Fluoride is an essential element needed for normal development and growth of animals and human beings. Exposure to fluoride is widespread and its main source in humans is drinking water, in which it is either present naturally in groundwater or due to community water fluoridation. In addition, fluoridated salt and fluoride-containing food, such as fish, may contribute to high-dietary fluoride intake. Dietary supplements are also available for use by persons living in nonfluoridated areas to increase their fluoride exposure. Another common source of fluoride is the use of toothpaste, mouthwash solutions, fluoride gels, and other topical sources [1,2]. Moreover, the atmosphere may contain airborne fluoride arising from soils, industry, coal fires, and volcanoes. It was estimated that the average intake of fluoride through food consumption is approximately 2 mg/day for adults. By the use of fluoridated water and salt, the fluoride intake could reach 6 mg/day, without taking into account toothpaste use .
After ingestion of fluoride, the majority is absorbed from the stomach and small intestine into the blood stream. It distributes evenly throughout intracellular and extracellular spaces. Fluoride has a high affinity for calcium and can form an insoluble salt within the gastrointestinal tract, which is not absorbed. Approximately 50% of the absorbed fluoride remains associated with hard tissues, whereas all of the remainder is excreted in urine [4–6].
Fluoride has long been recognized as one of the best public health measures in the prevention of dental caries. It can play an important role in the mineralization of bone and teeth with subsequent reduction of tooth decay and prevention of osteoporosis by facilitating the deposition of phosphate and calcium ions. The ability of fluoride to prevent dental caries may be also attributed to inhibition of bacterial metabolism [7,8].
Although fluoride is beneficial in recommended doses, excessive long-term fluoride intake can lead to adverse effects on health, such as skeletal and dental fluorosis as well as harmful effects on various body organs and genetic material. Fluoride toxicity has been associated with growth retardation and thyroid, kidney, and lung changes. In addition, oral mucosal tissue can serve as a long-term fluoride reservoir after topical application and retains a small amount of fluoride in oral environment. It was shown that fluoride concentration in saliva was increased after topical application of sodium fluoride mouthwash solution. The submandibular and sublingual glands are the major contributors of fluoride in the oral cavity [1,9].
The incidence and severity of fluorosis have become greater in the last decade in many countries due to excessive ingestion of fluoride, mainly from formulations intended for topical action. The increase of salivation in fluoride-intoxicated individuals and secretion of fluoride ion by salivary glands, led some researchers to examine the possible metabolic alterations in salivary glands due to fluoride action [9,10]. In addition, sodium fluoride was found to affect the morphogenesis of cultured fetal mouse submandibular gland . However, the detailed structural changes induced by sodium fluoride on the submandibular gland have not been entirely explained in previous researches.
Ginseng (the root of Panax ginseng) is one of the most commonly used herbal medicines in Asian and Western countries. Studies have shown a wide range of beneficial effects of ginseng against human diseases [12,13]. Ginseng roots contain multiple active constituents including ginsenosides, polysaccharides, peptides, and fatty acids. Ginsenosides are the major biologically active compounds of ginseng. They belong to a family of steroidal saponins that are believed to be responsible for the pharmacological effects of ginseng. Approximately 30 different ginsenosides have been isolated and identified from Panax ginseng. They have different effects on carbohydrate and lipid metabolism as well as on the function of neuroendocrine, immune, cardiovascular, digestive, and central nervous systems [14,15]. The potential therapeutic effects of ginseng have been attributed to its immunostimulatory, antioxidant, and anti-inflammatory activities [16,17].
On the basis of these facts, this study was conducted to investigate the structural changes that may occur in the submandibular gland of adult male albino rat after long-term treatment with sodium fluoride and to determine the possible protective role of ginseng using light and electron microscopy. Moreover, an immunohistochemical technique for α-smooth muscle actin (α-SMA) was used as a marker for identification of myoepithelial cells (MECs) .
