**1. Introduction**

Zearalenone (ZEN) and its metabolites (ZELs), α-zearalenol (α-ZEL) and β-zearalenol (β-ZEL), are the most ubiquitous mycotoxins in plant materials. They are frequently referred to as xenobiotics. These mycotoxins disrupt reproductive functions because they are structurally similar to oestradiol [1,2]. Alpha-ZEL is the main ZEN metabolite that affects pigs. Other animal species, including broilers, cows, and sheep, are more susceptible to β-ZEL, which is characterized by lower levels of metabolic activity [3]. Zearalenone's activity is determined by biotransformation processes in plants and animals as well as the immune status of the reproductive system (due to variations in steroid hormone levels

**Citation:** Mróz, M.; Gaj˛ecka, M.; Przybyłowicz, K.E.; Sawicki, T.; Lisieska-Zołnierczyk, S.; Zielonk, Ł.; ˙ Gaj˛ecki, M.T. The Effect of Low Doses of Zearalenone (ZEN) on the Bone Marrow Microenvironment and Haematological Parameters of Blood Plasma in Pre-Pubertal Gilts. *Toxins* **2022**, *14*, 105. https://doi.org/ 10.3390/toxins14020105

Received: 10 January 2022 Accepted: 27 January 2022 Published: 29 January 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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/).

during maturation, reproduction, and pregnancy) and the gastrointestinal system of the exposed individuals [4–7].

Different doses of mycotoxins induce various effects [7]. The symptoms and toxic effects of high mycotoxin doses have been extensively researched [8]. Low monotonic doses of mycotoxins are well tolerated by animals during prolonged exposure, and these compounds can meet the animals' life needs or exert therapeutic effects [9–11]. The adverse effects of low mycotoxin doses have also been well documented [12]. These effects can be attributed to hormesis, namely exposure to the highest doses below the No Observed Adverse Effect Level (NOAEL) that induce subclinical (asymptomatic) states or interact positively with the host organism in various stages of life [13–16]. In turn, doses corresponding to the Lowest Observed Adverse Effect Level (LOAEL) induce clear clinical symptoms of mycotoxicosis. The lowest measurable dose that interacts positively with the host body in different stages of life is known as the Minimal Anticipated Biological Effect Level (MABEL) [16].

The dose-response relationship has been also undermined by the low dose hypothesis. The above applies particularly to hormonally active chemical compounds, including mycoestrogens such as ZEN and ZELs, which disrupt the functioning of the endocrine system (ED), even when ingested in small quantities [5,17]. This ambiguous dose-response relationship does not justify direct analyses or meta-analyses of the risk (clinical symptoms or the results of laboratory analyses) associated with the transition from high to low doses [18]. The concept of the lowest identifiable dose that produces counter-intuitive effects is becoming increasingly popular in biomedical sciences. For this reason, the relevant mechanisms should be investigated to support rational decision-making in selected processes [7,19]. Substances that both disrupt and contribute to homeostasis have undermined the traditional concepts in toxicology, in particular "the dose makes the poison" adage. Zearalenone and ZELs induce different responses in mammals when administered in low doses [13]. Similar observations have been made by Knutsen et al. [12]. According to the Scientific Panel on Contaminants in the Food Chain (CONTAM), the influence of ZEN on animal health should be re-evaluated based on the animals' responses to the lowest detectable doses of ZEN (MABEL; the highest values of NOAEL and LOAEL), including the parent compound and its metabolites, in feed [7,12,20,21].

Hematopoietic stem cells (controlled primarily by the local bone marrow microenvironment as well as by long-range signals from, for example, the endocrine system [22], such as oestrogens or xenobiotics) cannot cross the bone marrow microenvironment barrier. Only mature blood cells containing specific membrane proteins are able to bind to the vascular endothelium and reach peripheral blood. Blood vessels act as a barrier that prevents immature blood cells from leaving the bone marrow microenvironment. Different types of haematopoietic cells have specific roles: red blood cells (RBC) contain haemoglobin, a specific protein that transports oxygen within the body [23], white blood cells (WBC) participate in immune functions and help fight infections, and platelets (PLT) participate in blood clotting at the sites of vascular injury [24].

The bone marrow microenvironment is an interesting object of scientific inquiry [25]. Red marrow is highly vascular, soft, gelatinous-spongy tissue, which fills the cavities of long bones and the spaces between trabeculae in cancellous bones, such as the wing of ilium. Red marrow is the primary producer of blood cells in the body, and it actively participates in haematopoiesis. Each day, red marrow produces 200 billion new morphotic elements in the blood. It is composed of delicate, highly vascular, fibrous tissue, and it contains hematopoietic stem cells, which give rise to various types of blood cells that are transported to the bloodstream upon maturation [26]. Yu and Scadden [27] described bone marrow as a carrier with sub-compartments that support different haematopoietic activities.

The aim of the present study was to determine whether exposure to low ZEN doses (MABEL [5 µg/kg body weight—BW], the highest values of NOAEL [10 µg/kg BW], and LOAEL [15 µg/kg BW]) administered *per os* to pre-pubertal gilts over a period of 6 weeks influences the concentrations of ZEN and ZELs in the bone marrow microenvironment and induces changes in selected haematological parameters.

#### **2. Results**

The presented results provide the basis for continuing research on the assessment of the impact of very low doses of ZEN on the macroorganism. There is a specific interaction between the mycotoxin level in the bone marrow microenvironment and the hematological values of the test animals. Preliminary data analysis documents that it is a set of highly individualized results for the parent substance and the absence of essential ZEN metabolites. The presented experimental design confirmed the correctness of the use of the method (chromatography), classified as precision medicine: medicine that allows the assessment of a single organism as well as the entire population. The existing values of the concentration of the undesirable substance in the bone marrow microenvironment contributed to the negative values of the Pearson's r coefficient for white and red blood cells and platelets.
