| Along with environmental pollutants accumulating, exposure to certain environmental chemicals can disrupt human endocrine system, and increase incidences of the diseases related with endocrine, and cause adverse effects to growth and development in both human and wildlife. Endocrine disruptors have emerged as an environmental issue and have become as the focus concerned by researchers around the world. Thyroid hormone disruptor is a kind of endocrine disruptors, but up to now, there are no feasible and effective screening and testing methods for them.In 1980, a sort of cell line were successfully established by COON and his group, and named FRTL-5 cell, which is hormone-dependent functional epithelial cells from rat thyroids. This cell line can concentrate iodine and produce thyroglobulin, and has been used as a tool to study mechanism of goitre, iodine uptake and thyroid neoplasm, so on.The purpose of the study is to use FRTL-5 cells to develop a sort of screening methods for chemicals that can affect thyroid on produce thyroglobulin and concentrate iodine, and explore mechanism of their thyroid disrupting.The study includes three parts:Part one: The study on the effects of amitrole, ethylenethiourea, pentachlorophenol and pendimethalin on cytotoxicity and cell proliferation of FRTL-5 cells. The purpose was to determine the dosages of four chemicals in the study have no cytotoxicity for FRTL-5 cells. The effects of four chemicals on proliferation and DNA synthesis of FRTL-5 cells were detected by the methods of MTT and ~3H-TdR integrated. The results showed that there were not obvious difference of the cell doubling time between all experimental groups treated with four chemicals and control group, which indicated that in the dose range set in the study, no chemical's cytotoxicity was found in FRTL-5 cells after treatment with four chemicals for 96h.The analysis of ~3H-TdR integrated showed that all four chemicals had no significant effects on DNA synthesis. But along with the increase of the concentration of ethylenethiourea and pentachlorophenol, a decreasing tendency was found on DNA synthesis in FRTL-5 cells, which hinted that the two chemicals have probably potential harmful effect on DNA synthesis of FRTL-5 cells.Part two: The study on the effects of amitrole, ethylenethiourea, pentachlorophenol and pendimethalin on thyroid hormone-associated gene transcription in FRTL-5 cells.The purpose was to understand how the four chemicals disrupted the thyroid hormone, and find they affected target genes, and study whether the synthesis-associated genes of thyroid hormone could be used to screen and test the chemicals that disrupt the synthesis of thyroid hormone. Tg, tpo, nis, tshr, ttf-1 and pax-8 genetic transcription were analyzed by RT-PCR. The results indicated that the level of tg genetic transcription was higher in all groups treated with amitrole than that of control, and the level of ttf-1 genetic transcription in the high dosage group was higher than that of control. By contraries, the level of tshr and pax-8 genetic transcription in the high dosage group was lower than that of control. No significant difference was found between the levels of tpo/nis genetic transcription and that of control.For ethylenethiourea, the level of tpo genetic transcription in the high dosage group was lower than that of control, while the level of nis genetic transcription in the low dosage group and high dosage group was lower than control group. No significant difference was found between the level of tg, tshr, pax-8 and ttf-1 genetic transcription and that of control.For pentachlorophenol, the levels of nis/tpo genetic transcription in the high dosage group were lower than that of control, but the level of nis in the low dosage group was higher than that of control. No significant difference was found between the level of other genes' transcription and that of control.For pendimethalin, the level of tpo genetic transcription in the high dosage group was higher than that of control. The level of Pax-8 genetic transcription in all dosage groups was higher than that of control. No significant difference was found between the level of other genes' transcription and that of control.The results RT-PCR suggested that the disrupting effect to thyroid hormone from the four chemicals were probably related to their effects on the synthesis-associated genes of thyroid hormone. However, their different effects to these genes' transcription indicated that their mechanisms of disrupting thyroid hormone maybe were different. These results also hinted that the synthesis-associated genes of thyroid hormone should be used as targets in sreening and testing methods.Part three: The study on the effects of amitrole, ethylenethiourea, pentachlorophenol and pendimethalin on thyroid hormone-associated protein and iodine uptake in FRTL-5 cells.The effects of the four chemicals on thyroid hormone-associated protein and iodine uptake were analyzed by isotope analytical method, RIA, ICC and IMF, to study whether the synthesis-associated proteins of thyroid hormone and iodine uptake of the cells could be used as targets in screen and test for the chemicals that disrupt the synthesis of thyroid hormone.The results of isotope analytical method indicated that, after 12 hours' treatment with all four chemicals, the iodine uptake of FRTL-5 cells increased significantly, comparing with that of control. However, after 24 hours' treat, except pentachlorophenol, the iodine uptakes of the cells in most treatment group were lower than that of control.The results of RIA analysis indicated that amitrole, ethylenethiourea and pentachlorophenol significantly decreased the concentration of TG in medium at middle and high dose groups, and there was dose-effect relation for ethylenethiourea and pentachlorophenol treatments. On the other side, pendimethalin increased significantly the concentration of TG in medium at middle and high dose groups.The results of ICC analysis indicated that for amitrol, IOD of TG was significantly decreased at the high level, but the IODs of TTF-1 in all dose groups were not different statistically from that of control. For ethylenethiourea and pentachlorophenol, the IODs of TG were not different statistically from that of control, but ethylenethiourea decreased significantly IOD of TTF-1 in all doses, and pentachlorophenol significantly decreasd IOD of TTF-1 at middle and high dose groups. For pendimethalin, no statistically effect on IOD of TG was found, but it was found decrease significantly for IOD of TTF-1 at the high dose.The results of ICC analysis indicated that amitrole decreased significantly IOD of TSHR at all doses. Ethylenethiourea increased significantly IOD of TSHR at the low dose, but decreased significantly IOD of TSHR at the high dose. For pentachlorophenol and pendimethalin, no statistically effects on the IOD of TSHR were found.The results of isotope, RIA, ICC and IMF analyses indicated that amitrole and ethylenethiourea could affect iodine uptake, and depress production of TG and expression of TSHR. Pentachlorophenol and pendimethalin could affect iodine uptake and production of TG These results also suggested that the iodine uptake, the production of TG and the level of TSHR expression could be employed in screening and testing methods for disrupting thyroid hormone.In conclusion, the synthesis-associated genes and proteins of thyroid hormone and the iodine uptake of the cells could be used as targets in screening and testing the chemicals disrupting thyroid hormone. In which, tpo, nis, TG, TSHR and iodine uptake were probably more significant. But the sensitivity, specificity and feasibility should be studied further in the future, and the methods and index combination should also be perfected and optimized further.
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