Fluoride is present in our environment and is added to drinking water supplies for cariostatic purposes as a prophylactic agent in dental caries, with a recommended dose between 0.7 and 1.2 mg/l (1 ppm) [13,18]. In this study, growing rats (30 days) were chosen as an experimental model. The intake of fluoride induced a significant change in the thyroid gland of growing rats and insignificant changes in nongrowing rats (120 days) [13,23]. This dose corresponds to the recommended daily physiological dose of fluoride. The chosen route in this study for exposure was through drinking water to mimic human exposure .
The biochemical assay of hormones in the Na–F-treated rats showed a significant and a highly significant decrease in serum T4 and T3, respectively. However, there was a highly significant increase in serum TSH. These results were similar to those reported by many researchers [18,19,24]. Serum levels of T3, T4, and TSH are commonly used as reliable indicators of the thyroid function in humans and experimental animals. All reactions necessary for the formation of T3 and T4 are influenced and controlled by TSH . The thyroid gland has a strong capacity for absorbing and accumulating fluoride [26,27]. Fluoride and iodine belong to the chlorine group element, but fluoride is more chemically active than iodine. The functional disorder of the thyroid caused by fluoride may be because of competition with iodine and also its effect on the absorption and condensation of iodine. Moreover, it can influence the biologic activity of the functional enzyme system and interferes with them (enzymes that catalyze the conversion of T4 into T3), thereby leading to a decrease in circulating thyroid hormone levels [19,26,28]. Also, fluoride disrupts sensitive G-proteins, which serve as the building blocks of our body's hormone receptors and switch off iodine uptake into follicular cells . Also, fluoride, by increasing the intracellular c-AMP concentration, causes desensitization of the TSH receptor .
The current study showed that fluorosis caused a significant reduction in body weight gain. These results were not in agreement with other studies [13,18,19,30]. This reduction could be because of atrophic gastritis, suppressed appetite, and disturbed nutrient digestibility. Furthermore, the dental lesions might impair the ability of animals to masticate food. A defect in motivated locomotor behavior may lead to suppression of eating . In addition, deficiency in thyroid hormone probably inhibits the synthesis of growth hormone releasing factor [31,32]. Also, fluorosis led to a highly significant increase in the weight of the thyroid gland, which has been confirmed by other studies [18,19,33]. Low T3 or T4 exerts a negative feedback on the pituitary. It releases more TSH to stimulate the thyroid gland, which in turn accelerates the production of the thyroid hormone. TSH stimulates the growth of the gland; thus, the gland becomes enlarged . Similarly, a close relationship was found between fluoride intake and the incidence of goiter in areas with high levels of fluoride in water. TSH is a major growth factor for thyroid. This gland under TSH undergoes enlargement, hyperplasia, neovascularization, and morphological alterations in the thyrocytes .
In the present work, the fluoride-treated group showed congested dilated blood capillaries heavily infiltrating the follicles. This could be attributed to a high level of TSH. This finding was in agreement with that of other studies  that reported better vascularization of the gland after methimazole treatment. Also, mast cells that were observed frequently in the interstitium were considered to release growth factors that modulate folliculogenesis and angiogenesis . They added that mediators including vascular endothelial growth and fibroblast growth factor induce chemotactic migration of mast cells to sites of neovascularization. Mast cell products such as tryptase degrade connective tissue matrix to provide spaces for neovascular sprouts. However, other researchers  attributed this vascularization to these growth factors and other vasoactive factors produced in the thyroid that were potent angiogenic proteins.
In the current work, alteration in the nuclear pattern and degenerative changes such as fusion of follicles and also expansion or dilatations of endoplasmic reticulum with loss of their lamellar arrangement were observed. In terms of nuclear changes, karyolysis, pyknosis, and karyorhexis were a result of glandular overstimulation. Nuclear changes were assumed to be one of these three patterns, all because of the breakdown of DNA and chromatins . The activation of mitogen-activated protein kinases and possibility C-jun-N-terminal kinase was involved in Na–F-induced apoptosis of epithelial lung cells . Also, Na–F-induced apoptosis by oxidative stress led to lipid peroxidation and the release of cytochrome C into the cytosol and further triggering of caspase cascades, leading to apoptotic cell death . According to previous studies , there are different possibilities to explain this dilation of endoplasmic reticulum. It may be because of the synthesis of secretory products greater than their removal by transport mechanisms. A defect in this transport system, such as some mechanical or enzymatic abnormalities in the endoplasmic reticulum that prevent the removal of the normal quantities of synthesized materials, is one theory. Synthesis of an abnormal secretory product that the transport mechanism cannot remove is another explanation or theory.
