-trichloro-1,1-bis (4-chlorophenyl)ethanol, analytical standard), L-tryptophan, L-phenylalanine, L-leucine, N-ethylmaleimide, phloretin, sodium taurocholate and choline chloride were purchased from Wako Pure Chemicals (Osaka, Japan). Ioxynil (3,5-di-iodo-4-hydroxybenzonitril, analytical standard) and acetochlor (2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-methylphenyl) acetamide) were purchased from Riedel-de Haën (Seeize, Germany). Methoprene ((E,E')-1-methylethyl-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate (9 Cl)) was from Ehrenstorfer Quality (Augsburg, Germany). 4-Nonylphenol, di-n-butyl phthalate and n-butylbenzyl phthalate were obtained from Nacalai Tesque (Kyoto, Japan). 2,4-Dichlorophenoxyacetic acid, di-2-ethylhexyl phthalate, diethyl phthalate and 2,4-dinitrophenol were from Kanto Chemicals (Tokyo, Japan), and diethylstilbestrol (DES) was from Tokyo Chemical Industry (Tokyo, Japan). Bromosulfophthalein was purchased from ICN Biochemicals (Irvine, CA, USA). All other chemicals used in this study were of chromatography grade or the highest grade available and purchased from Wako Pure Chemicals or Nacalai Tesque.
All chemicals tested as EDCs, except for 1-aminopyrene, benzo[a]pyrene and mirex, which were dissolved in benzene to a concentration of 10 mM, were dissolved in ethanol to concentrations of 5–10 mM. phloretin and T3 were dissolved in dimethylsulfoxide to concentrations of 0.6–200 mM. Monodansylcadaverine was dissolved in methanol to a concentration of 50 mM. Tryptophan, leucine, phenylalanine, bromosulfophthalein, BCH, sodium taurocholate, ouabain, N-ethylmaleimide and choline chloride were dissolved in frog Ringer solution (111 mM NaCl, 3.4 mM KCl, 2 mM CaCl2 and 2.4 mM NaHCO3) or 70% Leibovitz’s L-15 medium (Sigma). These chemicals were diluted with the frog Ringer solution or 70% Leibovitz’s L-15 medium to give less than 0.5% (v/v) organic solvent. Control assays were performed in the presence of the corresponding solvent only and at the same concentrations. The organic solvents added did not affect the assays for T3 uptake, binding or cellular response.
Preparations of RBCs
R. catesbeiana tadpoles at stages X–XV (Taylor and Kallros 1946) were collected from ponds in Saitama Prefecture and Shizuoka Prefecture, Japan. The tadpoles were maintained in aerated, dechlorinated tap water at 20– 25 °C, unless otherwise noted, and fed boiled spinach three times a week. They were anesthetized by immersion in 0.2% ethyl 3-aminobenzoate methanesulfonic acid (Sigma). RBCs were prepared as described previously (Yamauchi et al. 1989). In brief, the truncus arteries of the tadpoles were cut with scissors and blood samples were collected into tubes containing heparin or sodium citrate. The plasma was separated from the blood cells by centrifugation at 1500 g for 15 min at 4 °C. After removal of the plasma and the buffy coat, the RBC fraction was further washed at least seven times with the frog Ringer solution or 70% Leibovitz’s L-15 medium. The number of RBCs was estimated by counting under a microscope with the use of a hematocytometer. The RBC suspension was kept on ice and used within a day. For 2-day experiments, the RBC suspension was used at 25 °C immediately after preparation.
Uptake of [125I]T3 into tadpole RBCs
Tadpole RBCs were incubated in the frog Ringer solution or 70% Leibovitz’s L-15 medium at 23 °C. Uptake was initiated by mixing the RBC suspension (1.0x106 cells/ tube) with [125I]T3 solution at the same temperature, adjusting the final concentration of [125I]T3 to 0.1 nM in the presence or absence of 5 µM unlabeled T3. To examine the effect of chemicals, the RBC suspension was mixed with [125I]T3 solution containing each chemical at 23 °C without pre-incubation. After 2 min incubation at 23 °C, the cell-associated [125I]T3 and free [125I]T3 were separated by the oil-centrifugation method (Yamauchi et al. 1989, 2000). The cells were immediately separated from free [125I]T3 in the incubation medium by centrifugation at 9100 g for 2 min. The tip of the tube containing the cell pellet was cut off. The amount of [125I]T3 associated with cells and the amount of [125I]T3 remaining in the extracellular fraction were determined in a gamma scintillation counter (Auto Well Gamma System ARC-2000, Aloka, Japan).
