Fluoride is an essential trace element from the halogen group that is widely distributed in the environment . The widespread distribution of fluoride in nature and the chronic exposure of millions of individuals worldwide is an endemic problem in a number of countries . Foods rich in fluoride include sea food, bony meals, kale, barley, rice , and dark green vegetables such as the tea plant . Moreover, other sources of fluoride exposure include food additives, insecticides, anticarcinogenic, and some inhalational anesthetics such as methoxiflurane . Also, cooking in Teflon-lining cookware may increase the concentration of fluoride in food prepared in them . Sodium fluoride (Na–F) is the most commonly used compound in oral caries prevention in the form of fluorinated drinking water, salts, or milk, tooth pastes, mouth washes, and fluoride tablets .
Intoxication by fluoride results from extended periods of its action. This state is called fluorosis, which generally develops after 10–20 years in humans and 3–6 months in rats . There are two patterns of fluoride toxicity worldwide: endemic and industrial fluorosis. Endemic fluorosis is related to the high concentration of fluoride present in the drinking water, whereas industrial fluorosis is mainly because of air pollution of fluorine . The WHO recommends that the fluoride level in the water should not exceed beyond 1 mg/l . Egypt is one of top 21 countries that has problems with endemic fluorosis, where the main pathway of fluoride exposure is the ingestion of tap water from contaminated groundwater sources . The two governorates of Marsa Matrouh and Arish have a higher prevalence of fluorosis .
Fluoride is known to cross the cell membranes and to enter soft tissues . Fluorosis is a slow and progressive process that causes metabolic, functional, and structural damages, which have been reported in many tissues, particularly musculoskeletal, dental systems , kidney , liver , and brain . Correlations between fluorosis and oxidative stress in some organs have been proven [16,17]. Fluoride affects several parameters of thyroid activity and development in mice [18,19]. However, its effect on histological structures in rats has not been studied extensively. Therefore, this study aimed to examine the possible histological and biochemical changes in the thyroid gland induced by chronic Na–F exposure.
Materials and methods
Animals and housing
Twenty growing male albino rats (30 days, 40–45 g) were obtained from the Unit for Laboratory Animals at the Faculty of Veterinary Medicine, Zagazig University. They were housed in standard polypropylene cages with stainless-steel wire lids. Animals were maintained at a constant room temperature of 20–22°C and 60% humidity, with free access to feed (standard commercially available pellets for laboratory rodents) throughout the study. The experiment was conducted according to the norms of the Ethical Committee of the Zagazig University (Egypt).
Na–F was purchased from El-Gomhoria Company (Zagazig, Egypt). It was provided in a white powder form. It was dissolved in drinking water at an experimental dose.
Rats were allowed a 1-week acclimatization period, and then they were divided randomly into two groups (10 rats each).
Group I: This group served as a control and received distilled water orally daily by a gastric tube for 120 days.
Group II: This group included rats that received Na–F orally by a gastric tube dissolved in distilled water at a dose of 11 mg/kg/day  for 120 days.
General observations in rats
During the experimental period, clinical signs and general appearances, which included the amount of food and water consumed, were checked daily. Mortalities of the rats were recorded as it occurred.
The initial weights of all rats were recorded, and then all rats per group were individually weighed weekly afterwards throughout the study. The gain in body weight was then calculated.
At the end of the experiment, rats were anesthetized with ether inhalation. Venous blood samples were withdrawn from the retro-orbital sinus. The thyroid glands were dissected out in two stages to avoid tissue damage. First, the neck was opened by a longitudinal incision, the fascia was removed, and the trachea was cut by a horizontal plane superior and inferior to the thyroid . Then, subsequent dissection of the thyroid was carried out and used for the following studies.
The weight of the thyroid glands from all rats was recorded immediately after their excision.
