The Protective Effect of Selenium versus Ascorbic Acid on Potassium Dichromate-induced Thyroid Toxicity in Adult Male Albino Rats

Abstract

Background:

Potassium dichromate (PD) is considered a common inorganic chemical reagent, and it is considered one of the hexavalent chromium Cr (VI) compounds; these compounds have genotoxic, carcinogenic, and immunotoxic effects. It induces thyroid gland toxicity via oxidative stress. Selenium (Se) and ascorbic acid (Vitamin C) are known for their antioxidant role, and therefore, they are essential for humans and animals. Limited studies investigated the role of selenium versus ascorbic acid in protecting the thyroid gland from PD toxicity and so this study was designed.

Aim:

This study aims to assess the protective impact of selenium versus ascorbic acid against PD-induced thyroid toxicity in adult male albino rats.

Materials and Methods:

In this study, 42 adult male albino rats were randomized into six equivalent groups. Each animal received intraperitoneal injection of a treatment once daily for five successive days: The control group received distilled water; the selenium group received sodium selenite; the ascorbic acid group received Vitamin C; the PD group received potassium dichromate; the PD and selenium group received sodium selenite 1 h before PD; and the PD and Vitamin C group received Vitamin C 1 h before PD. By the end of the experiment, scarification of all animals was done, and their thyroid glands were extracted and processed for light microscopic examination after staining with hematoxylin and eosin, Masson’s trichrome, periodic acid–Schiff, and proliferating cell nuclear antigen (PCNA) immunostaining.

Results:

Thyroid gland sections of the PD group revealed loss of the thyroid general architecture. The follicles showed disrupted basal lamina, epithelial hypertrophy, hyperplasia, and sloughing. Colloid vacuolation was also seen. There was a significant decrease in the follicular diameter and area percentage of colloids. There was also an increase in the follicular cell height, area percentage of collagen, and number of PCNA immune-positive nuclei compared to the control group. Sections of the PD and selenium and PD and ascorbic acid groups showed a significant histological improvement compared to the PD group with no significant difference between both the groups.

Conclusion:

Both selenium and ascorbic acid had a similar partial protective effect on the thyroid gland against PD-induced toxicity in male adult albino rats.

INTRODUCTION

Potassium dichromate (PD) is a common inorganic chemical reagent, and it is considered one of the chromium hexavalent Cr (VI) compounds. Cr (VI) is a heavy metal that is widely used in several industries worldwide. However, the increased use of PD compounds and their improper disposal cause different health problems. These problems are not only restricted to workers’ exposure in industrial areas but also overreach the general population through contaminated food and water. PD was found to be highly carcinogenic and toxic to various body organs specifically the thyroid gland; it induces severe thyroid gland toxicity through oxidative stress and disruption of the redox balance of the thyroid gland tissues.[1] Interestingly, growing evidence revealed the protective role of some natural antioxidants against PD-induced thyroid gland toxicity.[2] Among the reported antioxidants are carotenoids, Vitamin E, selenium, and ascorbic acid.[3]

Selenium (Se) is a crucial micronutrient for the health of both humans and animals. It is known to be involved in several physiological and pathological processes, and it has antioxidant, anti-inflammatory, and anticancer effects. Selenium is required for optimal thyroid function.[4]

Ascorbic acid (Vitamin C) is one of the water-soluble vitamins, and it performs important metabolic functions in humans. It has potent antioxidant effects against oxidative damage triggered by different toxic heavy metals such as copper, cadmium, and PD.[5]

Selenium and ascorbic acid are responsible for protecting the animal tissues from PD-induced cellular injury, and modulate its toxicity through antioxidative mechanisms. They can scavenge the reactive oxygen species (ROS) resulting from PD reduction, thus preventing oxidative stress, tissue damage, and disruption of functional integrity of the thyroid gland.[6]

There are limited histological studies that showed the effect of Se and Vitamin C in protecting the thyroid gland against PD-induced thyroid toxicity.[7,8,9,10] Additionally, there are no available studies that compared their effects on ameliorating the thyroid damage induced by PD. Hence, this study is designed to demonstrate the protection role of Se versus Vitamin C against PD-induced thyroid toxicity in male adult albino rats, using different histological and immune histochemical techniques.

MATERIALS AND METHODS

Drugs

PD was obtained from El-Nasr Company for Pharmaceuticals ADWIC, Egypt, as 99% PD powder. Both selenium and ascorbic acid were obtained from Sigma-Aldrich, Gillingham, UK, in the form of sodium selenite powder and Vitamin C powder, respectively.

