Effects of excessive iodine intake during the perinatal period on thyroid function and higher brain functions in mouse offspring

Abstract

Iodine is an essential trace element crucial for thyroid hormone synthesis. While iodine deficiency has been recognized as a global health concern due to its association with hypothyroidism, certain regions may face challenges related to excessive iodine intake. The impact of excessive iodine intake during the perinatal period on higher brain functions remains unclear. To address this gap, we conducted a study using an animal model to elucidate the effects of perinatal iodine excess on higher brain functions. Dams received specific drinking water (control, ×20 iodine (KIO₃ 37.4 mg/L), ×200 iodine (KIO₃ 374 mg/L)) from prior to mating until weaning. Pups received the corresponding drinking water until the end of the experiment. Behavior test battery was utilized to investigate the behavioral outcomes associated with perinatal iodine excess. Excessive iodine intake increased learning acquisition in females whereas it decreased exploration of social novelty in males. Conversely, mRNA levels of several genes related to learning and memory in the hippocampus were rarely affected. Overall, the present study highlights the consequences of excessive iodine intake during developmental periods. However, these effects were mild and varied by sex, warranting the further investigation.

Introduction

Iodine is an essential trace element in Earth’s upper crust and is found primarily in or near coastal areas. In humans, 70–80% of iodine is found in thyroid, playing a crucial role in the synthesis of thyroid hormones. Iodine deficiency can lead to hypothyroidism, which, especially during the perinatal period, can result in delayed physical growth and neurodevelopmental abnormalities [1]. Worldwide efforts, such as iodine fortification of salt, have been made to prevent iodine deficiency actively. Nowadays, 88% of the global population use iodized salt, raising the number of countries with adequate iodine intake up to 118 in 2020 which is almost double from 67 in 2003 [2]. While only 21 countries remaining iodine-deficient, 13 counties are now reported to have excessive iodine intake and the number is expected to be increasing due to excess groundwater iodine or over-iodized salt [2]. For example, Japanese regularly consume iodine-rich seaweed, particularly kombu (Japanese kelp), leading to higher iodine intake. The recommended daily intake of iodine is 130 μg for adults and 110 μg for pregnant women in Japan [3]. However, actual iodine intake in Japan ranges from 1 to 3 mg/day, about ten times higher than the 138–353 μg/day in the United States [4]. Recent national survey targeting school-age children have reported that, while there are regional differences, iodine intake is generally appropriate [5]. However, the effects of excessive iodine intake remain unclear.

While it is known that excessive iodine intake can manifest in various systems, biological tolerance to amounts exceeding the physiological threshold is generally considered to be high [6]. However, there is an increased risk of developing iodine-induced thyroid dysfunction in individuals with a history of thyroid disease, the elderly, fetuses and newborns, or those with other risk factors. For instance, maternal excess iodine intake can cause fetal hypothyroidism and goiter development due to the inability of the fetus to escape the Wolff-Chaikoff effect [7]. While infants with congenital hypothyroidism are well known to face risk for some degree of impaired neurodevelopment, little is known about the long-term impacts of chronic excessive iodine intake on brain functional development. A recent cohort study reported a positive correlation between iodine intake during pregnancy and neurodevelopmental delay in infants [8]. In particular, a significantly increased risk of adaptive and language developmental delay was observed in infants born to mothers with iodine nutritional status above requirement. At the same time, higher thyroid-stimulating hormone (TSH) levels were also associated with fine motor developmental delay [8]. Another cohort study found that maternal serum iodine excess during pregnancy is slightly related to intellectual and motor developmental delay in infants [9]. A rodent study reported that excessive iodine intake from pre-pregnancy to weaning caused spatial learning deficits with altered in thyroid hormone status [10], whereas another study found deficient or excessive iodine intake during pregnancy impaired spatial learning [11]. This discrepancy warrants further investigation using animal models. Therefore, in this study, we created a chronic iodine excess mouse model to elucidate changes in thyroid function, as well as to evaluate neurobehavioral functions such as cognition and learning using touch panel-based learning devices, and to clarify the mechanisms involved using molecular biological methods.

