Sodium chlorate (NaClO3) has been identified as a byproduct of chlorine dioxide (ClO2). ClO2 is effective for killing most microorganisms and disinfection of drinking water. NaClO3 is also used as an oxidizing agent in the tanning and leather industry, in the manufacture of dyes, explosives, and matches, and as herbicide. The potential adverse health effects of NaClO3 are associated with blood oxidation such as increased methemoglobin formation, decreased hematocrit, and red blood cell membrane damage. Other subchronic toxicity tests have identified the rat thyroid as the primary target organ 1.
Curcumin is a plant polyphenol that has antioxidative, anti-inflammatory, cancer chemopreventive, antifungal, and antiparasitic properties 2. It was found that it protects the thyroid gland after exposure to toxicants and oxidative materials 3. However, its effect on the pituitary gland as well as on the pituitary–thyroid axis has not been clearly determined.
Caspases are a family of cysteine proteases that mediate signal transduction and execution of apoptosis. Members of the caspase family are subdivided into initiator caspases (caspase-1, -2, -4, -5, -8, -9, -10, -11, and -12), and executioner caspases (caspase-3, -6, -7, and -14) 4.
The S-100 protein family consists of 24 members that serve intracellular and extracellular regulatory functions. S-100 proteins are involved in the regulation of proliferation, differentiation, apoptosis, Ca2+ homeostasis, energy metabolism, inflammation, and migration/invasion of many cells 5. S-100 is a good marker used to define folliculostellate cells in the pituitary gland 6.
Because of the limited data on NaClO3-induced effects on the thyroid and pituitary glands, the present work was carried out to study the effects of NaClO3 exposure on rats’ thyroid and pituitary glands. Moreover, the current work studied the possible protective effect of curcumin against the suggested NaClO3-induced adverse effects.
Materials and methods
Thirty healthy male Wistar albino rats weighing 200–230 g were used in this study. They were obtained from the animal house of Faculty of Medicine, Cairo University. The animals were housed in standard stainless-steel cages on a 12 h light and dark cycle. Room temperature was maintained at 24±2°C and humidity was maintained at 50%.
All animal experiments were conducted in strict compliance with the guidelines of the Institutional Animals Ethics Committee on the care and use of laboratory animals. Rats were divided into three groups as follows:
Group I (control group): 10 rats received standard rat chow diet and water ad libitum for 4 weeks.
Group II (NaClO3-exposed group): 10 rats received standard rat chow diet and received NaClO3 (purchased from Sigma, Germany) in drinking water (0.2 g/l) for 3 weeks 1.
Group III (NaClO3+curcumin-treated group): 10 rats received curcumin (100 mg/kg/day) 1 week before receiving NaClO3 (0.2 g/l) and it was continued concomitantly with NaClO3 for 3 weeks. Curcumin powder was purchased from Sigma and suspended in corn oil (10 mg/ml). It was administered through a gastric tube 2,7.
No mortality was detected in any of the groups studied.
Detection of thyroid hormones: T3, T4, and thyroid-stimulating hormone (TSH) were measured using ELISA kits provided by Calbiotech Inc. (California, USA) 8. Superoxide anion was measured using the LumiMax superoxide anion detection kit supplied by Agilent Technologies (Canada) 9.
Specimens of thyroid and pituitary glands were fixed in 10% formol saline. Paraffin-embedded sections were cut into 7-μm thickness and were subjected to:
- H&E stain: thyroid gland sections 10.
- Azan trichrome stain: pituitary gland sections 10.
- Immunohistochemical stains:
- Caspase-3: thyroid sections were subjected to staining with the primary rabbit polyclonal anti-caspase-3 antibody (Thermo Scientific, Lab Vision, USA). Caspase-3 immunoexpression was detected in the cytoplasm of epithelial cells lining the follicles 11.
- S-100: anterior pituitary gland sections were incubated overnight with the primary rabbit polyclonal anti-S-100 antibody (Thermo Scientific, Lab Vision). S-100 immunoexpression was detected in the cytoplasm and nuclei of folliculostellate cells 12.
