The possible ameliorative role of Lycopene on Tributyltin induced thyroid damage in adult male albino rats (histological, immunohistochemical and biochemical study)

ABSTRACT Tributyltin is used in industrial applications. This current research aimed to study the effect of Tributyltin on the thyroid gland structure and function of adult male albino rats and the protective effect of Lycopene. Twenty-one male adult albino rats were classified into three groups: Control, treated that received Tributyltin, and protective that received Lycopene with Tributyltin. At the end of the experiment, blood samples were collected and T4, T3, and (TSH) were measured. Tissue superoxide dismutase (SOD) and malondialdehyde (MDA) were estimated. Thyroid gland specimens were processed for histological and immunohistochemical examination. Then morphometric and statistical analyses were done. The treated group showed affection in thyroid function and histological structure as vacuolated colloid and cytoplasm and dark nuclei. Ultrastructurally, follicular cells showed irregular shrunken nuclei, atrophied apical microvilli, vacuoles, multiple lysosomal granules, mitochondria with destructed cristae, and extensively dilated rough endoplasmic reticulum. There was increase in Caspase-3 immunoexpression and decrease in Beclin-1 immunoexpression. The thyroid structure and biochemical markers improved after Lycopene administration. The thyroid gland damage caused by Tributyltin is ameliorated by Lycopene.


Introduction
Tributyltin (TBT) is family member of organotins. It is identified as an endocrine disruptor. TBT can alter how hormones are produced, regulated, used or eliminated, as well as block hormonal activity. 1 Many researches studied the toxicity of organotin chemicals in mammals., TBT has been considered as immunotoxic and can cause apoptosis and thymic atrophy. It has been also recognized as a reproductive and developmental poison. 2 Exposure to organotin compounds has also been linked to a number of metabolic dysfunctions including early puberty, vascular and neuropathological alterations, and a rise in body weight. The organotin compounds are classified as obesogens, which produce metabolic anomalies leading to obesity. 3 The main route of organotin exposure of humans, especially TBT, is dietary through contaminated fish and shellfish ingestion. They have been applied as biocides in stainless steel production and food storage (in cans). 4 TBT has also been used in marine boat and fishing net antifouling coatings. It slowly leaks from the paint into the water and is a lipophilic chemical that tends to accumulate in shellfish. It is frequently used in boat paints, fungicides, wood preservatives, and other fields of industry. 5 The demand for alternative, safe and natural sources of dietary antioxidants has dramatically increased during the last few years. Natural antioxidants of plant origin stand out for being easily extracted from food products and underutilized plants, as well as for having biological value and economic advantages. Lycopene (LYC) is an unsaturated lipophilic carotenoid lycopene present in red fruits such as watermelon, papaya, pink guava, tomato, carrots, and grapefruit. 6 Lycopene is a potent antioxidant belonging to the carotenoid family. It may protect or reduce cellular oxidative damage because it fights biological reactive oxygen species. Tomatoes and tomato-related items provide the human diet with Lycopene and a considerable amount of carotenoids. 7 In addition to being an antioxidant, Lycopene possesses biological features including protecting DNA and regulating gap junctions in between cells. Lycopene has been shown to lower the risk of some chronic diseases as cardiovascular disease, liver dysfunction, and gastropathy. It has also been shown to decrease tumor cell hyperplasia and cellular DNA damage. 8 Thyroid hormones are essential for proper function of all organs because of their role in normal adult physiology including healthy metabolism regulation, brain development, and other aspects. Therefore, undesirable consequences may have an influence on metabolism or development if the thyroid gland's function changes or the effectiveness of thyroid hormone is impaired. 9 The exchange of the molecules with the neighboring cells or the extracellular environment occurs through channels in the plasma membrane by transmembrane proteins called Connexins. For cells to survive, they must be able to communicate with one another and exchange physiologically active chemicals, ions, and nutrients with the external environment. Abnormal transmembrane protein expression (up-or down-regulation) is frequently linked to malignant phenotypes. A sizable amount of cellular communication is mediated by gap junction signaling, in which cells distribute and exchange endogenous nucleic acids, peptides, small molecules, and ions across gap junctions. As a result, abnormal gap junction expression may change cellular metabolism. 10,11 Other than estrogenicity and obesity, there are many investigations to detect endocrine disruption caused by organotin compounds. Thyroid gland and hormones are significant objectives of organotin chemical activities. 12 The aim of our study is to detect if TBT is hazardous to the thyroid gland and also to determine whether Lycopene protects against thyroid gland affection.

