Ebrahim Rahmani Moghadam1, Elham Nadimi2, Afsoon Bordbar3, Nazanin Ayareh4, Ehsan Bordbar5 and Hossein Bordbar6,*
* Corresponding author: Hossein Bordbar, Histomorphometry and Stereology Research Center, Department of Anatomical Sciences, Medical School, Shiraz University of Medical Sciences, Shiraz, Iran. Tel/Fax: +987132304372; Email: bordbarh@sums.ac.ir
Received 2021 April 04; Revised 2021 October 25; Accepted 2021 November 05.
Background: Bisphenol-A (BPA) is one of the hazardous chemicals, which is extensively used. BPA can cause oxidative stress. Resveratrol (RES) is a natural polyphenol that possesses several health benefits, such as antioxidant effects.
Objectives: This study aimed to investigate the protective effects of RES against BPA-induced oral mucosa and tongue toxicity.
Methods: A total of five groups of Sprague-Dawley male rats (n=30) were used in this study. Group 1 was the control group, and group 2 received 50 mg /kg BPA by gavage. Group 3 was given 100 mg/kg of RES, and group 4 received 50 mg/kg BPA plus 100 mg/kg RES. In group 5, the sham group, the volume of the injectable drug received from olive oil was given as gavage for eight weeks. Following that, the paraffinized samples were sectioned in 5µm thickness to estimate the volume.
Results: The results showed that BPA had a different effect on tissues. Moreover, the total volume of lamina propria, mucosal glands, the total glands of the tongue, volumetric density of the epithelium, and glands of the oral mucosa increased in the BPA group, while in the BPA+RES group, these structures reduced, compared to the BPA group. In addition, the total volume of the epithelium of the tongue decreased in the BPA group, whereas this structure increased in the BPA+RES group, compared to the BPA group.
Conclusion: BPA has different effects on the oral mucosa and tongue. These effects can exert influence on the normal function of the cells in these areas. RES, with its antioxidant properties, had a protective effect on these structures against the BPA.
Keywords: Bisphenol-A, Oral mucosa, Resveratrol, Tongue, Toxicity
Humans and wildlife are negatively affected by hazardous chemicals and increasing environmental pollution (1) . Bisphenol-A, 2,2-(4,4-dihydroxy phenyl, BPA) is one of the widely used chemicals which is extensively used in polycarbonate bottles, resins, plastics, fungicides, heat resistant materials, and the epoxy layer of canned foods (2-4). Application of BPA is on the rise, such that each year, more than 2.2 million tons of these products are manufactured worldwide (5). BPA-based coatings are forbidden in the US since BPA can leach to food and water under normal conditions (4, 6, 7). It has been indicated that BPA can be released in human tissues, such as the hair (5, 8), and it could be accumulated in the human body (1). BPA is a biologic toxin that can be released, independent of their pH temperature, into food, air, saliva, and blood (9); moreover, it has been reported to act as a hepatotoxic agent that leads to elevated levels of alanine transaminase, aspartate transaminase, and lactate dehydrogenase, as well as defects in the liver morphology (5, 10). Furthermore, it can cause oxidative stress, inflammation, endocrine disturbance, and genome/transcriptome modifications in different tissues, such as the brain, liver, and kidney (4, 7, 11-13). Folia et al. showed that oral exposure to BPA in the rat disrupts the thirst and buccal homeostasis and raises questions about the salivary gland secretions (14). In another study, Seki et al. reported that oral epithelial cells exposed to BPA showed a significant gene expression alteration. In these cells, several genes related to the acceleration of cell death processes were significantly up-regulated, indicating the detrimental effect of BPA on cell survival (15). Natural antioxidants, such as vitamins and polyphenols, play an essential role in preventing diseases associated with oxidative stress and inflammation (16).
Resveratrol (RES) is one of the natural polyphenols commonly found in various plants, especially in grapes (17). RES possesses several health benefits, including antioxidant (18, 19), anticarcinogenic (20), antibacterial (21), antifungal (22), antiviral (23), cardioprotective (24), hepatoprotective (18), and neuroprotective (25) effects. Although the exact mechanism of the action of RES is not well-understood, it is supposed that RES exerts its protective effect mainly through free radical scavenging and up-regulation of antioxidant genes, such as heme oxygenase-1 (26, 27). There is evidence indicating the protective effects of RES against BPA-induced toxicity. It has been shown that RES can reverse the BPA-induced metabolic syndrome and endothelial dysfunction by regulating insulin signaling and antioxidant properties (28, 29). Furthermore, liver damage caused by BPA was attenuated by the effect of RES on the extent of oxidative status and improved enzymatic/non-enzymatic antioxidant defense system (30). In addition, research suggests that RES can increase endogenous antioxidant biosynthesis, inhibit membrane lipids peroxidation, decrease interleukins and TNF-α, and increase endothelial nitric oxide (eNOS) expression, followed by increasing blood supplying the damaged tissues (31-33). Despite extensive studies on the toxicity of BPA on various tissues, there is no research about the stereology parameters of oral mucosa and tongue toxicity as part of the gastrointestinal tract by BPA.
