| ObjectivesTo discuss the effects of high concentrations of iodide on relative cell viability,LDH release, mitochondrial superoxide production, Prx 3 and cleaved caspase 3 and9 expression and cell apoptosis on Fisher rat thyroid cell line(FRTL), and reveal the development process of oxidative stress on FRTL cells caused by 100 μM iodide.We investigate the effect of DETC, SOD and mitochondrial ETC complex I inhibitor Rotenone and complex III inhibitor Antimycin A in 100 μM iodide induced oxidative stress on FRTL cells. Use methallothionein I/II knockout mice(MT-I/II KO mice) to set up short-term iodide excess modle, discuss the effects of short-term iodine excess in the spleen cells with or without methallothionein I/II and investigate the effects of high concentrations of iodide on cell viability, LDH release,superoxide production and Prx 3 expression in the spleen.Methods Effects of high concentrations of iodide on oxidative stress in FRTL cells1.After exposure to 100 μM KI, 2 mM DETC, 1000 unit/ml SOD, 0.5 μM Rotenone and 10 μM Antimycin A for 2h, changes of cell viability were evaluated by methyl thiazolyl tetrazolium(MTT) colorimetry.2.Lactate dehydrogenase(LDH) leakage in the supernatant following different treatment was measured using a cytotoxicity detection kit(LDH).3.Changes of superoxide production were assayed by flow cytometry and fluorescence microscope using MitoSOX.4.Methods of immunofluorescence staining and Western blot technology were used for detecting Prx 3 expressing in FRTL cells.5.Cleaved caspase 3 and cleaved caspase 9 were tested using ELISA kits.6.TUNEL kit was used for detecting cell apoptosis.Effects of short-term iodide excess on oxidative stress in the spleen of Methallothionein I/II knockout miceIn vivo study:8 weeks male MT-I/II KO mice and Wildtype mice(WT mice) were divided randomly to three groups, NI(normal iodide intake group), 10HI(10 times high iodine intake group) and 100HI(100 times high iodide intake group), each group consisted of ten mice. They all have normal iodine chow(containing 300μg/kg iodine) and deionized water(containing different concentration of iodide) for 14 days. Iodide intake each day was 1.5 μg, 15 μg and 150 μg. Animals were sacrificed respectively at the 14 days, took the body weight and spleen weight analyzed the ratio. Cell relative viability was meassured by MTT method. LDH release was detected by LDH kit. Mitochondrial superoxide production in the spleen cells was messured by flow cytometry using MitoSOX. Western blot technology was used to investigate the expression of Prx 3.In vitro study:Spleen cell suspensions were prepared from six to eight-week old and healthy male methallothionein I/II knockout mice(MT-I/II KO mice) and wildtype(WT)mice, cells were exposed to various concentrations of KI( 0, 10-4, 10-3, 10-2 mol/L KI)and 10-3 mol/L H2O2 respectively for two hours. Mitochondrial superoxide production in the spleen cells was meassured by flow cytometry using MitoSOX. Cell viability was assayed by methyl thiazolyl tetrazolium(MTT) colorimetric method. LDH release was detected by LDH kit. Mitochondrial superoxide production in the spleen cells was meassured by flow cytometry using MitoSOX. Western blot technology was used to investigate the expression of Prx 3.Results Effects of high concentrations of iodide on oxidative stress in FRTL cells1.100 μM iodide induced oxidative stress on FRTL cellsAfter exposure to 100 μM KI for 2h, relative cell viability of FRTL cells decreased(P < 0.05), LDH release increased(P < 0.05), mitochondrial superoxide production increased(P < 0.05), Prx 3 protein expression increased(P < 0.05),cleaved caspase 3 and cleaved caspase 9 expression increased(P < 0.05), TUNEL positive cells increased(P < 0.05).2. Effect of SOD in oxidative stress on FRTL cells induced by 100 μM iodide(1) DETC(2 mM) enhanced the decreased relative cell viability(P < 0.05), the increased LDH release(P < 0.05), the increased mitochondrial superoxide production,and the increased Prx 3 protein( P < 0.05) and cleaved caspase 3 and cleaved caspase9 expression(P < 0.05) and TUNEL positive cells induced by 100 μM KI(P < 0.05).