Materials and Methods

Animals and treatment

C57BL/6J mice were used, and breeding was carried out at the Gunma University Biological Resource Center. All experiments were performed in accordance with the relevant guidelines and regulations. Mice were housed in a room with a temperature of 24°C and a 12-hour light-dark cycle (light period: 7:00 to 19:00) and provided with free access to water and food.

According to the Ministry of Health, Labour and Welfare, Japan, the recommended iodine intake for adults in Japan is 130 μg/day, with actual intake reported to be 1–3 mg/day [3]. Additionally, in potassium iodide (KI) therapy for Graves’ disease, doses of 5–100 mg/day are administered. Therefore, in this study, two experimental groups were established: one receiving 20 times the normal iodine intake and the other receiving 200 times the normal iodine intake. The control group received normal drinking water. The feed used (CE-2, CLEA Japan, Tokyo, Japan) contained 0.17 mg/100 g of iodine, and it was estimated that the mice consumed 6 μg/day of iodine. For the experimental groups, potassium iodate (KIO₃, Sigma-Aldrich, St Louis, USA) was administered to the mice via drinking water at doses of 37.4 mg/L (×20 group) and 374 mg/L (×200 group). The experimental groups received the administration continuously throughout the entire experimental period.

Adult male and female C57BL/6J mice were purchased from Japan SLC, Inc (Hamamatsu, Japan). After administering KIO₃ solution or water for 4 weeks or more, mating was conducted. Offspring were weaned at postnatal day 21, and mice from each group were raised until 10 weeks of age before behavioral battery experiments were conducted. After completion of the behavioral experiments or at the timing of weaning, mice were sacrificed under an i.p. administration of a ketamine-xylazine mixed anesthetic (100 mg/kg + 10 mg/kg, respectively) to collect tissues. RNA was extracted from the hippocampus, and mRNA expression levels were analyzed using reverse transcription followed by real-time PCR. Blood and urine samples were also collected for measurement of thyroid hormone levels and urinary iodine levels. Schedule of the experiment is described in Fig. 1A. All experiments comply with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines 2.0 and were approved by the Gunma University Animal Experimentation Facility Operating Committee (14-016).

Fig. 1  Administration of iodine and its effect on pup’s growth

A) Schematic illustration of experimental schedule. After administering KIO₃ solution or water for 4 weeks or more, C57BL/6 J female mice were mated. Offspring were weaned at postnatal day 21. Dams were sacrificed at weaning day for collecting plasma and/or thyroid. Behavioral battery was conducted after 10 weeks of age. After completing battery, tissues were collected.

B) The change in pups’ body weight that was measured every week. There was no significant difference between groups by two-way ANOVA.

(male: control; n = 17, ×20; n = 17, ×200; n = 15, female: control; n = 21, ×20; n = 18, ×200; n = 14)

Data are presented as the mean ± SEM.

Urinary iodine measurement

Urinary iodine measurement was described previously [12]. In brief, urine iodine concentrations were determined by inductively coupled plasma mass spectrometry (ICP-MS) (ELAN DRC II, Perkin Elmer, Waltham, USA). Sample and standard solutions were prepared by alkaline digestion with 25% tetramethyl ammonium hydroxide. Iodine was detected at mass ¹²⁷I, with ¹³⁰Te as the internal standard.

Thyroid hormone measurement

Thyroid hormones were measured as described previously. Briefly, blood samples were centrifuged (8,000 g) to separate plasma. The concentrations of total triiodothyronine (T₃) and thyroxine (T₄) in the plasma were measured by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) (QTRAP 5500 LC-MS/MS System; AB SCIEX, Tokyo, Japan). 200 μL of plasma was mixed with internal standard (10 μL of a 25 ng/mL solution of T₃-¹³C₆). Thyroid hormones were extracted from plasma using the EVOLUTE EXPRESS AX (Biotage AB, Uppsala, Sweden). Then, the extracts were subjected to LC-ESI-MS/MS analysis. MS/MS analysis was performed in multiple reaction monitoring and positive ionization mode, using mass transition m/z 651.8 > 605.8 for T₃, m/z 777.8 > 731.8 for T₄, and m/z 657.8 > 611.8 for T₃-¹³C₆.