All chemicals were prepared by dissolving in distilled water (DW) (1 mg/mL).[2,7,11]

Animals and study design

This study was performed on 42 healthy adult male albino rats of identical age and weight (200–230 g). All experimental animals were purchased from the Faculty of Veterinary Medicine’s animal shelter, Suez Canal University. Animals were adapted for 5 days prior to the experiment with free use of food and water and kept in sanitary cages at a standard room temperature. The Faculty of Medicine, Suez Canal University, adhered to the National Institutes of Health’s guidelines for the usage of lab animals (NIH Publications No. 8023, amended 1978) in reviewing and authorizing this study’s methodology. After obtaining the approval from the Research Ethics Committee of the Faculty of Medicine, Suez Canal University (approval No. 3883) at 24/6/2019 six groups of animals were randomly assigned, each of seven animals. Each animal received intraperitoneal injection of a treatment once daily for 5 successive days as follows:

The control group received DW (2 mL/kg-BW). The selenium (Se) group received sodium selenite (0.5 mg/kg-BW). The ascorbic acid (Vitamin C) group received Vitamin C (120 mg/kg-BW). The PD group received PD (2 mg/kg-BW). The PD + selenium (Se) group received sodium selenite 1 h before PD. The PD + Vitamin C group received Vitamin C 1 h before PD.[7,8,9,10]

Specimen collection and the used histological techniques

On the 6^th day of the experiment, all animals received anesthesia in the form of diethyl ether inhalation and then they were sacrificed by cervical decapitation. From each animal, the thyroid gland was extracted for paraffin section preparation (5 μm thickness) for light microscopic (LM) examination. Sections were stained with hematoxylin and eosin (H and E), Masson’s trichrome, periodic acid–Schiff (PAS), and proliferating cell nuclear antigen (PCNA) immune staining.

For proliferating cell nuclear antigen immune staining

PCNA is considered as a helper protein of DNA polymerase enzymes, essential for DNA synthesis and it serves as a common indicator of cells proliferation. Labeled streptavidin-biotin, horseradish peroxidase technique (UltraVision) was used in this study for the detection of PCNA in the nuclei of the epithelial cells of the thyroid gland follicles. PCNA antibody is considered a mouse monoclonal antibody raised against rat PCNA (Novus Biologicals, USA). Immunostaining preterement was done by antigen retrieval method using a microwave oven; sections were exposed to 750 W of radiation in a 0.01 M citrate buffer (PH 6.0) for 10 min, then left to cool in room temperature for 20 min, and then they were rinsed using phosphate-buffered saline. Sections were then incubated for an hour with the primary antibody. Immunostaining process was accomplished by UltraVision detection system. Then counterstain was done with Mayer’s hematoxylin. Lab Vision Thermo Scientific provided the Mayer’s hematoxylin, citrate buffer, and UltraVision detection equipment (Fremont, California, USA).

Tonsil tissue was stained following the same protocol as a positive control, and the brown coloration of the nucleus indicates a positive reaction. The same protocol was followed with the omission of the primary antibody for negative control.

Histopathological and morphometric study

Qualitative assessment

For each experimental animal, three H- and E-stained sections and five fields/each section were examined using a light microscope to assess the following general histopathological changes, and the frequency distribution of each change was computed:[8,9,10]

• Loss of architecture of the thyroid gland

• Widening of the interfollicular connective tissue spaces

• Dilatation and/or congestion of the interfollicular blood vessels.

Twenty random thyroid follicles/animal were examined at high-power field (×400) and the following follicular histopathological changes were assessed, then the percent of each change was computed:[7,8,10]

• Disruption of basal laminae and coalescence of thyroid follicles

• Hypertrophy of follicular cells

• Stratification (hyperplasia) of follicular cells

• Sloughing of follicular cells into the lumen of the follicles

• Vacuolation of colloid

• Retraction of colloid.

Quantitative assessment

For each experimental animal, three sections and five fields/each section were analyzed using computer-assisted image analysis, the pro-plus software, at the Histology Department of the Faculty of Medicine, Suez Canal University. The following parameters were assessed, and the mean and standard deviation of each parameter were calculated:[2,8]

• Thyroid follicle diameter in H- and E-stained sections (×100)

• Height of follicular epithelium in H- and E-stained sections (×400)

• Mean color area percentage (mean CA%) and mean optical density (mean OD) of magenta-colored colloid in PAS-stained sections (×400)

• Mean CA% of green-colored collagen in Masson’s trichrome-stained sections (×100 and × 400)

• Number of PCNA immune-positive nuclei of the thyroid follicular cells in immune-stained sections (×400).

Statistical analysis

Quantitative variables were presented as mean and standard deviation and qualitative variables were presented as frequency and percentage, and the difference among the studied groups was presented by tables and figures and graphs.