Protective agents seem urgent regarding the increasing use of BPA and its serious harmful effects on biological systems. Therefore, this study aimed to investigate the possible protective effect of RES against BPA-induced oral mucosa and tongue toxicity.
3.1. Animals
A total of 30 male Sprague Dawley rats weighing 200-250 g were purchased from the comparative and experimental medical center of Shiraz University of Medical Sciences, Shiraz, Iran (SUMS). Animals were kept in the standard conditions (ambient light: 12:12 light-dark cycle) and free access to water and food were provided to them for eight weeks to maintain their environmental compatibility. The research was performed with the approval and based on the guidelines of the Animal Care and Ethics Committee (IR.SUMS.REC.1398.392) of the SUMS, Shiraz, Iran.
3.2. Methods of drug administration for all groups
The rats were randomly divided into five groups of six animals per group. Group 1 received distilled water as control, and group 2 received 50 mg /kg BPA. Furthermore, group 3 was given 100 mg/kg of RES, and group 4 received 50 mg/kg BPA plus 100 mg/kg RES. In group 5, the sham group, the volume of injectable drugs taken from olive oil (BPA and RES solvent) was given as oral gavage. This process was performed for the groups for eight weeks (34).
3.3. Tissue preparation
The mucosal tissue of the oral cavity with the tongue was removed for histological and stereological studies. The tissue preparation steps were performed according to the usual method. After that, the paraffinized tongue and oral mucosa were sectioned in 5µm thickness (H&E staining) for volume estimation.
3.4. Sampling of the tongue
To determine the shrinkage and volume density of the tongue structures, isotropic uniform random sections were used in this study. For this determination, the tongue was randomly placed on the φ clock. After selecting a random number from 1 to 10, an opposite cut was made along with the selected number, which caused two pieces of the tongue. The first piece was then located on the θ clock along with its previous cut surface on the 0-0 axis. Following that, a random number was selected again, and a similar cut was made along with the selected number. The other piece that caused from the cut made on the φ clock was placed on the θ clock steeply so that its cut surface overlay the 0-0 axis. The slab thickness was chosen to guarantee that a total of 8-12 slabs would be obtained (Figure 1 B, C). To estimate the shrinkage, a tissue cylinder was punched out from the tongue and slab by a trocar (diameter 3 mm), and the trocar radius was considered the “area” before (πr2). After tissue processing, the area was calculated using a microscope (40× magnification).
With the following formula, the number of wrinkles related to the tongue of each rat was calculated:
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Figure 1. A summary of stereological techniques: A. The tongue was separated from other parts of the mouth. B. To obtain isotropic uniform random sections of the tongue according to the orientation method, the tongue was placed on an equally divided circle and sectioned into two parts in a random direction (here 4). Other parts of the tongue were sectioned in the new random directions (here 5 and 1) after being placed on the cosine-weighted divided circle. C. The slabs were collected, and a circle was punched out. D. A-stained section on one microscopic slide. E. Point-counting method to estimate the volume density on the H&E staining sections. (Scale bar: 5µm) |
Volume Shrinkage=1- {(area after/area before)1.5
3.5. Sampling of the oral mucosa
To determine the density of the structures of the oral mucosa, vertical uniform sections (VUR) were needed, and VUR sampling (VURS) was used in this study. The oral mucosa was laid parallel to the horizontal plane and then cut vertically in a random manner. The resulting vertical sections were located on a transverse plane that was parallel to the horizontal plane. The object had to be positioned in the same direction (0°) as the previously detected internal marker. The VUR sections were obtained by uniformly, randomly, and systematically rotating a knife (Figure 2) (35)
3.6. Estimation of the volume density
The stereological counting equipment consisted of a Nikon E-200 microscope (Nikon, Japan) was connected to a computer, and the image of each section was assessed at a final magnification of 152×. According to the point-counting method and using the stereological software (Stereo Lite, SUMS, Shiraz, Iran), the stereological probe (point grid) (1E, 2E) was superimposed on the images of the tissue sections viewed on the monitor. Briefly, each section of the microscopic fields of view was systematically sampled in a random manner (fixed equal distances in x- and y-axes) on each histological slide. The right upper corner of each cross point was considered a point, and the volume density (VV) of the epithelium and glands of the oral mucosa and epithelium, lamina propria, total glands, serous glands, and mucous glands of the tongue were estimated using the point-counting method and the following formula: Vv=ΣP structure/ΣP reference,
where Vv is the volume density and ΣP signifies the total number of points composed on the structure and the reference (36).
