**1. Introduction**

Oestrogens and oestrogen-like substances found in the natural environment including the mycoestrogen ZEN, affect the developing reproductive and non-reproductive tissues [1,2]. Oestrogens are synthesised by the body, but they are also present in the environment, in the form of xenobiotics and naturally occurring compounds (undesirable substances) [3]. Most of these substances (not necessarily pollutants) are known as endocrine disruptors (EDs) [4], and they are usually found in soil, air, water, food and feed (i.e., the environment) [5,6]. Phytoestrogens (genistein, coumestrol) and the mycoestrogen ZEN (fungal metabolite) are naturally occurring EDs [7–9].

Zearalenone and α-zearalenol (α-ZEL) have an oestrogen-like structure. However, they are not steroids and do not originate from sterane structures [10]. EDs such as zearalenone are involved in several processes [11,12] that influence the endocrine system [13] and induce side effects [14]: (i) in prepubertal gilts, EDs compete with endogenous oestrogens for the binding sites of oestrogen receptors (ERs), which can alter mRNA expression

**Citation:** Gaj˛ecka, M.; Otrocka-Domagała, I.; Brzuzan, P.; D ˛abrowski, M.; Lisieska-Zołnierczyk, ˙ S.; Zielonka, Ł.; Gaj˛ecki, M.T. Immunohistochemical Expression (IE) of Oestrogen Receptors in the Intestines of Prepubertal Gilts Exposed to Zearalenone. *Toxins* **2023**, *15*, 122. https://doi.org/10.3390/ toxins15020122

Received: 21 December 2022 Revised: 29 January 2023 Accepted: 30 January 2023 Published: 2 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

levels and protein synthesis and reduce the efficacy of endogenous steroids [10,15–17]; (ii) EDs can bind to the inactive receptor (i.e., blocking it), thereby preventing the binding of natural hormones to that receptor (antagonistic effect) [11,17]; (iii) EDs reduce the levels of circulating natural hormones because they bind to blood transporting proteins, [2]; and (iv) EDs can also affect the body's metabolism by influencing the rates of synthesis, decomposition, and release of natural hormones [10,18–20].

When ingested, ZEN can prevent or delay the clinical and subclinical spread of oestrogen-dependent tumours [2,21,22]. Sex hormones and exogenous oestrogen-like chemicals are frequently implicated in the aetiology of tumours in various tissues [8]. Many oestrogen-sensitive tumours are termed oestrogen receptor-positive tumours because ERs are mediators of oestrogens or oestrogen-like substances that cause cancer [14,21]. Zearalenone may be a selective oestrogen receptor modulator, but its binding affinity for ERs is 10,000 times lower than that of 17-oestradiol (E2) [2]. Zearalenone has agonistic or antagonistic effects on target tissues, depending on the type of ER [1,2]. The chemopreventive effect of ZEN can be attributed to its antagonistic influence on ERs [18]. There is evidence that ZEN can inhibit circulating oestrogen precursors and slow the development and progression of oestrogen-dependent tumours by binding to ERs, and ERs can probably also inhibit the activity of steroid hormones that convert circulating hormones to E<sup>2</sup> [18,23].

Elements of the oestrogen response have been investigated in studies involving endogenous oestrogens and oestrogen-containing drugs [12,13,18]. When endogenous oestrogens exert genomic effects via ERs, oestrogen response elements bind with ERs or other response elements in the neighbouring genes that respond directly to oestrogens [3]. The resulting bonds influence the transcription of oestrogen-responsive genes. Mycoestrogens trigger similar responses by binding to ERs and initiating molecular cascades that alter gene expression [8]. Zearalenone is involved in molecular mechanisms, but its oestrogenic activity remains insufficiently investigated. Previous research has demonstrated that the presence of ZEN in feed or food affects the mRNA expression of ERs [8,24] and the activity of other genes encoding metabolic processes in enterocytes [25,26]. Subclinical symptoms of ZEN mycotoxicosis can cause changes in hormonal signalling when enterocytes in different intestinal segments are exposed to this mycotoxin [19]. The role of zearalenone in the digestive system should be evaluated to determine possible risks for gilts before puberty [2,27–30]. Therefore, this experiment aimed to find out whether a low monotonic dose of ZEN affects the immunohistochemical expression (IE) of ERα and ERβ in the gut of prepubertal gilts. The findings may contribute to a mechanistic understanding of changes in ERα and ERβ expression.

#### **2. Results**