The difference among the six groups considering the frequency distribution of histopathological changes of the thyroid gland was tested for significance using the Chi-square test, and then, pairwise comparisons were performed using Fisher’s exact test.

Furthermore, the difference among different groups including the qualitative and quantitative assessment of the thyroid follicles was tested for significance using Kruskal–Wallis test, then performing post hoc pairwise comparisons used adjusted Bonferroni approach. Statistical significance was considered at P < 0.05.

RESULTS

Hematoxylin and eosin results

Group I: Control group showed normal thyroid gland architecture in 100% of the animals [Table 1]. The capsule was seen enclosing a number of varied-sized thyroid follicles, separated by narrow spaces of interfollicular connective tissue [Figure 1a]. Blood vessels were seen between the follicles [Figure 2a]. Almost all the thyroid follicles in this group were normal. The follicles appeared as rounded or oval cyst-like compartments of variable sizes with larger follicles at the periphery and smaller ones in the center. Each follicle was formed of a simple cubical epithelium with basophilic cytoplasm and rounded central nuclei resting on a well-defined basal lamina, and the follicular lumen was filled with colloid [Figures 1a, 2a and Table 2]. In 11.98% of the examined follicles, the colloid was vacuolated [Table 2].

Table 1.

The frequency distribution of thyroid gland histopathological changes among different experimental groups

Change	Group (%)						P
	Control	Se	Vitamin C	PD	PD + Se	PD + Vitamin C
Loss of architecture	0	0@	0@	71.42#	0@,$	0@$	<0.001*
Widening of interfollicular spaces	0	0@	0@	85.71#	28.57@,$	14.28@,$	<0.001*
Congestion of blood vessels	0	0@	0@	85.71#	28.57@,$	14.28@,$	<0.001*

*Statistically significant at P<0.05, ^#Statistically significant compared to control group, ^@Statistically significant compared to PD group, ^$No statistically significant difference between PD + Se and PD + Vitamin C groups. Chi-square test, pairwise comparisons using Fisher’s exact test. PD: Potassium dichromate

Figure 1.

Photomicrographs of thyroid gland sections from different groups stained with H and E, ×100. (a) (Control group) showing normal appearance of the thyroid gland, the capsule (thick arrow) enclosing thyroid follicles (F) filled with colloid (C), and separated by little interfollicular C.T. (*). (b) (PD group) showing distorted architecture of the thyroid gland, thickened capsule (thick arrow) enclosing varied-sized follicles (F) which appeared smaller in size compared to the control group. Some follicles show retracted colloid (R). The follicles are separated by wide interfollicular C.T. spaces (*). PG is shown. (c and d) (PD + Se group) and (PD + Vitamin C group) respectively showing almost preserved architecture of the thyroid gland. The capsule (thick arrow) appears thin. Most of the follicles (F) appear regular, large, filled with colloid (C), and separated by narrow interfollicular C.T. (*). Few follicles show retracted colloid (R). Dilated and congested BV, in addition to widening of interfollicular spaces in some parts (*) are still shown in D. PG: Parathyroid gland, BV: Blood vessel, PD: Potassium dichromate, H and E: Hematoxylin and eosin

Figure 2.

Photomicrographs of thyroid gland sections from different groups stained with H and E, ×400. (a) (Control group) showing varied-sized follicles (F) separated by scanty interfollicular C.T. (*) And a BV (red arrow) is shown. Each follicle is lined with simple cubical epithelium (arrowheads) with basophilic cytoplasm and rounded central nucleus resting on a regular and well-defined basal lamina. The follicular lumen is filled with colloid (C). In some follicles, the colloid is vacuolated (V). (b and c) (PD group); (b) showing shrunken follicles (F), some of them are lined with high cubical to columnar cells (arrowheads). Other follicles show stratified epithelium (ST). Some follicles are filled with vacuolated colloid (V), while others show retracted colloid (R). The follicles are separated by wide interfollicular C.T. spaces (*) occupied in some areas with dilated and congested BVs; (c) showing loss of architecture of the thyroid gland and coalescence of the thyroid follicles. It shows thyroid follicles with disrupted basal lamina (arrows) and sloughed follicular epithelium into the lumen (SL). (d and e) (PD + Se group) Showing evidence of improvement. (d) Showing follicles (F), lined with cubical follicular cells resting on a regular and well-defined basal lamina, nearly similar to the control. Few follicles still have high cubical to columnar follicular cells (arrowheads). Most of the follicles are filled with colloid (C), and some follicles show retracted (R) or vacuolated (V) colloid. The follicles are separated by narrow interfollicular C.T. (*). A congested BV is shown. (e) Showing follicles (F), lined with cubical follicular cells (arrowheads) resting on a regular and well-defined basal lamina, nearly similar to the control. The colloid is vacuolated (V) in many follicles. The follicles are separated by wide interfollicular C.T. spaces (*) compared to control. (f and g) (PD + Vitamin C group) Showing evidence of improvement as in the previous group. (f) Showing one follicle that has high cubical or columnar cells (arrowheads). (g) Showing follicles with ST. The follicles are still separated by wide interfollicular C.T. (*) compared to control. A dilated and congested BV is also shown. BV: Blood vessel, ST: Stratified epithelium, H and E: Hematoxylin and eosin, PD: Potassium dichromate

Table 2.

