Prophylactic effect of selenium against buspirone induced gestational pancreatic damage in fetuses

Introduction
Pregnancy has an effect on psychological state of pregnant women, with an increase in anxiety and discomfort[1]. Psychotropic drugs prescription is limited during pregnancy due to the potential risks risks on offspring’s. However, stopping medication exhibits new dangers [2].

Buspirone is a strong anti-anxiety drug. It works on the serotonin 5-HT1A receptors. Clinically, buspirone is not accompanied by adverse effects of sedation, addiction, or cognitive impairment [3]. 

Selenium has antioxidant roles. The thioredoxin reductase group of selenium has numerous biochemical reactions such as thiol redox regulation, and DNA synthesis [4].

In this study, we aim to investigate the effect of Buspirone administration during pregnancy on the pancreas of  fetuses and the possible protective influence of selenium administration along with the drug

Material and Method
Buspar as pills From Beecham pharmaceutical, Cairo, Egypt. It was given orally by the nasogastric tube as a single oral doses daily 4.1 mg /kg/day[5], to the buspirone group. Selenium (Sigma-Aldrich, Cairo, Egypt) was co-admini-stered with buspar we orally by nasogastric tube at a dose of 0.3mg/kg/day[6].

Animals and treatments:
The Ethical Committee  of  Minia University (Approval no. 691:11/2020) gave its consent to the experiment.   Fifty-four adult female albino rats and 27 adult male albino rats we brought them from the Minia University, Laboratory Animals Unit, and were maintained in appropriate to be mated. Their weights were between 200-250g. The animals were divided into three groups each 18 female albino rats with 9 male albino rats. We searched for the plug in the vagina every day. The first day of pregnancy considered when vaginal plug was seen. Pregnant animals were treated from 6th day until 20th day and assigned into three groups: 18th  pregnant rats in each group 
Group I (control): received refined water equivalent to the dose of the drug given to other groups.
Group II (Buspar): Buspirone hydrochloride 4.1 mg/kg/day was given orally by nasogastric tube[5].
Group III (Buspar-Se): Buspar along with selenium were given, selenium was given orally by nasogastric tube at a dosage of 0.4 mg/kg/day[6].

Histological Study: At the end of the experiment, the pregnant rats were anesthetized, then the fetuses were obtained and fetal pancreases were harvested and, and cut apart to obtaine the pancreas which immersed in ice-cold 0.15 ml NaCl and dried by filter paper, then it was preserved in formalin, a buffered isotonic solution of 37% formaldehyde. Pancreases were then dried out by ascending concentration of alcohol, then washed with xylol. Samples were processed for paraffin embedding. Paraffin blocks were sectioned and stained by hematoxylin and eosin for histopatho-logical analysis.
Immunohistochemically study (caspase-3): In brief, paraffin-immersed fetal pancreas was treated with 0.3% hydrogen peroxide in Phosphate-Buffered Saline (PBS) for 30 minutes to prevent the endogenous peroxidase. In the primary anti-rat antibody against caspase (cysteine-aspartic proteases) we put the parts of pancreas. The tissues were then bathed three times in PBS before being kept for 1 hour at 20 to 25°C with goat anti-rat peroxidase-combined secondary antibody. The immune reaction was detected using chromogen 3,3′diaminobenzidine hydrogen peroxide, then parts were stained with hematoxylin.
Method for morphometric analysis :the photos of slides enter into imag j programme to estimate the discolored area and its density.

Results
 Pancreatic tissues stained with in Hematoxylin and Eosin (H&E): 
Group I (Control group): fetal pancreases showed   normal architecture of pancreatic acini and duct system. Acinus cells were seen cuboidal in shape and contained eosinophilic cytoplasm and basal active basophilic nucleus. (Fig. 1-A)
Group II (Buspar group): The fetal pancreases showed less developed pancreatic tissue fetuses. Many of acini exhibited failure of development, deep stained basophilic nucleus in addition to necrotic changes in numerous cells. (Fig. 1-B)
Group III (Buspar – selenium group): Pancreas showed a a variable degree of histopathological changes, some normal pancreatic tissues with normal acini and nucleus. There was some acini revealed failure of the development, and a deep basophilic nucleus. Occasionally degenerative and necrosis in some cells. (Fig. 1-C)

Discussion  
Anxiety and stress during pregnancy are linked with spontaneous abortion, preterm delivery, fetal malformation, and delivery difficulties[2]. Buspirone is an effective anxiolytic medication [3]. It is classified as group B in pregnancy, which means that it is classified as safe to use in pregnancy [11]. In the research of Zhao, X., et al., study [12], declared that strict selenium insufficiency, caused atrophy of the pancreas and poor growth. 

The development of exocrine part of the fetus of rats is noticed to be functioned by Direnzo, D., et al.,[13], at almost the age of 19th day of intrauterine life in the form of elaborated zymogens granules.

In our study, we conducted to assess the effect the usage of Buspar while women is pregnant on the fetus pancreas and the shielding effect of selenium to improve the damage produced by buspirone.

In the Hematoxylin and Eosin staining, the findings were the same as El-Shaer et al.,[14] in the study of control group, it exhibited normal structure of acini and ducts with active nucleus and in the 

Buspar group, many acini exhibited failure 
of formation of pancreatic tissues, uniform strong eosinophilic cytoplasmic ingre-dients, as well as necrosis of many cells. The similar damage of the pancreas was also found in Zhao et al., study [12], in the form of pancreatic deterioration due to lack of selenium.

Buspar had been noticed to influence other organs. In the kidneys, Buspar displayed degeneration in the kidney tissues in the form of destruction of proximal and distal convoluted tubules[15]. Also, in the cerebellum, there were obvious deterio-ration in the cerebellar tissues in the form of deformity in the granular and Purkinje cells layer[5].

Depletion of carbohydrates in the cells of the fetus organs due to Buspar was documented in El-Shaer et al., study, in the pancreatic cells compared  to control group agreed with same findings we found[14], in the cells of kidneys[15], and in the cerebeller cells[5]. The partial restoration of the normal amounts of polysaccharides stained by PAS in the buspirone-selenium group was decum-ented that the selenium regulated carbohydrate enzymes activities agreed with the same findings we found in third group[16].

In the immunohistochemical study, we found observable reaction of caspase-3 in the control group which was unlike the El-Shaer et al., finding[14], who stated that sections were negatively stained with caspase-3. Existence of little proportion of programmed cell death in the living tissues was recorded by Jakubowska et al., agreed with our findings[17], also documented that in the control group existed a low percentage of active caspase-3 agreed with our finding[10]. Programmed cell death in control pancreatic tissue ranges from 3% to 7%,[18]. Apoptotic level elevates in drugs that induces destruction in the pancreas agreed with our finding [9]. Some of the malformations caused by the buspirone in the fetus of the albino rats were documented in the form of some fetuses were born died, and decrease in the weight of fetuses[19].