2022 年 2 巻 p. 54-59
The human thyroid receptor (hTR)-agonist activities of 796 compounds were evaluated using a yeast two-hybrid assay in this study. A total of 17 compounds exhibited agonist activity at tenfold the effective concentration (EC×10) [0.0083–7,500 nM]. Additionally, TRIAC, TETRAC, and GC-1, which exhibited 30–200 times higher activities than T3 and T4, may cause thyroid-hormone receptors (TR) activity at extremely low concentrations in real environmental water. Moreover, 3-chloro-3’,5,5’-triiodo-L-thyronine exhibited high TR-agonist activity for the first time. Chemicals with a structure equivalent to thyroid hormones exhibited TR-agonist activity regardless of the TR type. Information on hTR-agonist activity is important because previous studies, such as those involving EDSP21, have focused exclusively on rTR-agonist activity. Therefore, the knowledge gained from the present study will boost chemical regulation strategies benefiting human and wildlife.
Chemical compounds are extremely important for the development of human life, and it is suspected that numerous chemicals of anthropogenic origin exist in the environment (Binetti et al., 2008). These chemicals have an effect on human health via the endocrine system (Harrison et al., 1997), specifically through estrogen-hormone receptors (ERs), androgen-hormone receptors (ARs), and thyroid-hormone receptors (TRs). The thyroid hormones (THs) 3,5,3’-triiodothyronine (T3) and 3,3’,5,5’-tetraiodothyronine (T4) are crucial for development, metabolism, and homeostasis in vertebrates (Yen, 2001), and their disruption in humans can cause adverse effects such as tachycardia, atrial arrhythmias, and heart failure.
The ER- and AR-agonist activities of 583 chemicals were measured previously, using a yeast two-hybrid assay system (Shiraishi et al., 2018). However, the use of the assay on environmental samples revealed that only a few chemicals exhibited ER-agonist activity (Yagishita et al., 2019). In this context, research using similar recombinant receptor-reporter gene assays on TR activity has been limited to a small number of compounds, viz. a few TH metabolites and halogenated phenols (Freitas et al., 2011; Terasaki et al., 2011). Correspondingly, TR activity in environmental samples was reported by various in vitro assays (Ishihara et al., 2009; Jugan et al., 2009; Kassotis et al., 2016). However, exploring chemicals with TR activity in the environment has not progressed. It could be due to a lack of knowledge about chemicals with TR activity. Chemical impacts on the environment were evaluated overall, using Whole Effluent Toxicity methods. Understanding the various receptor-binding activities of chemicals is important for developing engineering technology and human health risk assessment strategies for remediating environmental pollution; for example, this information is necessary to develop adverse outcome pathways (AOPs) (Browne et al., 2017). AOP information is crucial for developing integrated approaches to testing and assessment, which aim to reduce the use of in vivo assays in the regulation of chemicals (OECD, 2017).
In this study, we aimed to determine the hTR-agonist activity of 796 compounds using a yeast two-hybrid assay (TR yeast-cell assay). The 796 compounds selected as test chemicals were suspected to be endocrine disruptors and include all compounds with rat TR (rTR)-agonist activity that has been published in EDSP21. Additionally, we consulted the TR information in the 2016 Extended Tasks on Endocrine Disruption (EXTEND, 2016). Accordingly, the majority of compounds suspected to have TR-agonist activity were evaluated, and useful knowledge about the hTR-agonist activity was obtained in this study.
All solvents were of the same grade as those used for testing pesticide residues and polychlorinated biphenyls (Nacalai Tesque, Inc., and FUJIFILM Wako Pure Chemical Corporation). Native standards were purchased from a wide range of chemical suppliers with a minimum purity of 95%. The stock solution of each test chemical was prepared in dimethyl sulfoxide (DMSO) and stored at −30°C in the dark.
