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.