Influence Of Chronic Iodine Excess On Apoptosis Of Thyrocytes In Wistar Rat

OBJECTIVEWith the elimination of iodine deficiency disorders, higher incidences of thyroid disorders have been reported in many countries. Based on the epidemio-logical criteria set by WHO, UNICEF and ICCIDD in 2001 for the evaluation of the iodine status from the urinary iodine levels of school children, the adequate intake of iodine is 100 - 199 μg/L in median urinary iodine, more than adequate for 200 - 300 μg/L in MUI; and excessive for 300 μg/L. Epidemiology study in 2001 showed that the iodine intake in Chinese population already reached the excess level ( MUI 306 μg/L). Epidemiology study carried out in our group suggests that the incidence of clinical and subclinical hypothyroidism in areas with excessive iodine ( MUI 374μg/L and 614 μg/L) is much higher than that in areas with adequate intake ( MUI 103μg/L). The major cause of clinical hypothyroidism is autoimmunity, whereas, 70% of subclinical hypothyroidism is related to excessive intake of iodine.Studies have shown that the toxic effect of iodine excess on thyrocytes in unsusceptible animals is related to apoptosis, not immunological injuries. Up to date, the molecular mechanism of thyrocyte apoptosis induced by iodine excess has not been clarified. In 2000, Vitale identified that the apoptosis of thyrocytes by iodine was mediated by free radicals when he studied the influence of iodine excess on TAD - 2 and primary thyrocytes apoptosis. At the same time, Tamura found the increase in apoptosis of thyrocytes and Fas expression during goiter formation and involution in wistar rats, which was the main factor for cell loss.In 2002, Zhu HM found that thyrocytes apoptosis induced by iodine excess through upregulation of expression of bax and downregulation of expression of bcl -2. In addition, Smerdely P. reported that iodine excess arrested the cell cycles , inhibited the proliferation of FRTL - 5 cells through the regulation of GoGl and G2M, leading to apoptosis.Therefore, the debate is still focused on the apoptotic effect of iodine on normal thyrocytes and its molecular mechanism. Previous animal studies had some weak aspects e. g. high dose of iodine intake, and short experimental duration. These conditions are greatly improved in the present study. The current study used animals with no background in inheritable thyroid and mimicked the iodine status in normal population. By investigating the effect of median and severe iodine excess on apoptosis of thyroids in animals, apoptotic pathway, and the changes occurring after restriction of iodine intake, we could explain the phenomenon found in epidemiology studies and provide experimental evidence for the USI program.METHODS1. Animals:720 wistar rats were used in the present study. They were randomly divided into five groups and were fed with common chow of our animal center of China Medical University and double distilled water containing different concentration of iodine. NID; ddw; HID1: lOOfxg/L iodine in ddw (more than adequate a-mount) ; HID2; 400 |xg/L iodine in ddw ( three times excessive amount) ; HID3 ; 1000|j,g/L iodine in ddw ( six times excessive amount) ; HID4; 3000fxg/ L iodine in ddw (20 times excessive amount). The rats were killed at 0 month, 1 month, 2 months, 4 months and 8 months after treatments. The rats treated for 8 months were fed ddw without iodine for another 3 months before they were killed.2. Methods;To qualify and quantify the apoptosis of thyrocytes, electron microscopy, PI staining, annexin - V FITC binding with PI staining and detection by flow cy-tome try, and TUNEL were used. The ROS generated by thyrocytes was detected by flow cytometry. Both flow cytometry and immunohistochemistry analysis were used to measure the expression of fas/fasL, TRAIL. The serum TSH, FT4, FT3, TT4 and TT3 were measured by RIA. The urinary iodine concentration was detected by the standard method ( GB/T6682 - 1992). The SPSS professional version 11.5 was used for statistic analysis. One - way analysis of variance was used in comparison among groups, t test was used to compare the difference between two groups. The P value less than 0.05 is considered to be significant.RESULTSThe mean MUI in NID group was 152.7jxg/L. There was positive correlation between urinary iodine concentration and water iodine concentration ( r = 0. 815 ~ 0.949, P =0.000). No significant changes in every aspect were detected after 0. 1 and 2 months of treatments.The early apoptotic rate of thyrocytes was low [range from (2. 27 ± 1. 48) % to (3. 83 ± 1. 88 ) % ]. It was slightly increased after 2 months [ range from (7.08 ± 1.76) % to (9. 32 ±2. 28) % ]. At 4 months, the early apoptotic rates in HID2 (10.75 ± 3.03) % and HID3 (10. 