Materials and methods
In this study, 40 adult male albino rats (180–200 g each) were used. They were kept in clean, properly ventilated cages and were maintained on a standard laboratory diet. Free access to food and water was allowed throughout the study period. All experimental procedures were conducted with approval of the Research Ethics Committee, Faculty of Medicine, Tanta University and followed the Guide for the Care and Use of Laboratory Animals . The animals were divided into four main groups (10 animals each).
Group I (the control group)
This was subdivided into two subgroups; five animals were left untreated, whereas the others were given 1 ml of distilled water orally once daily for 4 weeks.
The animals were treated by Panax ginseng at a dose of 10 mg/kg body weight (dissolved in 1 ml of distilled water) orally by a gastric tube once daily for 4 weeks. This dose was similar to that used in previous studies . Ginseng was available in the form of capsules containing the dried roots of Panax ginseng. Each capsule contained 100 mg and was purchased from Pharco Pharmaceuticals, Alexandria, Egypt.
The animals were treated by sodium fluoride at a dose of 5 mg/kg body weight (dissolved in 1 ml of distilled water) orally by a gastric tube once daily for 4 weeks. The dose was determined according to that used in previous studies . Sodium fluoride was available in the form of a white powder and was purchased from El-Gomhoria Chemical Company, Tanta, Egypt.
The animals were concomitantly treated by both Panax ginseng and sodium fluoride at the same doses and duration as in groups II and III, respectively.
At the appropriate time, all animals were anesthetized by an intraperitoneal injection of sodium pentobarbital (40 mg/kg body weight). Then, the animals were perfused through the heart with 2% paraformaldehyde and 1.25% glutaraldehyde solution . The submandibular glands were removed and dissected.
For light microscopy
The specimens were fixed in 10% neutral-buffered formalin, dehydrated through alcohols, cleared in xylene, and embedded in paraffin wax. Then, sections (5 μm thick) were stained with hematoxylin and eosin . Moreover, immunohistochemistry for α-SMA was performed on formalin-fixed paraffin-embedded specimens.
The sections were first deparaffinized, rehydrated, and then incubated for 30 min with 3% hydrogen peroxide to block endogenous peroxidase. After three rinses with phosphate-buffered saline (PBS), the sections were incubated overnight in a humid chamber with monoclonal antibodies to α-SMA at 4°C. Sections were rinsed three times with PBS, incubated for 1 h with biotinylated peroxidase-conjugated secondary antibody, and washed again with PBS. The immunoreaction was visualized with 3-3′-diaminobenzidine hydrogen peroxide as a chromogen for staining the α-SMA-bound complex. After three rinses with PBS, the sections were counterstained with hematoxylin, dehydrated, and mounted. The negative control sections were incubated with normal serum instead of the primary antibody, whereas other steps of the procedure were the same [23,24]. Cytoplasmic stain of the adult vascular smooth muscle cells was used as a positive control .
For electron microscopy
The specimens were immersed for 2 h in 2.5% phosphate-buffered glutaraldehyde solution (pH 7.4) at 4°C. After washing with phosphate buffer, the specimens were postfixed for 1 h in 1% buffered osmium tetroxide solution. Subsequently, the specimens were dehydrated in ethanol, treated with propylene oxide, and embedded in epoxy resin. Ultrathin sections were cut, double-stained with uranyl acetate and lead citrate, and were examined with a JEOL electron microscope at the Electron Microscopy unit, Tanta University .
With regard to α-SMA immunoreactivity, 10 different fields containing no blood vessels were randomly examined from each animal at a magnification of ×400 and the optical density of the immunoreaction was measured using the image analyzer at Faculty of Medicine, Cairo University.
All quantitative data were expressed as means±standard deviation and were compared using unpaired Student's t-test. P value was calculated using Minitab software and P value of less than 0.05 was considered statistically significant, whereas P value of less than 0.001 was highly significant.