3.7. Estimation of the total volume of the tongue
The tongue was cleaned, and its total volume was estimated using the Scherle method (37). Briefly, a jar filled with isotonic saline was placed on a scale and weighed. Subsequently, the tongue was suspended by thin cotton in the jar, and the tongue volume was estimated by the immersion method. Afterward, the tongue “V (tongue)” was isolated and weighed, and the volume of the tongue was estimated. In the end, the final volume was calculated using the following formula:
V final tongue = V primary× (1-Volume Shrinkage)
Ultimately, the total volume associated with each component of the tongue was estimated through the multiplication of the volume density by the total volume per animal (34):
V structure=V final tongue ×Vv structure
3.8. Statistical analysis
The data were analyzed through one-way ANOVA, and a p-value less than 0.05 was considered statistically significant. The statistical analyses were performed in SPSS software (version 15), and the chart was drawn in GraphPad Prism, Version 8.1 (IBM, Armonk, NY, USA program).
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Figure 2. A summary of the stereological techniques. A. The oral mucosa was separated from other parts of the mouth. B. To obtain vertical uniform sections of the oral mucosa. C. The slabs were collected for blocking. D. A stained section on one microscopic slide. E. Point-counting method to estimate the volume density on the H&E staining sections. (Scale bar: 5µm) |
4.1. Total volume of the tongue structures
The results showed that BPA had different effects on the total volume of the epithelium, lamina propria, total glands, serous glands, mucous glands of the tongue, volume density of epithelium, and glands of the oral mucosa. Moreover, RES prevented these changes and acted as a protective agent against changes in BPA-induced toxicity.
The results of the study on the tongue showed that the total volume of the epithelium decreased significantly in the BPA group, compared to the control and sham groups (P<0.001), while in the BPA+RES group, compared to the BPA group, the total volume of the tongue epithelium increased significantly (P<0.05) (Figure 2A). The results regarding the volume of the lamina propria showed that the volume of lamina propria in the BPA group increased significantly, compared to the control group (P<0.05). However, in the BPA+ RES group, the volume of lamina propria reduced significantly, compared to the BPA group (P<0.05) (Figure 2B).
The study results on the total volume of the tongue total glands showed a significant increase in the BPA group, compared to the control and sham groups (P<0.01). However, the total gland volume decreased significantly in the BPA+RES group, compared to the BPA group (P<0.01). Furthermore, the total volume of the serous glands decreased in the BPA group, compared to the control group; however, the difference was not statistically significant (P>0.05). In the BPA+RES group, the serous gland volume increased significantly, compared to the BPA group (P<0.05). Based on the results of the study, the total volume of the mucosal glands increased in the BPA group, compared to the control and sham groups (P<0.05). The total volume of the mucosal glands in
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Figure 3. Dot plots showing stereological findings in the tongue structures in different groups. The total volume of A. epithelium B. lamina propria C. serous glands D. mucous glands E. total glands. The lines over each dot plot represent mean±SEM (a standard deviation of the mean). (CON: Control / OO: Olive Oil / BPA: Bisphenol A / RES: Resveratrol) |
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Figure 4. Dot plots showing stereological findings in the oral mucosa structures in different groups. The volume density of A. epithelium B. total glands. The lines over each dot plot represent mean±SEM (a standard deviation of the mean). (CON: Control / OO: Olive Oil / BPA: Bisphenol A / RES: Resveratrol) |
the BPA+RES group decreased, compared to the BPA group (P<0.05) (Figure 2).
4.2. Volume density structures of the oral mucosa
The results showed that the volumetric density of the epithelium and glands increased significantly in the BPA group, compared to the control group (P<0.05). In the BPA+RES group, the volume density of the epithelium and glands reduced significantly, compared to the BPA group (P<0.05).