Histology

Thyroid gland in continuity with the larynx and trachea were removed and placed in 4% paraformaldehyde solution. Thyroid was embedded in paraffin and then sectioned at 5 μm. The sections were stained with hematoxylin and eosin (H&E) and evaluated by microscope (BZ-9000, Keyence, Osaka, Japan). Uniform tissue sections were selected, and individual follicular areas were measured using the BZ-X analyzer (Keyence, Osaka, Japan). The average follicular area for the left and right thyroid glands was calculated, and the mean of these values was determined to obtain the individual’s average follicular area.

Behavioral battery tests

The methods for each behavioral test were described previously [13-16]. Procedure information for each experiment is explained briefly below.

1. Open field test

Open field test was carried out to assess locomotor activity levels and exploration in a novel environment. The mice were put in an open field (45 × 45 × 20 cm) detected by 16 × 16 crossed infrared beams at intervals of 2.5 cm on the sides (LE 8811; Panlab, S.L.U., Barcelona, Spain). Acti-Track program (Panlab, S.L.U.) was used for data analysis. Travelling distance and time in the center area was calculated.

2. Rotarod test

Motor coordination was evaluated using rotarod (LSI-Letica Scientific Instruments, Barcelona, Spain). Five rotarod trials with increasing speed from 4 rpm to 40 rpm in 5 min were performed. The average time on the rotarod was used for analysis.

3. Object in-Location Test / Object Recognition Test

To examine memory, OLT and ORT were conducted. All the tests were performed in an open-field arena (30 × 30 × 39 cm). Before tests, we habituated the mice to the arena without objects for 30 min/day for five consecutive days. The recognition test consisted of a sample exposure and a test phase. In the sample-exposure test, mice spent 5 minutes in the open-field arena with two identical objects. The 7-minute test session was conducted 10 minutes (short) or 24 hours (long) after the sample-exposure test. In the test session of ORT, one object was replaced by a new object. In the OLT session, we changed the location of one object. Objects were cleaned using 70% ethanol between sessions to avoid olfactory influence. We manually assessed exploration times to objects using recorded videos. Each test was conducted with at least a three-day interval between them.

4. Pairwise visual discrimination task

Pairwise visual discrimination task was conducted using Touch Panel Operant System (O’hara & Co., Ltd., Tokyo, Japan). Following 5 days of food restriction (approximately 90% of free feeding body weight), mice were trained to learn a task procedure. In the training session, the machine presented identical stimuli (windmill) at both windows. The mouse was rewarded a pellet via the pellet dispenser when it touched either stimulus. This training session was conducted for 30 min each day for at least 3 days until the mice could complete task minimum 20 times.

After the completion of the training sessions, mice were trained to discriminate between two different novel stimuli that were vertical and horizontal stripes. One of these stimuli was assigned as the correct stimulus, whereas the other was the incorrect. Assignment of correct stimulus is counterbalanced. A response to the correct stimulus resulted in a reward pellet, whereas a response to the other resulted in no reward. The left–right presentation of the correct stimuli was randomized across a daily session, which consisted of 50 trials. This task was conducted for 9 days.

5. Three-chamber social interaction test

The three-chamber social interaction test was conducted to examine the social behavior. Mice were habituated to the three-chamber box (39 × 20 × 30 cm) for five minutes. In the session 1, a tested mouse was introduced to a novel mouse (“Stranger 1,” age- and sex-matched) in a small box in the chamber 1. The box in the chamber 2 was empty. Time the tested mouse approached to each box was counted for 10 min. In the session 2, another novel mouse (“Stranger 2,” age- and sex-matched) was placed in the chamber 2. Again, the approaching time to each chamber was counted for 10 min. The sessions were recorded by the video camera located over the apparatus and analyzed by ANY-maze ver. 6.3 (Stoelting Co., Wood Dale, USA).

