4. Discussion
In order to provide hopeful evidence for a link between exposure to EMF and an increased prevalence of health risks, possible impacts of EMF on biological systems were widely examined.28 Numerous in-depth studies have been conducted over the last few decades to examine the potential biological effects of electromagnetic fields (EMF) on various human systems. The endocrine system, namely the pineal gland, has received the most attention out of all the researched systems.29 Effects from low radiation doses are more challenging to forecast and analyse. There is no limit to how efficient adaptive mechanisms promote cellular protection. As a result, it is impossible to draw a firm judgement concerning exposure to low radiation levels.30,31 Due to the sensitivity of the thyroid gland to EMFs, this exposure led to morphological modifications and a decrease in blood T4 and T3 levels. These alterations remained over the whole trial, proving that normal thyroid function takes more time to rebound after exposure to EMFs.
Loss of weight is typically linked to hyperthyroidism.32 Our findings were consistent with previous studies in that UVB irradiation resulted in considerable thyroid and body weight loss. 33,34 The thyroid gland is impacted by UV radiation, and long-term radiation exposure predominantly targets many genes, proteins, and lipids and leads to cancer.35 We discovered that UVB radiation-induced oxidative stress produced free radicals and induced hyperthyroidism. T3, T4, FT3, and FT4 were all considerably higher in hyperthyroidism, whereas TSH was much lower. By causing damage to crucial components, including body protein and thyroid tissue, UVB irradiation was shown to dramatically diminish the body weight and thyroid weight of the experimental mice. Naringin was utilised as an antioxidant in our analysis. Additionally, compared to the UVB-irradiated group, the administration of naringin resulted in a substantial drop in T3, T4, FT3, and FT4 levels and a large increase in TSH levels. In vivo cellular oxidative stress is thought to be modulated by any fluctuation in circulatory thyroid hormones. Reactive oxygen species are produced due to increased mitochondrial respiration, which is thought to be the primary mechanism of this physiological/pathological alteration.36
We observed that Swiss albino male mouse thyroid glands exposed to UVB radiations experienced a heightened rise in ROS. Other investigations have revealed elevated levels of oxidative stress in several organs, including the testis, skeletal muscle, heart, pancreas, and brain, which is mediated by the hyperthyroid condition.37,38 Physiological levels of ROS are created during typical cell metabolism. However, excessive ROS production can result in cell apoptosis, necrosis, or autophagy in several pathological circumstances, including inflammatory bowel disease, diabetes, cancer, or obesity.39 Organisms have complete, integrated endogenous enzymatic repair mechanisms to deal with ROS damage. Important non-enzymatic antioxidants include glutathione (GSH), vitamin E, vitamin C, h-carotene, and uric acid, which are either ingested with food or produced endogenously. The endogenous enzymatic antioxidants are represented by Cu2+, Zn2+ and MnSODs, catalase, and GPx.40 The decrease in antioxidant capacity in hyperthyroid patients is most likely due to increased free radical production. Increased enzyme activity in hyperthyroidism patients is likely due to increased ROS production.41 In the mouse thyroid, exposure to UVB significantly increased the level of lipid peroxidation. Earlier research on lipid peroxidation showed a similar impact.42,43 Lipid peroxidation is inversely related to oxidative stress, reducing some defensive systems effectiveness. Exogenous chemicals may be added to boost defence systems. Treatment with NG dramatically decreased the production of TBARS in the mouse thyroid. According to reports, NG prevents lipid peroxidation by H2O2.44
Additionally, it has been demonstrated that melatonin, ascorbic acid, and Ocimum flavonoids guard against lipid peroxidation in mice.45 There are several potential strategies for reducing the damage that oxidative stress causes to macromolecular structures. The preventive effectiveness of NG against radiation-induced lipid peroxidation was further evaluated by detecting some antioxidants in the thyroid of mice exposed to UVB radiation. Whole-body UVB exposure reduces an organism’s potential for general antioxidant defence and depletes known antioxidants like GSH.39 Mice exposed to UVB radiations exhibited significantly reduced thyroid GSH activity. It has been observed that GSH depletion enhances lipid peroxidation since it is known to impair glutathione peroxidase activity both in vitro and in vivo. The current study shows a similar relationship between the decrease in GSH and the rise in lipid peroxidation. Treatment with NG considerably slowed the reduction in thyroid gland GSH content in mice. According to reports, NG’s antioxidant activity is comparable to GSH.43 According to our study, thyroid Glutathione Reductase (GR) activity significantly increased due to UVB exposure. According to some authors, the thyroid hormone impact directly contributes to the enhanced activity of glutathione reductase, and administration of peroxidative products to rats has been shown to dramatically promote the activity of several detoxification enzymes.46 Catalase, hydrogen peroxide, and superoxide dismutase activity were significantly increased throughout our research. As per the previous study, hyperthyroid rats’ hearts and erythrocytes had greater SOD, CAT, and H2O2 activity levels.47 The expression of antioxidant enzymes may be impacted by hyperthyroidism depending on the cell type, mitochondrial activity, and ROS concentration inside the cell.48 The lipid-soluble antioxidant probucol has been shown in both in vivo and in vitro studies to halt oxidative damage caused by gamma radiation.49 Supplemental NG and probucol have been demonstrated to boost antioxidant reserve in rabbits fed a high cholesterol diet.50
The alterations in thyroid gland function in the exposed group were further supported by the histological examination of the thyroid follicles in the current investigation, which exhibited glaring light microscopic and ultrastructural abnormalities. In many instances, these follicles were disorganised and lost. While some follicles seemed to have involuted walls, others had fragmented follicular walls. While other cells had vacuolated cytoplasm, the follicular cells lost their epithelial covering in the lumina. Additionally, some follicular cells possessed darkly coloured nuclei, and interfollicular septa had cellular infiltration. The efficiency of TSH’s gland-stimulating actions and hyperactivity might be employed to explain the thyroid follicles’ histological alterations. Morphometrical data supported the changes in epithelial height. Our histological findings agree with those of other investigations. Because low thyroid hormone levels weaken the feedback inhibition of TSH and induce the thyrotrophs to secrete more TSH, chronic stimulation of the pituitary can give rise to thyroid gland malignancy, which manifests as follicular cell proliferation, colloid region shrinkage, and thyroid gland enlargement.51 Yu highlighted that the reactive oxygen species (ROS) and lipid peroxidation produced by chlorpyrifos may have contributed to the death and deterioration of follicular cells.52 Furthermore, nuclear alterations are a marker of Necrosis and apoptosis of cells, according to previous study.53 The thyroid follicles in the group treated with naringin (80 mg/kg) were healthy and free of any pathological alterations. The study suggests that under typical circumstances, naringin has no negative consequences. The results suggest that the test flavonoids may be useful in managing hyperthyroidism.
