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Protective Effect of Resveratrol | Study

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Chapter 3

5.3. Experiments Following Fluoride Exposure and Resveratrol Supplementation

 

5.3.5. Effect of resveratrol on fluoride-induced alteration of thyroid metabolic activity

The changes in tissue DNA and RNA level of rat thyroid have been represented in Figure 23 and 24. Resveratrol supplementation in fluoride-intoxicated rats restored the decreased DNA content of thyroid by 15.46% (p<0.001) as compared to the treated group. Vitamin C exhibited better beneficial effects than resveratrol to protect against fluoride-induced DNA damage in thyroidal tissue.

Figure 23: Effect of resveratrol on fluoride-induced alteration of DNA level in thyroid

[Values are Means ± S.D. pa compared with control group, pb compared with F-treated group group, *** indicates p<0.001, Figure in the parentheses indicate the number of animals]

RNA content of fluoride-exposed animals was also decreased significantly in thyroid gland of rat. The decrease was found to be 33.08% (p<0.001) from control value. Administration of resveratrol partially counteracted the decreased RNA content in thyroid gland by 33.98% (p<0.001) as compared to that of fluoride-intoxicated group. Vitamin C also exhibited partial (35.22%; p<0.001) ameliorative effects in restoration of RNA content in the thyroid gland.

Figure 24: Effect of resveratrol on fluoride-induced change in RNA level of rat thyroid

[Values are Means ± S.D. pa compared with control group, pb compared with F-treated group group, *** indicates p<0.001, Figure in the parentheses indicate the number of animals]

Figure 25: Effect of fluoride on thyroidal Na+-K+ ATPase activity with or without resveratrol and vitamin C supplementation

[Values are Means ± S.D. pa compared with control group, pb compared with F-treated group group, *** indicates p<0.001, Figure in the parentheses indicate the number of animals]

Changes in the activity of Na+-K+-ATPase and 5' deiodinase I are represented in Figure (25 and 26). Na+-K+-ATPase activity in the fluoride-exposed group decreased by 40.59% (p<0.001) as compared to the control group. Resveratrol along with sodium fluoride showed significant (p<0.001) ameliorative effect to restore the enzyme activity towards the control value. Similarly, vitamin C supplementation also counteracted the enzyme activity by 26.67% (p<0.001). Additionally, resveratrol checked the inhibitory effects of fluoride on 5' deiodinase I enzyme activity and counteracted its activity by 58.93% (p<0.001). Vitamin C also exhibited partial protective effect against fluoride-induced alteration of 5' deiodinase I enzyme activity.

Figure 26: Effect of fluoride on thyroidal 5' deiodinase I activity with or without resveratrol and vitamin C supplementation

[Values are Means ± S.D. pa compared with control group, pb compared with F-treated group group, *** indicates p<0.001, Figure in the parentheses indicate the number of animals]

Table 26: Effect of co-administration of fluoride with resveratrol on thyroid parameters

Groups of animals

TPO

(ΔOD/min/mg of protein)

T3

(ng/ml)

T4

(µg/dl)

Control (6)

0.39±0.04

1.53±0.10

5.83±0.06

F-treated (6)

0.23±0.02, pa***

0.65±0.07, pa***

4.13±0.14, pa***

F-treated + resveratrol (6)

0.34±0.02, pb***

0.97±0.09, pb***

4.91±0.19, pb***

F-treated + vit. C (6)

0.33±0.03, pb***

1.1±0.07, pb***

5.09±0.16, pb***

[Values are Means ± S.D. pa compared with control group, pb compared with F-treated group group, *** indicates p<0.001, Figure in the parentheses indicate the number of animals]

Fluoride exposure significantly inhibited the activity of TPO in thyroid tissue by 42.06% (p<0.001) (Table 26). Resveratrol counteracted the TPO activity by 48.89% (p<0.001) as compared with the fluoride-intoxicated group, whereas vitamin C supplementation exhibited better protective effects than resveratrol against fluoride-induced decreased serum T3 level by restoring 43.7% (p<0.001) compared to that of fluoride intoxicated group.

Moreover, sodium fluoride significantly decreased T3 level in serum by 57.47% (p<0.001) (Table 26). Resveratrol counteracted fluoride-induced alteration of T3 level by 48.97% (p<0.001), whereas vitamin C supplementation exhibited better protective effect than resveratrol against fluoride-induced decreased serum T3 level by restoring 69.23% (p<0.001) as compared to that of fluoride-exposed group.