Blood samples were collected in both types of tubes with and without EDTA and were centrifuged at 3000 rpm for 20 min to separate the serum and plasma. Clear sera were separated into small glass tubes and stored at −20°C until analysis. The following markers were measured in the serum and plasma:
- A hormonal assay was carried out to determine the serum levels of T3, T4, and thyroid-stimulating hormone (TSH) using commercially available Chemiluminescence Immunoassay (CLIA, catalogue no. ABIN504750; ABIN, Canoga Park, C.A. USA) following the manufacturer's instructions.
- The oxidative marker plasma superoxide dismutase (SOD) was assessed colorimetrically (absorbance 450 nm) using a commercially available kit (catalogue no. K335-100; Biovision, San Francisco, USA) following the manufacturer's instructions.
- Lipid peroxidation marker malondialdehyde (MDA) was assessed colorimetrically (absorbance 532 nm) using a commercially available kit (catalogue no. K739-100; Biovision) following the manufacturer's instructions.
For light microscopy study, specimens were fixed in 10% neutral-buffered formalin, dehydrated, embedded in paraffin, and then the sections were cut at 4 μm and stained with H&E for a routine histological examination .
For the immunohistochemical study, the paraffin sections were processed using the streptavidin–biotin complex (Strep ABC) immunohistochemical method following the manufacturer's instructions. The sections of 4 μm paraffin sections were deparaffinized in xylene and rehydrated in a descending series of ethanol. The specimens were subjected to antigen retrieval in a citrate-buffered solution (pH 6.0) for 10 min by a microwave. Endogenous peroxidase was eliminated by incubation in 10% H2O2 in PBS (pH 7.4) for 10 min. After washing, the specimens were blocked in ready-to-use normal goat serum for 20 min at room temperature. The sections were incubated with polyclonal antibody (case no. C8198; Sigma Aldrich Co., Cairo, Egypt). Primary antisera were diluted in antibody diluents (1:10000). The peroxidase activity was observed using the AEC (3-amino-9-ethyl carbazole) substrate kit (TA-004-HAC; Labvision). The sections were rinsed in PBS. The negative control was obtained when the primary antibody was replaced with PBS. The sections were counterstained with Mayer's hematoxylin .
For electron microscopy study, small tissue biopsies (1 mm3) were taken from the thyroid, fixed in 2% glutaraldehyde, and postfixed in 1% osmium tetroxide, dehydrated, and embedded in epoxy resin. Thick 1 μm sections were mounted on glass slides and stained with toluidine blue. Ultrathin sections of the thyroid (guided by a semithin section) were cut and double stained with uranyle acetate and lead citrate  and examined using a JEOL 1010 electron microscope (Tokyo, Japan) at the Mycology and Regional Biotechnology Center, Al Azhar University, Cairo, Egypt.
The image analyzer computer system Leica Qwin 500 (Cambridge, UK, Leica Microsystems Imaging Solutions Ltd) in the image analyzing unit of the Pathology Department, Faculty of Dentist, Cairo University, Egypt, was used to determine the height of the follicular cells, the number of calcitonin-immunoreactive C cells, and the area % of colloid. It was measured using the interactive measure menu. The area % and standard measuring frame of a standard area were 118 476.6 mm2 were chosen from among the parameters measuring 10 readings from five sections from each rat of the randomly chosen five rats. In each randomly chosen field, the section of the thyroid was enclosed inside the standard measuring frame; then the area % of the colloid and number of calcitonin brown immune-positive reaction C cells were masked by a blue binary color to be measured. These measurements were carried out using total magnification × 100 with area % of colloid and total magnification × 400 with the height of follicular cells and area % of a calcitonin-positive immune reaction.
Statistical analysis was carried out on all parameters. The data obtained were expressed as mean values ± SD and analyzed using an unpaired Student's t-test where the level of significance (P) was set at 0.05 (one-way analysis of variance).
General observations in rats
In the fluoride-treated group, lassitude and anorexia in addition to a reduction in feed and water intake were observed during the experiment. One rat died in the same group.