Kinetic studies were carried out at 23 °C for 2 min. Kinetic parameters were determined by fitting the plot of initial velocity (Vi) versus T3 concentration (S) to the Michaelis–Menten equation: Vi=Vmax/(1+Km/S), where Vmax is the maximum uptake rate and Km is the Michaelis constant.
RNA analysis by real-time PCR
Tadpole RBCs (5.0x106 cells/35 mm dish) were incubated in 70% of Leibovitz’s L-15 medium for 2 days at 25 °C with solvent only or each chemical in the presence or absence of 10 nM T3. The medium did not change during the incubation. Total RNA was extracted from tadpole RBCs using a QIAamp RNA Blood Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s directions. The RNA (5 µg per lane) was electrophoresed in 1% agarose gel containing 2.6 M formaldehyde to check the integrity of the RNA samples. Amounts of specific RNA species were estimated by real-time PCR using SYBR Green Master Mix and an ABI Prism 7000 thermocycler (Applied Biosystems, Foster City, CA, USA) after the RNA samples were treated with reverse transcriptase (TaqMan Reverese Transcription Reagents; Applied Biosystems). Each PCR was run in triplicate to control for PCR variation. The endpoint used in real-time PCR quantification, Ct, is 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 staring material. Quantification was determined by applying the 2–Ct formula and calculating the average of the three values obtained for each sample. To standardize each experiment, the amount of bullfrog TR (bTR) transcript was divided by the amount of 18 S rRNA in the same samples. Primer sequences used were as follows: bTR transcript sense 5'-GCGTCGGAAGGA GGAGATG-3' (nucleotides 229–247) and antisense 5'-TCCCACTCCTCGCTGCTT-3' (294–277; Schneider & Galton 1991); and 18 S rRNA sense 5'-TGTGC CGCTAGAGGTGAAATT-3' (908–928) and anti-sense 5'-TGGCAAATGCTTTCGCTTT-3' (970–952), which were designed by comparing the corresponding Xenopus laevis and human sequences (Maden 1986).
[125I]T3-binding assay
The ligand-binding domain of bTR fused with glutathione S-transferase (GST-bTR LBD) was expressed in Escherichia coli and then purified from the cell extract as described previously (Ishihara et al. 2003a). Competitive [125I]T3 binding was performed with solvent only or each chemical, which was diluted with a buffer (10 mM Tris/HCl, pH 7.5, 1.5 mM EDTA, 1 mM dithiothreitol and 10% (v/v) glycerol) containing [125I]T3, at defined concentrations. [125I]T3 binding was initiated by mixing 50 µl GST-bTR LBD (150 ng/tube) in the buffer with 200 µl of the above buffer containing [125I]T3 with or without each chemical at 4 °C, adjusting the final concentration of [125I]T3 to 0.1 nM. After 1.5 h incubation at 4 °C, protein-bound [125I]T3 was separated from free [125I]T3by the Dowex method (Lennon et al. 1980, Lennon 1992). Radioactivity was measured in a gamma scintillation counter. The amount of [125I]T3 bound non- specifically was derived from the radioactivity of the samples incubated with 1 µM unlabeled T3. The non- specific binding value was subtracted from the amount of total bound T3 to give the value of specifically bound [125I]T3.
Statistical analysis
The data are presented as means ± S.E.M. 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. The number of observations (n) is shown in parentheses.
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Results |
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[125I]T3 uptake into tadpole RBCs reached equilibrium by 10 min at 23 °C. As the initial uptake was approximately linear for at least the first 3-min period, the Vi of uptake was routinely measured after 2 min of incubation. Saturability of T3 uptake was demonstrated by measuring [125I]T3 uptake in the presence of 5 µM unlabeled T3. Approximately 85% of the VI of [125I]T3 uptake was inhibited by 5 µM unlabeled T3 (data not shown). These experiments suggest that a saturable uptake system is responsible for transporting most of the circulating T3 into RBCs.
Saturable uptake conformed to Michaelis–Menten kinetics. RBCs were incubated with 0.1 nM [125I]T3 and various concentrations of unlabeled T3 (S, ranging from 10–8 to 10–6 M). The ratio of S/ Vi was calculated and plotted as a function of S according to the Hanes representation (Fig. 1
). The plots are linear and repeated experiments gave a Km of 110 ± 10 nM and a Vmax of 2.5 ± 0.3 pmol/min per 106 cells in the frog Ringer solution (n=3) and a Km of 170±20 nM and a Vmax of 2.3 ± 0.2 pmol/min per 106 cells in 70% Leibovitz’s L-15 medium (n=5). The Km value was 1.5 times greater when 70% Leibovitz’s L-15 medium was used instead of the frog Ringer solution. It is believed that this culture medium may have contained some competitors that interfered with the saturable T3-uptake system of tadpole RBCs.