DNA microarray

Total RNA was extracted from dorsal hippocampus after behavioral test battery using the RNeasy Lipid Tissue Mini kit (QIAGEN, Hilden, Germany) followed by manufacturer’s protocol. The quality of RNA was checked by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA) as RNA Integrity Number (RIN) >8. Total RNA from female control group and ×20 group (n = 2/group) were hybridized to the SurePrint G3 Mouse GE 8 × 60K Microarray (Agilent Technologies). Microarray signals were detected using the Agilent DNA Microarray Scanner. The raw signal intensity of each spot was normalized by substitution with the background signal intensity. The intensities of the detected signals for each gene were normalized by a global normalization method. The RankProd (version 3.16.0) package on R (version 4.0.3) was used to identify genes with increased or decreased expression. A significance level of false-discovery rate (FDR) = 0.05 was set in all the analyses.

Real-time PCR

Total RNA was prepared from dorsal hippocampal tissues using RNeasy Lipid Tissue Mini Kit (QIAGEN) same as above (n = 6/group). 500 ng of total RNA was reverse-transcribed by ReverTra Ace^®️ qPCR Master Mix with gDNA Remover (FSQ-301, Toyobo Co., Ltd. Life Science Department, Osaka, Japan) to synthesize cDNA. Real-time PCR was performed using Thunderbird^®️ Sybr qPCR Mix (Toyobo) and the Step One Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Levels of glyceraldehyde 3-phosphate dehydrogenase (Gapdh) and cyclophilin b (Cypb) were used for the housekeeping gene. All the relative mRNA levels were normalized to the geometric mean of the two internal controls as previously described [13]. The primer sequences of each gene are shown in Supplementary Table 1.

Statistical analysis

GraphPad Prism version 8.4.3 for Windows (GraphPad Software, San Diego, USA) was used for all statistical analyses and graphing data. All values are presented as mean ± standard error of the mean (SEM). Statistical comparisons were performed by either t-test, one-way or two-way analysis of variance (ANOVA), followed by the Dunnet’s test or Bonferroni’s test for post-hoc analysis. Differences were considered significant at p < 0.05.

Results

Impact of excessive iodine on growth and thyroid function

The trend of body weight of pups during the experimental period is shown in Fig. 1B. No significant differences were observed between any of the groups, regardless of gender. Additionally, to confirm the excessive iodine model, urinary iodine concentration was measured using ICP-MS. The results showed approximately 33 times higher iodine concentration in the ×20 group and approximately 493 times higher concentration in the ×200 group compared to the control group (Table 1). This confirmed that the intended iodine administration levels were achieved in this model. LC-ESI-MS/MS revealed a significant decrease in plasma T₄ levels in ×200 group of female offspring. On the other hand, no significant differences were observed in T₄ and T₃ of the male group and T₃ of the female group. (Fig. 2A). In addition, thyroid enlargement tendencies were observed in the dose-dependent manner with the significant enlargement in ×200 group compared to the control group in both sexes (Fig. 2B). Thinning of thyroid follicular cells was also observed, suggesting thyroid dysfunction (Fig. 2B). mRNA levels of thyroid hormone responsive genes in hippocampus of adult offspring were not changed (Fig. 2C). In dams, thyroid enlargement was observed in the same manner as pups, and T₄ was significantly increased in ×200 group (Supplementary Fig. 1).

Table 1 Urine iodine concentration: Dam (n = 7/group), Pup (n = 10/group)

The total is calculated from the individual values of 27 animals, including dams and pups.

(ng/mL)	Dam	Adult pup male	Adult pup female	Total
control	637.7 ± 114.7	474.5 ± 37.57	432.6 ± 47.35	501.3 ± 38.96
×20	13,626 ± 4,271	16,789 ± 3,305	19,043 ± 3,140	16,804 ± 1,982
×200	214,239 ± 85,616	198,612 ± 46,424	319,484 ± 122,332	247,431 ± 52,463

Fig. 2  The effects of excessive iodine on thyroid system

A) The concentrations of total triiodothyronine (T₃) and total thyroxine (T₄) in adult offspring. (n = 10/group)

B) Representative H&E staining of the thyroid in adult offspring. The average follicular areas were measured. (n = 5/group)

C) The mRNA levels of thyroid hormone-regulated genes in adult offspring hippocampus, measured by qPCR. (n = 6/group)

Data are presented as the mean ± SEM. * p < 0.05 ** p < 0.01 compared to those of the control group, determined by Dunnett test

Excessive iodine does not affect motor function

Motor function was assessed by open field and rotarod tests. Significant differences were detected in travel distance between male groups (Fig. 3A), whereas there were no differences in rotarod test and grip strength test (Fig. 3B, C).