- Tissue sections were counterstained with Mayer’s hematoxylin.
- Morphometric study using Leica Qwin 500 LTD (England) software image analysis: the mean area percentages of caspase-3 and S-100 were measured at ×400 magnification. Counting of pituitary basophils stained with azan trichrome was performed at ×1000 magnification. All measurements were performed within 10 nonoverlapping randomly chosen fields for each animal.
- Statistical analysis of serum hormones and superoxide anion levels, and the morphometric results was carried out using the analysis of variance test 13.
Serum hormones and superoxide anion levels
Serum T3 and T4 levels decreased significantly (P<0.05) after NaClO3 exposure (group II) compared with the control group and increased significantly (P<0.05) in the curcumin-treated group (group III) compared with group II, but still decreased significantly (P<0.05) compared with the control group. However, the TSH level increased significantly (P<0.05) in both groups II and III compared with the control group, but decreased significantly (P<0.05) in group III compared with group II. The superoxide anion level showed a significant increase (P<0.05) in group II compared with both the control and the curcumin-treated groups (Table 1, Histograms 1 and 2).
Histological and immunohistochemical results
H&E results: thyroid sections from the control group showed follicles lined by single uniform layers of flattened or cubical epithelial cells and contained abundant densely stained colloid material within the lumen (Figs 1 and 2).
Sections from rats exposed to NaClO3 (group II) showed evidence of focal follicular cells hyperplasia that was indicated by the presence of some follicles lined by two or more layers of epithelial cells (Fig. 3). Focal cellular hypertrophy was also observed by the presence of tall cuboidal or columnar follicular epithelial cells with cytoplasmic vacuolation in some of them. Loss of epithelial cells was observed in certain areas in some follicles. Moreover, blood extravasation and lymphocytic infiltration were observed in some thyroid follicles (Figs 3–5). In addition, some follicles were completely empty of colloid with or without detached cells in the lumen (Figs 3 and 4) whereas others had decreased colloid substance, with some follicles containing pale pink, lacy material (Fig. 6).
Sections of the thyroid gland from rats exposed to NaClO3 and rats that received curcumin (group III) showed apparently normal follicular epithelium (flat to cubical cells) but contained less densely stained colloid substance, with no inflammatory changes detected. Some cells showed apoptotic changes in the form of small dark nuclei and deep acidophilic cytoplasm, and appeared detached from the follicles (Figs 7 and 8).
Caspase-3 immunostaining results: thyroid sections from the control group showed a moderate distribution of caspase-3-positive immunoreactive cells with brown cytoplasmic deposits (Fig. 9).
Thyroid gland sections from rats exposed to NaClO3 (group II) showed decreased distribution of caspase-3 immunoreactivity (Fig. 10).
Thyroid sections from rats exposed to NaClO3+curcumin (group III) showed widely distributed caspase-3-positive immunoreactivity (Fig. 11).
Azan trichrome stain results: anterior pituitary gland sections from the control group showed few dispersed basophils between other pituitary cells (Fig. 12).
Examination of the anterior pituitary gland sections from rats exposed to NaClO3 (group II) showed an increase in the number of basophils (Fig. 13).
Sections of anterior pituitary glands from rats that received curcumin and NaClO3 (group III) showed dispersed, few basophils (Fig. 14).
S-100 immunohistochemical stain results: pituitary sections from control rats showed widely distributed S-100-positive folliculostellate cells. Folliculostellate cells appeared as branched cells with S-100 immunoreactivity in nuclei, cytoplasm as well as cytoplasmic processes (Fig. 15).
Pituitary gland sections from rats exposed to NaClO3 (group II) showed dispersed S-100 immunoreactive folliculostellate cells (Fig. 16).
The pituitary sections examined from rats that received curcumin and NaClO3 (group III) showed a moderate distribution of S-100 immunoreactive folliculostellate cells (Fig. 17).