Light microscopic results
The submandibular gland from both control animals (group I) and ginseng-treated animals (group II) showed numerous lobules containing closely packed serous and mucous acini as well as intralobular ducts. Connective tissue septa radiate between the lobules conveying blood vessels and large excretory ducts (Fig. 1). The serous acini appeared lined by pyramidal cells containing darkly stained cytoplasm and pale rounded nuclei. The mucous acini appeared lined by low cuboidal cells containing poorly stained cytoplasm and dark flattened nuclei with some acini appeared capped by serous demilunes. The intralobular intercalated ducts were lined by simple cuboidal epithelium, whereas the striated ducts were lined by high cuboidal cells with basal cytoplasmic striations. Both types of acini and the intercalated ducts appeared embraced by the flattened MECs with large flattened nuclei (Fig. 2). In toluidine blue-stained semithin sections, the serous cells were easily distinguished by their apical darkly stained secretory granules. Mucous cells were identified by the pale mucous granules filling their cytoplasm (Fig. 3). The intralobular ducts were observed consisting of the intercalated, the striated, and the granular convoluted ducts of rodent submandibular glands, which were identified by the apical dark secretory granules (Fig. 4). Moreover, minimal immunoreaction to α-SMA was demonstrated in the cytoplasm of the star-shaped MECs at the periphery of the acini and the intercalated ducts, whereas no immunoreaction was observed at the periphery of the striated ducts. The smooth muscle cells of the vascular walls also showed positive immunoreaction (Fig. 5).
Examination of specimens from fluoride-treated animals (group III) revealed marked structural changes in the acini, mainly in the mucous type that appeared destroyed and were widely separated by connective tissue containing some infiltrating cells and interstitial hemorrhage (Fig. 6). Some dilated congested blood vessels were also observed in between the acini (Fig. 7). Many acinar cells showed cytoplasmic vacuolation and irregular displaced nuclei that were compressed by the variable-sized vacuoles (Figs 6–8). Some cells showed lysis of nuclear chromatin. Disarrangement of acinar cells and marked disturbance of acinar architecture were also observed in focal areas (Fig. 7). Some intralobular ducts showed widening and irregularity of their lumina (Fig. 8) as well as disorganization of the lining epithelial cells with vacuolated cytoplasm and some pyknotic nuclei (Figs 7 and 8). However, no apparent structural changes were observed in the interlobular large excretory ducts. Semithin sections showed lysis and destruction of some acinar cells with congested blood vessels in between the acini (Fig. 9). Widening and irregularity of some acinar lumina were also observed (Fig. 10). Many acinar cells showed hyperchromatic nuclei (Fig. 9) as well as variable degrees of cytoplasmic vacuolation and basal migration of secretory granules (Figs 9–11). The intralobular ducts also showed destruction and vacuolation of the lining epithelial cells (Fig. 10). Considering immunohistochemistry, there was an apparent increase in the actin-positive immunoreaction that was observed at the periphery of the acini, intercalated ducts as well as the striated ducts (Fig. 12).
Specimens from animals concomitantly treated with both ginseng and fluoride (group IV) showed mild congestion of blood vessels in between the acini. Some acinar cells showed basal migration of secretory granules. However, most acinar and ductal cells appeared more or less normal (Fig. 13). In addition, a minimal increase in α-SMA immunoreactivity was observed at the periphery of the acini and the intercalated ducts, whereas no immunoreaction was observed at the periphery of the striated ducts (Fig. 14).
Electron microscopic results
Examination of ultrathin sections from both control animals (group I) and ginseng-treated animals (group II) showed normal morphological features of acinar cells. Serous cells were identified by the supranuclear homogenous electron-dense secretory granules. The basal part of cells contained regular euchromatic rounded nuclei, mitochondria, and numerous parallel cisternae of rough endoplasmic reticulum (RER). Mucous cells showed electron-lucent secretory granules of variable sizes filling most of the cytoplasm, whereas the basal part of cells was occupied by the oval nuclei, mitochondria, and RER. The acinar cells were embraced by the flattened MECs located between the basal cell membranes of secretory cells and the basement membrane (Fig. 15).