Assessment of qualitative parameters of the thyroid follicles (%) among different experimental groups presented as mean±standard deviation

Group	Parameter						P
	Control	Se	Vitamin C	PD	PD + Se	PD + Vitamin C
Disruption of basal laminae	1.74±0.37	2.02±0.31@	1.97±0.21@	16.21±3.04#	3.05±0.83@,$	2.38±0.64@,$	<0.001*
Follicular cell hypertrophy	6.24±1.49	6.74±1.86@	6.43±1.56@	40.6±6.31#	12.91±3.24#,@,$	12.05±2.95#,@,$	<0.001*
Follicular cell hyperplasia	3.52±0.97	3.86±0.67@	3.67±1.14@	20.18±2.32#	7.67±1.65#,@,$	6.98±1.75#,@,$	<0.001*
Follicular cell sloughing into lumen	1.48±0.31	1.66±0.52@	1.54±0.57@	15.52±2.63#	4.19±0.78#,@,$	3.95±1.06#,@,$	<0.001*
Colloid vacuolation	11.98±3.78	12.37±3.56@	12.06±3.89@	28.38±4.58#	12.85±1.86@,$	12.28±2.05@,$	<0.001*
Colloid retraction	1.84±0.55	2.03±0.43@	1.92±0.48@	18.42±2.19#	4.13±0.75#,@,$	3.64±1.12#,@,$	<0.001*

*Statistically significant at P<0.05, ^#Statistically significant compared to control group, ^@Statistically significant compared to PD group, ^$No statistically significant difference between PD + Se and PD + Vitamin C groups. Kruskal–Wallis test. Post hoc pairwise comparisons used adjusted Bonferroni approach. PD: Potassium dichromate

The mean follicular diameter was 9.47 ± 1.79 μm. The mean height of follicular epithelial cells was 1.62 ± 0.42 μm [Table 3].

Table 3.

Assessment of quantitative parameters of the thyroid follicles (µm) among different experimental groups presented as mean±standard deviation

Parameter	Group						P
	Control	Se	Vitamin C	PD	PD + Se	PD + Vitamin C
Follicular diameter	9.47±1.79	9.24±1.47@	9.13±1.55@	4.35±0.72#	8.32±1.49#,@,$	8.51±1.53#,@,$	<0.001*
Epithelial cell height	1.62±0.42	1.76±0.34@	1.64±0.38@	4.21±0.45#	2.16±0.31#,@,$	2.08±0.28#,@,$	<0.001*

*Statistically significant at P<0.05, ^#Statistically significant compared to control group, ^@Statistically significant compared to PD group, ^$No statistically significant difference between PD + Se and PD + Vitamin C groups. Kruskal–Wallis test. Post hoc pairwise comparisons used adjusted Bonferroni approach. PD: Potassium dichromate

Both Groups II and III: Selenium and ascorbic acid groups, respectively, were similar to the control group as seen with different stains (H and E, Masson’s trichrome, PAS, and PCNA immune stain) [Tables 1–3 and Graphs 1–4].

Graph 1.

Mean CA% of green-colored collagen in Masson’s trichrome-stained sections among different experimental groups. CA: Color area, PD: Potassium dichromate

Graph 4.

The mean number of PCNA immunopositive nuclei in immunostained sections among the different experimental groups. PD: Potassium dichromate, PCNA: Proliferating cell nuclear antigen

Group IV: PD group showed a loss of architecture of the thyroid gland in 71.42% of animals. The capsule enclosing the thyroid follicles was thickened. Widening of the interfollicular connective tissue spaces [Figure 1b] and dilated, congested blood vessels [Figure 2b] were noticed in 85.71% of the animals [Table 1].

Most of the thyroid follicles appeared shrunken and irregular. In this group, 16.21% of the counted follicles had disrupted basal laminae. The follicular epithelium was disorganized and, in some areas, detached from the basement membrane and sloughed into the lumen in 15.52% of the examined follicles. Among the examined follicles, 40.6% showed hypertrophy, as their epithelia were ranging from high cubical cells with basally shifted nuclei to tall columnar cells with oval nuclei. In 20.18% of the examined thyroid follicles, there was stratification (hyperplasia) of the lining epithelium. Colloid retraction was seen in 18.42% of the follicles. In 28.38% of the examined follicles, colloid was vacuolated [Table 2 and Figure 2b, c].