The number of pituitary basophils was assessed in the anterior pituitary gland sections stained with azan trichrome stain. Sections from rats that were exposed to NaClO3 (group II) showed a significant increase (P<0.05) in basophils compared with both group I and III. Group III showed no significant difference versus group I (Table 2, Histogram 3).
The mean area percentage of caspase-3 and S-100 was assessed in immunostained sections (Table 3, Histogram 4).
Caspase-3: sections of thyroid glands from rats exposed to NaClO3 (group II) showed a significant decrease (P<0.05) in the mean area% of caspase-3 compared with groups I and III, whereas sections from rats that received curcumin+NaClO3 (group III) showed a significant increase (P<0.05) in the mean area% of caspase-3 compared with the control group.
S-100: sections of thyroid glands from rats exposed to NaClO3 (group II) showed a significant decrease (P<0.05) in the mean area% of S-100 compared with both group I and III. Also, sections from rats that received curcumin+NaClO3 (group III) showed a significant decrease (P<0.05) in the mean area% of S-100 compared with the control group.
Humans are exposed to NaClO3 daily as it is used in drinking water and herbicides. Moreover, NaClO3 abuse could lead to many health hazards. The current work aimed to study the effects of NaClO3 exposure on rats’ thyroid pituitary axis and the possible protective effect of curcumin against these effects.
In the current study, NaClO3 was used at a dose of 0.2 g/l for 3 weeks. Investigators considered this as a moderate dose that could initiate thyroid alterations in a rat model 1.
Starting with the results of serum hormones and superoxide anion levels, this study showed that exposure to NaClO3 (group II) caused a significant reduction in serum levels of T3 and T4, with a significant compensatory increase in TSH. Researchers have reported that NaClO3 affects pituitary–thyroid homeostasis by competitively inhibiting iodide uptake at the sodium iodide symporter of the thyroid gland, which results in decreased synthesis of thyroglobulin and reduced circulating thyroid hormone levels. As a consequence, there may be persistent stimulation of the pituitary–thyroid axis 14. Also, there was a marked increase in the level of superoxide anion, which can be explained by the fact that NaClO3 is a thyroid toxicant and a strong chemical oxidant that has the potential to react violently with many organic chemicals 15.
Thyroid gland sections from rats that received NaClO3 (group II) showed focal follicular cell hyperplasia and hypertrophy, vacuolated cytoplasm, decreased or absent colloid substance, blood extravasation, and lymphocytic infiltration. The findings of this work are supported by other studies that investigated the effects of exposure to NaClO3 in drinking water 1,14.
The focal follicular cell hyperplasia detected in the present study could be a direct result of decreased apoptosis as indicated by the significant decrease in caspase-3 immunoreactivity (caspase-3 is known to be an apoptotic protease) compared with the control group. However, hypertrophy of the follicular cells could be attributed to the marked increase in serum TSH that stimulated the follicular cells.
Many scientists have proved that TSH is the main regulator of thyroid function. TSH action is initiated by its binding to the G protein-coupled TSH receptor located at the surface of thyrocytes that regulates thyroid cell proliferation and differentiation 16. Moreover, thyroid cell differentiation induced by TSH involves the expression of the different genes required for thyroid hormone synthesis such as the sodium iodide symporter, thyroglobulin, and thyroperoxidase 17.
The decreased colloid substance observed in thyroid sections in rats exposed to NaClO3 in this study was explained by previous researchers who found that NaClO3 inhibited iodide uptake at the sodium iodide symporter of the thyroid gland, which resulted in decreased synthesis of thyroglobulin 14.
The lymphocytic infiltration detected in the present study could point to the inflammatory effect of NaClO3. Moreover, other studies have suggested that the lymphocyte infiltrate could secrete growth factors that stimulate the proliferation of thyrocytes 18.