Animals
At the Animal House, Faculty of Medicine, Zagazig University, twenty-one healthy male adult albino rats with typical body weights (200-230 g) were employed and housed in cages made of stainless steel. They received a typical, well-balanced diet and unrestricted access to water while being kept at room temperature. This investigation was carried out according to the Guide for Laboratory Animals use and care. The experimental procedure received permission from the Zagazig University Ethical Committee Experimental design.

Experimental design
Before the experiment began, the rats spent a week getting used to their new environment. The experiment was conducted for one month.
These rats were divided into 3 experimental groups (7 animals each): Group I (Control group): Included 7 rats, subdivided into three subgroups: Subgroup Ia (2 rats): Each rat received 3 ml of distilled water orally through orogastric tube.
Subgroup Ib (2 rats): Each rat received 0.4 ml of corn oil orally through orogastric tube.
Subgroup Ic (3 rats): Each rat received Lycopene dissolved in corn oil as a vehicle orally by gastric tube. The dose of Lycopene was 10 mg/kg body weight. 13 Group II (Tributyltin-treated group): Included 7 rats, each rat received Tributyltin chloride dissolved in corn oil in a dose of 5 mg/kg for 30 days. 14 Group III (Protective group: Tributyltin and Lycopene group): Included 7 rats that received Tributyltin chloride dissolved in corn oil in a dose of 5 mg/kg with concomitant administration of Lycopene at a dose of 10 mg/kg for 30 days. 13

General observations of animals
Clinical indications and overall appearances, including the quantity of consumed water and food were monitored every day during trial period. Rat deaths were kept track of as they happened. Rats exposed to TBT may experience changes in their overall body composition. 15 Rats were given sodium pentobarbital (Nembutal, 30 mg/kg body weight) through injection intraperitoneally to make them unconscious before being sacrificed. The retro-orbital sinus was used to collect venous blood samples (2 ml each) for hormonal analysis.
To prevent tissue damage, removal of thyroid glands occurred in two stages of dissection. The trachea was severed by a horizontal plane superior and inferior to the thyroid after opening the neck by a longitudinal incision. Removal of fascia occurred. On either side of the trachea, it was visible as two little reddish oval masses. 16 Each rat's thyroid gland was rapidly removed to get specimens.

Light microscopic study
One thyroid gland's lobe was fixed in 10% of neutral buffered formalin, dehydrated, as well as embedded in paraffin. Then the 5-μm-thickness paraffin sections were cut and stained with hematoxylin and eosin (H&E) stains. Also, the Periodic Acid Schiff (PAS) and Masson's trichrome staining procedures were used according to 17 .

Immunohistochemical study
Caspase-3 and Beclin-1 were detected immunohistochemically using the streptavidin-biotin complex immunoperoxidase technique. On charged slides, serial sections of specimens with paraffin embedding were deparaffinized. To block endogenous peroxidase, the sections were incubated in 0.1% hydrogen peroxide for 30 min. The sections were incubated for 30 minutes at room temperature with 1.5% nonimmunized goat serum and diluted primary antibodies for rabbit polyclonal Beclin-1 antibody (ab62557) and rabbit polyclonal active Caspase-3 antibody (ab13847). 18 Following three rounds of washing with PBS (pH 7.4) for 30 minutes, the sections were incubated for 60 minutes with goat anti-mouse immunoglobulin serum that had been biotinylated. The sections were treated with avidin-biotin peroxidase complex after being gently rinsed with PBS. Ultimately, using the DAB (3,30-diaminobenzidine) substrate, peroxidase binding sites were found. 19 Then, after being counterstained with hematoxylin, tissue sections were examined by light microscope and given morphometric measurements. The kits were bought from St. Louis-based Sigma-Aldrich.

Electron microscopic study
Thyroid specimens of three rats from each group were processed for EM examination. Specimens were fixed in 2.5% glutaraldehyde buffered with 0.1 M phosphate buffer at pH 7.4, then post-fixed in 1% osmium tetroxide in the same buffer, before being examined under an electron microscope. After dehydrating them, epoxy resin was used to embed them. Uranyl acetate and lead citrate were used to double-stain ultrathin sections. 20 The Electron Microscope unit of Agriculture Faculty at University of El Mansoura used a JEOL JEM 2100 electron microscope to examine and take photographs of the stained sections.

