ABSTRACT
Bisphenol A [2,2-bis(4hydroxyphenyl) propane, BPA], one of the endocrine disruptors, is of great concern due to its widespread use throughout the world. Several studies have shown that BPA has toxic effects when ingested, making contact with this substance a risk factor for the development of diseases in various organs, including the intestine and associated lymphoid tissue. Therefore, the objective of this work was to study the morphology of Peyer’s patches in young rats supplemented with selenium and exposed to BPA. The study was approved by the UFPI Animal Use Ethics Committee, with protocol number 583/19. 28 male Wistar rats (Rattus novergicus albinus ) were randomly divided into 4 groups: Control (CT), BPA, Se and BPA+Se. The pups were weaned on the 21st day and, from the 22nd postnatal day, the animals in the BPA group received daily doses of 5 mg/kg of BPA diluted in 0.3 ml of corn oil, administered orally. The Se group received 10 μg/kg of Se, the BPA+Se group received 5 mg/kg of BPA and 10 μg/kg of Se and the CT group did not receive any substance but was subjected to the oral gavage process. After 4 weeks of exposure, the puppies were anesthetized and euthanized for intestinal collection and subsequent histological, immunohistochemical and morphometric analysis. Our results showed that BPA caused damage to the epithelial layer of Peyer’s patches, there was a structural disarray in the architecture of the follicular region of the BPA and BPA+Se groups, zones of inflammation with the presence of vacuoles in the tissue. A reduction in Ki-67 expression in Peyer’s patches was also observed in the BPA group, as well as a significant reduction in the number of defense cells in the group. Animals exposed to BPA, but supplemented with Se, showed no damage to the epithelial layer and there was no reduction in Ki-67 expression. BPA, when ingested above the safe level, altered Peyer’s patches tissue, and reduced cell proliferation. Additionally, BPA reduced the immune cell population in Peyer’s patches. Se was able to reverse the observed damage, suggesting antioxidant potential.
Keywords: Gastrointestinal tract, Immune system, Intestinal mucosa, Bisphenol A.
1. INTRODUCTION
The gastrointestinal tract is made up of several cell types such as epithelial cells, goblet cells, and immune cells that play a significant role in gut homeostasis. Since the gastrointestinal tract is exposed to a significant number of pathogens, structures such as Peyer’s patches (PP´s) act in the identification of antigens and in the production of antibodies (Okumura; Takeda, 2017; Zhang; Wu, 2020; Reboldi; Cyster, 2016; Panneerselvam; Budh, 2020).
Chemical compounds such as endocrine disruptors are substances capable of interfering with the synthesis, secretion, and metabolism of hormones. In addition, these chemicals can modulate the body’s immune response. Among them, Bisphenol A (BPA) stands out, known to bind to estrogen receptors and thus interfere with their actions (MALOY; POWRIE, 2011; VANDENBERG et al., 2012; Ratajczak-Wrona et al., 2020; MILANO et al., 2022).
Several routes of exposure to BPA have been described, vertical transmission (maternal-fetal), the respiratory system (inhalation) and the integumentary system (skin contact). However, the food route is the main form of contamination of this compound. (Almeida et al., 2018; López-Rodríguez et al., 2021; Faheem et al., 2021; Jang et al., 2022). The European Food Safety Authority has carried out a reassessment of the risks to public health related to the presence of BPA in foodstuffs. This organization established a reduction of the tolerable daily intake (TDI) of BPA from 4 μg/kg of body weight per day to 0.2 ng/kg of body weight per day (EFSA, 2023).
Selenium (Se) has become an element of great interest in view of its antioxidant effect. Critical functions of selenoenzymes include participation in the regulation of thyroid hormone synthesis, increased male fertility, and anti-inflammatory effects. Among the 25 selenoproteins identified in humans, GPxs and TrxRs are well-recognized antioxidant enzymes (Hariharan; Dharmaraj, 2020; Minich et al., 2022; Labunskyy; Hatfield; Gladyshev, 2014).
Previous studies have shown that using selenium alone ameliorated BPA toxicity in the liver, testicles, and lungs (Abedelhaffez et al. 2017; Amraoui et al., 2018; Khalaf et al., 2019; Ahmed Zaki et al., 2021; Bashir et al. 2022). Kaur et al. (2021) found that Se supplementation considerably restored the activities of antioxidant enzymes and reduced the expressions of stress-activated kinases, which further decreased apoptosis. Thus, Se supplementation has been shown to be effective against testicular damage caused by BPA.
In this sense, Se supplementation can reduce the risk of damage to various organs, such as the intestine, heart, lungs, and kidneys, as well as determine a protective effect against toxicity by substances such as BPA, which has already been demonstrated in some studies (Wetherill et al., 2007; Wang et al., 2015; Al-Amoudi, 2018). Thus, the objective of this study was to study the morphology of Peyer’s plaques in young rats supplemented with selenium exposed to BPA.