The current immunohistochemical staining of calcitonin-secreting cells (parafollicular or C cells) in the Na–F group showed a highly significant increase in the number of C cells in comparison with the control group. Ultrastructurally, the secretory granules of C cells were numerous, but small, with reduced electron density. Evidence of hyperactivity was found in C cells such as follicular cells. This might be because of the high level of TSH, leading to hyperplasia and hypertrophy of C cells. These findings were in agreement with those of other researchers [47,48], who have reported that a hyperactive thyroid showed the presence of enlarged C cells distributed either in small groups or even singly. These observations might point to the possibility of a relationship between the functional state of the thyroid gland and the activity of C cells. Also, they suggested that the possible mechanisms involved in the changes in C cell with the thyroid status were in line with the changes in follicular cells. Considering that the TSH level is increased in hypothyroid rats, three possible explanations were partially related to thyrotropin functions: (a) TSH directly regulates C cells, (b) follicular cells regulate the C cells, and (c) C cells regulate follicular cells. The first hypothesis is supported by different reports  that have described the appearance of reactive C-cell hyperplasia when TSH levels were increased in rats. The second hypothesis clarified that the regulation of C cells by follicular cells could be carried out by either a local elevation in T3 and T4 or through the release of regulatory substances such as FGF and IGF, and other products such as thyroglobulin play a decisive role in the autocrine regulation of TSH-stimulated follicular cells growth, differentiation, and synthesis of thyroid hormones. According to this hypothesis, those substances could also exert a possible paracrine influence on C cells . Moreover, other studies  have reported a third hypothesis: C cells were probably involved in the intrathyroidal regulation of secretion and growth processes by secreting numerous regulatory peptides usually defined as paracrine factors that were found in the C cells. Some regulatory peptides such as calcitonin and somatostatin are considered as inhibitors of thyroid hormone secretion, whereas gastrin-releasing peptides and helodermin are stimulators of thyroid hormone secretion.
Acute or chronic intoxication with Na–F in experimental animals, as well as in humans who live in zones of fluorosis, has been reported to lead to oxidative stress in several organs, such as the kidney, brain, liver, and gonads. In such patients, the signs of fluorosis appear accompanied by alterations in the enzyme activity of SOD, catalase, in addition to an increase in MDA levels in the serum or tissues . Oxidative stress is defined as a disruption in the equilibrium between the pro-oxidant [reactive oxygen species (ROS)] and the antioxidant system. However, the antioxidative defense system, which includes SOD, catalase, and glutathione peroxidase, confers protection to cells against ROS. Excessive generation of ROS in cells is known to damage DNA, proteins, and lipids, resulting in several biological effects ranging from alterations in signal transduction, gene expression, mutagenesis, and apoptosis .
In the current study, there was a significant increase in MDA, which is a lipid peroxidation marker, and a significant decrease in the level of serum antioxidant enzyme (SOD) in the Na–F-treated group. These findings were in agreement with other reported results . The mechanism by which Na–F may induce oxidative stress in these organs is not known with certainty. Nevertheless, studies carried out in vitro show that Na–F may interact with enzymes that contain a transition metal as part of their cofactors or in their active site. Fluoride, because of its chemical nature, is capable of inter-relating with metal and can thus exert activating or inhibitory influences on enzyme activity [52,54].
Fluoride induced proliferative changes in both follicular and C cells. Also, it induced degenerative changes in follicular cells with decreased colloid production as well as decreased secretion of the thyroid hormones, indicating a functional interaction between follicular and C cells. Moreover, the biochemical results indicated that the generation of ROS and lipid peroxidation seemed to play an important role in the mechanisms of Na–F toxicity. We recommend re-examination of the safety profile of the Na–F ratio used in fluorination of water.
There is no conflict of interest to declare.
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