56 ±0.91) % groups were significantly higher than that in NID group(7. 04 ± 1. 87 ) % ( P < 0. 05 ). Likewise, at 8 months, the early apoptotic rates in HID3 ( 11. 72 ± 0. 87) % and HID4(13 ±2.48)% groups were significantly enhanced compared to other groups{ P <0.05). In addition, the apoptotic rate in HID2(7. 1 ±2.92)% was apparently increased compared to those in NID (4. 14 ± 1. 08) % and HID1 (4.03 ±0.95) % groups ( P <0. 05). The late apoptotic rate of thyrocytes at 0 and 1 month was low [ range from (2. 13 ± 1. 74) % to (3.45 ±2.46) % ] and slightly increased at 2 months [ range from ( 5. 56 ± 5. 84 ) % to ( 7. 6 ± 3. 02) % ]. At 4 months, the late apoptotic rate in HID3 group (12.51 ± 1.52) % was significantly higher than that in NID group (5. 92 ± 2) % ( P < 0. 05 ). As expected, the late apoptotic rates in HID3 (11. 58 ± 1. 33) % and HID4 (12. 16 ± 1. 52) % groups at 8 months were also much higher than those in other groups ( P <0.05).No apparent difference in the percentage of cells in all cell cycle phases was detected at 0, 1 and 2 months. The Sub - GOG1% was low [range from (8.49 ±5.41) % to (16.76 ± 5. 21)%] at 0 and 1 month, and slightly elevated at 2 months [ range from (12. 99 ± 3. 71) % to (19.25 ±5.03) % ]. The increase in Sub - GOG1% between HID3 (27. 01 ± 10. 19)% group and other groups is significant at 4 months ( P < 0. 05 ). After 4 more months of treatments, Sub - G0Gl% in HID3 (18. 6 ± 4. 85 ) % and HID4 (24 ± 10. 51) % groups were considerably higher than those in other groups ( P < 0. 05 ). The G0Gl% [range from ( 68. 59 ± 3. 74) % to ( 79. 79 ± 5. 43 ) % ] and G2M% [range from (4.79 ±0.66)% to (8. 14 ± 1. 16)% ] were high at 0, 1 and 2 months, while both of these parameters in HID3 groups significantly decreased at 4 months compared to NID group( P <0.05). In addition, G0Gl% in both HID3 and HID4 had further decrease at 8 months ( P < 0. 05). The S% was low [range from (3. 14 ±0.89)% to (6.51 ±2.45)%] at 0 and 1 month. It was greatly increased in HID3 group (5. 09 ± 1.54) % at 4 months compared to NID(2. 81 ± 0. 87) % and HID1 (2. 86 ± 0. 45 ) % groups ( P < 0. 05 ). The similar changing profile was also found in HID3 (5. 87 ± 1.76) % and HID4 (5. 5 ±2. 56) % groups compared to NID(2.81 ± 1.05) % group at 8 months ( P < 0.05).The ROS level [range from (49. 18 ± 14. 22) % to (103.42 ±35.73)%] was low at 0, 1 and 2 months, while the parameter in HID3 group(171.9 ±82. 93)% significantly elevated at 4 months compared to NID group (95. 8 ±40. 8)%{ P <0.05). In addition, ROS level in both HID3(160.61 ±38.64)% and HID4(185. 36 ± 64.71) % had further increase at 8 months( P < 0. 05 ).Within the same time frame of 8 months, the thyroid weight in HID4 group was significantly lower than in Nil) group ( P <0. 05). No obvious changes were found in the expression of fas/fasL, TRAIL, bcl - 2 and bax after 0,1,2 and 4 months of treatments, however, the expression of fas/fasL, and TRAIL were upregulated at 8 months in HID2, HID3 and HID4 groups, bcl -2 and bax were not.Urinary iodine concentration was restored to normal level after iodine restriction for 3 months. The ROS level, apoptotic rate, thyroid weight, thyroidfunction and thyroid morphological changes were restored to normal level in HID1, HID2 and HID3 groups, while in HID4 group, only ROS level and early apoptotic rate returned to normal, not the late apoptotic rate, thyroid weight, thyroid function and thyroid morphological changes. The expression level of fas/ fasL, TRAIL, bcl - 2 and bax remained the same compared with the status before iodine restriction in all groups.CONCLUSIONS1. Chronic iodine excess increased the apoptotic rate, production of ROS, elongated S and sub -G0G1 phase, and shortened G0G1, G2M phase in thyro-cytes in wistar rats. Iodine excess induced thyrocytes apoptosis through ROS production, which in turn affected the cell cycle.2. Chronic iodine excess increased the expression of fas/fasL and TRAIL, but had no effect on the expression of bcl - 2 and bax.3. Chronic iodine excess doesn t lead to the formation of thyroid goiter in wistar rat.4. Iodine restriction treatment for 3 months in rats in HID1, HID2 and HID3 groups had the ROS level, apoptotic rate, thyroid weight, thyroid function and thyroid morphological changes restored to normal level, while rats in HID4 group only restored the ROS level and early apoptotic rate, not the late apoptotic rate, thyroid weight, thyroid function and thyroid morphological changes.5. It causes dangerous and toxic effects to normal animals with treatments of more than 3 to 20 times more of normal iodine intake. Iodine restriction could recover the toxic effects on thyroids with treatments of 3 to 6 times of normal iodine intake, but not 20 times of normal dose within a short period of time.