The mean follicular diameter in this group was 4.35 ± 0.72 μm. The mean height of follicular epithelial cells was 4.21 ± 0.45 μm [Table 3].

Group V: PD + Se group showed improvement evidence of the thyroid follicles. All animals’ sections showed almost preserved architecture of the thyroid gland. The capsule appeared thin compared to the PD group. Most of the animals showed narrow interfollicular connective tissue spaces between the follicles, except for 28.57% of the animals which still showed widening of the interfollicular connective tissue spaces with dilatation and congestion of blood vessels [Figures 1c, 2d, e and Table 1].

Most of the follicles appeared regular and large, filled with colloid, with cubical follicular cells resting on a regular and well-defined basal lamina, and only 3.05% showed disruption of their basal laminae. Follicles were lined with follicular epithelium almost identical to that of the control group. Only 12.91% of the examined follicles showed hypertrophy of the follicular cells. Follicles with stratified epithelium represented 7.67% of the examined follicles. Very few follicles (4.19%) showed sloughing of follicular cells into their lumina. Colloid filled the follicles with vacuolation in 12.85% of the examined follicles. Only 4.13% of the follicles showed colloid retraction [Figure 2d, e and Table 2].

The mean follicular diameter in this group was 8.32 ± 1.49 μm. The mean height of follicular epithelial cells was 2.16 ± 0.31 μm [Table 3].

Group VI: PD + Vitamin C group showed improvement evidence of the thyroid follicles similar to the improvement observed in the PD + Se group. However, 14.28% of the animals still showed widening of the interfollicular connective tissue spaces with dilatation and congestion of blood vessels [Figures 1d, 2g and Table 1]. Only 12.05% of the examined follicles showed hypertrophy of the follicular cells. Follicles with stratified epithelium represented 6.98% of the examined follicles. Very few follicles (2.38%) showed disruption of their basal laminae and 3.95% showed sloughing of follicular cells into their lumina. Colloid filled the follicles with vacuolation in 12.28% of the examined follicles. Only 3.64% of the follicles showed colloid retraction [Figure 2f, g and Table 2].

The mean follicular diameter in this group was 8.51 ± 1.53 μm. The mean height of follicular epithelial cells was 2.08 ± 0.28 μm [Table 3].

Masson’s trichrome results

Group I: Control group showed normal green-colored stained collagen fibers in the capsule, interfollicular spaces, and walls of blood vessels [Figure 3a and b].

Figure 3.

Photomicrographs of thyroid gland sections from different groups stained with Masson’s trichrome L. column ×100 and R. column ×400. (a and b) (control group) (a) Showing the normal green-colored collagen fibers in the capsule (thick arrow) and interfollicular C.T. (*). (b) Showing increased collagen in the walls of BVs. (c and d) (PD group) showing increased green-colored collagen fibers in the thickened capsule (thick arrow), interfollicular C.T. (*), and walls of BVs. Increased collagen is also shown in blood vessel wall in d. (e and f) (PD + Se group) showing decreased green-colored collagen fibers in the capsule (thick arrow) and interfollicular C.T. (*) and also in the walls of BVs compared to the PD group. (g and h) (PD + Vitamin C group) showing the same findings as in the PD + Se group. BV: Blood vessel, PD: Potassium dichromate

The mean CA% of the green-colored collagen was 6.03 ± 1.56 [Graph 1].

Group IV: PD group showed increased green-colored collagen fibers in the thickened capsule, interfollicular connective tissue spaces, and walls of blood vessels [Figure 3c and d].

The mean CA% of the green-colored collagen in this group was 15.93 ± 1.99 [Graph 1].

Group V: PD + Se group showed decreased green-colored collagen fibers in the capsule, interfollicular connective tissue spaces, and walls of blood vessels compared to the PD group [Figure 3e and f].

The mean CA% of the green-colored collagen was 13.06 ± 1.62 [Graph 1].

Group VI: PD + Vitamin C group showed evidence of improvement in the form of decreased green-colored collagen fibers as in the PD + Se group [Figure 3g and h].

The mean CA% of the green-colored collagen was 12.53 ± 1.24 [Graph 1].

Periodic acid–Schiff results

Group I: Control group showed strong PAS +ve colloid. The basal laminae of the follicular epithelium also showed PAS +ve reaction which appeared as thin magenta lines [Figure 4a].

Figure 4.