Antioxidant activity and lipid peroxidation level
According to instructions of manufacturers, spectrophotometric measurements of antioxidant enzyme Superoxide dismutase (SOD) activity was made in the thyroid homogenate using (Biodiagnostic kit) (CAT.NO. SD 25 21) and Lipid peroxidation process indicator, which is Malondialdehyde (MDA), was estimated using (Biodiagnostic kit) (CAT.NO. MD 25 29). 21,22

Hormonal analysis
Following the manufacturer's recommendations, serum samples were produced and stored at 20°C for measurement of T3, T4, and TSH, which were quantified using ELISA test kits (MyBiosource kits) with CAT. NO. MBS2000350, MBS580037, and MBS729687 respectively.

Gene expression analyses
Thyroid tissues were homogenized and total RNA was extracted from tissue homogenate using (RNeasy Mini Kit, Qiagen), following the manufacturer's instructions. Total RNA purity was determined through the absorption ratio (260/280 nm). It was between 1.8 and 2.0 for all preparations. cDNA was reverse transcribed using the QuantiTect Reverse Transcription Kit as recommended by the manufacturer. The gene expression analysis was evaluated by qRT-PCR using 5 uL of the cDNA, 10 uL of SYBR Green 2× Master Mix Green (QuantiTect SYBR Green PCR Kits,Qiagen) and 10 pmol/uL of each primer. The primers of BAX, BCL-2, and Connexin43 are listed in Table 1. RT-qPCR was performed by M×3005P (Stratagene, CA, USA). Thermal cycling was performed as the following conditions: first denatured at 94°C for 5 min, 40 cycles of 95•C for 15 sec; annealing at 60•C for 1 minute and elongation at 72•C for 30 sec, and extension for 10 min in the last cycle. The data were normalized against GAPDH transcript level and 2-ΔΔCt method was used to calculate the relative expression. 23

Morphometric study
The image analyzer computer system Leica Qwin 500 (Cambridge, UK, Leica Microsystems Imaging Solution, Ltd) is used in pathology department's image analysis lab at Dentistry Faculty, Cairo University.
•Mallory's trichrome sections for detection of area percentage of collagen fibers.
•Periodic acid Schiff (PAS)-stained sections for detection of the area percentage of colloid in thyroid follicles.
•Immune-stained sections were used for the detection of percentage of positive cells for Caspase-3 and Beclin-1. This was performed in 5 nonoverlapping fields of 5 different sections of 5 rats in every group at × 400.

Statistical analysis
The biochemical and morphometric measurements were calculated as mean ± standard deviation (SD) and were analyzed by one-way analysis of variance ANOVA, then post hoc test. Results were estimated as significant when P value was<0.05. SPSS software version 16 (SPSS Inc., Chicago, Illinois, USA) was used. 24

General observation
There were no rat deaths during the experiment.

Histological results
Data obtained from all subgroups (Ia, Ib,& Ic) of control group regarding light and electron microscopic results were nearly similar, so, results of subgroup (Ia) were selected to be compared by other groups.

Light microscope results
H&E stained sections of the thyroid gland of the control group showed thyroid follicles lined by cuboidal follicular cells with central rounded nuclei. There was acidophilic and homogenous colloid filling in the luminae of the follicles. The parafollicular cells were small oval with dark stained nuclei and situated between the follicular cells on the basement membrane ( Figure 1a). The TBT-treated group showed some follicles lined by cuboidal cells with rounded nuclei and others lined by flat cells with flat nuclei. Numerous follicular cells showed vacuolated cytoplasm. In some follicles, multiple layers of follicular cells were seen on one side. There were large interstitial spaces between some follicles. Some follicles were dilated while others were involuted. There was a large congested blood vessel. There were numerous darkly stained nuclei. The capsule was noted. Some follicles had no colloid (Figure 1b). The protective (TBT-Lycopene) group showed nearly normal thyroid architecture. Thyroid follicles were of variable sizes. Almost all the follicles were     group (Figure 5b), and brownish reaction in cytoplasm of few follicular cells of protective group, which increased compared to the treated group. (Figure 5c).

