**1. Introduction**

Raw materials and feed components of plant origin are often contaminated with undesirable substances such as mycotoxins [1], which pose a health risk to humans [2] and various livestock species, pigs in particular [3]. The symptoms and health (toxicological) risks associated with exposure to high doses of these compounds have been investigated with regard to a limited number of mycotoxins, including ZEN and its metabolites: α-ZEL and β-ZEL [4–7]. In light of the hormesis paradigm, which posits that low doses of undesirable substances exert beneficial effects on the body [8,9], the consequences of prolonged exposure to low concentrations of mycotoxins (which are frequently found in animal feed) should be studied. Numerous studies involving mammals have been undertaken to identify potential physiological dysfunctions resulting from exposure to

**Citation:** Gaj˛ecka, M.; Zielonka, Ł.; Babuchowski, A.; Gaj˛ecki, M.T. Exposure to Low Zearalenone Doses and Changes in the Homeostasis and Concentrations of Endogenous Hormones in Selected Steroid-Sensitive Tissues in Pre-Pubertal Gilts. *Toxins* **2022**, *14*, 790. https:// doi.org/10.3390/toxins14110790

Received: 20 October 2022 Accepted: 9 November 2022 Published: 11 November 2022

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**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/).

**<sup>\*</sup>** Correspondence: mgaja@uwm.edu.pl

the pure parent compound [10–15] without metabolites, or to modified mycotoxins [9], particularly in the reproductive [16] and hormonal systems [10].

The existing research indicates that exposure to low ZEN concentration may cause side effects that are hard to predict [17]. The observed changes are influenced by the administered dose and duration of exposure [18]. Low mycotoxin doses can elicit surprising effects: for example, the body's failure to detect undesirable substances such as mycotoxins [19]. Long-term exposure to orally administered ZEN leads to increased mycotoxin accumulation in target cells [13,14] and induces the compensatory effect [20] by altering the analyzed indicators, for example, in the reproductive system [21], changing the activity of the hypothalamic-pituitary-gonadal (HPG) axis [22,23] and disrupting hormonal homeostasis in pre-pubertal animals. Homeostasis is restored [24] in subsequent stages of exposure [10]. These factors, as well as the promiscuity [25] of ZEN and its known metabolites [17], and the type and intensity of physiological reaction in gilts exposed to this mycotoxin, point to the need for further study into the effects of low dietary zearalenone doses.

Based on the results of our prior studies [10,14,15], a low ZEN dose was defined by examining whether clinical symptoms of ongoing mycotoxicosis were present. Three ZEN doses were proposed based on our previous work and a review of the literature: (i) the lowest dose which elicits clinical symptoms [3] (>10 µg ZEN/kg of body weight, BW), defined as the lowest observed adverse effect level (LOAEL) [19]; (ii) the highest dose which does not elicit clinical symptoms (subclinical states) (= 10 µg ZEN/kg BW), defined as the no observed adverse effect level (NOAEL) [26]; and (iii) the lowest measurable dose which enters into positive interactions with the host organism in different stages of life (<10 µg ZEN/kg BW), defined as the minimal anticipated biological effect level (MABEL) [6,27,28].

Since zearalenone is a mycoestrogen, the dose-reaction paradigm has been subverted and replaced with the low dose hypothesis [17]. This applies, in particular, to hormonally active chemical compounds [29]. The ambiguous dose-response relationship prevents a direct, monotonic extrapolation or meta-analysis of the risks (including clinical symptoms and the results of laboratory analyses) associated with the transition from a high to a low dose [3,24]. On the other hand, ZEN's toxicity can be attributed to its chemical structure and ability to interact with steroid hormone receptors in many internal organs [30]. Zearalenone can also cross the barrier between the cerebral capillary blood and the interstitial fluid of the brain and affects neurons in the central nervous system [31,32]. Recent research has shown that exposure to ZEN disturbs the synthesis of neuronal factors and enzymes in brain neurons. In pigs (including pre-pubertal gilts), reproductive functions are controlled by complex regulatory networks which integrate peripheral and internal signals, thus affecting brain regions that control, e.g., the HPG axis [33]. By binding to specific receptors on gonadotropic cells in the pituitary gland, ZEN and/or its metabolites block these signals, which inhibits the biosynthesis and release of two gonadotropins—the luteinizing hormone (LH) and the follicle-stimulating hormone (FSH) [23]—and decreases the amplitude of LH pulsation [34]. LH and FSH are essential for gonadal development and fertility, and they bind to gonadal receptors to regulate gametogenesis and steroidogenesis [35,36].

Based on the above observations and a review of the literature, we hypothesized that ZEN present in feed materials at very low concentrations is accumulated in the reproductive systems, hypothalamus, and pituitary glands of pre-pubertal gilts. Thus, the aim of this study was to determine whether exposure to low doses of zearalenone (MABEL dose [5 µg/kg BW], the highest NOAEL dose [10 µg/kg BW], and the lowest LOAEL dose [15 µg/kg BW]) administered per os to sexually immature gilts over a period of 42 days affects the levels of zearalenone, alfa-ZEL, and beta-ZEL in selected reproductive system tissues (ovaries, uterine horn—ovarian and uterine sections, and the middle part of the cervix), the hypothalamus and pituitary gland, along with whether it affects the peripheral blood levels of two steroid hormones: estradiol and progesterone.