(2) SOD(1000 unit/ml) attenuated the decreased relative cell viability(P < 0.05),the increased LDH release(P < 0.05), increased mitochondrial superoxide production,and increased Prx 3 protein expression(P < 0.05) and cleaved caspase 3 and cleaved caspase 9 expression(P < 0.05) and TUNEL positive cells(P < 0.05) induced by 100μM KI.3.Effect of mitochondrial ETC(electron transport chain) complex inhibitor in oxidative stress on FRTL cells caused by 100 μM iodideBoth Rotenone(0.5 μM) and Antimycin A(10 μM) enhanced the decreased relative cell viability(P < 0.05), the increased LDH release(P < 0.05), increased mitochondrial superoxide production, and increased Prx 3 protein expression(P <0.05) and cleaved caspase 3 and cleaved caspase 9 expression(P < 0.05) and TUNEL positive cells(P < 0.05) induced by 100 μM KI.Effects of short-term iodide excess on oxidative stress in the spleen of Methallothionein I/II knockout mice In vivo study:In MT-I/II KO mice and WT mice, compared to the control group and 10 HI group, 100 HI group showed a significant decline in organ / body weight ratio(P <0.05); MTT showed a significant decline in cell viability(P < 0.05); LDH release was significantly increased(P < 0.05); Mitochondrial superoxide production was significantly decreased(P < 0.05); Prx 3 expression was significantly increased(P <0.05). Compared to the WT mice, in both 10 HI and 100 HI groups of MT-I/II KO mice, the decline of organ/ body weight ratio was even higher(P < 0.05); the decrease of the cell viability was much remarkable(P < 0.05); LDH release was significantly increased(P < 0.05); mitochondrial superoxide production was significantly increased(P < 0.05); the expression of Prx 3 was increased significantly(P < 0.05).In vitro study:In both MT-I/II KO mice and WT mice, cell viability, LDH release and mitochondrial superoxide production and Prx 3 expression of spleen were different among the treatment groups(all P < 0.05). Compared to the control group, cell viability of 10-4, 10-3, 10-2 mol/L KI treatment groups and 10-3 mol/L H2O2 group decreased(P < 0.05), LDH release increased significantly(P < 0.05), mitochondrial superoxide production in the spleen cells increased significantly(P < 0.05), Prx 3expression in 10-3, 10-2 mol/L KI and 10-3 mol/L H2O2 treatment groups significantly increased(P < 0.05). In 10-4, 10-3, 10-2 mol/L KI and 10-3 mol/L H2O2 treatment groups, cell viability of MT-I/II KO mice spleen were lower than WT mice; LDH release were higher than WT mice(all P < 0.05); mitochondrial superoxide production were higher than WT mice(all P < 0.05); Prx 3 expression were higher than WT mice(all P < 0.05).Conclusion Effects of high concentrations of iodide on oxidative stress in FRTL cells1.Exposure to 100 μM iodide for 2h induced oxidative stress on FRTL cells,which lead to decreased relative cell viability of FRTL cells, increased LDH release,increased mitochondrial superoxide production, increased Prx 3 protein and cleaved caspase 3 and cleaved caspase 9 expression, induced cell apoptosis.2. 2 mM DETC further enhanced oxidative stress on FRTL cells induced by iodide excess, effected as a damage factor.3. 1000 unit/ml SOD attenuated oxidative stress on FRTL cells induced by iodide excess, effected as a protective factor.4. 0.5 μM Rotenone and 10 μM Antimycin A both further enhanced oxidative stress on FRTL cells induced by iodide excess, both effected as harmful factors.Effects of short-term iodide excess on oxidative stress in the spleen of Methallothionein I/II knockout mice1. 100 times high iodide intake for 14 days may induce oxidative stress in the spleen cells of MT-I/II KO mice and WT mice, declined the organ / body weight ratio and the cell viability, increased LDH release and increased mitochondrial superoxide production and Prx 3 expression, which were much more significant in MT-I/II KO mice, suggesting the antioxidative effect of MT-I/II in short-term iodide excessinduced oxidative stress in the spleen.2. High concentrations of iodide(10-4 mol/L KI, 10-3 mol/L KI and 10-2 mol/L KI)may induce oxidative stress in the spleen cells of MT-I/II KO mice and WT mice,two hours exposure to 10-4 mol/L KI, 10-3 mol/L KI and 10-2 mol/L KI may decline the cell viability, increase LDH release and increase mitochondrial superoxide production and Prx 3 expression; Which were much more significant in MT-I/II KO mice, suggesting the antioxidative effect of MT-I/II in high concentrations of iodide induced oxidative stress in the spleen. |
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