| The goal of eliminating iodine deficiency disease (IDD) has been achieved since Universal Salt Iodization (USI) policy has been widely carried out in many nations including China. On the other hand, reports are increasingly appearing on the side effects caused by excessive iodine intake. Iodine-induced goiter has been reported in many nations and areas resulted from ingestion of excessive iodine in foods, in drinking water and in medications, or resulted from excessive iodine supplementation in iodated salt. Previous studies reveal that iodine deficiency during pregnancy may result in stillbirths, abortions and congenital abnormalities such as cretinism, a grave, irreversible form of mental retardation. However, it is not well documented if iodine excess also has an effect on embryonic development as iodine deficiency. Microelement selenium is essential to thyroid hormone metabolism and thyroid function by the selenocysteine-containing enzymes-glutathione peroxidase (GSH-Px) system and deiodinase system. Several studies have been carried out to establish the role of combined selenium and iodine deficiency. However, few researches were carried out to study the influence of different dose of selenium on iodine excess-induced disorder. In this study, on the basis of the investigation of embryo toxicity induced by excessive iodine intake in mice, we observed the changes of antioxidant ability in maternal mice, maternal-fetal thyroid hormone metabolism, and mRNA and protein expression of TRβ1 and Hoxc8 in fetus by RT-PCR ,Western-blot and immunohistochemistry to explore the mechanisms of embryo toxicity induced by excessive iodine intake. Meanwhile, intervention of different dose of selenium on the embryo toxicity induced by excessive iodine intake also was investigated. The results were summarized as follow:Part one The study of embryo toxicity induced by excessive iodine intake in miceObjective To estabolish the animal model of embryo toxicity induced by different doses of excessive iodine intake in mice and observe the embryo toxicity induced by excessive iodine intake.Methods 195 weanling BABL/C mice were assigned into 6 groups and given taper water or drinking water including different doses of iodine. The iodine concentrations were 1500μg/L, 3000μg/L, 6000μg/L, 12000μg/L and 24000μg/L respectively. Four months later, urinary iodine concentration of female mice was determined. Then the mice were mated (female:male=2:1) and the morning of appearance of a vaginal plug was taken as 0.5 day(0.5d) of pregnancy. Then 10 pregnant mice in control group were given 15000IU/kgbw.(ig.)VitA on 5.5d～15.5d of pregnancy as the positive control group. On the 19.5d of pregnancy, pregnant mice were sacrificed and the uteri were cut open to take out the fetuses immediately. The body weight, body length and tail length of the fetuses were measured; the malformation of external appearance, internal organs and skeleton of the fetuses were examined. Meanwhile, serum of maternal mice was used for thyroid hormone level determination and thyroid used for histological examination.Results In excessive iodine intake groups, mice showed diffused colloid goiter and a significant increase in urinary iodine level. Serum TT4 level was significantly higher, and TT3 level lower than control group when iodine level reached or exceeded 3000μg/L. An obvious reduce of average food intake and weight gain was observed in 12000μg/L and 24000μg/L groups. Average placenta weight was marked lower in 3000μg/L or above groups than that in negative control group. There was no significant difference in rate of pregnancy and miscarriage in excessive iodine intake groups. However, excessive iodine intake resulted in a dose-dependent increase rate of fetal resorption and stillbirth. An obvious external appearance malformation (such as anencephaly or encephalocele) was found in positive control group, but not observed in excessive iodine intake groups. The incidence of skeleton anomalies increased dose-dependently in excessive iodine intake groups. The skeletal abnormalities were including an extra or pair ribs, transformation of vertebrae and agenesis of sternbrae, metacarpi, fontanesl.Conclusion Excessive iodine intake induces embryo toxicity, mainly results in skeletal malformation. The threshold level of iodine induced embryo toxicity in this study is 3000ng/L (0.75mg/kgbw.).Part twoThe study of the mechanisms of embryo toxicity induced by excessive iodineObjective To observe the effect of excessive iodine intake on the maternal antioxidant ability, maternal-fetal thyroid hormone metabolism and the expression of TRβ1 and Hoxc8, to explore thoroughly the mechanisms of embryo toxicity induced by excessive iodine.Methods Animal treatment was the same as the part one. Serum, liver, thyroid, kidney and placenta of 12.5d and 19.5d pregnant mice were used to determine the oxidant/antioxidant level, selenium content and the activity and mRNA expression of deiodinase. 