2. MATERIALS AND METHODS
2.1 Ethical aspects
The research design is experimental. All the procedures described were approved by the Ethics Committee on the Use of Animals (CEUA), of the Federal University of Piauí, with protocol number 583/19. The procedures carried out in this study are in accordance with the ethical guidelines for animal experimentation recommended in the ”European Communities Council Directive” of November 24, 1986 (86/609/EEC) on the recommendations for the care and use of laboratory animals.
2.2 Animals and Application of Bisphenol A
Twenty-eight young male Wistar rats (Rattus novergicus albinus ) (n=7/group studied) were used, obtained from the Vivarium of the Center for Agrarian Sciences (CCA) of the Federal University of Piauí (UFPI). The animals were fed feed and water ad libitum.
The animals were kept with their sows until the 21st postnatal day in the Vivarium of the Department of Biophysics and Physiology of the Health Sciences Center (CCS) and randomly divided into four groups: Control Group (TC); BPA Group, BPA+Se Group and Se Group.
From the 22nd postnatal day, the male pups in the BPA group (n=7) received daily doses of 5 mg/kg of BPA (a dose already used by the research group, Chem Service Inc., West Chester, PA) diluted in 0.3 ml of corn oil administered orally with gavage. ® The BPA+Se group (n=7) received daily doses of 5 mg/kg of BPA + 10 μg/kg of Se dissolved in water (used as sodium selenite - Na2SeO3 - Dynamic).® The Se group (n=7) received 10 μg/kg of Se dissolved in water (used in the form of sodium selenite - Na2SeO3 - Dynamic)® and the TC group (n=7) did not receive any substance, but underwent the oral gavage process (Al-Amoudi, 2018).
After 4 weeks of exposure and supplementation, the animals were taken to the Morphological Sciences Research Laboratory (LABCIM-UFPI), where the euthanasia protocol was performed in which they received a dose of analgesic (Tramadol 4mg/kg - intraperitoneally) and after 5 minutes they were anesthetized with an anesthetic combination of xylazine (10 mg/kg) and ketamine (80 mg/kg) administered by deep intramuscular route.
Then, the animals were euthanized using an anesthetic overdose, and then an incision was made in the abdominal region of the animal, exposing the intestinal loops to remove the material to be studied. The tissues were stored in 10% buffered formalin solution.
2.3 Histological Analysis
Samples of small intestine from the control (n = 7), BPA (n =7), BPA+Se (n = 7) and Se (n = 7) groups were immersed in 10% paraformaldehyde overnight. Then, the samples were treated with different concentrations of alcohol and xylene and were embedded in paraffin (Sigma). Samples from all groups were cut into 5 μm sections and stained with hematoxylin-eosin (H & E), as well as Masson’s trichrome.
2.4 Immunohistochemistry
Immunostaining was performed with anti-Bcl-2 antibody (CONFIRM anti-bcl-2 (124) - Mouse Monoclonal Primary Antibady, Ventana, Roche, United States) and anti-Ki-67 (CONFIRM anti-Ki-67 (30-9) - Rabbit Monoclonal Primary Antibody, Ventana, Roche®®, United States) expressed in the cytoplasm and nucleus, respectively.
The tissues were fixed, embedded in paraffin, and cut to approximately 4 μm thickness. They were then placed on positively charged TOMMO (VWR®) slides for IHC analysis. The slides containing the samples of interest were deparaffinized in a xylol bath, hydrated in ethyl alcohol, then subjected to automated reaction in the BENCHMARK IHC/ISH equipment (Ventana, Roche®, United States).
The ultraView DAB Detection Kit was used, the antibodies evaluated, the reagents, the contrast agents, and the contrast agents (Hematoxilin II and Bluing Reagent), as provided by the manufacturer.
2.5 Morphometric Analysis
The number of leukocytes was counted by examining all assembly preparations under a light optical microscope with a magnification of 1000 ×. All well-defined cells in each fragment were counted. For quantification, counts were made in five microscopic fields alternating for each animal in the region of the germinal center of the PP´s.
2.6 Image Analysis
The photos of the tissues were obtained using a light microscope and the Xiaomi 11 mobile phone camera, Mi 11 Lite 5G version, (9216x6912). The boards were made using GIMP software version 2.10.34.
2.7 Statistical analysis
The data were statistically compared by analysis of variance (ANOVA), followed by Tukey’s test, with a significance level of p < 0.05. The analysis was performed using GraphPad Software, San Diego, CA, USA.
3. RESULTS
PP´s are in the mucous layer of the small intestine. Macroscopically, there was no qualitative morphological difference between the groups. Regarding the number of follicles per plaque, it was observed that it was variable in all groups, with the presence of two to three in each of them being more frequent (Figure 1).