The thyroxine (T4) level was also reduced significantly in the serum samples of rats following exposure to fluoride (Table 26). The decrease was found to be 29.14% (p<0.01) compared to the control group. Administration of resveratrol appreciably counteracted fluoride-induced change in serum T4 level. Vitamin C supplemented group also exhibited 23.24% (p<0.01) restoration against fluoride-induced alteration of serum thyroxine level.

Table 27: Alkaline comet assay parameters (tail length, tail intensity and tail moment) in fluoride-exposed thyroid cells with or without resveratrol with the help of CASP

Assay Parameters

Control (6)

F-treated (6)

F-treated + resveratrol (6)

F-treated + vit. C (6)

Tail length (µm)

3.42±1.24

19.26±2.32

15.73±2.11

14.82±2.51

Tail intensity (µm)

0.35±0.05

1.71±0.52

1.46±0.49

1.39±0.62

Tail moment

0.04±0.006

0.21±0.04

0.18±0.006

0.17±0.009

[Values are Means ± S.D.]

Both qualitative and quantitative analyses of ethidium bromide stained comets on the basis of DNA damage are represented in Table 27. In comparison with the fluoride intoxicated group, there was a reduction in the tail length, tail intensity and tail moment of thyroid cells in the resveratrol supplemented group (Figure 27).

Figure 27: Comet assay micrograph

[A: undamaged and damaged thyroid cells of control, B: fluoride-treated, C: fluoride plus resveratrol-supplemented and D: fluoride plus vitamin C -supplemented group of animals. Cells were photographed under the fluorescence microscope using a 40x objective equipped with a 515-560 nm excitation filter and a 590 nm barrier filter.]

Figure 28: Histological changes in the rat thyroid after fluoride treatment with or without resveratrol

[(A) indicates normal architecture of follicular epithelial cell, (B) indicates flattened follicular epithelial cell in the enlarged follicle with over filling colloid, (C) and (D) indicate regaining of proper shape of follicular epithelia towards normalcy in “NaF+Res.” and “NaF+Vit.C” supplemented groups; Images were captured at 400 magnification]

Histological observations reveal that, the areas of thyroid follicles were significantly increased in NaF-intoxicated group. The follicular epithelia appeared flat in the enlarged follicles for being crushed over by filling colloid and hyperplastic nodules, consisting of thyroid parafollicular cells. Resveratrol markedly inhibited fluoride-induced damage on thyroid follicle and restored the changes near to the control group in respect of follicular size, colloidal material and epithelial tissue architecture (Figure 28).

5.3.6. Discussion

The present study further elucidates protective effects of resveratrol on fluoride-induced alteration of thyroid metabolic activity in male Wistar rats. Fluoride exposure at the present dose and duration significantly decreased the gain in body weight of rats which is consistent with our earlier observations. Alteration in body weight is supposed to be due to less intake of food after fluoride exposure or increased breakdown of muscle and tissue proteins. Moreover, suppressed activity of growth hormone on overall body growth due to less free thyroxine level in fluoride-intoxicated animals as found in the present study as well as earlier (Sakamoto et al., 2001), may be regarded as another causative factor for decrease in whole body weight. Gain in organ (thyroid) weight in relation to body weight has been expressed in terms of organo-somatic index, which shows that fluoride exposure significantly increased the organo-somatic index. This may be due to enlargement of thyroid gland by fluoride either by hypertrophy or hyperplasia of thyroidal cells in experimental rats as thyroid gland has a strong capacity for absorbing and accumulating fluoride (Ge et al., 2005). Earlier reports revealed that structural alteration of thyroid follicle by fluoride has been associated with induction of cytoplasm reduction, karyopycnosis of follicular epithelial cells and also reduction in the number of microvilli on the cristae of epithelial cells leading to swelling of vacuoles in follicular epithelial cells (Bouaziz et al., 2005). The present findings reveal that sub-acute fluoride exposure significantly alters the histological architecture of thyroid gland characterized by larger follicles, compressed follicular epithelia, over filling of colloid and reduction in inter-follicular space (Figure 28). Resveratrol supplementation in fluoride-intoxicated rats almost completely restored the altered organo-somatic index by preventing the changes in thyroid tissue architecture caused by fluoride. Resveratrol is thus assumed to protect thyroid tissue from oxidative stress-mediated cell damage.