This study shows that BPA has systemic toxic effects on the stereology parameters of the tongue and oral mucosa as part of the gastrointestinal tract. This is the first stereology study on the toxicity induced by BPA on oral toxicity, and other studies have confirmed the cellular and molecular oral toxicity of BPA (14, 15). Various animal studies, in vitro cellular investigations, and molecular assessment, could demonstrate the beneficial effects of RES on modeling health-related physiological processes and pharmacological activities (38). In this regard, its protective effects against oxidative and inflammatory reactions, as well as carcinogen processes, have been recently shown (39). Furthermore, its preventive effects on atherosclerotic pathways by inhibiting the production of reactive oxygen species (ROS) have been recently indicated as the main components of the stress oxidative process (40). Additionally, mediated by its anti-carcinogenic properties, RES has an effective anti-mutagenic effect by suppressing the tumor promoter-induced cell proliferation and regulating tumor cell apoptosis phenomenon (41). In this regard, the protective effects of this agent on arresting the tongue squamous carcinoma cells have also been revealed (42). Based on such evidence, it was first hypothesized that applying RES might protect the oral mucosa and tongue against the toxicity induced by BPA. The first evidence related to the cellular protective role of RES is that it has been recently shown that RES can significantly reduce ROS production, cellular apoptosis reaction, as well as caspase 3 and caspase 9 enzymes activation, all induced by BPA on the cellular levels (43). In other words, one of the main protective mechanisms of RES to neutralize BPA toxicity was demonstrated to be the deactivation of oxidative stress. As the second protective role, RES can modify mitochondrial membrane depolarization, and therefore, regulate the mitochondrial function, thereby increasing the viability of the normal cells in different tissues (44). Considering the toxic damaging effects of BPA via the activation of stress oxidative pathways, it seems that its main protective effects on the cellular toxicity induced by this compound can be mediated by the antioxidant effects of RES (45). Along with the antioxidant properties of RES, cell regenerative effects of this agent via its vasodilatory effect have been demonstrated as well (46). In this regard, RES can increase the expression of eNOS, leading to improved vascular nitric oxide responses, induced endothelial-dependent vasorelaxation, as well as decreased superoxide production in the vessels that all lead to tissue regeneration (47).
The key finding of the present study was that the use of RES could lead to a significant increase in the total volume of the tongue epithelium and normalize the oral serous glands that were disturbed by BPA induction. Although the protective effects of RES on the malignant cells of the tongue had been previously assessed (42), the present study was the first that investigated its beneficial effects on regenerating the buccal and tongue cells damaged by BPA. As indicated previously by Mikami et al. (48), RES could significantly inhibit cell growth, enhance chemo‑ and radio-sensitivity, and block the cancer invasion of the head and neck squamous cell carcinoma cells by the up regulation of the regenerating gene (REG) Ⅲ expression. Since REG expression is significantly associated with the progression of oral and gastrointestinal inflammatory damages, it is possible that down-regulation of REG expression in such tissues by RES could be other mechanisms explaining the protective effects of RES.
As another mechanism, Quagliariello et al. (49) showed that BPA could stimulate lipid peroxidation. Since one of the main protective effects of RES is based on inhibiting the membrane lipid peroxidation, it seems that such effects can also be considered another main regenerative role of RES on the damaged buccal and tongue cells by BPA induction. Moreover, the effects of BPA on the activation of inflammatory cascade by the induction of producing and secreting inflammatory cytokines, such as TNF-α or IL-6, have been also demonstrated (50). In this regard, due to inhibiting effects of RES on such pro-inflammatory biomarkers, such effects can also be suggested as the protective role of RES on the oral tissues damaged by BPA.
In summary, different cellular and molecular pathways can be described as the arms of RES to generate its protective roles in the regeneration of the oral and buccal cells induced by toxic agents, such as BPA that includes reducing free radicals and ROS, as well as increasing endogenous antioxidant biosynthesis, 2) inhibiting membrane lipids peroxidation, 3) decreasing intracellular pro-inflammatory mediators, such as interleukins and TNF-α, 4) increasing eNOS expression and NO synthesis in vascular beds (followed by increasing blood supplying the damaged tissues), and 5) preventing free radical-mediated damage through SIRT1 pathway activation (31-33, 51, 52). Finally, due to the carcinogenic effects of BPA, it seems that RES can deal with this carcinogenic effect by inducing apoptosis and inhibiting proliferation in the tumor cells, which was not considered in the present study.
In conclusion, BPA has different effects on the oral mucosa and tongue. These effects can influence the normal function of the cells in these areas. RES, with its antioxidant properties, has a protective effect on these structures against the BPA.
The authors would like to thank the Student Research Committee of Shiraz University of Medical Sciences, Shiraz, Iran, and Dr. Laleh Khojasteh for editorial assistance.
Authors' Contribution: None
Conflict of Interests: The authors report no conflicts of interest.
Ethical Approval: All animal procedures were approved by the local Ethics Committee of Shiraz University of Medical Sciences, Shiraz, Iran (IR.SUMS.REC.1398.392).
Funding/Support: The present study was financially supported by Shiraz University of Medical Sciences, Shiraz, Iran (No. 97-01-21-18338).
Financial Disclosure: None
Informed consent: None
