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).