| The goal of elimination iodine deficiency disease (IDD) has been achieved sinceUniversal Salt Iodization (USI) policy has been widely carried out in many nationsincluding China. On the other hand, reports are increasingly appearing on the sideeffects caused by excessive iodine intake. There are about 60 millions people who livein the areas where drinking water contains excessive iodine. In addition, there aremany people having iodine-rich food and taking iodine-rich medicine such asaminodarone. Previous study reveals that the most ordinary damage resulted fromexcessive iodine is goiter. There are many theories to explain the mechanism, however,none could explain it perfectly. Microelement selenium, as the form of selenocysteine,composes glutathione peroxidase (GSH-Px) and deiodinase and plays a veryimportant role in thyroid hormone metabolism. Previous studies on selenium andiodine focused on their deficiency. There was no report about the intervention effectof selenium on the disordered thyroid hormone metabolism resulted from excessiveiodine. Our study observed the change of oxidation and anti-oxidation, deiodinase,cathepsin and cell apoptosis resulted from excessive iodine in mice and thyroid cell.We also detected the intervention effects of selenium on the damage of these keypoints of thyroid hormone metabolism resulted from excessive iodine by real-timePCR, Western blot and flow cytometry, et al. The results were summarized asfollows: Part one Effect of excessive iodine on thyroid and intervention with selenium in mouseObjective To establish the mouse model of excessive iodine and observe the damage of thyroid damage induced by excessive iodine and the intervention effects of different doses of selenium. To study the mechanisms of damage resulted from excessive iodine on thyroid hormone metabolism and the intervention with selenium. The goal of our study is to select appropriate selenium intervention dosage.Methods 320 weanling female Balb/c mice were randomly divided into 8 groups by weight. Every group included 40 mice. NI group was normal iodine control group and given tap water. EI group was excessive iodine control group and given drinking water containing 3000μg/L iodine. IS groups were selenium supplement groups and given drinking water containing 0.1, 0.2, 0.3, 0.4, 0.5 and 0.75mg/L selenium, respectively, besides 3000μg/L iodine. After four months, the thyroid was removed and fixed in 10% formalin or 2.5% glutaral for histological or electron microscope measurement. Such indexes were detected as the iodine in urine and thyroid, urinary and hepatic selenium, the oxidative/anti-oxidative levels in serum, liver, kidney and thyroid, the activities of type 1 deiodinase (D1) in liver, kidney and thyroid, the activities of thyroid peroxidase (TPO). Thyroid hormone in plasma was determined by radioimmunoassay. The mRNA levels of TPO in thyroid and D1 in liver, kidney and thyroid were determined by RT-PCR.Results There were no significant differences among the eight groups in weight, thyroid weight and the ratio of thyroid weight to body weight. The concentrations of iodine in urine and thyroid increased significantly in EI group mice. The urinary and hepatic selenium decreased significantly. Thyroid pathology showed that thyroid follicles increased significantly in size and were full of red colloid in the chamber, and follicular epithelial cells were thin and flat in the EI group. It showed that goiter occurred. Electron microscope showed that karyon is thin and flat, mitochondrion is swelling and lysosome is rare in the EI group. Serum tetraiodothyronine (T4) level was significantly higher, and triiodothyronine (T3) level was significantly lower thanNI group. The activities of GSH-Px and SOD decreased significantly in liver, kidneyand thyroid while the level of MDA increased significantly in EI group. Compared toNI group, the activities and the mRNA levels of D1 in liver, kidney and thyroiddecreased significantly. The activities and the mRNA levels of TPO decreasedsignificantly. Selenium had intervention effects on the damage resulted fromexcessive iondine: ①The urinary iodine increased significantly, while theconcentrations of iodine in thyroid decreased than NI group, especially in 0.2mg/Lselenium group.② Most of the follicular chamber size of the IS group were similar tothe NI group, but there were some cells which sizes range from NI to EI groups. Thefollicular cells of Se group were similar to the NI group. Electron microscope showedthat the lesion relieved in IS group. ③ The levels of urinary and hepatic seleniumand serum thyroid hormone restored to normal levels in 0.1～0.5mg/L seleniumsupplement group. ④ The activities of GSH-Px and SOD and the levels of MDA inliver, kidney and thyroid restored to normal levels in 0.2～0.3mg/L seleniumsupplement groups. ⑤ The mRNA levels of D1 restored to normal levels in all ofthe IS groups, while only 0.1～0.4mg/L selenium supplement groups had normalactivities of D1 in liver, kidney and thyroid. (6) The mRNA levels of TPO restored tonormal in all of the IS groups, while only 0.1～0.3mg/L selenium supplement groupshad normal activities of TPO.Conclusion The damage resulted from excessive iodine showed as follows: disequilibrium of the levels of oxidation/anti-oxidation, decreased activities and mRNA levels of D1 and TPO. Excessive iodine resulted in disordered thyroid hormone and goiter through decreasing the activities of GSH-Px, D1 and TPO. Selenium supplement leads to an elevation of selenium level and exhibits protective effects against its toxicity induced by excessive iodine. The optimal dose of selenium, which can protect against disordered thyroid hormone metabolism induced by excessive iodine in mice, ranges from 0.2mg/L to 0.3mg/L. (0.05 mg/kg·bw ～0.075mg/kg·bw). Part two Effect of