Body weight and thyroid weight
Oral administration of Na–F led to a significant reduction in body weight gain (Table 1 and Histogram 1) and a highly significant increase in the weight of the thyroid gland compared with the controls (Table 2 and Histogram 2).
Group I (control)
H&E-stained sections from the thyroid glands showed that the glands were surrounded by connective tissue capsules and divided into lobules by trabeculae. The thyroid parenchyma was composed of different-sized follicles, where large follicles were present, especially at the periphery (Fig. 1). The follicular walls were lined by a single layer of flattened to cuboidal follicular cells, with oval to round nuclei. The follicular lumens were filled with homogenous acidophilic colloids that had peripheral small vacuoles. An apparent few number of interfollicular cells and blood capillaries were observed in between the follicles (Fig. 2).
Immunohistochemical study showed a few calcitonin-immunoreactive C cells forming a part of the lining cells of the follicles and also in between the follicles (Fig. 3).
Toluidine blue-stained semithin sections showed that the thyroid follicles were lined by a single layer of follicular epithelium. The lining follicular cells appeared with rounded to oval nuclei. Also, large pale C cells with small granules were observed. Fibroblasts with dark elongated nuclei and also small blood capillaries appeared in between the follicles (Fig. 4).
Ultrastructurally, some follicular cells were more or less inactive and low in height or flat. They had oval and irregular nuclei with clumps of heterochromatin. Their cytoplasm had rough endoplasmic reticulum (rER), a few vacuoles, and also dense lysosomal granules. Their apical border showed a moderate or a small number of microvilli projecting into the colloid. Blood capillaries were observed beneath the follicular basal laminae (Fig. 5). Other follicular cells were active and high in height or cuboidal with rounded euchromatic nuclei resting on the basal laminae. Their cytoplasm showed numerous regular paralleled cisternae of rER, dense polymorphic lysosomal granules and also vacuoles. Their apical borders had a huge number of microvilli projecting into the colloid (Fig. 6). Less common cells were C cells with rounded indented euchromatic nuclei. These cells were separated from the luminal colloid by a part of the cytoplasm of follicular cells with rER. Their cytoplasm had many electron small-dense secretory granules, mitochondria, and also tubular cisternae of endoplasmic reticulum (Fig. 7).
Group II (Na–F treated)
H&E-stained sections from the thyroid glands showed that most of the follicles were apparently enlarged and distended with vacuolated colloid whereas others were atrophied. Congested blood capillaries were also observed (Fig. 8). Some follicles appeared with no colloid in their lumens and were lined by cuboidal vacuolated cells with rounded nuclei. Other follicles were lined by multiple layers of vacuolated follicular cells. Signs of mitosis were observed in the follicular epithelial lining (Fig. 9). Some follicles appeared fused with extensive vacuolated colloid and others with loss of their nuclei. Microfollicles with narrow lumens and also an apparent increase in the interfollicular cells were observed (Fig. 10).
Immunohistochemical study showed an apparent increase in the number of calcitonin immunoreactive C cells forming a part of the lining cells of the follicles and also in between the follicles (Fig. 11).
Toluidine blue-stained semithin sections showed that the thyroid follicles were large, inactive, and lined by flattened follicular cells, with flat dark nuclei and a vacuolated cytoplasm (Fig. 12). Other active follicles with an apparent increase in the height of their lining cells were observed. These cells were high cuboidal with rounded pale and darkly stained nuclei and also an extensive vacuolated cytoplasm (Fig. 13). Other hyperactive follicles were also observed. These follicles had multiple layers of follicular cells on one side. These cells had vesicular nuclei and a vacuolated cytoplasm (Fig. 14). Numerous large C cells with rounded pale nuclei and a pale cytoplasm were observed within the follicular epithelium and in the interfollicular connective tissue. Also, mast cells were observed (Fig. 15). Numerous blood capillaries, some of them indenting the follicular epithelium, were observed (Figs 12–14).