Fig. 3  The effects of excessive iodine on motor function

A) Open field test: Travel distance (left) and time in the center area (right) in 30 minutes. Two-way ANOVA in group factor gave *p < 0.05.

B) Rotarod test: average latency to fall from the rotarod was described.

C) Grip strength test

(male: control; n = 17, ×20; n = 17, ×200; n = 15, female: control; n = 20, ×20; n = 18, ×200; n = 14)

Data are presented as the mean ± SEM.

Excessive iodine intake enhanced learning ability in female mice

To evaluate memory, object in-location test (OLT) and object recognition test (ORT) were conducted at 10–11 weeks of age. Discrimination rates indicating the proportion of exploration time for novel objects or locations showed no significant differences among the groups in either OLT or ORT (Fig. 4A).

Fig. 4  The effects of excessive iodine on cognitive function

A) The object location recognition memory test (OLT) and object recognition memory test (ORT): The test began with a 7-min sample exposure phase followed by a 7-min test phase for both OLT and ORT, after a 10-min or 24-hr interval in the home cage. Schematic illustrations of test objects were shown under each graph.

B) The learning curves of pairwise visual discrimination task. Two-way ANOVA in group factor gave **p < 0.01. † p < 0.05, ††p < 0.01 compared to those of the control group, determined by Bonferroni test.

C) Three-chamber social interaction test: *p < 0.05, **p < 0.01, ***p < 0.001 determined by unpaired t-test.

Data are presented as the mean ± SEM.

(male: control; n = 17, ×20; n = 17, ×200; n = 15, female: control; n = 20, ×20; n = 18, ×200; n = 14)

D) Summary of behavioral battery test

Results of the visual discrimination test conducted using a touchscreen-operated learning apparatus are shown in Fig. 4B. In males, both the control and ×20/×200 groups were at chance levels on the first day of testing but showed an increase in correct response rates with learning acquisition from the second day onwards, reaching 80% on the fifth day. In contrast, in females, while the control group reached 80% correct response rate on the seventh day, the ×20 and ×200 groups reached 80% on the fourth day, indicating an increase in learning acquisition.

Excessive iodine intake altered social behavior in ×200 male group

A three-chambered social interaction test was conducted to evaluate social behavior (Fig. 4C). This test consisted of habituation, social evaluation (Session 1), and evaluation of social novelty exploration (Session 2). In Session 1, significant increases in exploration time towards novel individuals (Stranger Mouse 1) were observed in both male and female groups, indicating no abnormalities in social behavior. In Session 2, significant increases in exploration time towards Stranger Mouse 2 were observed in all female groups, while only the ×200 male group showed no significant difference in exploration time towards Stranger Mouse 1 and 2, suggesting a potential abnormality in social novelty exploration in the ×200 male group. All the results of behavioral tests were summarized in Fig. 4D.

mRNA levels in the adult offspring’s hippocampus were not affected

Among brain regions, the hippocampus is known to be highly sensitive to thyroid hormones. While no abnormalities were observed in OLT and ORT, which are known to be involved in the hippocampus (Fig. 4A), the results of the visual discrimination test (Fig. 4B) suggested an influence on the hippocampus. Therefore, we investigated the mRNA levels of several genes related to learning and memory in the adult hippocampus using qPCR. Although several genes, such as Grin2a in the male ×20 group and Camk2a in the male ×200 group, were significantly altered, no changes supporting the behavioral findings were observed (Fig. 5A). Based on these results, we performed a genome-wide gene expression analysis using a microarray, comparing the female control group and the female ×20 group, which showed differences in the visual discrimination test (Fig. 4B). Unexpectedly, only four genes showed significant changes according to the RankProd analysis (Fig. 5B). Compared to the control group, Scgb1a1 and Etnppl were downregulated in the ×20 group, while Cyp2g1 and Fam187b were upregulated. However, these genes are unlikely to affect behavioral outcomes.

