Metallothionein-I/II Knockout Mice Aggravate Mitochondrial Superoxide Production and Peroxiredoxin 3 Expression in Thyroid after Excessive Iodide Exposure

Abstract

Purpose: We aim to figure out the effect of metallothioneins on iodide excess induced oxidative stress in the thyroid.

Methods: Eight-week-old MT-I/II knockout (MT-I/II KO) mice and background-matched wild-type (WT) mice were used. Mitochondrial superoxide production and peroxiredoxin (Prx) 3 expression were measured.

Results: In in vitro study, more significant increases in mitochondrial superoxide production and Prx 3 expression were detected in the MT-I/II KO groups. In in vivo study, significantly higher concentrations of urinary iodine level were detected in MT-I/II KO mice in 100 HI group. Compared to the NI group, there was no significant difference existing in serum thyroid hormones level in either groups (P > 0.05), while the mitochondrial superoxide production was significantly increased in 100 HI groups with significantly increased LDH activity and decreased relative cell viability. Compared to WT mice, more significant changes were detected in MT-I/II KO mice in 100 HI groups. No significant differences were detected between the NI group and 10 HI group in both the MT-I/II KO and WT mice groups (P > 0.05).

Conclusions: Iodide excess in a thyroid without MT I/II protection may result in strong mitochondrial oxidative stress, which further leads to the damage of thyrocytes.

Figure 1

Oxidative and antioxidative effect of high concentrations of KI on the mitochondria of thyrocytes in MT-I/II KO mice and WT mice. (a) Decreased relative viability induced by high concentrations of KI (10⁻⁴ M, 10⁻³ M, and 10⁻² M) or 10⁻³M H₂O₂ in the thyroid cells of MT-I/II KO mice by MTT assay (N = 8). (b) Representative western blot of Prx 3 (27 kDa), β-actin (45 kDa) was used as a loading control. Densitometric analysis showed a significant increase (^* P < 0.05) in Prx 3 expression compared to untreated control in the thyroid cells of either WT or MT-I/II KO mice. (c) Increased LDH release following 2 h of high concentration of KI or H₂O₂ exposure in the thyroid cells of MT-I/II KO mice (N = 8). (d) High concentration of KI or H₂O₂ induced increased mitochondrial superoxide production in the thyroid cells of WT and MT-I/II KO mice. Histogram analysis was performed on the mean fluorescence intensity of MitoSOX Red as measured by flow cytometry. Experiments were repeated 3 times with similar results. Data are represented as mean ± SD. One-way ANOVA with the LSD test was used. ^* P < 0.05 compared with the control group of WT or MT-I/II KO mice, respectively; ^# P < 0.05 WT mice versus MT-I/II KO mice under the same treatment.

Figure 2

Mitochondrial superoxide production, Prx 3 protein expression, and LDH release and relative viability in the thyroid of WT and MT-I/II knockout mice following NI, 10 HI, or 100 HI diet for 14 days. (a) 100 HI intake decreased the relative viability in thyroid of MT-I/II KO and WT mice. (b) Representative western blot of Prx 3 (27 kDa), β-actin (45 kDa) was used as a loading control. Densitometric analysis showed a significant increase in Prx 3 expression in 100 HI group in the thyroid of either WT or MT-I/II KO mice, especially in MT-I/II KO mice. (c) 100 HI increased the LDH release in the thyroid of MT-I/II KO and WT mice. (d) 100 HI intake increased the mitochondrial superoxide production in thyroid of WT and MT-I/II KO mice. Histogram analysis was performed on the mean fluorescence intensity of MitoSOX Red as measured by flow cytometry. Experiments were repeated 3 times with similar results. Data are represented as mean ± SD (N = 10/group). One-way ANOVA with the LSD test was used. ^* P < 0.05 compared with the control group of WT or MT-I/II KO mice, respectively; ^# P < 0.05 WT mice compared with the MT-I/II KO mice under the same treatment.