Effects of Long-Term In Vivo Exposure to Di-2-Ethylhexylphthalate on Thyroid Hormones and the TSH/TSHR Signaling Pathways in Wistar Rats
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. Animals and Treatments
2.3. Sample Collection and Preparation
2.4. Iodine Level Detection
2.5. Haematoxilin-Eosin (HE) Staining and Histological Evaluation
2.6. Radioimmunoassay RIA
2.7. Enzyme-Linked Immunoabsorbent Assay
2.8. RNA Extraction and Real-Time Quantitative PCR
2.9. Western Blot Analysis
2.10. Statistical Analysis
3. Results
3.1. Body Weight (BW)
3.2. DEHP Elevated Relative Thyroid Weights and Reduced Urine Iodine Levels of Treated Rats
3.3. Histological Changes in the Thyroid and Liver
3.4. DEHP Altered Serum Levels of Thyroid Hormones
3.5. DEHP Influenced mRNA Expressions of Hypothalamus TRHr, Pituitary TSHβ, Thyroid TPO, TSHr, NIS, Tg, TTf-1, D1 and Liver Deiodinase 1
3.6. DEHP Influenced Protein Expressions of NIS, TSHr, TPO, TG, TTf-1 and TSHβ
4. Discussion
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Primer | Type | Primer Sequence | GenBank |
---|---|---|---|
TSHβ | Forward | 5‘-TACTGCCTGACCATCAACACC-3’ | NM_013116.2 |
Reverse | 5’-GGTAGGAGAAATAAGGAGCAACAT-3’ | ||
NIS | Forward | 5’-CAGTTCTGGAATGGACACGG-3’ | NM_052983.2 |
Reverse | 5’-TCTTGGTCACAGCAGGGATG-3’ | ||
TSHr | Forward | 5’-GTGGGAATAAGCAGCTACGC-3’ | NM_012888.1 |
Reverse | 5’-GGATTTCGGACGGTGATGT-3’ | ||
TTF-1 | Forward | 5’-GCACTTGGAGTAAGGCAGAAA-3’ | XM_006224320.2 |
Reverse | 5’-ACCCCACGATACACGAACC-3’ | ||
TPO | Forward | 5’-ATGAGGCTGTGACTGAAGATGA-3’ | NM_019353.2 |
Reverse | 5’-GTGGTCCGTGAGGAGTTTGA-3’ | ||
TG | Forward | 5’-GCCCTAACTCATCCGTCCA-3’ | NM_030988.2 |
Reverse | 5’-TGTTGATAAGCCCATCGTCCT-3’ | ||
D1 | Forward | 5’-GCAGACCCCTGGTGTTGAA-3’ | NM_021653.3 |
Reverse | 5’-GGTCCTGGAGGCTTCGGT-3’ | ||
TRHr | Forward | 5’-AGGAGTCAGACCGCTTTAGCA-3’ | NM_013047.3 |
Reverse | 5’-GAACTGGGTCCATTCTTCTCG-3’ | ||
β-actin | Forward | 5’-CCGTAAAGACCTCTATGCCAACA-3’ | NM_013116.2 |
Reverse | 5’-GGGGCCGGACTCATCGTA-3’ |
Primary Antibodies | Primary Source | Dilution | Brand | Second Source | Dilution |
---|---|---|---|---|---|
GAPDH | Rabbit | 1:800 | Santa Cruz, USA | Anti-Rabbit | 1:800 |
NIS | Goat | 1:800 | Santa Cruz, USA | Anti-Goat | 1:800 |
TSHr | Goat | 1:200 | Santa Cruz, USA | Anti-Goat | 1:800 |
TTf-1 | Rabbit | 1:500 | Santa Cruz, USA | Anti-Rabbit | 1:800 |
TPO | Rabbit | 1:200 | Abcam, USA | Anti-Rabbit | 1:800 |
TG | Goat | 1:500 | Santa Cruz, USA | Anti-Goat | 1:800 |
TSHβ | Goat | 1:250 | R&D Systems, USA | Anti-Mouse | 1:800 |
Group | Liver/Weight (g/100 g) | Thyroid/Weight (g/100 g) | Urine Iodine (μg/L) | |||
---|---|---|---|---|---|---|
3 M | 6 M | 3 M | 6 M | 3 M | 6 M | |
C | 2.29 ± 0.73 | 2.46 ± 0.17 | 8.19 ± 1.61 | 8.27 ± 2.63 | 160.82 ± 43.56 | 95.73 ± 14.47 |
L | 2.69 ± 0.32 a | 2.55 ± 0.34 | 8.51 ± 1.46 | 10.48 ± 2.85 | 147.90 ± 31.79 | 70.92 ± 17.27 b |
M | 2.79 ± 0.17 b | 2.71 ± 0.25 a | 8.55 ± 1.76 | 10.59 ± 2.00 | 133.36 ± 24.58 | 72.29 ± 18.97 b |
H | 3.78 ± 0.46 b | 3.00 ± 0.30 b | 10.87 ± 2.63 b | 12.50 ± 6.65 | 79.04 ± 15.46 b | 71.24 ± 13.90 b |
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Dong, X.; Dong, J.; Zhao, Y.; Guo, J.; Wang, Z.; Liu, M.; Zhang, Y.; Na, X. Effects of Long-Term In Vivo Exposure to Di-2-Ethylhexylphthalate on Thyroid Hormones and the TSH/TSHR Signaling Pathways in Wistar Rats. Int. J. Environ. Res. Public Health 2017, 14, 44. https://doi.org/10.3390/ijerph14010044
Dong X, Dong J, Zhao Y, Guo J, Wang Z, Liu M, Zhang Y, Na X. Effects of Long-Term In Vivo Exposure to Di-2-Ethylhexylphthalate on Thyroid Hormones and the TSH/TSHR Signaling Pathways in Wistar Rats. International Journal of Environmental Research and Public Health. 2017; 14(1):44. https://doi.org/10.3390/ijerph14010044
Chicago/Turabian StyleDong, Xinwen, Jin Dong, Yue Zhao, Jipeng Guo, Zhanju Wang, Mingqi Liu, Yunbo Zhang, and Xiaolin Na. 2017. "Effects of Long-Term In Vivo Exposure to Di-2-Ethylhexylphthalate on Thyroid Hormones and the TSH/TSHR Signaling Pathways in Wistar Rats" International Journal of Environmental Research and Public Health 14, no. 1: 44. https://doi.org/10.3390/ijerph14010044