YEAST-TWO-HYBRID ASSAYWe used a yeast two-hybrid thyroid-hormone assay system using yeast cells (Saccharomyces cerevisiae Y190) in which the human thyroid receptor hTRα and the coactivator, transcriptional intermediary factor-2 (TIF2), were introduced. The conjugation procedure was adapted to use a 96-well culture plate with a chemiluminescent reporter gene (for β-galactosidase) (Shiraishi et al., 2000, 2003). Yeast cells were preincubated for 24 h at 30°C with shaking in a modified SD medium (lacking tryptophan and leucine, 0.88% dextrose). The cell density was adjusted to achieve an absorbance of 1.75–1.84 at 595 nm. The medium (60 μL) was placed in the wells of the first row of a black 96-well culture plate for chemiluminescence measurement. All wells were loaded with the medium (60 μL, containing 0.2% DMSO). A solution of the test compound (1 mM in DMSO, 20 μL) was added to 480 μL of the medium, and the aliquots of this mixture (60 μL) were added to the wells of the first row in the plate. The test solution was serially diluted from rows 1 to 7 (each duplicate), and then the yeast-cell suspension (60 μL) was added to each well (including those in row 8, which served as the solvent control). Thus, the first row contained a 10,000 nM solution of the test chemical, the second row a 2,500 nM solution, and so forth. After adding the yeast suspension and vortex mixing, the plates were incubated at 30°C with high relative humidity for 4 h. Subsequently, a total of 50 μL of lysis solution, prepared using 2.0 mg of zymolyase 100T (from Arthrobacter luteus, Nacalai Tesque, Kyoto, Japan) in 7 mL of buffer solution, was added to each well of the plate for enzymatic digestion. The plate was then incubated at 37°C for 1 h after agitation. Following that, a total of 80 μL of the substrate (Tropix Gal-Screen Substrate, Applied Biosystem) and enhancer (Sappire-II, Applied Biosystem) solution in phosphate buffer for inducing chemiluminescence from the released β-galactosidase was added to each well, and the plate was incubated at 30°C for 10 min after agitation. The plate was placed in a 96-well plate luminometer (Luminescencer JNRAB2100, Atto Corp., Tokyo, Japan). The chemiluminescence produced by β-galactosidase in each well was measured. The agonist activity was then recorded as the ten-fold effective concentration (EC×10), which is defined as the concentration of the test solution that produces a chemiluminescent signal intensity 10 times that of the blank control, i.e., pure DMSO aqueous solution. In brief, activity values were determined from the linear regressions of the dose-response curve, which was obtained by plotting the luminescence intensity against the concentration of the test chemical. The hTR-agonist activity of test compounds exhibiting it in the first assay was evaluated in three replicates on different days to ensure precision.
DATA ANALYSISAll data analyses were performed with R software ver. 3.6.1 and the extension package drc. Dose-response curves were obtained by plotting the luminescence intensity against the applied concentration of each compound for each assay and were calculated by the logistic model (equation 1).
| (1) |
In this study, the hTR-agonist activities of 796 compounds were evaluated using the TR yeast-cell assay (Table S1). In total, 17 compounds exhibited hTR-agonist activity in this study (Table 1). Except for the 5 phenol compounds and triclabendazole, 11 of those compounds had distinct structures similar to T3 and T4 THs. These 11 compounds exhibited greater agonistic activity than the remaining 6 compounds (Table 1), while some TH-analogs exhibited greater agonistic activity than T3 and T4. Dose-response curves of the 17 compounds are shown in Fig. S1. Moreover, the order of their hTR-agonist activities is shown in Fig. S2, and the hTR-agonist activities of monohydroxy polychlorinated biphenyls (OH-PCBs) are included in this figure because their activities were evaluated using the same assay and calculated at the same endpoint as EC×10 (Shiraishi et al., 2003).