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). Of the three amino acids tested, tryptophan was the most powerful inhibitor while phenylalanine was a weak inhibitor and leucine had no effect, at 1 mM. The effect of BCH, a prototypical substrate of the amino acid transporter System L, was weak even at 5 mM. Organic anions, bromosulfophthalein and taurocolate were strong inhibitors. Bromosulfophthalein, at the concentration of 0.3 mM, inhibited [125I]T3 uptake by more than 90%. Choline chloride (111 mM), substituted for sodium chloride in the frog Ringer solution, showed no effect. N-Ethylmaleimide (1 mM), a thiol reagent, inhibited only 20% of the saturable T3 uptake. Ouabain (1 mM) did not inhibit, but phloretin (0.1 mM) and monodansylcadaverine (0.5 mM) inhibited the saturable T3 uptake by 80%.
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shows dose-dependent curves of eight selected compounds, including unlabeled T3, on the saturable initial uptake of [125I]T3 into tadpole RBCs in the frog Ringer solution. The most effective compound was unlabeled T3. The rank order affnity was T3 (the concentration of T3 required for 50% inhibition of saturable [125T3I]T3 (IC50)=0.14 µM)> phloretin (52 µM)=monodansylcadaverine (59 µM)>bromosulfophthalein (75 µM)>taurocholate (270 µM)>tryptophan (470 µM)>> leucine=BCH (Table 2). Leucine and BCH had no effect at all concentrations tested.
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Saturable initial uptake of [125I]T3 into the tadpole RBCs was examined in the presence of various chemicals (8 µM) at 23 °C (Fig. 3). As unlabeled T3 inhibited completely the saturable initial uptake of [125I]T3 at 5–8 µM, a concentration of 8 µM for the chemicals was chosen to determine their relative potencies. T4 inhibited the saturable initial uptake of [125I]T3 by 60%, suggesting that this uptake system can recognize both T3 and T4 but is more specific for T3 than for T4. Of the five xenoestrogens, ethinylestradiol, DES, p-t-octylphenol, 4-nonylphenol and bisphenol A, ethinylestradiol and DES were the most potent inhibitors: approximately 50% of the saturable initial uptake of [125I]T3 was inhibited. Of the two pyrene compounds, benzo[a]pyrene activated the uptake up to 150%. Among the five phthalates, di-n-butyl phthalate and n-butylbenzyl phthalate were the most potent inhibitors: approximately 70–80% of the saturable initial uptake of [125I]T3 was inhibited. Of the other chemicals, including agricultural chemicals, dicofol was the most powerful inhibitor: more than 80% of the saturable initial uptake of [125I]T3 was inhibited. Mirex and tributyltin accelerated the saturable initial uptake of [125I]T3 up to 150 and 290%, respectively.
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shows dose-dependent curves of the five selected chemicals, including unlabeled T3, on the saturable initial uptake of [125I]T3 into tadpole RBCs in 70% Leibovitz’s L-15 medium. The IC50 values of di-n-butyl phthalate, n-butylbenzyl phthalate and dicofol were all 2.2 µM (Table 2
). The concentration of tributyltin required to accelerate the saturable initial uptake of [125I]T3 up to 200% (ED200) was 3.2 µM (Table 2). These effective concentrations were one order of magnitude greater than the IC50 of T3 (0.24 µM), but may be less than the IC50 T4 of (approximately 8 µM; see Fig. 3) although we did not determine the accurate IC50 value of T4.
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transcript by T3 in tadpole RBCs
To evaluate the biological effect of the inhibitors of the saturable T3-uptake system on T3 signaling in tadpole RBCs, we examined their interference with the induction of the early primary T3-response gene, tr gene, by 10 nM T3. The addition of T3 increased the amount of the TR transcript by 6–7 fold. Further addition of mono-dansylcadaverine (final concentration of 60 µM), phloretin (50 µM), bromosulfophthalein (1 µM), DES (8 µM), ethinylestradiol (8 µM) and dicofol (0.4 µM), depressed significantly the T3 response (Fig. 5). Di-n-butyl phthalate, but not n-butylbenzyl phthalate, had a signifi-cant synergic effect on the induction of the TR transcript by T3 at 8 µM, the concentration at which the saturable initial uptake of [125I]T3 was strongly inhibited by these phthalates (Fig. 3). This effect was abolished in the absence of T3. Bisphenol A (8 µM) and ioxynil (0.2 µM) inhibited the induction of the TR transcript by T3 although they did not inhibit the saturable initial uptake of [125I]T3 at the same or higher concentrations (Fig. 3). We could not evaluate the effect of tributyltin on the induction of the TR transcript by T3 because of its severe cytotoxity at 10–7-10–5 M.
