2.1. Preparation of a stably transfected HeLa cell line.
In the previous study, we constructed a TH-responsive dual-reporter cassette using retrovirus to establish a stably transfected cell line. The TH-responsive dual-reporter cassette consisted of a TRE region, a minimal 15-bp TATA box, secNluc luciferase, and IRES-EGFP. Correct construction of the TH-responsive dual-reporter cassette was confirmed by DNA sequencing 10. TRs are expressed in several human organ tissues, including brain, breast, cervix, kidney, liver, lung, pancreas, and stomach tissues 10. To develop a human cell-based TH agonist TA assay, we used TH-responsive dual reporter assay system based on previously established A549 system to transfect HeLa cells. This human cell-based assay used an innate transcription system controlled by TH. Therefore, we selected and used HeLa cells, a human cervix adenocarcinoma cell line with a higher TR expression level than the previously used A459 cell line. To establish stably transfected HeLa cells, we used the TH-responsive dual-reporter cassette, since stably transfected A549 cells showed stable T3-mediated EGFP and secNluc luciferase gene expression in the previous study.
2.2. Confirmation of T3-mediated EGFP and secNluc luciferase gene expression in stabilized HeLa cells with the transfected dual-reporter system.
HeLa-TRE-Nluc-EGFP cells were treated for 24 h with (3,3′,5-triiodo-L-thyronine, +T3) or without (-T3) 100 nM T3. Nucleus: Hoechst 33342 (blue); EGFP: expressed in Hela-TRE-Nluc-EGFP cells (green).
A retrovirus harboring the TRE-secNLuc-IRES-EGFP reporter cassette was used to transfect the TH-responsive dual-reporter system into the HeLa cell line. We treated each clone with 100 nM T3 and evaluated T3-mediated EGFP expression to select HeLa cells transfected with TH-responsive dual-reporter system. The T3-mediated EGFP expression in HeLa cells was induced by treatment with 100 nM T3. These results established that the TH-responsive dual-reporter system in HeLa cells was controlled by T3 (Fig. 1).
The HeLa cells stably transfected were exposed to T3 (10 pM to 10 µM). The mean induction-fold in comparison with the vehicle control (VC) is presented (n = 3).
To evaluate TH agonistic properties of the test chemicals, we measured the T3-mediated secNluc luciferase gene expression. The HeLa cells were treated with T3 in a dose-dependent manner at seven concentrations (each concentration diluted 10-fold, from 10− 5 ~ to 10− 11 M) to evaluate the effect of T3 on TRE-induced secNluc luciferase gene expression. As the concentration of T3 increased, secNluc luciferase activity increased in a dose-dependent manner compared to the activity of vehicle control. The increase ratio of secNluc luciferase activity with 100 nM T3 was consistently 6-fold or higher compared to the vehicle control (Fig. 2). The mean value of log[EC10(M)] was − 9.84 and that of log[EC50(M)] was − 8.71. In the “Rat pituitary tumor cell (GH3) reporter gene assay” in OECD GD 207 ‘Ranking Parameter Analysis’ of TR transactivation, the log[EC10(M)] and log[EC50(M)] values induced by T3 were − 11 and − 10, respectively 11. Thus, this stabilized HeLa cell line transfected with a thyroid-responsive dual-reporter system showed sensitivity to T3 similar to that of the rat pituitary cell line. The stably transfected A549 cells required incubation for 72 h after T3 treatment until sufficient amounts of luciferase were secreted in the supernatant 10. In contrast, the stably transfected HeLa cells required incubation for only 48 h after T3 treatment until sufficient amounts of luciferase were secreted in the supernatant. Thus, the TH agonist TA assay using stably transfected HeLa cells required less time for measurement of luminescence than the assay using A549 cells. This advantage suggests that the assay with HeLa cells offers more speed and sensitivity than the assay with A549 cells.
2.3. Transactivation assay with potential TH agonistic chemicals.
The secNluc luminescence units induced by the 17 chemicals are represented as the % activity observed with reference to the activity of 100 nM T3 (n = 3).
To evaluate the developed TA assay, 17 chemicals were selected for testing their potential TH agonistic activity based on the OECD GD 207 12. Five of these 17 chemicals (T3, T4, Triac, Tetrac, GC-1) showed TH agonist activity based on the log[EC10(M)] and log[EC50(M)] values and showed the same results as GD 207 (Fig. 3). The mean values of log[EC10(M)] and log[EC50(M)], the standard deviation of triplication, and the coefficient of variation (%CV) are summarized in Table 1.
Table 1
Log[EC10(M)] and Log[EC50(M)] values from the TH agonist TA assay
No.
|
Test chemicals
|
Log[EC10(M)]
|
Log[EC50(M)]
|
#1
|
#2
|
#3
|
Avg.