Photomicrographs of thyroid gland sections from different groups stained with periodic acid–Schiff (PAS) ×400. (a) (Control group) Showing strong PAS +ve colloid and basal lamina of the follicular epithelium. (b) (Potassium dichromate [PD] group) Showing decreased amount of PAS +ve colloid compared to the control group. The vacuolated areas of colloid are PAS −ve. (c and d) (PD + Se group) and (PD + Vitamin C group) Respectively showing increased PAS +ve colloid with few areas of vacuolations, compared to the PD group

In this group, the mean CA% of the PAS +ve material of the colloid and basal laminae was 19.41 ± 2.15 [Graph 2]. The mean OD of the PAS +ve material was 0.46 ± 0.08 [Graph 3].

Graph 2.

Mean CA% of magenta color in PAS-stained sections among different experimental groups. CA: Color area, PD: Potassium dichromate, PAS: Periodic acid–Schiff

Graph 3.

Mean OD of magenta color in PAS-stained sections among different experimental groups. PD: Potassium dichromate, PAS: Periodic acid–Schiff, OD: Optical density

Group IV: PD group showed a decreased amount of PAS +ve colloid compared to the control group. Vacuolated areas of colloid were PAS −ve. The basal laminae showed PAS +ve reaction similar to the control group [Figure 4b].

The mean CA% of the PAS +ve material of the colloid and basal laminae was 12.18 ± 2.84 [Graph 2]. The mean OD of the PAS +ve material was 0.23 ± 0.02 [Graph 3].

Group V: PD + Se group showed an increased amount of PAS +ve colloid with few areas of vacuolation, compared to the PD group. The basal laminae also showed PAS +ve reaction [Figure 4c].

The mean CA% of the PAS +ve material of the colloid and basal laminae was 17.95 ± 2.57 [Graph 2]. The mean OD of the PAS +ve material was 0.38 ± 0.04 [Graph 3].

Group VI: PD +Vitamin C group showed evidence of improvement in the form of increased PAS +ve structures [Figure 4d] compared to the PD group.

The mean CA% of the PAS +ve material of the colloid and basal laminae was 18.21 ± 2.98 [Graph 2]. The mean OD of the PAS +ve material was 0.39 ± 0.05 [Graph 3].

Proliferating cell nuclear antigen immune staining results

Group I: Control group showed almost negative immune staining of the follicular cell nuclei [Figure 5a].

Figure 5.

Photomicrographs of thyroid gland sections from different groups stained with proliferating cell nuclear antigen (PCNA) immunostaining ×400. (a) (Control group) Shows negative PCNA immunostaining of the nuclei of the follicular cells. (b) Potassium dichromate (PD) group showing strong PCNA immunostaining of almost all nuclei of the follicular cells in the form of brownish color reaction. (c and d) (PD + Se group) and (PD + Vitamin C group) respectively showing decreased number of brown immunopositive nuclei of the follicular cells, compared to the PD group

The mean number of PCNA immune-positive nuclei for this group was 12.28 ± 2.61 [Graph 4].

Group IV: PD group showed many follicular cells with positive nuclear reaction [Figure 5b].

The mean number of PCNA immune-positive nuclei for this group was 57.46 ± 10.62 [Graph 4].

Group V: PD + Se group showed decreased immune reactivity of follicular cell nuclei, compared to the PD group [Figure 5c].

The mean number of PCNA immune-positive nuclei for this group was 32.24 ± 5.04 [Graph 4].

Group VI: PD + Vitamin C group showed evidence of improvement in the form of decreased immune reactivity [Figure 5d].

The mean number of PCNA immune-positive nuclei for this group was 31.76 ± 4.66 [Graph 4].

DISCUSSION

In this study, Selenium (Se) and ascorbic acid (Vitamin C) were used to protect against thyrotoxicity induced by PD which is considered one of the hexavalent chromium Cr (VI) compounds. Both Se and Vitamin C were reported to be essential for optimal thyroid function[4] and have many protective effects against oxidative stress triggered by different toxic heavy metals such as copper, cadmium, and PD.[5]

H- and E-stained thyroid gland sections of the PD group showed significant loss of architecture of the thyroid gland. Thyroid follicles appeared irregular with disrupted basal laminae. A significant reduction in the diameter of the thyroid follicles was seen in this group. Other researchers described similar results,[9] they used PD, in the same dose as that of our study for 2 weeks in adult male albino rats. These results supported other studies’ results[7,8] which reported that short-term treatment of rats with intraperitoneal injection of PD in a dose of 60 μg/kg B. W. caused a reduction in the size of the thyroid follicles and formation of small densely packed thyroid follicles with shrunken colloid. It was assumed that the size of a follicle largely depends on the amount of colloid in its lumen. Consequently, decreased colloid content found in our results is most probably the cause of reduced follicular diameter.[2]