Electron microscope results
Electron micrograph of ultrathin sections in the thyroid gland of control rat showed thyroid follicular cells with apical microvilli, oval euchromatic nucleus, rough endoplasmic reticulum and mitochondria. Follicle was surrounded by thin regular basal lamina and blood capillary. Junctional complexes between adjacent cells appeared intact. Homogenous colloid was noticed ( Figure 6). Tributyltin treated group showed follicular cells with irregular shrunken nuclei and atrophied apical microvilli. The cytoplasm contained vacuoles, multiple lysosomal granules and extensively dilated rough endoplasmic reticulum (Figure 7a,b,c). A congested blood capillary was seen (Figure 7a). There were mitochondria with destructed cristae (Figue 7b,c). The interfollicular space contained fibroblast with heterochromatic nucleus and collagen fibers deposition (Figure 7c). Tributyltin & Lycopene group showed thyroid follicle lined by more than one layer of follicular cells, one cell had rounded euchromatic nucleus and other exhibited irregular nucleus. Follicular cells had apical microvilli border, rough endoplasmic reticulum, electron dense lysosomes and numerous mitochondria. Junctional complexes between adjacent cells, regular basal lamina and homogenous colloid were noticed (Figure 8).

Biochemical results
There was statistically significant increase in MDA level in the Tributyltin-treated group (II) compared to the control group (I), while in the TBT+ Lycopene-treated group (III) there was statistically significant decrease compared to group (II), though it remained statistically higher than the MDA level in group (I). There was statistically significant decrease in SOD level in the Tributyltintreated group (II) compared to the control group (I), while in the TBT+ Lycopene-treated groups there was statistically significant increase compared to group (II), though it remained statistically lower than the SOD level in group (I) ( Table 2).
There was statistically significant increase in TSH level in the Tributyltin-treated group compared to control group, which became reversed in the TBT+ Lycopene-treated group, though it remained statistically higher than the level in the control group. There was statistically significant decrease of T3 and T4 in the Tributyltin-treated group compared to control group, which has been improved in the TBT+ Lycopene-treated group, though it remained statistically lower than the level in control group (Table 3).  There was statistically significant decrease in mRNA expression level of Connexin 43 and BCL-2 in the Tributyltin-treated group compared to control group, although there was significant increase in the TBT+ Lycopene-treated group compared to group (II) but Connexin 43 remained statistically lower than control group. There was statistically significant increase in mRNA expression level of Bax in the Tributyltin-treated group compared to control group although, it was significantly decreased in the TBT+ Lycopene-treated group compared to group (II) but remained statistically higher than the control group (Table 4).

Morphometrical and statistical results
Statistical analysis of collagen fiber area percent showed a highly significant increase in the Tributyltin-treated group (II) compared to control group (I). However, there was a nonsignificant increase in the Tributyltin-Lycopene group (III) compared to control group (I) ( Table 5).
Statistical analysis of colloid area percent showed a highly significant decrease in the Tributyltin-treated group (II) compared to control group (I). However, there was a non-significant decrease in the Tributyltin-Lycopene group (III) compared to control group (I) ( Table 5).
Statistical analysis of percentage of positive cells for Caspase 3 showed a highly significant increase in the Tributyltin-treated group (II) compared to control group (I). However, there was a nonsignificant increase in the Tributyltin-Lycopene group (III) compared to control group (I) ( Table 5).
Statistical analysis of percentage of positive cells for Beclin 1 showed a highly significant decrease in the Tributyltin-treated group (II) compared to control group (I). However, there was a nonsignificant decrease in the Tributyltin-Lycopene group (III) compared to control group (I) ( Table 5).