From the previously discussed results, NaClO3 was not only disturbing the thyroid gland follicular functions but also producing histological changes such as hyperplasia, which could be a very important factor in progression to precancerous lesions. These findings were supported by other researchers who reported that proliferation, inflammation, and oxidative stress in the thyroid gland are very important causes of precancerous lesions 19. Moreover, Lukas et al. 20 found that prolonged increase in TSH levels induced thyroid cancer.
The pituitary gland sections in rats exposed to NaClO3 in the present work showed a significant increase in the number of basophils at the expense of other pituitary cells. This could be attributed to T3 and T4 deficiency and subsequently increased number of basophils that secrete TSH as a compensatory mechanism. This is consistent with what was found by other scientists who reported an apparent increase in the number of basophils in the examined pituitary sections of rats exposed to NaClO314. Furthermore, others have reported that thyroid hormone is important for pituitary development and maintenance. They reported that in a mouse model with hypothyroidism, the thyrotrophs showed hyperplasia and hypertrophy, whereas lactotrophs showed a drastic reduction in number 21.
Moreover, Meikle 22 found alterations in all basophils, not only thyrotrophs in hypothyroidism, as altered thyroid hormone was usually associated with changes in serum concentrations of other glycoprotein hormones secreted from the anterior pituitary. In addition, other investigators found thyroid hormone receptors in corticotropes, gonadotropes, and somatotropes that indicated that changes in these cells on the basis of a direct action of thyroid hormone 23.
In addition, the pituitary gland sections in rats exposed to NaClO3 (group II) in the present study showed a significant decrease in S-100 immunostained folliculostellate cells compared with the control group. This increase in the folliculostellate cells was accompanied by a significant increase in the number of basophils; this could point to the differentiation of the folliculostellate cells into basophils as reported previously by investigators that folliculostellate cells can act as stem cells in the anterior pituitary 24 and even as multipotential stem cells 25.
Pituitary folliculostellate cells are epithelioid cells that are organized into three-dimensional networks capable of intercellular communication through gap junction. Folliculostellate cells have an autocrine/paracrine regulation of anterior pituitary cell function through cytokines and growth factors 6.
Moreover, in the current work, the significant decrease in the folliculostellate cells was not only accompanied by a significant increase in the number of basophils but also a significant increase in TSH hormone. This could highlight the role of folliculostellate cells in regulating TSH hormone and hence T3 and T4 regulation.
These results are more clearly explained by the findings of the researchers that folliculostellate cells play an important role in thyroid hormone activation and thus suppression of TSH secretion. They supposed that about 50% of T3 was produced locally in the pituitary gland from thyroxine (T4) by the action of the deiodenase enzyme (D2) in folliculostellate cells 23. Moreover, another study recorded the expression of TSH receptors in folliculostellate cells of normal human pituitary. Thus, the folliculostellate cells could play an important role in TSH 26.
In group III (rats received NaClO3+curcumin), pretreatment with curcumin caused a significant increase in serum T3 and T4 levels, with a significant reduction in TSH and superoxide anion levels compared with group II; however, T3 and T4 levels still decreased significantly compared with the control group. The increased levels of T3 and T4 suggested the stimulating effect of curcumin on the thyroid gland and subsequently its regulating function on the pituitary gland as confirmed by the significant decrease in TSH. However, the reduction in the superoxide anion may point to the antioxidant and scavenger effect of curcumin on many free radicals such as superoxide.
This was supported by other studies that found that the levels of T3 and T4 increased significantly after the administration of curcumin 27. Furthermore, some studies found that the polyphenolic compound curcumin provided considerable protection against thyroid dysfunction in rats exposed to oxidative chemicals 3. Moreover, curcumin exerted a slight stimulating effect that did not lead to hyperthyroidism in rats. This stimulating activity of curcumin may counteract the hypothyroidism, whereas its anti-inflammatory activity is assumed to prevent the autoaggressive diseases that affect the thyroid gland 2.