| Chemical names | CAS | M.F. | Y2H (nM) | EDSP21 (nM) |
|---|---|---|---|---|
| 3,3’,5-Triiodothyroacetic acid (TRIAC) | 51-24-1 | C14H9I3O4 | 0.0083 (0.0065–0.01) | 0.010 |
| 3,3’,5,5’-Tetraiodothyroacetic acid (TETRAC) | 67-30-1 | C14H8I4O4 | 0.014 (0.012–0.016) | 0.93 |
| Sobetirome (GC-1) | 211110-63-3 | C20H24O4 | 0.051 (0.047–0.055) | — |
| 3,3’,5,5’-Tetraiodothyronine (T4) | 51-48-9 | C15H11I4NO4 | 1.7 (1.3–2.1) | 6.1 |
| 3,5,3’-Triiodothyronine (T3) | 6893-02-3 | C15H12I3NO4 | 1.7 (0.93–2.5) | 1.1 |
| 3,3’,5-Triiodo-l-thyronine sodium salt (T3Na) | 55-06-1 | C15H11I3NNaO4 | 1.9 (1.7–2.1) | 0.17 |
| 3,5-Diiodothyropropionic acid (DITPA) | 1158-10-7 | C15H12I2O4 | 3.0 (2.9–3.1) | — |
| 3-Chloro-3’,5,5’-triiodo-L-thyronine (Cl-3-T3) | 909279-46-5 | C15H11ClI3NO4 | 4.8 (4.2–5.4) | — |
| 3,3’,5’-Triiodo-L-Thyronine (rT3) | 5817-39-0 | C15H12I3NO4 | 7.0 (6.4–7.7) | 218 |
| N-Acetyl L-Thyroxine | 26041-51-0 | C17H13I4NO5 | 24 (13–35) | — |
| 3-Iodo-L-thyronine (T1) | 10468-90-3 | C15H14INO4 | 135 (132–139) | — |
| Tetrachlorobisphenol A | 79-95-8 | C15H12Cl4O2 | 177 (135–219) | 1.0×109 |
| Tetrabromobisphenol A | 79-94-7 | C15H12Br4O2 | 530 (416–643) | 1.0×109 |
| Triclabendazole | 68786-66-3 | C14H9Cl3N2OS | 2,272 (1,987–2,556) | 514 |
| 3,3’,5-trichlorobisphenol A | 40346-55-2 | C15H13Cl3O2 | 3,544 | — |
| 2-chloro-4-octylphenol | 79162-47-3 | C14H21ClO | 3,921 | — |
| 2,6-dichloro-4-tert-butylphenol | 34593-75-4 | C10H12Cl2O | 7,513 | — |
Except for 3,3’,5-trichlorobisphenol A, 2-chloro-4-octylphenol, and 2,6-dichloro-4-tert-butylphenol, all activity values obtained using the TR yeast-cell assay are presented as the mean of three replicates. M.F. indicates molecular formula. A hyphen indicates “no evaluation in the EDSP21.” The activities of the TR yeast-cell assay were calculated as EC×10 (nM), and those of EDSP21 were evaluated as AC50 (activity concentration at half-maximal response) (nM). 95% confidence intervals (95% CIs) for 14 compounds evaluated using the TR yeast-cell assay are indicated in the parentheses.
TRIAC, TETRAC, and GC-1 exhibited extremely strong agonistic activity. TRIAC is a substrate for deiodinase, which is a primordial bioactive TH found in the protochordate amphioxus (Klootwijk et al., 2011; Köhrle, 2019). TETRAC is a naturally occurring T4 metabolite found in human serum and a precursor to TRIAC (Köhrle, 2019). GC-1, also known as sobetirome and QRX-431, is a synthetic thyroid-hormone analog that acts as a full agonist for both TRs, leading to a decrease in cholesterol, lipoprotein, and triglycerides in the plasma (Chiellini et al., 2002; Baxter et al., 2004; Scanlan 2010; Coppola et al., 2014). These are potent TR ligands that have been regarded as major concerns (Sorimachi and Yasumura, 1981; Köhrle, 2019), and their TR-agonist activity has been reported (Freitas et al., 2011; Gierach et al., 2012; Leusch et al., 2018). The order of TR-agonist activities in this study is consistent with previous studies, as is the fact that TRIAC exhibited the highest activity level of the chemicals tested. The TR-agonist mechanisms of the analogs, such as TRIAC and GC-1, have been determined, and some parameters, such as molecular size, are strongly related to their TR-agonist activity (Wagner et al., 2001; Martínez et al., 2009; Gierach et al., 2012).