Discussion
Thyroid gland is an exceptional endocrine gland. 25 There is scanty information about the effect of TBT on thyroid tissues. Hence, our study goal is to detect the effect of Tributyltin on the follicular  cells of thyroid gland and also the effect of Lycopene as a strong antioxidant. According to our biochemical results we have found that there was statistically significant increase in the level of (MDA, TSH) and significant decrease of (SOD, T3, and T4) in the Tributyltin-treated group when compared to the control and TBT+ Lycopene-treated groups. These results are in Table 5. Statistical analysis of morphometric results of thyroid gland specimens of the studied groups.  agreement with Badr El Dine et al., (2017) as they claimed that compared to control and TBT+ green tea-treated groups, there had been a significant decrease in T3 and T4 levels, following 30 days of TBT treatment. 26 The TBT-treated group's serum TSH level significantly increased. Additionally, they show that when compared to the other two groups, the TBT-treated group's serum MDA level increased significantly. Shruti et al. (2014) assumed that following 45 days of Tributyltin treatment, the mean serum T4 content in Swiss albino mice decreased markedly in a dose-dependent manner from 5 to 50 g/kg/day. At the highest dose of 50 g/kg/day, serum T3 decreased markedly. Forty-five days of exposure to TBT Cl also resulted in a dose-dependent rise in serum TSH levels when compared to control, in addition to its effects on T3 and T4 levels. 27 Andrade et al. (1987) stated that among the two groups of rats given TBT and the control group, there were no appreciable variations in total T3 serum levels. The group that received the greatest dose of TBT, however, showed a propensity toward having higher total T3. Additionally, when compared to controls, TBT1000 rats had lower serum levels of total T4 than usual. TBT 200 animals showed a rise in serum TSH levels that was almost 83% higher than that of control rats. Despite the lowered T4 levels, no appreciable alterations in TSH were seen in TBT 1000 rats. 28 All cells in the human body have gap junctions, which are made of connexins, a protein. There are at least 21 connexin isoforms in humans, and they combine to form homomeric channels or heteromeric hemichannels with other connexin isoforms. Connexin 43, named after its molecular weight of 43 kDa, is the most thoroughly researched connexin. 29 With respect to the gene expression results, we have detected a statistically significant decrease in mRNA expression level of (Connexin 43 and BCL-2) and significant increase in mRNA expression level of Bax in the Tributyltin-treated group when compared to the control and TBT+ Lycopene-treated groups. Some researchers have assessed the level of gene expression of Connexin 43 in thyroid gland and evaluated its role in development of thyroid diseases like cancer.