DNA damage by fluoride is found in various cell types and oxidative stress is recognized as a causative factor of it (Wang et al., 2004b). Over ingestion of fluoride may result in alteration of functional status of the hypothalamo-pituitary thyroidal system and adversely affecting the synthesis of cellular metabolites such as DNA and RNA in thyroidal cells. Marked increase in DNA damage of thyroidal cells due to high fluoride and low iodine uptake has been reported (Ge et al., 2005). In the present study, sub-acute exposure to fluoride caused damage to the cellular components like DNA, confirming the earlier observation. Fluoride may directly damage cells by altering membrane structure and induce rupture of DNA strands as indicated by increased tail length and tail intensity of DNA in thyroidal cells as evident from the comet assay (Figure 27). DNA damage is supposed to be one of the reasons for high morbidity rates among those afflicted with hypothyroidism goiter and subcretinism in high fluoride and low iodine areas (Yaming et al., 2005). Alteration of nucleic acid metabolism in thyroidal cells might be involved in fluoride-induced functional disorders of thyroid.

Moreover, fluoride exposure at the present dose and duration significantly reduced the RNA content in thyroid tissue. Fluoride-induced depression of RNA level in other tissue was reported earlier (Sarkar et al., 2014) which might be due to inhibition of nucleic acid synthesis and improper attachment of m-RNA to ribosome after fluoride exposure. Fluoride, being an inhibitor of calcium and magnesium, the co-factors of certain metalloenzymes involved in nucleic acid biosynthesis reduces their synthesis (Verma and Guna Sherlin, 2002). Disturbed nucleic acid metabolism in thyroid tissue upon fluoride exposure is indicative of transcriptional and translational imbalance and chromosomal abnormalities. Resveratrol supplementation in fluoride-exposed rats appreciably reversed the changes in nucleic acid contents that were perturbed due to fluoride toxicity and also checked DNA strand breaks in thyroid cells. It is assumed that beneficial effects of this antioxidant may be due to its detoxifying ability to eliminate toxic fluoride from the tissue and to check free-radical mediated damage of cellular components like DNA, RNA and proteins. Trans-resveratrol itself exerts antioxidant effects likely due to the redox properties of the phenolic hydroxyl groups in its structure and thereby imparts in scavenging highly toxic free radicals that can damage cellular components (Leonard et al., 2003).

Hormone synthesizing capability of thyroid was seriously affected by fluoride exposure at the present dose and duration. Disturbed synthesis and secretion of thyroid hormones by fluoride and its interference in activity of enzymes that catalyze the conversion of thyroxine (T4) to active triiodothyronine (T3) as observed presently were also previously reported (Bouaziz et al., 2005). The reasons for decreased level of T4 during fluoride intoxication might be due to inhibition of absorption of iodine through the interaction of fluoride and/or insufficient synthesis and secretion of thyroglobulin and oxidized iodides from the thyroid gland owing to the structural changes of the thyroid follicle injured by the excessive intake of fluoride. Earlier observation also suggested that fluoride inhibits the activity of TPO in the thyroid gland (Zhan et al., 2006c). TPO, the main regulatory enzyme for thyroid hormone biosynthesis showed positive correlation with T3 and T4 level in hypothyroid patients (Singla and Shashi, 2013). The inhibition of TPO-catalyzed reaction as evident from present findings results in decrease in serum level of thyroid hormones. The suggested mechanism of action for enzyme inhibition may involve the conversion of thyroid peroxidase to a free radical that reacts with resorcinol moiety to produce a flavonoid radical (Chandra et al., 2011) that could covalently bind to the catalytic amino acid residues on the enzyme, leading to enzyme inactivation (Chandra et al., 2011). Similar explanation can also be suggested for the present study where fluoride-induced conversion of TPO to free radical may cause inhibition of this enzyme activity. In animal model, Boas et al. (2012) reported that fluorinated compounds such as perfluorooctane sulfonate and perfluorooctanoic acid also inhibited TPO activity in rats, with reductions in T4 and T3. It is assumed that iodide groups on TPO molecule may attract fluoride that causes decrease in the free active site on TPO molecule, leading to inactivation of this enzyme. The present study further reveals that resveratrol counteracted fluoride-induced changes in serum T3 and T4 level as well as TPO activity towards normalcy. Resveratrol administration effectively elevated serum T3 and T4 level by increasing the activity of TPO. As resveratrol is suggested to scavenge toxic free radicals generated due to fluoride toxicity and may also eliminate those harmful metabolites from the tissue, thus reducing the adverse effects of fluoride on thyroid metabolic functions.