Two TH metabolites, T3Na and 3-Cl-T3, showed high hTR-agonist activity in this study; this is the first time the TR-agonist activities of both chemicals have been reported. T3Na, also known as liothyronine, has been used as a therapeutic agent in conditions such as hypothyroidism and myxedema coma. In this study, T3Na exhibited the same agonistic activity as T3. This result contradicts the one published by EDSP21 (EPA, 2021). T3Na may be chemically similar and pharmacologically equivalent to T3 because it is a salt of T3; therefore, the TR activities of T3 and T3Na are theoretically equivalent. However, the results of this study and those of EDSP21 were quite dissimilar. This could be a result of the assay systems used, such as the test solution. The assay system, especially the composition of the test solution, may affect the number of hydrogen bonds formed between the ligands (T3Na and T3) and TR. It has been suggested that 3,5-dimethyl-3’-isopropylthyronine (Dimit), a T3 analog, forms a hydrogen bond with the amino acids in the TR via its carboxylate group (Wagner et al., 1995). Moreover, the same type of linking was confirmed in an interaction between TRIAC and TR, implying that only the oxygen in the TRIAC carboxylate group interacted with the amino acids in the TR via the hydrogen of the amino acids (Martínez et al., 2009). This interaction between T3 and TR may be due to the fact that Dimit and TRIAC are T3 analogs with carboxylate groups. As a result, the hydroxyl group in the T3 carboxylate must be charged. Without a charged hydroxyl group, the interaction is weakened. The interaction is inversely proportional to the TR-agonist activity (Wagner et al., 1995, 2001; Martínez et al., 2009). In T3Na, sodium and oxygen form an ionic bond, whereas the oxygen and hydrogen in T3 carboxylate form a covalent bond. In terms of the bonding power, an ionic bond has a lower bonding strength than a covalent bond. This difference in bond type could be related to the rate of oxygen dissociation in T3Na and T3 during exposure in those bioassays. The hypothesis, however, could not be confirmed in this study, and no supporting studies for our hypothesis were included. Therefore, further study on this subject is necessary.
3-Cl-T3 is a chlorinated derivative of 3,3’,5’-triiodo-L-thyronin (rT3). In this study, 3-Cl-T3 exhibited TR-agonist activity equivalent to that of T3 and T4, implying that chlorine substitution at position 3 of the inner ring of rT3 leads to increasing rT3 activity. The outer ring perpendicular to the inner ring is maintained by the iodine located at positions 3 and 5 in the inner ring, and the TH activity of the chemicals is closely related to its molecular structure (Shiraishi et al., 2003). Therefore, the chlorine substitution in rT3 is expected to induce an optimal molecular structure for TR-agonist activity.
Although the five phenol compounds and triclabendazole were found to be TR-agonistic, their activity was relatively low. TBBPA and TCBPA, which are halogenated derivatives of bisphenol A, were found to exhibit TR-agonist activity (Kitamura et al., 2002; Freitas et al., 2011; Terasaki et al., 2011). Additionally, the molecular structure of the compounds affecting these TR-agonist activities was determined (Arulmozhiraja et al., 2005). Therefore, the positive results obtained with the five phenolic compounds were not surprising.
DIFFERENCE IN TREND WITH THE rTR-AGONIST ACTIVITY IN EDSP21The Endocrine Disruptor Screening Program (EDSP) was established by the US EPA in 1998 to determine potential chemical endocrine disruptors in humans and animals (Browne et al., 2017). The EDSP21 project aims to support the advancement of EDSP by integrating information from various activities and conducting rapid in vitro assays using computational modeling and robotics. EDSP21 has a dashboard that publishes related information such as the ER, AR, and TR of approximately 10,000 chemicals.
The rTR-agonist activities of 8,305 compounds evaluated in “Tox21_TR_LUC_GH3_Agonist” were published in the EDSP21 dashboard; however, hTR activity was not evaluated. Therefore, the hTR-agonist activities in the TR yeast-cell assay were compared to the rTR-agonist activities in EDSP21 to determine the difference in the trends of compounds with TR-agonist activities (Fig. 1). Among the 8,305 compounds, 893 compounds were identified as having an rTR-agonist activity (EPA, 2021). Among the 796 chemicals tested in this study, 492 compounds were included in the EDSP21 test compound list (Table S2). Seven compounds demonstrated TR-agonist activity in both this study and EDSP21 (Fig. 1), and all compounds, except triclabendazole, had a structure equivalent to TH (thyroid structure); therefore, it was hypothesized that compounds with a thyroid structure would exhibit TR-agonist activity regardless of the TR type. Additionally, two compounds, TBBPA and TCBPA, exhibited activity only for hTR, while 77 compounds exhibited activity for rTR; 406 compounds were inactive in both tests. Moreover, 349 compounds were evaluated in this study, and eight of these compounds exhibited hTR-agonist activity.