The follicles in this group were found to have scanty and/or vacuolated colloid. It was suggested that one of the immediate reactions of activated thyroid follicular cells is engulfing colloid from the follicular lumen with peripheral colloid vacuolation. The colloid endocytosis rate is higher than its synthesis rate, resulting in progressive depletion of colloid which may explain the reduction, we found in, the amount of colloid in the follicular lumen on using PD.[9]

Some follicles showed sloughing of their follicular epithelium into the lumen. These results are in accordance with other researchers’ results.[2,10] The detachment of the follicular cells from the basement membrane could be resulted from loss of the apical tight junctions and adherens junctions attaching follicular cells to each other, as a result of increased ROS generation. In addition, the induction of oxidative stress caused impairment of cytoskeleton. Both junctional loss and cytoskeleton impairment might have led to the separation and sloughing of the follicular epithelial cells.[8,9]

The thyroid follicles also showed significant areas of follicular cell hyperplasia, as well as follicular cell hypertrophy. This is consistent with the results described in other studies.[2] It was suggested that the follicular hyperplasia could be related to a series of cellular events following PD exposure in the form of; oxidative stress, DNA injury, and alteration of genes that control apoptosis as P53.[8] In addition, most of the follicular cells were hypertrophied, with increased epithelial cell height which agreed with a study[9] that reported increased number and height of follicular cells. Follicular cell hypertrophy and hyperplasia were reported to be a consequence of the thyroid-stimulating hormone (TSH) stimulation in an experimental model of hypothyroidism.[7] The follicular cell hypertrophy and hyperplasia caused by TSH stimulation lead to disorganization of the architecture of the thyroid gland including its follicles.[9]

The widening of the interfollicular connective tissue spaces, dilatation, and congestion of the interfollicular blood vessels, found in this group are in accordance with the results of other investigators[9] who used a similar dose of PD as that of our study. This widening could be resulted from either collapse or necrosis of some follicles leaving such widening spaces.[10] The dilatation and congestion of the interfollicular blood vessels could be attributed to the inflammation associated with PD toxicity. This inflammation may be related to elevated levels of NF-κB, which was proven to be increased with Cr (VI) compounds toxicity.[12] The NF-κB increase was attributed to the oxidative stress that stimulates transcription factors, including NF-κB.[13,14,15]

Masson’s trichrome-stained sections of the PD group showed thickening of the connective tissue capsule, interfollicular connective tissue, and walls of blood vessels. These findings were similar to findings reported by another study.[8] The increase in deposition of collagen fibers could be explained by the enhancement of P38 mitogen-activated protein kinase by ROS, which in turn triggers fibrosis caused by the transforming growth factor-β1 (TGF-b-1).[16,17,18] A previous study[19] attributed the increased collagen deposition to the generation of inflammatory cytokines such as tumor necrosis factor- and interleukin-1 by different inflammatory cells.

PAS-stained sections of the PD group showed a decreased amount of PAS +ve colloid inside the follicular lumen with PAS −ve vacuoles. The mean CA% and mean OD of the PAS +ve material were significantly decreased compared to the control. Similar findings were described by others[9] that attributed the decreased PAS reaction to the engulfment of the colloid from the follicular lumen with peripheral vacuolation.

PCNA immune-stained sections of the PD group showed a significant increase in the number of PCNA immune-positive nuclei compared to the control group. This indicates the increased proliferation of the follicular epithelial cells induced by PD. Similar results were found in a previous study[2] that reported increased PCNA immune reactivity in the thyroid tissues of rats given PD injections and referred follicular cell proliferation to increased TSH stimulation. These results are in concordance with the increased immune reactivity of Ki-67 proliferation marker noticed by other researchers[8] in thyroid glands of PD-treated male adult albino rats.

The overall, histopathological alterations noticed in the LM examination could be attributed to oxidative stress caused by PD through increasing the levels of ROS and free radicals that could destruct cellular proteins, lipids, and genomic DNA, and promote necrosis,[20,21] in addition to its role in decreasing the activities of antioxidants in the thyroid gland such as glutathione and catalase.[22,23] Furthermore, the cytotoxic effects of PD could be attributed to its binding to genomic DNA forming intra-strand crosslinks and adducts, that may prevent DNA replication and/or transcription and trigger a number of signal transduction pathways that initiate the apoptotic process.[24]

H- and E-stained thyroid gland sections of the PD + Se group showed evidence of improvement as the thyroid architecture was preserved with a significant decrease in all histopathological parameters compared to the PD group. Moreover, there were a significant decrease in the follicular epithelial cell height and an increase in the diameter of the thyroid follicles which is consistent with less follicular epithelium loss and colloid resorption. However, most of the improved parameters did not return significantly to the control levels indicating partial protection. Other researchers reported similar results[8,10] who used another form of Se, which is Nano Se, in the same dose and duration as that of our study against Cr (VI)-induced thyroid damage. Another study[25] that used Se in the form of sodium selenite in a dose of 10 μg/kg B. W for 6 weeks against chlorpyrifos-induced thyroid toxicity in adult male albino rats found similar results.