The present study further reveals that fluoride exposure inhibits Na+-K+-ATPase activity that plays an important role in active transport of Na+ and K+ ions across the plasma

membrane. This finding is in conformity with the earlier observation (Zhan et al., 2006c). The decreased activity of Na+-K+-ATPase could adversely affect the accumulation of iodide in the thyroid, which is opposite to stimulation of Na+-K+-ATPase induced by hypothyroidism (Le Grow et al., 1999). This effect might be due to accumulation of fluoride in the thyroid which directly inhibits the Na+-K+-ATPase (Murphy and Hoover, 1992) or the combined activity of fluoride and high TSH on activation of the protein kinase C, which decreases the activity of Na+-K+-ATPase (Bocanera et al., 2001). Fluoride may cause internal injury to the cell membrane and affect the activity of membrane bound enzymes like Na+-K+-ATPase by disturbing membrane fluidity and membrane integrity and thus altering its permeability (Chinoy et al., 1994). Both thyroidal and extra-thyroidal tissues, like liver and kidney, are the main site of generation of circulating T3, which is produced by peripheral deiodination of T4 to T3. 5′-monodeiodinase I enzyme is responsible for this deiodination reaction. Sodium fluoride exposure at the present dose and duration reduces significantly the activity of 5′-monodeiodinase I, suggesting that fluoride decreases the rate of conversion of T4 to T3. All of these adverse effects of fluoride may be due to interference with synthesis or secretion of thyroid hormones by various mechanisms such as blockage of iodine uptake by the thyroid follicular cells, by inhibiting Na+-K+-ATPase activity, organification defect due to inhibition of thyroid peroxidase and suppression of thyroid hormone release. The study further demonstrates that relative adverse effects of fluoride on thyroid functions are appreciably counteracted by resveratrol supplementation. Moreover, resveratrol elevates antioxidant power of the thyroidal cell by inducing the expression of a set of cytoprotective genes through an antioxidant responsive element (Iwasaki et al., 2013). Remarkable beneficial effects of resveratrol were found against fluoride-induced alteration of thyroid functions. Although no group of animals treated with resveratrol alone was included in the present investigation, studies by Duntas (2011) demonstrated that resveratrol influences thyroid metabolic function by enhancing iodide trapping and, by increasing TSH secretion via activation of sirtuins and the phosphatidylinositol-4-phosphate 5 kinase γ (PIP5Kγ) pathway. Resveratrol administration was also demonstrated to increase iodide trapping in thyroid cell line (Sebai et al., 2010), thus preserving the functional status of thyroid. These studies clearly indicate promising effects of resveratrol on thyroid metabolic functions. Resveratrol has been recognized as a powerful antioxidant and also used in earlier occasions to combat against oxidative stress-induced cytotoxicity (Iwasaki et al., 2013). Antioxidative effects of resveratrol have already been established against other environmental toxicants like arsenic trioxide (Zhang et al., 2008). The studies revealed that pre-treatment with resveratrol resulted in a significant increase in the activities of GSH, GPx, CAT and SOD in plasma, thus supporting the antioxidant property of resveratrol. The protective efficacy of resveratrol in fluoride induced thyrotoxicity has been demonstrated in in vivo rat model in terms of histological changes, activities of thyroid metabolic enzymes like TPO and 5′-monodeiodinase, iodide trapping mechanism by Na+-K+-ATPase and changes in thyroid hormone secretion.

5.3.7. Summary

The present study elucidates the protective effect of resveratrol, a natural antioxidant against fluoride-induced alteration of thyroid functions, possibly mediated by the anti-oxidative property of resveratrol. Sub-acute fluoride exposure through drinking water imposes stress on biosynthetic machinery of thyroid hormones, as indicated by depletion of serum T3 and T4 and decreased activities of TPO, Na+-K+-ATPase and 5′ deiodinase I enzymes. Additionally, perturbation and damage of the nucleic acids of the thyroid gland by fluoride are also observed in the present study. These metabolic changes were associated with tissue architectural alteration in fluoride-intoxicated thyroid gland. The efficacy of resveratrol to attenuate the adverse effects of fluoride on thyroidal metabolic function, DNA damage and ultra-structural disorganization could provide insight into whether abnormalities in metabolic function are linked to the development of early stages of fluoride induced thyroidal tissue damage. The present study thus establishes the new role of resveratrol as a promising protective agent against fluoride-induced thyroidal toxicity.


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