Fig. 5  The effects of excessive iodine on gene expression profiles in the hippocampus

A) mRNA levels of several genes related to learning and memory in the hippocampus were analyzed by qPCR (n = 6/group). Data are presented as the mean ± SEM. *: p < 0.05 compared with the control group, determined by the Dunnett’s test.

B) Scatter-plot analysis of the microarray data compared between control female and ×20 female group (n = 2/group). This graph shows similar gene expression patterns from the hippocampus of each group, except for four genes (Scgb1a1, Etnppl, Cyp2g1 and Fam187b), which are unlikely to influence the behavioral outcomes in the female ×20 group.

Discussion

The present study demonstrated the long-term effects of chronic excess iodine intake on thyroid function, somatic growth, and neurodevelopment in mouse offspring. There was a tendency of thyroid enlargement with an increase of iodine intake in both male and female. Male iodine excess displayed significant decreases in exploration in an open field and social activity without changing in thyroid hormone levels. On the other hand, female iodine excess mice presented a better cognitive performance compared to the control mice with decrease of T₄ levels in ×200 iodine group (Graphical Abstract). There was no remarkable change in gene expression profiles following the exposure to excess iodine. While excess iodine intake leads to alterations in thyroid function and changes in brain phenotypes, the extent to which each factor contributes to the observed effects remains uncertain.

Graphical Abstract 

Coastal goiter, linked to excessive iodine intake in Japan, was observed in some coastal areas of Hokkaido, manifesting as colloid follicular adenoma with normal thyroid function [17]. Similarly, thyroid changes resembling colloid follicular adenoma were observed in the excessive iodine intake group in this study (Fig. 2B). On the other hand, Teng et al. reported hypothyroidism in individuals with excessive iodine intake [18]. In a rodent study, Zhang et al. reported an increase in total T₄ and free T₄ levels in mothers administered three times the normal iodine dose, while only mild elevation of thyroid-stimulating hormone (TSH) was observed in neonatal rats, with other parameters remaining normal [10]. Xia et al. reported that iodine doses over eight times normal increased TSH, decreased total T₃, and increased total T₄ in female adult mice [19]. Consistently, this study observed elevated total T₄ in mothers of the ×200 group (Supplementary Fig. 1B), while female pups in this group showed reduced total T₄ (Fig. 2A). Unlike iodine deficiency models, thyroid hormone levels in iodine excess models vary depending on species, dose, duration, and blood collection timing, requiring careful confirmation in each experiment. Furthermore, since T₃ that binds to nuclear receptors in the brain is largely formed locally from T₄ by type 2 iodothyronine deiodinases, it is important to note that the amount of thyroid hormones, especially T₃, in the plasma does not necessarily correspond to the levels present in the brain [20].

The effects of iodine excess on cognitive learning and memory tests were evaluated using OLT, ORT, and visual discrimination test. OLT and ORT capitalize on rodents’ preference for novelty; with OLT assessing spatial memory primarily mediated by the hippocampus, and ORT assessing non-spatial memory primarily mediated by the olfactory cortex [21, 22]. Previously, we demonstrated differential effects of thyroid hormone on various brain regions using OLT/ORT in hypothyroid mouse models [16]. Therefore, it was anticipated that different behavioral experiment results would be obtained depending on the brain region, but no significant differences were observed among groups or genders in each experiment (Fig. 3, 4A). Additionally, we conducted learning acquisition assessments using operant tasks in visual discrimination test (Fig. 4B). We performed experiments using an operant box with a touch screen, similar to those used for human cognitive function assessments. This experiment reflects hippocampal function [23]. Similar to the results of OLT and ORT, no differences in learning acquisition were observed in males, but females in the excessive iodine intake groups showed increased learning acquisition ability (Fig. 4B). In the three-chamber social interaction test to evaluate sociability, no changes were observed in females, whereas a decrease in social novelty exploration was observed in the ×200 group of males (Fig. 4C). Although the mechanisms of these sex-dependent differences have not yet been understood, previous studies report sex-dependent thyroid hormone action in brain [24]. Results of present study may also reflect such differential effects during brain development.