excessive iodine on FRTL Cell and intervention with seleniumObjective To detect the damage on the activities and mRNA of cathepsin B and D, the oxidative/anti-oxidative level, cell apoptosis, and the mRNA and protein levels of bcl-2 and bax resulted from excessive iodine, meanwhile to observe effects of the intervention of different dosages of selenium To explore the mechanism of goiter based on the above-mentioned results.Methods FRTL cell was cultured in Kaighn's modified Ham's F-12 medium supplemented with 5% calf serum and a six-hormone mixture of 1 mU/ml TSH, 0.01 mg/ml insulin, 10 nmol/L hydrocortisone, 0.005 mg/ml transferrin, 10 ng/ml somatostatin, and 10 ng/ml glycil-L-histidyl-L-lysine acetate. The items were detected as follows: ①The DNA damage was detected by agarose gel electropherosis. The cells were divided into 4 groups: The medium of NI group did not contain selenium or iodine. The medium of EI group contained 10mmol/L potassium iodide. The medium of Se group contained 0.1μmol/L sodium selenite and the IS group contained 10mmol/L potassium iodide and 0.1μmol/L sodium selenite. The DNA was extracted and electrophoresed on agarose gel. ②The reactive oxygen species (ROS) were detected by flow cytometry. The cells were divided into 16 groups: The medium of NI group did not contain selenium or iodine. The medium of EI group contained different dosages of potassium iodide and the concentrations were 1,5, 10, 50 and 100mmol/L. The medium Se group contained different dosages of selenium and the concentrations were 0.1, 0.5, 1.0, 2.0 and 5.0μmol/L sodium selenite. The medium of IS group contained 10mmol/L potassium iodide and different dosages of selenium and the concentrations were 0.1, 0.5, 1.0, 2.0 and 5.0μmol/L sodium selenite. The cells were collected and detected by flow cytometryer in 530nm wavelength. ③ Cell apoptosis was detected by flow cytometry. The cells were divided into 10 groups: The medium of NI group did not contain selenium or iodine. The medium of EI group contained different dosages of potassium iodide and the concentrations were 1, 5, 10, 50 and 100mmol/L. The medium of IS group contained 10mmol/L potassium iodide and different dosages of selenium and the concentrations were 0.1, 0.5, 1.0 and 5.0μmol/L sodium selenite. The cells were collected and detected by Annexin V -FITC/7-AAD staining. ④ The mRNA levels of cathepsin B and D, bcl-2 and bax were detected by real-time PCR. The cells were divided into 4 groups: The medium of NI group did not contain selenium or iodine. The medium of EI group contained 10mmol/L potassium iodide (bcl-2 and bax) or 50mmol/L potassium iodide (CB and CD). The medium of Se group contained 0.1μmol/L sodium selenite and the medium of IS group contained 10mmol/L potassium iodide (bcl-2 and bax) or 50mmol/L potassium iodide (CB and CD) and 0.1μmol/L sodium selenite. The cells were collected and detected by real-time PCR. ⑤ The protein concentrations of bcl-2 and bax were detected. The cells were divided into 4 groups: The medium of NI group did not contain selenium or iodine. The medium of EI group contained 10mmol/L potassium iodide. The medium of Se group contained 0.1μmol/L sodium selenite and the medium of IS group contained 10mmol/L potassium iodide and 0.1μmol/L sodium selenite. The cell was collected and detected by Western blot. ⑥ The activities of cathepsin B and D were detected. The cells were divided into 20 groups: The medium of NI group did not contain selenium or iodine. The medium of EI group contained different dosages of potassium iodide and the concentrations were 1, 5, 10, 50 and lOOmmol/L. The medium Se group contained different dosages of selenium and the concentrations were 0.1, 0.2, 0.5, 1.0, 2.0, 5.0 and 10.0 μmol/L sodium selenite. The medium of IS group contained 10mmol/L potassium iodide and different dosages of selenium and the concentrations were 0.1, 0.2, 0.5, 1.0, 2.0, 5.0 and 10.0 μmol/L sodium selenite. The cells were collected and detected the activities of CB and CD.Results The effects of iodine and selenium on FRTL cell were described as follows: ①The DNA strap of EI group on agarose gel was ladder-shaped, while the strap of IS group was weaker than EI group and the NI, Se groups had no ladder-shaped straps. (2) The levels of ROS increased in the 5, 10, 50 and 100mmol/L EI groups and restored to normal levels in 0.1～1.0μmol/L selenium supplement groups. ③ The apoptosis increased in 10, 50 and 10mmol/L iodine groups. It was restored to normal level in 0.1～0.2 μmol/L selenium supplement groups, but had the elevation tendency compared with NI group. ④In EI group, the mRNA levels of CB and CD decreased after treated by 50mmol/L iodine. The mRNA levels of CB and CD were normal in IS group. ⑥ The mRNA and protein levels of bcl-2 decreased, while bax increased in EI group. In IS group, the mRNA and protein levels were improved. ⑥ The activities of CB decreased after 50mmol/L possium iodide treated 12 hours, while the activities of CD decreased after 24 hours. There were negative correlations between CB, CD and iodine. The activities of CB and CD decreased in 2.0, 5.0 and 10.0μmol/L Se groups and there were negative correlations between their activities and selenium level. Their activities improved in 0.1～0.5μmol/L selenium supplement groups.Conclusions Excessive iodine resulted in goiter through decreased the levels of activities and mRNA of cathepsin B and D. One of the mechanisms of intervention effect of selenium on goiter was its amelioration effect on CB and CD. The rate of apoptosis increased through the changes of oxidative/anti-oxidative disequilibrium and apoptosis related gene bcl-2 and bax. Selenium supplement could improve the damages resulted from excessive iodine and the optimal dosage ranges from 0.1μmol/L to 0.2μmol/L.
|
PDF Full Text Request