Several studies have shown that the phenolic moiety of curcumin is an important part that is involved in the antioxidative mechanism of the curcumin compound and it derivatives. The mechanism of the antioxidant activity of phenols is widely known to involve their ability to act as free radical scavengers, leading to the formation of phenoxyl radicals 28.
Thyroid sections from rats treated with curcumin in the present study showed apparently normal thyroid follicles lined with flattened or cubical epithelium, some apoptotic cells with no evidence of hyperplasia, accompanied by a significant increase in caspase-3 immunoreaction compared with both group I and II. These results could reflect the role of curcumin in inducing apoptosis and subsequently preventing hyperplasia in thyroid follicles.
The apoptotic role of curcumin was proved previously in several studies that used it in different animal tumors, including colon, duodenal, stomach, prostate, liver, blood, submandibular gland, and breast carcinogenesis, both in vitro and in vivo as it blocks both tumor initiation (by its anti-inflammatory and antioxidative effects) and suppresses tumor proliferation and progression (by its apoptotic effects) 19.
In terms of the mechanism by which the curcumin could initiate apoptosis, most studies have reported that curcumin activates caspase-8 (a proapoptotic protein), causes mitochondrial cytochrome C release, and induces caspase-3 activation. Also, curcumin induces changes in the mitochondrial membrane potential that lead to the release of cytochrome C from the mitochondria, thus leading to the sequential activation of caspase-3 29.
However, the marked increase in caspase-3 immunoreactivity in the curcumin-treated group in the present work was much more marked than the apoptotic changes detected in H&E-stained sections in this group. This finding could highlight the nonapoptotic role of caspases especially in the regulation of cells’ proliferation. Recently, investigators have reported the other functions of caspases rather than apoptosis as they found that caspases play an important role in immunity, regulating the proliferation and differentiation of cells as they found that loss of caspase-3 and caspase-8 in mice results in hyperproliferation of both B and T lymphocytes 4.
In the present study, no inflammatory changes could be detected in the curcumin-treated group. Several studies have shown that curcumin can modulate the production of various inflammatory mediators, inflammatory cytokines such as interleukin-6, and inflammatory enzymes such as cyclooxygenase-2, thereby showing a potent anti-inflammatory activity 30.
Although in the current work curcumin produced a significant improvement in the thyroid functions and histology, the thyroid follicles contained less densely stained colloid compared with the control group. The less densely stained colloid could explain the significant decrease in T3 and T4 compared with the control group.
In the pituitary gland sections, pretreatment with curcumin led to a significant reduction in the number of basophils compared with group II, but with no significant difference from the control group. Thus, it could be suggested that curcumin ameliorated the effects exerted by NaClO3 on the pituitary gland.
In addition, pituitary gland sections showed a significant increase in S-100-positive folliculostellate cells compared with rats that received NaClO3, but showed a significant decrease in S-100-positive folliculostellate cells compared with the control group. This could reflect, to some extent, the regulatory role of curcumin in folliculostellate cells and therefore its regulatory effect in hormone-producing cells in pituitary glands, including thyrotrophs. However, the significant decrease in folliculostellate cells compared with the control could be attributed to the apoptotic effect of curcumin.
Recently, researchers found that curcumin inhibited the proliferation of folliculostellate cells in a monolayer cell culture in soft agar. They added that curcumin-induced cell cycle arrest at G (2)/M was accompanied by stimulation of cleaved caspase-3 and induction of DNA fragmentation. They also reported that curcumin exerts therapeutic effects on the intrapituitary regulation of hormone production and release, as well as on pituitary tumor pathogenesis 31.
The data of the present work showed that curcumin has protective actions (antiproliferative, apoptotic, and anti-inflammatory effects) against NaClO3-induced adverse effects on thyroid glands. In the pituitary gland, to some extent, folliculostellate cells and subsequently basophils were preserved. Moreover, the present work showed that curcumin has pa artial hormonal regulatory action on the thyroid–pituitary axis in rats exposed to NaClO3.
Conflicts of interest
There is no conflict of interest to declare.
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