Immunohistochemistry and immunofluorescence revealed increased amounts of cells positive for THRβ-1, Caspase and NOS-2 after UVB exposure. In the present study, the expression of THRβ-1 was lowered down in UVB exposed group while enhanced Caspase-3 and NOS-2 expression was detected in thyroid follicular cells in the UVB exposed group. In UVB irradiated group thyroid showed reduced expression and fluorescence of TRHβ-1 in thyroid follicular cell. The administration of naringin in UVB irradiated group showed enhanced expression and fluorescence in thyroid follicles. Thyroid hormones regulate mammals’ growth, development and differentiation. This is because as the oxidative stress generated by UVB radiations breaks the thyroid hormones receptors, as such the breakdown of receptors did not allow the antibodies to bind over the receptors, thus the expression got decreased.54Thus, the study showed the degenerative effect of UVB radiations which lowered down the expression of TRHβ-1 in UVB exposed group as compared to control group. To date, little information exists on the effect of UVB radiation on caspase-3 expression as well as the effect of naringin in correction of the UVB radiation’s alteration. In the present study, UVB radiation induced significant increase in caspase-3 expression indicating UVB provokes apoptosis in the mice thyroid cells. Interestingly, the addition of naringin improved the condition by lowering the caspase-3 expressions. Apoptosis is a physiological process of selected cell deletion. As an antagonist of cell proliferation, apoptosis contributes to keeping the cell number in thyroid tissue and helps to remove superfluous and damaged cells, but excessive apoptosis could cause destruction of thyroid tissue.55 In a similar study in China to explore the effects of expressions of caspase-3 in mice testes at different concentrations and time of lead acetate, it increased the expressions of caspase-3, which induces apoptosis of germ cells.56 The occurrence of thyroid cell apoptosis and the expression of caspase-3 in the adult male mice following UVB radiation administration were investigated. Compared with the control group, the protein levels of caspase-3 were significantly higher in UVB exposed group. Excessive NO production due to elevated NOS-2 causes cytotoxicity and induces germ cell apoptosis.57Furthermore, NO is a signalling molecule that has an important role in the pathogenesis of inflammation.58 In UVB irradiated group, NOS-2 expression was detected. These results suggested that thyroid follicular cells are also affected by UVB radiation. Furthermore, UVB exposure led to inflammation in the thyroid follicular cell apoptosis, as demonstrated in the DAB and DAPI-FITC assay.
Nevertheless, it is necessary to establish therapeutic dosages for human treatment. At dosages of 1 g/day or greater, naringin is widely considered safe, and no side effects have been noted. Because the concentration of naringin utilised in this investigation equates to 88.09 mg/kg in a 70-kg human, based on a comparison of body surface area, the flavonoid dosages employed in this study are thus more or less realistic for people.59
There have been reports of NG’s ability to scavenge free radicals. In vitro studies have shown that it can scavenge the free radicals OH, O2-, DPPH, and ABTS.60 When O2- is inhibited by NG, less hydrogen peroxide and hence less OH is produced. While oxygen receives one electron and generates superoxide at a rate constant of 1.9X1010M-1s-1, NG rapidly accepts electrons at a rate of (1.0±0.1)X1010M-1s-1.61 As a result, oxygen and NG continually conflict with one another for available electrons. By removing the radiation-induced electrons that oxygen molecules compete with, NG molecules may limit potential harm. One of NG’s key characteristics in its defence against oxidative stress is its capacity to scavenge free radicals. Earlier investigations, found that NG provided radioprotection by scavenging free radicals and improving the antioxidant state of radio-exposed mice.60 Numerous medications, including felodipine, cyclosporine, simvastatin, and lovastatin, are affected by NG and other grapefruit flavonoids. According to reports, NG contains antiestrogenic, antiatherogenic, anti-apoptosis62 and cholesterol-lowering properties9. According to reports, up to 2 g in humans, it is non-toxic. NG may function as a radioprotector in individuals undergoing radiation because its protective dosage is 2 mg/kg, and the human tolerance dose is larger than 2 g.63 Human radioprotection of normal tissue is a promising way to advance radiotherapy. The key characteristic of a radioprotector that only protects normal tissue is the exclusion of tumour tissue. It might not be impossible for NG to protect tumours in some cases. As a result, further study is required in this area before NG may be used in radiation for cancer. More research is planned to determine if NG is specifically rejected from neoplastic cells and whether it could offer distinct radiation protection.