Considering fluoride-treated animals (group III), marked ultrastructural changes were observed mainly in the mucous acinar cells in the form of focal separation of some cells with widening of the intercellular spaces, disruption of the cell membranes, condensed nuclear chromatin as well as focal areas of cytoplasmic rarefaction (Fig. 16). The cytoplasm showed disarrangement and fragmentation of RER, huge secondary lysosomes as well as coalescence and pooling of the secretory granules and basal migration of some of them (Figs 16 and 17). In some acinar cells, the secretory granules compressed the nuclei causing their indentation and irregularity. The mitochondria showed marked abnormalities in the form of swelling and ballooning, destroyed cristae, mitochondrial vacuoles, and dense bodies (Fig. 18).
Examination of specimens from animals concomitantly treated with both ginseng and fluoride (group IV) showed that the majority of acinar cells appeared normal with no apparent structural changes. However, few cells showed coalescence and basal migration of secretory granules and few swollen mitochondria with destroyed cristae, as well as mitochondrial vacuoles. The secretory granules compressed the nuclei causing their indentation and irregularity in few acinar cells (Fig. 19).
The mean optical density of α-SMA immunoreactivity in the glandular tissue of control group (group I) was measured as 0.168±0.02 and that of ginseng-treated group (group II) was 0.176±0.01 with no significant difference from the control group. With regard to fluoride-treated group (group III), there was a highly significant increase (P<0.001) in the mean optical density, which was measured as 0.262±0.05 compared with the control group. However, there was a nonsignificant increase (P>0.05) in α-SMA immunoreactivity in animals treated with both ginseng and fluoride (group IV), which was measured as 0.183±0.01 when compared with the control group (Table 1).
This study showed that long-term administration of sodium fluorid induced marked structural changes in the acinar and intralobular ductal cells of rat submandibular gland in the form of disorganization, cellular destruction, cytoplasmic vacuolation, and irregular displaced or pyknotic nuclei, which were features suggestive of degenerative changes.
It was reported that fluoride may affect the cells by crossing the plasma membrane, or may interact with certain cell surface molecules . At the cellular level, some studies pointed to its action on the salivary glands by adenyl cyclase . Moreover, fluoride could inhibit magnesium-dependent adenosine triphosphatase (ATPase) with an indirect effect on cellular metabolism .
Fluoride was shown to stimulate the activities of hexokinase, phosphofructokinase-1, and glucose-6-phosphate dehydrogenase enzymes in the submandibular gland with subsequent glucose utilization by the cell in glycolysis. It was also suggested that fluoride acts on the membrane transport system through inhibition of ATPase/ATP synthesis. In this condition, only a small amount of ATP would be formed at this level, requiring, therefore, a greater action by the glycolytic pathway. The disturbance of cell metabolism and failure of sodium pump at the plasma membrane may lead to cloudy swelling and cytoplasmic vacuolation, as well as disarrangement of cells .
Examination of ultrathin sections revealed ultrastructural changes in acinar cells of fluoride-treated animals, such as disorganization and fragmentation of RER, marked swelling of mitochondria, lysosomal dense bodies as well as nuclear indentation and irregularity. In agreement with these findings, fluoride was found to induce endoplasmic reticulum stress and DNA fragmentation in enamel cell lines .
The marked swelling and ballooning of mitochondria as well as destruction of their cristae could result from structural changes of the internal membrane with subsequent disturbance of pump mechanism and decreased retention of calcium. With fluoride, all the ATPase activity is coupled to Ca2+ transport, and therefore most of the energy is converted into work and little is dissipated as heat .
This study also showed cellular infiltration and interstitial hemorrhage in between the acini. These stromal inflammatory changes may restrict diffusion of nutrients, essential minerals, and oxygen to parenchymal cells, and thus may adversely affect late attempts of regeneration and function by the surviving parenchymal cells .