Bonacquisti et al. (2019)
proved that it has been demonstrated that abnormal connexin expression, particularly that of C×43, plays a role in the development and spread of cancer. Connexin functions as a tumor suppressor or an oncogene, depending on the type and stage of the cancer. 30 Dominguez et al. (2011) also suggested that papillary thyroid cancer frequently has abnormal Connexin 43 pattern of expression. These changes are also found in around 50% of benign lesions (multinodular goiter and follicular/oncocytic adenomas) that demonstrate dysfunctional cell proliferation. 31 TBT affects the apoptotic machinery of m NSCs in vitro, leading to apoptotic cell death. Lui et al. (2020) also have proved that the mRNA expression was significantly lower for Bcl-2 in the high dose group of nickel sulfate exposure in thyroid gland, but the test groups did not show changes in mRNA expression of the Bax gene. 32 In comparison with the control groups, Pb (group receiving sublethal dose of lead acetate), ZnO-NPs (zinc oxide nanoparticles), and Pb + ZnO-NPs groups showed significant Bax expression elevation and Bcl-2 expression reduction. Comparing the Pb + ZnO- NPs group with the Pb and ZnO-NPs groups revealed significant Bax expression elevation and Bcl-2 expression reduction in Pb + ZnO-NPs. 33 Examination of TBT treated rats by light microscope revealed that the normal structure of thyroid was greatly affected. Wang et al, (2008) claimed that TBT can cause severe thyroid tissue damage in the form of decrease in the follicular region, depletion of colloid, and abnormal follicles. 34 Organotin compounds can induce cellular damage due to oxidative stress caused by reactive oxygen species (ROS) overproduction. 35 Darkly stained nuclei are signs of apoptosis. Widening of the interstitial spaces between some follicles was observed due to collapse or necrosis of some follicles. 16 Some follicles showed hyperplasia. Chiamolera and Wondisford (2009) explained that reduced thyroid hormone levels stimulate the pituitary, causing the thyrotrophs to generate more TSH, which causes hyperplasia of follicular cells and colloid area shrinkage. 36 Congested blood vessels were explained by Shady and Noor El-den (2010), who stated that lipid peroxidation and oxidative stress cause congestion and dilatation of blood vessels by promoting their wall lesion. Also, hyperemia is a mechanism for removal of the toxin. 37 The protective group (TBT-Lycopene-treated group) showed nearly normal thyroid structure. Cavusoglu et al. (2009) stated that lycopene can decrease cellular oxidative damage. 38 Ultrastructurally, the follicular cells showed irregular shrunken nuclei and vacuoles. These results were in keeping with Sharan et al. (2014), who documented the TBT effect on thyroid gland. They stated that TBT had an adverse thyroid effect. It affected the thyroid hormone receptors transcription through altering the physiological levels of thyroid hormones. 12 Marked dilatation of RER was explained by Dabrowska et al. (2005) who stated that it may be a compensating strategy, since oxidative enzymes were essential for the detoxification of damaged cells and RER responded quickly to the toxin's effect. The presence of aberrant protein within the cisternae caused the RER to enlarge. 39 Nakazawa et al. (2008) explained the presence of few microvilli that poor thyroid hormones synthesis might interfere with colloid transport between follicular cells and the follicular lumen. 40 Multiple lysosomal granules were noticed in the follicular cells. This was attributed to enhanced phagocytic activity of the damaged follicular cells. Also, it was due to increased secretory activity of the cells as a result of high levels of circulating TSH. 41 Mitochondria with destructed cristae were seen as TBT mainly affects the follicular cells through inducing oxidative stress due to reactive oxygen species (ROS) overproduction 35 .
PAS-stained thyroid sections of the TBT-treated group showed weak positive reaction in the follicular cell basement membrane and in the colloid due to hypothyroidism, which increased the need for thyroid hormones stored in the colloid, which caused more colloid to be extracted from storage. 16 Mallory's trichrome sections of the TBT-treated group showed deposition of marked collagen fibers due to lipid peroxidation, which prompts the satellite cells to boost collagen synthesis. 42 Caspase-3-immunostained thyroid sections of the TBT-treated group showed immunoreaction in many thyroid follicular cell nuclei, while TBT-Lycopene-treated group showed diminished immunoreaction in few thyroid follicular cell nuclei. Caspase-3 is the most significant apoptosisregulating protein activated by apoptotic stimuli. Oxidative stress is a major stimulus causing proapoptotic factors release into cytosol then activation of procaspases. 43 Caspase-3 is primarily found in cytoplasm. Caspase-3 is cleaved, activated, then translocated into the nucleus during apoptosis causing apoptotic nuclear changes. 44 Beclin-1-immunostained sections of the TBTtreated group showed immunoreaction in cytoplasm of scanty follicular cells of the treated group, while the TBT-Lycopene-treated group showed immunoreaction in cytoplasm of few follicular cells, which increased compared with the treated group.
A physiological catabolic process called autophagy aims to recycle damaged organelles and cellular parts. In order to preserve cell homeostasis, it protects organisms under cellular stress. Excessive inflammatory cytokines and ROS are considered as negative autophagy regulators. Autophagy is composed of sequestration of cellular cytoplasmic components in double-membrane vesicles called autophagosomes that break down their contents by lysosomal proteolysis to produce energy. 45 Beclin-1 is an autophagy key regulator. It is localized in the cytoplasm and its mutation leads to impaired autophagic activity. It can be cleaved by Caspases-3, 8, and 7 and prevented from inducing autophagy. 46 Under stress, autophagosomes engulf the ROS-producing mitochondria and apoptotic proteins to help cell survival. So, if autophagy is inhibited, ROS production and apoptosis will increase. 47 This can illustrate the results in the TBT-treated group where increased Caspase-3 immunoexpression was accompanied by decreased Beclin-1 immunoexpression. Levels of Beclin-1 were increased after treatment with Lycopene due to modulating autophagy. Lycopene decreased the apoptosis of cells by increasing the autophagy. 48 Recent researches have shown the relation between apoptosis and autophagy, as autophagy comes before apoptosis. Many stimuli induce autophagy and then induce cell death pathways when the autophagic process is dysregulated. 49

Conclusion
Our study provides information about TBT as it results in major thyroid changes ultrastructurally and biochemically. Its use should be seriously restricted. Lycopene might be a preventive agent against TBT toxicity through its potent antioxidant activity.

Disclosure statement
No conflict of interest was reported by the authors.

Funding
According to the author(s), the work of this article is not supported financially.