The protective effect of Se may be correlated to its strong antioxidant features. In addition, Se enhances the ability of the cells to resist oxidative damage by promoting the expression of selenoprotein genes. Se has a vital role in antioxidant defense mechanisms and in degrading the free radicals inside the cells. Se could block the apoptosis induced by PD via inhibition of ROS overproduction.[10] Moreover, Se was found to increase expression of free radical scavengers that protect against oxidative stress and also, have strong antioxidant properties.[26,27] Apart from its antioxidant activity, Se can repair DNA adducts caused by PD. It exhibits protection from DNA damage by boosting the pathways and enzymes responsible for DNA repair activities.[28,29]

Masson’s trichrome-stained sections of the PD + Se group showed that the mean CA% of collagen was significantly decreased compared to the PD group but still significantly more than that of the control group. This means decreased deposition of collagen in the capsule, interfollicular spaces, and walls of blood vessels, contrary to the PD group. These results are similar to a previous study[8] that reported decreased collagen deposition on using Nano Se against PD-induced thyroid toxicity in adult male albino rats. It also agreed with other researchers[30] who reported that Se supplementation protected the thyroid gland from TGF-b-1-induced fibrosis.

PAS-stained sections of the PD + Se group showed that the follicles of the thyroid were filled with PAS +ve colloid with few PAS −ve vacuoles. These results were explained by a previous study which reported that Se causes restriction of colloid resorption and this leads to an increased amount of PAS +ve colloid.[25]

PCNA immune-stained sections of the PD + Se group showed weak PCNA immune reactivity confirmed by a decreased number of PCNA immune-positive nuclei as compared to the PD group. This could be due to the antioxidant effect of Se and blockage of the PD-induced cellular proliferation. These results are in concordance with the decreased Ki-67 immunoreactivity noticed by other researchers[8] in the thyroid glands of adult male albino rats on using Nano Se against PD-induced thyroid toxicity.

H- and E-stained thyroid gland sections of the PD + Vitamin C group revealed the same evidence of improvement as that of the PD + Se group. The ameliorative effect of Vitamin C on histopathological changes in rat thyroid glands caused by PD was described by previous investigators[6,7,9] who used a similar dose of Vitamin C like that of our study (120 mg/kg B. W.) in adult male albino rats.

The ability of Vitamin C to ameliorate the histopathological changes induced by PD could be attributed to its antioxidant features and hydroxyl radical-scavenging activity. It also participates in the synthesis of glutathione, which serves as an effective cellular antioxidant. Vitamin C was found to inhibit enzymes that catalyze the peroxidation of endogenous lipids.[31]

Besides, Vitamin C was reported to increase the antioxidant capacity of tissues by increasing the activity of several antioxidant enzymes.[3,32]

Masson’s trichrome-stained sections of the PD + Vitamin C group showed the same improvement noticed in the PD + Se group. The amelioration of collagen deposition by Vitamin C was also noticed by another investigator that used Vitamin C in a dose of 250 mg/kg/day in combination with Vitamin E and Se orally for 8 days against renal toxicity caused by cadmium in adult male Sprague–Dawley rats. Blocking of TGF-b-1 expression by vit. C suppresses the production of collagen which consequently reduces the fibrosis.[33]

PAS-stained sections of the PD + Vitamin C group showed the same findings as that of the PD + Se group. This was confirmed by previous studies that used Vitamin C in the same dose as that of our study against thyroid toxicity induced by PD in adult male albino rats.[7,9]

PCNA immunostained sections of the PD + Vitamin C group showed the same improvement as that shown in PD + Se group. This was similarly reported in a study that used Vitamin C supplementation and proved its significant impact on minimizing the proliferating cell number and its protection against Monosodium glutamate-induced thyroid toxicity in adult male albino rats.[34]

All assessed histopathological changes showed partial improvement in the PD + Vitamin C group and the PD + Se group compared to the PD group, with no statistically significant difference between the two groups. This could be because both Se and Vitamin C share some common properties, as they both exert antioxidant effects via different mechanisms.

CONCLUSION

Selenium and ascorbic acid had nearly similar partial protective effect on the thyroid gland against PD toxicity in adult male albino rats.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

This study was partially funded by the Faculty of Medicine, Suez Canal University, and the rest of the funding required was paid by the authors.