|
SD
|
%CV
|
#1
|
#2
|
#3
|
Ave
|
SD
|
%CV
|
1
|
T3
|
-9.89
|
-9.72
|
-9.69
|
-9.76
|
0.11
|
1.12
|
-8.73
|
-8.45
|
-8.45
|
-8.54
|
0.16
|
1.87
|
2
|
T4
|
-7.83
|
-7.77
|
-7.75
|
-7.78
|
0.04
|
0.55
|
-6.96
|
-6.75
|
-6.74
|
-6.82
|
0.12
|
1.77
|
3
|
Glycitein
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
4
|
BP-2
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
5
|
E2
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
6
|
BPA
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
7
|
Tetrac
|
-7.62
|
-7.63
|
-7.60
|
-7.61
|
0.02
|
0.23
|
-6.18
|
-6.29
|
-6.24
|
-6.24
|
0.05
|
0.86
|
8
|
GC-1
|
-8.91
|
-8.94
|
-8.99
|
-8.95
|
0.04
|
0.48
|
-6.87
|
-6.94
|
-7.46
|
-7.09
|
0.32
|
4.52
|
9
|
Triac
|
-9.68
|
-9.78
|
-9.86
|
-9.77
|
0.09
|
0.94
|
-8.14
|
-8.27
|
-8.46
|
-8.29
|
0.16
|
1.91
|
10
|
Amiodarone hydrochloride
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
11
|
Hispidulin
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
12
|
Procymidone
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
13
|
Bithionol
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
14
|
Closantel
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
15
|
Rafoxanide
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
16
|
4,4'-Diiodobiphenyl
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
17
|
BDE No 28
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
T3: 3,3′,5-Triiodo-l-thyronine sodium salt; T4: Levothyroxine sodium; BP-2: 2,2′,4,4′-Tetrahydroxybenzophenone; E2: 17β-Estradiol; BPA: Bisphenol A; Tetrac: 3,5,3′,5′-Tetraiodo Thyroacetic Acid; GC-1: Sobetirome; Triac: 3,3′,5-Triiodothyroacetic acid
T3 and T4 are the major forms of human TH. Although T4 is secreted in relatively higher amounts than T3 in the human body, the TH agonist TA activity of T3 was higher than that of T4 because the binding affinity of T3 to TR is 10-fold higher than that of T4 and because T3 primarily plays the role of a transcription ligand 13. Other chemicals that showed TH agonist TA-positive activity (Triac, Tetrac, and GC-1) show structural similarities as natural TH derivatives. Thus, Triac, Tetrac, and GC-1 showed relatively weak TH agonist TA activity. We also measured %CV for triplicates was measured to confirm the accuracy and repeatability of the assessments. Very low %CV values were obtained, suggesting good reproducibility of the TH agonist TA assay using stably transfected HeLa cells.
Table 2
Log[EC10(M)] and Log[EC50(M)] values from the TH agonist TA assay (subdivided concentrations)
No.
|
Test chemicals
|
Log[EC10(M)]
|
Log[EC50(M)]
|
#1
|
#2
|
#3
|
Ave
|
SD
|
%CV
|
#1
|
#2
|
#3
|
Ave
|
SD
|
%CV
|
1
|
T3
|
-9.31
|
-9.25
|
-9.39
|
-9.32
|
0.07
|
0.75
|
-8.11
|
-7.86
|
-8.01
|
-7.99
|
0.13
|
1.58
|
2
|
T4
|
-8.45
|
-8.77
|
-8.33
|
-8.52
|
0.22
|
2.64
|
-7.73
|
-7.94
|
-7.59
|
-7.75
|
0.18
|
2.28
|
3
|
Tetrac
|
-7.55
|
-7.61
|
-7.51
|
-7.56
|
0.05
|
0.71
|
-6.53
|
-6.68
|
-6.57
|
-6.59
|
0.08
|
1.15
|
4
|
GC-1
|
-9.16
|
-9.11
|
-9.03
|
-9.10
|
0.07
|
0.73
|
-8.20
|
-7.99
|
-7.92
|
-8.04
|
0.14
|
1.80
|
5
|
Triac
|
-9.36
|
-9.24
|
-9.33
|
-9.31
|
0.07
|
0.70
|
-8.48
|
-8.52
|
-8.62
|
-8.54
|
0.07
|
0.83
|
T3: 3,3′,5-Triiodo-l-thyronine sodium salt; T4: Levothyroxine sodium; BPA: Bisphenol A; Tetrac: 3,5,3′,5′-Tetraiodo Thyroacetic Acid; GC-1: Sobetirome; Triac: 3,3′,5-Triiodothyroacetic acid |
Furthermore, the 5 positive chemicals were further tested with additional concentrations to provide more detailed data (Fig. 4). The mean values of log[EC10(M)] and log[EC50(M)], the standard deviation, and %CV of triplication are summarized in Table 2. Similar to the tests for the 17 chemicals, the %CV values of 5 selected chemicals were very low values. These results indicate that the TH agonist TA assay using stably transfected HeLa cells developed in this study showed very high reproducibility for screening potential TH agonistic chemicals. Furthermore, in a comparison of the dose-dependent responses of each positive chemical in the TH agonist TA assays using A549 and HeLa cells, the TH agonist TA assay using HeLa showed higher induction levels than the assay using A549 cells at all concentration, indicating the thyroid TA activity of each chemical. Higher level of induction indicates that the stably transfected HeLa cells responded more sensitively to chemicals mimicking thyroid hormones. These results indicated that the TH TA assay using HeLa could provide more sensitive thyroid TA activity information of test chemicals than the TH agonist TA assay using A549 cells. Moreover, the TH agonist TA assay using A549 cells required 4 days to yield results, which is a relatively long time for high-throughput screening. Therefore, we developed the TH TA assay using HeLa cells, which redressed some of the limitations of the test method using A549. The TH agonist TA assay using HeLa cells could provide accuracy, sensitivity, and specificity.
With the increasing risks associated with EDCs, the development of appropriate test methods for management and assessment of these EDCs has become important. This is especially pertinent because the thyroid hormone is involved in energy metabolism and homeostasis, necessitating the development of practical test methods to assess EDCs that disrupt thyroid hormones. The TH TA assay in this study was suitable for assessment and analysis of EDCs that mimic TH in the human body because of the use of the using human HeLa cell line.