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transcript by T3 interact directly with TR to compete with T3 binding, we next examined their effect (at the same concentrations) on [125I]T3 binding to purified GST-bTR LBD (Fig. 6). Phloretin, bromosulfophthalein and DES inhibited [125I]T3 binding to GST-bTR LBD; however, mono-dansylcadaverine, ethinylestradiol, bisphenol A, dibutyl phthalate, ioxynil and dicofol showed no effect on [125I]T3 binding to the GST-bTR LBD at concentrations that affected the induction of the TR transcript by T3. The T3-binding activity of GST was negligible when purified GST, which was expressed from pGEX-6P-3 in E. coli, was substituted for the GST-bTR LBD fusion protein in the assay.
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Discussion |
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). These were consistent with the inhibitory mode of the T3-uptake system of rat and rainbow trout RBCs (Zhou et al. 1990, McLeese & Eales 1996a) and rat liver (Blondeau et al. 1988), some of which were characterized as System T-linked T3-uptake system (Osty et al. 1988, Samson et al. 1992). The fact that the T3-uptake system in tadpole RBCs was sensitive to taurocholate and bromo-sulfophthalein but was insensitive to choline suggests the possibility that some of Na+-independent organic anion transporters (Abe et al. 1998, 1999, Fujiwara et al. 2001) are involved in the tadpole T3-uptake system. The Km values obtained from our studies were within the same range of those values obtained for the System T-linked T3-uptake system (Blondeau et al. 1988, Osty et al. 1988, Zhou et al. 1990, McLeese & Eales 1996b), but one order of magnitude lower than those values obtained for System L (Blondeau et al. 1993, Ritchie et al. 1999, Ritchie & Taylor 2001, Friesema et al. 2001) and the organic anion transporters (rat oatp2 and oatp3, human LST-1 and OATP-E; Abe et al. 1998, 1999, Fujiwara et al. 2001). Therefore, the T3-uptake system that is responsible for a major part of saturable T3 uptake into tadpole RBCs may be the System T-linked uptake system and may be a common feature for rat, bullfrog and rainbow trout RBCs. This is the first report demonstrating the existence of the System T-linked T3-uptake system in frogs. The expression of this T3-uptake system in RBCs has probably been conserved during vertebrate evolution. Tadpole RBCs (larval type) are specifically removed from the bloodstream during metamorphic climax stages by apoptotic cell death under the influence of THs (Hasebe et al. 1998). Therefore, the system T-linked T3-uptake system on the plasma membrane would play an important role in T3-induced apoptosis in tadpole RBCs. The Km value determined in 70% Leibovitz’s L-15 medium was 1.6 times greater than that determined in the frog Ringer solution. As Leibovitz’s L-15 medium contains 1.5 mM phenylalanine and 0.1 mM tryptophan, it is likely that these amino acids compete with saturable T3 uptake into tadpole RBCs.
The effect of N-ethylmaleimide on System T- linked T3-uptake systems was contradictory. N-Ethylmaleimide, at a concentration of 1 mM, hardly inhibited saturable [125I]T3 uptake into tadpole RBCs (Table 1). Our result was consistent with the report for rat hepatocytes (Blondeau et al. 1988). Like us, Blondeau et al. examined [125I]T3 uptake in the presence of N-ethylmaleimide without preincubation with it. The saturable [125I]T3 uptake into rat and rainbow trout RBCs was strongly inhibited by N-ethylmaleimide when the cells were pretreated with it at 0.5–1.0 mM for 10–15 min (Zhou et al. 1990, McLeese & Eales 1996a). However, Kemp & Taylor (1997) found that approximately 40% of the saturable [125I]T3 uptake into sinusoidal membrane vesicles obtained from rat liver homogenate, corresponding to the System T-linked uptake system, was N-ethylmaleimide-resistant after preincubation with 1 mM N-ethylmaleimide even for 0.5 min. cDNA for Na+-independent aromatic amino acid transporter was cloned from rat small intestine and its nucleotides were sequenced. However, T3 and T4 did not exhibit significant effects on this transporter-mediated [14C]tryptophan uptake, and the transporter hardly accelerated uptake of [125I]T3 and [125I]T4into cells (Kim et al. 2001). Very recently, Friesema et al.(2003) cloned a cDNA for System T-related transporter from rat liver and characterized it. This transporter, termed monocarboxylate transporter 8, induced [125I]T3 uptake in a Xenopus oocyte expression system with a Km of 4 µM, but did not transport tryptophan and phenylalanine. Therefore, several types of System T- and/or System T-linked T3 uptake system may exist.