Since behavioral experiments indicated the involvement of the hippocampus, we conducted an analysis of hippocampal mRNA levels in adult offspring using real-time PCR and microarray (Fig. 5). Unlike the previous study, no remarkable changes were observed in microarray possibly because of the age we performed the analysis. Whereas the previous study reported the change in RNA transcription in the brain due to perinatal iodine excess at PD 20 [25], during which a series of genes are dynamically regulated for neurodevelopment [26], we used hippocampal tissues from PD 90 mice. In particular, Bdnf was expected to be changed as previous studies found the downregulation of BDNF-TrkB signaling and expression of Bdnf in the hippocampus following iodine excess intake [10, 11, 27]. However, no consistent trends were observed in the expression levels of genes known to be affected by thyroid hormone (Nrgn, Hr, Ntf3, Nr1d1, Klf9, Mbp, Bdnf, Itpr1, Thra, and Thrb) among the experimental groups (Fig. 2C). One reason for this discrepancy could be that genes regulated by thyroid hormone exhibit varying effects depending on brain region and age [26], but a major contributing factor is believed to be the lack of dynamic changes in thyroid hormone levels in the experimental groups. On the other hand, no significant changes were observed in the expression levels of genes related to synapses which constitute neural circuits and are involved in the transmission and processing of information. NMDA receptors, AMPA receptors, and presynaptic and postsynaptic membrane-related genes were not affected, except for a decrease in Grin2a (cording NMDA receptor type 2) in male ×20 group and an increase in Camk2a in male ×200 group (Fig. 5A). CAMK2α is known to play an important role in learning and memory by phosphorylating enzymes, synaptic vesicle binding proteins, ion channels, and neurotransmitter receptors [28]. Considering no improvement in learning and rather impairment in social recognition in male ×200 group, this increase in Camk2a expression could be a part of compensating effects. Also, the downstream of the pathway should be examined. The microarray results identified four genes that were significantly altered (Fig. 5B). However, there is insufficient evidence to support their involvement in behavioral changes. Etnppl has recently been reported to be specifically expressed in astrocytes within the central nervous system, including the adult hippocampus [29]. Given that its expression fluctuates in various neurological disorders, its biological significance is suggested [29, 30]. However, many aspects remain unclear, making it difficult to directly link these findings to our results. To elucidate the detailed mechanism, additional experiments are required.

This study has several limitations. First, changes in thyroid hormone levels in pups were only examined in adult blood samples, and we were unable to assess thyroid hormone levels in the blood and brain during early development. Additionally, gene expression profiling was conducted only in the hippocampus of adults. Notably, the microarray analysis was performed with a small sample size (n = 2 per group), and increasing the sample size would be necessary to obtain more accurate data. To achieve a more comprehensive understanding of the underlying mechanisms, it would also be essential to examine other brain regions and different developmental stages. These limitations highlight the need for further research to fully elucidate the developmental impact of excessive iodine intake. Future studies addressing these gaps will be crucial in deepening our understanding of its long-term effects on brain function and behavior.

Funding

This work was supported by a grant from Yazuya Co. Ltd. (challenge category, 2015) to IA.

Disclosure

None of the authors have any potential conflicts of interest associated with this research. Noriyuki Koibuchi is a member of Endocrine Journal’s Editorial Board.

Author Contributions

Izuki Amano: Conceptualization, funding acquisition and resources, data curation, formal analysis, and wrote manuscript.

Ayane Ninomiya: Data curation, formal analysis, reviewed manuscript.

Hiroyuki Yajima: Data curation.

Machiko Suda-Yajima: Data curation.

Michifumi Kokubo: Data curation.

Miski Aghnia Khairinisa: Data curation.

Yusuke Takatsuru: Data curation.

Reika Kawabata-Iwakawa: Data curation.

Satomi Kameo: Data curation.

Shogo Haraguchi: Data curation.

Asahi Haijima: Data curation.

Noriyuki Koibuchi: Supervision of project and review and editing manuscript.

Ethics Approval

All experiments comply with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines 2.0 and were approved by the Gunma University Animal Experimentation Facility Operating Committee (14-016).

Data Availability

Microarray data has been deposited in the National Center for Biotechnology Information’s Gene Expression Omnibus database, URL accession number GSE270137: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE270137. The data in support of the results are available from the corresponding author on reasonable request.

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