Comparison of human thyroid receptor (hTR)-agonist activity by yeast two-hybrid assay (TR yeast-cell assay) and rat TR (rTR)-agonist activity by the Endocrine Disruptor Screening Program for the 21st Century (EDSP21)
If the agonist activities were not identified in each test’s concentration range, the compound was indicated as inactive for convenience in this study. The maximum chemical concentration tested in the TR yeast-cell assay was 10,000 nM, and that in the EDSP21 assay was 1×109 nM.
Triclabendazole exhibited TR-agonist activity in both tests. This compound is a TH analog because it contains two benzene rings connected by an oxygen atom and three halogens (chlorines), which stimulate the ligand-protein binding (Lu et al., 2010). On the other hand, because it lacks a phenol or carboxyl group in its structure, its TR-agonist activities are relatively lower than those of TH and its metabolites in both tests. Additionally, TBBPA and TCBPA were evaluated as TH-analogs because their structures are more similar to those of TH and the metabolites than triclabendazole. However, they did not exhibit TR-agonist activity for rTR. This could be due to the differences in the bioassays as it is reported that TBBPA and TCBPA have slightly agonistic activities toward rTR (Kitamura et al., 2002; Freitas et al., 2011). However, it is suggested that it has antagonistic properties toward hTR, as they inhibited the interaction of T3 and TR (Kitamura et al., 2005; Sun et al., 2009). These findings are not contradictory, because a compound with weak TR-agonist activity exhibits antagonist activity in the presence of a compound with strong agonist activity.
Most of the 492 compounds exhibited a distinct pattern of TR-agonist activity mediated by hTR and rTR; this might have been caused by the difference in the designs of the two tests. The TR yeast-cell assay evaluates the binding affinity of hTRα to a test compound, whereas the EDSP21 assay quantifies the binding affinity of both subtypes, rTRα and rTRβ. The affinity of compounds for TR varies depending on the subtypes of TR (Martínez et al., 2009; Gierach et al., 2012). The amino acid sequences of hTR and rTR subtypes were determined (Wagner et al., 2001), and some differences between TRα and TRβ were found. On the other hand, the difference between hTRα and rTRα in those amino acid sequences was observed only at a single amino acid residue. Moreover, the contacts between each TR and its ligands (TRIAC, GC-1, and T3) were determined, and the differences in those amino acid sequences above did not affect the TR’s binding to its ligands (Wagner et al., 2001). This is consistent with the results for TRIAC and T3 in Fig. 1, and the remaining five of the seven compounds may exhibit the same binding behavior to both TRs due to their distinct structures that are similar to those of TRIAC and T3. If the contacts between TRs and compounds vary according to their chemical structures, the results of Fig. 1 may be accurate, as the TR yeast-cell assay used in this study only included hTRα, whereas the EDSP21 test included rTRα and rTRβ.
In this study, the hTR-agonist activities of 796 compounds were evaluated using the TR yeast-cell assay, and 17 of them showed agonistic activity. These results were compared to those published by EDSP21 for rTR-agonist activity, and a distinct trend was observed: Compounds with a thyroid structure, such as TH metabolites, exhibit strong TR-agonist activity for both hTR and rTR, whereas many compounds showed agonist activity for only rTR but not hTR. Additionally, it was determined that 349 compounds evaluated in the TR yeast-cell assay were not evaluated in the EDSP21, and the TR-agonist activity of 3-Cl-T3 was reported for the first time in this study. Therefore, this report will be an invaluable resource for understanding TR activity as an endocrine disruptor in processes such as the regulation of chemical compounds.
This research was conducted by the Environment Research and Technology Development Fund (JPMEERF20195053) of the Environmental Restoration and Conservation Agency of Japan. We would like to express our deepest thanks to Mr. Takeshi Oyama, Ms. Mayumi Masuda, Hisae Kawakita, and Maki Ochi for their technical assistance.
Table S1, Human thyroid receptor (hTR)-agonist activities of 802 chemical compounds evaluated by a yeast two-hybrid assay (Y2H); Table S2, TR-agonist activities of 492 chemical compounds evaluated in both tests; Fig. S1, Dose-response curves of the 17 compounds with the human thyroid receptor (hTR)-agonist activity; Fig. S2, Order of hTR-agonist activities of 17 compounds and OH-PCBs.
This material is available on the Website at https://doi.org/10.5985/emcr.20210016.