In this study, the MECs could be demonstrated by immunohistochemistry for α-SMA that has been considered as a specific marker expressed by the smooth muscle cells early during their differentiation. It is expressed by MECs and the mesenchymal vasculature, and so it is a useful tool for identification of the MECs as they have structural features of both epithelial and smooth muscle cells. By exclusion of blood vessels, the expression of α-SMA was restricted to MECs. It was reported that the MECs are normally situated at the periphery of the acini and intercalated ducts of human and rat salivary glands extending their long processes around them [23,29].
In fluoride-treated animals, the MECs were located around the residual acini, intercalated ducts, and striated ducts in contrast to their normal distribution in control animals. This finding could be explained by the report that under unusual conditions such as massive parenchymal destruction, the acinar progenitor contributes to the maintenance of the larger ducts that result in the occurrence of striated ducts with MECs .
By quantitative analysis, a highly significant increase in α-SMA immunoreactivity was observed in this study in fluoride-treated animals. It was reported that the physiological regeneration of the acini and intercalated ducts is based on a low baseline proliferation of mature acinar cells, intercalated ductal cells, and MECs. In the presence of chronic noxious stimuli, the proliferation indices were increased approximately two-fold in acinar cells, four-fold in intercalated ductal cells, and more than 10-fold in MECs. These cells can regenerate independently without cellular transition and without the participation of an additional population of cells . Similarly, other studies suggested that the mature MECs have proliferative ability that is especially active under some experimental conditions. The active proliferation phase of MECs was almost coincident with that of acinar cells . In line with these findings, the MECs were observed to undergo morphological and proliferative changes during both atrophy and regeneration of acinar cells with subsequent increase in the size and number of these MECs .
The MECs are known to assist salivary secretion by compressing the underlying parenchyma and by providing tensile support for the basis of the acini. These functions are probably important for the viscous saliva to pass through narrow and tortuous channels in the submandibular glands . Under stressful events, restoration of gland function seems to require increasing the secretory capacity of the surviving cells . In this regard, the highly significant increase in α-SMA immunoreactivity that was observed in this study in fluoride-treated animals could be explained by the capacity of MECs to be activated in the presence of parenchymal injury to increase the secretory function of these cells. This finding could be also in line with the coalescence and pooling of secretory granules as well as their accumulation and basal migration that were observed in this study in fluoride-treated animals, which could be related to the increase in the secretory capacity of the acinar cells.
As regards animals concomitantly treated with both ginseng and fluoride, this study showed that the majority of parenchymal cells appeared normal and some acinar cells showed mild structural changes with a nonsignificant increase in α-SMA immunoreactivity, indicating that ginseng could reduce the degenerative changes induced by sodium fluoride.
The diverse biological effects of ginseng may be attributed to multiple effects of the ginsenosides or its other active components acting as potent anti-inflammatory and immunomodulatory agents . It was shown that ginseng acts through suppression of proinflammatory cytokines or mediators including tumor necrosis factor-α and interleukins [31,32]. It regulates cytokine production and phagocytic activities of macrophages and dendritic cells. However, it acts as an immunostimulant activating T-lymphocytes and B-lymphocytes [15,33]. In addition, ginseng has been shown to ameliorate myocardial fibrosis  as well as renal interstitial fibrosis . The protective effects of ginseng were also attributed to induction of cytoprotective heat-shock proteins that may contribute to prevention of tissue injury .
In summary, this study showed that long-term exposure to sodium fluoride could induce marked structural changes in rat submandibular gland. These changes could be partially reduced by concomitant administration of ginseng. It is recommended that special precautions must be taken to limit the level of fluoride exposure. Fluoridation of public water supplies should be cautiously performed according to the health-care standards. Fluoride concentration must be included in the labels of bottled water and inadvertent use of highly mineralized water should be avoided. The intake of fluoride through consumption of toothpaste must be considerable.
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