12.5d embryos were used for the analysis of mRNA and protein expression of TRβ1 and Hoxc8 by RT-PCR, Western blot and immunohistochemistry.Results ①Excessive iodine intake decreased the antioxidant ability and increased the MDA content in serum, liver and thyroid of maternal mice in a dose-dependant manner. Compared with the control group, serum GSH-Px activity reduced in all excessive iodine groups, serum SOD activity and hepatic GSH-Px and SOD activity decreased significantly in 3000μg/L or above groups, GSH-Px activity in thyroid dramatically decreased in 6000μg/L or above groups, and MDA level in serum, liver and thyroid elevated obviously in 3000μg/L or above groups. (2) Excessive iodine intake related to a decrease of selenium content in liver. Hepatic selenium content reduced significantly in 3000μg/L or above groups and had a obvious negative correlation with the dose of iodine. No change in placental selenium content was observed among groups.③ Excessive iodine intake induced an obvious depression of D1 activity and mRNA expression in liver and kidney. Hepatic and renal D1 activity and hepatic D1 mRNA expression reduced in 3000μg/L or above groups, and renal D1 mRNA level decreased in 12000μg/L and 24000μg/L groups. ④ A dose-dependent fall in 12.5d placental D2 activity was found in 3000μg/L or above groups. The change of mRNA expression was parallel with that of the activity. No change was found in D3 activity of 12.5d placenta, D2 and D3 activity of 19.5d placenta. However, for 19.5d uterus, D2 activity decreased and D3 activity increased. ⑤ mRNA and protein expression of TRβ1 and Hoxc8 was down-regulated by excessive iodine. The result of immunohistochemistry analysis demonstrated that Hoxc8 protein expression in nuclei of spinal ganglion also down-regulated in 3000μg/L or above groups.Conclusion Oxidative stress and relative selenium deficiency play an essential role in excessive iodine induced disorders. Excessive iodine results in embryo toxicity by the approach of influencing thyroid hormone level of maternal mice through the regulation of hepatic and renal D1 activity at transcription or translation level, and modulating the fetal thyroid hormone level through the regulation of the activities of D2 and D3 in placenta and uterus. The down-regulation of fetal TRβ1 and Hoxc8 expression at mRNA and protein level is an important molecular mechanism of embryo toxicity induced by excessive iodine intake.Part three Intervention of different doses of selenium on the embryo toxicity induced byexcessive iodine intakeObjective To observe the intervention of different doses of selenium on the embryo toxicity induced by excessive iodine intake, to determine the optimal dose of selenium intervention and to explore the mechanisms of selenium intervention on embryo toxicity induced by excessive iodine.Methods 240 weanling BABL/C mice were divided into 8 groups. A group was normal control group(NC) and given taper water; B group was excessive iodine(EI) control group and given drinking water containing 3000μg/L iodine; C～H groups were selenium supplementation(SS) groups and given drinking water containing 0.1, 0.2, 0.3, 0.4, 0.5, 1.0mg/L selenium, respectively, besides 3000μg/L iodine. Animal treatment and indices analysis were the same as the part one and part two.Results ①Selenium nutritional status of maternal mice was improved in SS groups. Compared with EI group, selenium level in liver and placenta increased in 0.2mg/L～1.0mg/L or 0.3mg/L～1.0mg/L SS groups, respectively. ②Average placenta weight was significant higher in 0.3mg/L SS group than that in EI group. Selenium supplementation resulted in a decrease in the rates of resportion and stillbirth, but this decrease was not significant. Selenium supplementation reduced the incidence of skeletal malformation at a certain range of doses. This reduce was significant at 0.3mg/L selenium, and above this level, incidence of anomalies increased on the contrary. ③In SS groups at the doses range from 0.1 to 0.5mg/L, activity of GSH-Px in serum, liver and thyroid of maternal mice elevated dose-dependently, the content of MDA reduced significantly. ④Selenium supplementation decreased serum TT4 and increased TT3 at the doses range from 0.1 to 0.5mg/L. No change was found in 1.0mg/L SS group.⑤ A dose-dependant increase of D1 activity in liver and kidney was observed in SS groups.Compared with EI group, D1 mRNA expression in liver markedly increased in 0.2-0.5mg/L SS groups, and in kidney increased in 0.3-1.0mg/L SS group. ⑥ Selenium supplementation enhanced D2 activity in placenta and uterus at a certain range of doses, but had no effect on the D3 activity in placenta. D3 activity in 19.5d uterus decreased obviously in 0.2-1.0mg/L SS groups. ⑦ Selenium supplementation resulted in an up-regulation of TRβ1 and Hoxc8 expression at mRNA and protein level at a certain range of doses.Conclusion Selenium supplementation leads to an elevation of selenium level in excessive iodine intake mice and exhibits a protective effect against embryo toxicity induced by excessive iodine intake. Selenium supplementation can ameliorate oxidative stress of thyroid by strengthening the antioxidant ability, influence maternal-fetal thyroid hormone level by regulating activity of deiodiase and up-regulate TRβ1 and Hoxc8 expression. Under the conditions of this study, the optimal dose of selenium, which can protect against embryo toxicity induced by excessive iodine, is 0.3mg/L (0.075mg/kgbw.).
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