We found that di-n-butyl phthalate, n-butylbenzyl phthalate, dicofol and tributyltin, when added to 70% Leibovitz’s L-15 medium, affected the Vi of the saturable [125I]T3 uptake with IC50 (or ED200 for tributyltin) of 2.2–3.2 µM (Fig. 4 and Table 2). Tributyltin accelerated the initial uptake of [125I]T3 into tadpole RBCs at 10 –6 M in a 2-min treatment; however, it showed severe cytotoxity with hemolysis at 10–7-10–5 M in a 2-day treatment. Therefore, it is doubtful that tributyltin exerts EDC activity mediated by the T3-uptake system at these concentrations in vivo. These phthalates have been produced abundantly as plasticizers. Dicofol is a chlorinated miticide, which has been applied at high doses to defined agricultural land. Tributyltin has been used in antifouling paints. All of these chemicals are distributed at high concentrations in their special environments. Our results propose a novel mechanism that some chemicals interfere with the thyroid system owing to inhibition or activation of the activity of the T3-uptake system on the plasma membrane. However, from the above result alone, we cannot simply explain to what degree the suppression or acceleration of saturable [125I]T3 uptake influences the T3-signaling pathway in target cells.
To facilitate our understanding of the effect of the chemicals used in this study on TH signaling in tadpole RBCs, their possible target sites are summarized in Table 3, where the chemicals are divided into five groups according to their mode of action. The first group includes the chemicals that inhibited both the T3-uptake system and T3 binding to TR: phloretin and DES. The second group consists of the chemicals that inhibited the T3-uptake system but not T3 binding to TR: monodansylcadaverine and ethinylestradiol. Conversely, the chemical belonging to the third group, bromosulfophthalein, did not inhibit the T3-uptake system but did inhibit T3 binding to TR. The fourth group includes the chemicals that inhibited neither the T3-uptake system nor T3 binding to TR: ioxinyl, dicofol and bisphenol A. All chemicals belonging to the first four groups suppressed the induction of the TR transcript by at the indicated concentrations. Therefore, the chemicals in the fourth group would target some process other than the two processes we investigated here. The fifth group contains those chemicals that inhibited T3 uptake but not T3 binding to TR and that did not suppress the induction of TR transcript by T3. Di-n-butyl phthalate, a chemical belonging to the fifth group, activated synergically the induction of the TR transcript with T3 by an unknown mechanism. These results indicate that thyroid-disrupting chemicals target several processes within the thyroid system in a chemical-dependent manner, although at present we cannot elucidate which process when inhibited by the chemicals exerts the most profound effect on the thyroid system. The present study raises the possibility that the T3-uptake system could be a candidate target for these chemicals belonging to the first and second groups.
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gene in 2-day cultured RBCs, in the presence of EDCs. The difference in the cellular responses to EDCs between the 2-min and 2-day treatments needs to be considered.
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Acknowledgements |
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LBD. We are also grateful to Ms Julie Monk for a thorough and critical reading of the manuscript. This work was supported by a Grant-in Aid for Scientific Research on Priority Area (2) (no. 14042223) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a Grant-in Aid for Scientific Research (B) (nos 13559001 and 16310038) from the Japan Society for the Promotion of Science. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work. |
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Received 5 July 2004
Accepted 25 August 2004
Made available online as an Accepted Preprint 9 September 2004
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) or the frog Ringer solution (•) containing 0.1 nM [125I]T3 and various concentrations (S) of unlabeled T3 to determine initial velocity (Vi). Values are means of triplicates. Kinetic constants were obtained by linear regression. The abscissa intercepts of the lines correspond to –Km, the ordinate intercepts to Km/Vmax and the slopes to 1/Vmax. The graphs were drawn after subtraction of nonsaturable uptake, as described in the Materials and Methods section.
), leucine (
) or BCH (
) in (A), and phloretin (