Ultrastructurally, some follicular cells were flat, with irregular heterochromatic nuclei. Dense polymorphic lysosomal granules, vacuoles, numerous markedly dilated irregular cisternae of rER, and also large apical colloidal vacuoles were observed. Their apical borders had a large number of long microvilli, and many mast cells with dense granules were also observed (Fig. 16). Other follicular cells were columnar or high cuboidal follicular cells with irregular heterochromatic nuclei. Numerous dilated irregular cisternae of rER, vacuoles, and dense polymorphic lysosomal granules with a large number or aggregated microvilli were observed. Blood capillaries indenting the epithelial lining of follicles were observed (Fig. 17). Some follicular cells were arranged in layers with variable forms of nuclei. A large number of aggregated microvilli, numerous dilated irregular cisternae of rER, vacuoles, and also dense lysosomal granules were observed (Fig. 18). C cells with multiple rounded euchromatic nuclei, numerous very small, low-density secretory granules, and also small mitochondria occupied a large portion of their cytoplasm. Numerous dense lysosomal granules were also observed in the apical portion of some follicular cells (Fig. 19). Abundant collagen fibers, mast cells with dense granules, and also macrophage cells were observed in the interfollicular connective tissue (Fig. 20).
In the group that received an oral administration of Na–F, as compared with the control group, there was a highly significant increase in the height of follicular cells (Table 3 and Histogram 3), number of C cells (Table 4 and Histogram 4), and a highly significant decrease in the area % of colloid (Table 5 and Histogram 5).
The Na–F-treated rats showed a significant and a highly significant decrease in serum T4 and T3, respectively, compared with the controls (Tables 6 and 7 and Histograms 6 and 7). However, there was a highly significant increase in serum TSH (Table 8 and Histogram 8). The MDA activity increased significantly in Na–F, whereas the SOD content decreased significantly compared with the controls (Table 9 and Histogram 9).
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 current work, alterations in the thyroid gland function in the fluoride-treated group were further confirmed by histological examination of both follicular and C cells, which showed evident light microscopic and ultrastructural changes. Manifestations of hyperactivity were found in some thyroid follicles as extensive vacuolations of colloid and colloidal droplets in follicular cells. These vacuolations were as attributed to an increased endocytotic activity to release the stored hormones as a compensatory mechanism to fluoride-mediated suppressive effects on follicular cells. They also hypothesized that these vacuolations were under the influence of increased TSH . This explanation was in agreement with others , who reported that during the huge demand for thyroid hormones, follicular cells extend pseudopods into the follicular lumen to envelop and absorb the colloid. Furthermore, fluoride-treated thyroid showed an increase in the height of follicular epithelium that was confirmed statistically. This was in agreement with the results of many researchers , who confirmed a significant increase in the height of the epithelium in hypothyroidism. Also, hyperplasia, epithelial stratification, microfollicles, and also hypertrophy of follicular cells were observed. These could be attributed to an increased TSH level, which was responsible for the proliferative activity of follicular cells [19,36]. Also, fluoride itself is mutagenic; it can cause uncontrolled proliferation of cells . Hypertrophy is an increase in cell size and functional capacity; when trophic signals or the functional demand increase, adaptive changes occur to fulfill these needs . This hypertrophic change is because of the hydropic swelling resulting from impairment in cellular volume regulation. The injurious agent may interfere with membrane-regulated processes by increasing the permeability of the plasma membrane to sodium or damaging the pump directly or interfering with ATP synthesis [38,39]. Also, the proliferation of microvillous borders and increased number of lysosomes with their apical condensation were observed. These ultrastructural findings of hyperactivity were in agreement with those reported by other researchers . This proliferation of microvillus border facilitates the transport and iodination of thyroglobulin across the cell membrane .
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.
Conflicts of interest
There is no conflict of interest to declare.
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