#### **2. Results**

The presented results were obtained as part of a large-scale experiment which did not reveal clinical signs of ZEN mycotoxicosis. However, differences were frequently observed in the values of the carry-over factor (CF) of zearalenone and its metabolites in intestinal tissues, in CYP1A1 and GSTπ1 expression in the large intestine, in selected serum biochemical profiles, in the myocardium and the coronary artery, in cecal water genotoxicity, in selected steroid concentrations, in intestinal microbiota parameters, and in the weight gain of animals. Samples gathered from the same animals were analyzed. Previous findings were published in several publications [10–15,17,37].

#### *2.1. Experimental Feed*

Experimental diets did not contain any mycotoxins, or their amounts were below the limit of detection (LOD). The concentrations of masked and/or modified mycotoxins were not determined.

#### *2.2. Results of Laboratory Analyses*

2.2.1. Concentrations of ZEN and Its Metabolites in Selected Tissues

The CF values of ZEN and its metabolites differed considerably (see Tables 1–3, Figures 1–3, Figures S1–S3) not only on different exposure dates, but also between groups and the analyzed tissues. These differences are evident in the resulting tree maps, where hierarchical data are presented by a series of nested rectangles (see Figures S1–S3).

In all groups, mean ZEN concentrations decreased in all analyzed tissues on successive exposure dates (see Table 1). In group ZEN5 (MABEL dose), significant differences were found in the uterine horn (ovarian and uterine sections) and the middle part of the cervix on D2 and D3 relative to D1. In group ZEN10 (NOAEL dose), significant differences were observed in the uterine section of the uterine horn on D2 and D3, and in the ovarian section of the uterine horn only on D2, in comparison to D1. In group ZEN15 (LOAEL dose), significant differences were noted in the ovarian section of the uterine horn on D2 and D3 vs. D1, and in the middle part of the cervix on D3 vs. D1.

The statistical analysis of ZEN concentrations (see Table 1) in the analyzed tissues, on different exposure dates and in different groups, revealed that ZEN levels increased with a rise in the administered ZEN dose. On D1, significant differences in the ovarian and uterine sections of the uterine horn, in the middle part of the cervix, and in the hypothalamus were observed in group ZEN15 vs. groups ZEN5 and ZEN10. Groups ZEN5 and ZEN10 also differed in ZEN concentrations in the middle part of the cervix. On D2, significant differences were also found between group ZEN15 and groups ZEN5 and ZEN10, excluding both sections of the uterine horn in group ZEN5. On D3, significant differences were observed only in the ovaries, the ovarian section of the uterine horn, and the middle part of the cervix.

The hierarchical analysis revealed that ZEN levels were highest on exposure date D1 in all analyzed tissues and in all groups. In all groups and on all exposure dates, the evaluated parameter was highest (see Figures S1–S3) in the ovaries (proportional to the administered dose; Figure 1), followed by the middle part of the cervix, and, interestingly, the pituitary gland.

Insignificant differences were noted between groups on D1, and between exposure dates in group ZEN5 (MABEL dose) (see Table 2). A comparison of α-ZEL levels in groups and on different exposure dates indicates that mean α-ZEL concentrations increased in proportion to the ZEN doses administered in groups, and to exposure dates, which probably could be attributed to the biotransformation of ZEN. The concentrations of the parent compound (see Table 1) in groups and on different exposure dates followed the opposite trend.


**Table 1.** The CF and the mean (±) concentrations of ZEN (ng/g) in the reproductive system tissues, hypothalamus and pituitary glands of sexually immature gilts.

Abbreviations: D1—exposure day 7; D2—exposure day 21; D3—exposure day 42. Experimental groups: Group ZEN5—5 µg ZEN/kg BW; Group ZEN10—10 µg ZEN/kg BW; Group ZEN15—15 µg ZEN/kg BW. In the pituitary gland, ZEN concentrations were assayed in aggregate samples. The differences were regarded as statistically significant at <sup>a</sup> , b , <sup>x</sup> *<sup>p</sup>* <sup>≤</sup> 0.05 and aa , xx , yy *<sup>p</sup>* <sup>≤</sup> 0.01; <sup>a</sup> , aa significant difference between exposure date D1 and exposure dates D2 and D3; <sup>x</sup> , xx significant difference between group ZEN15and groups ZEN5 and ZEN10; yy significant difference between group ZEN5 and group ZEN10.

In group ZEN10 (NOAEL dose) (see Table 2), significant differences in α-ZEL concentrations were observed in the ovaries and the uterine section of the uterine horn on D2 and D3, and in the middle part of the cervix on D3 relative to D1. Significant differences in the examined parameters were also noted in the uterine section of the uterine horn and in the middle part of the cervix between D2 and D3. In group ZEN15 (LOAEL dose) (see Table 2), α-ZEL levels in the ovaries and the hypothalamus differed significantly between D1 and D2. Significant differences in this parameter were found in the ovaries and in the ovarian section of the uterine horn between D1 and D3. In the ovarian section of the uterine horn, significant differences in α-ZEL concentrations were determined between D2 and D3.


**Table 2.** The CF and the mean (±) concentrations of α-ZEL (ng/g) in the reproductive system tissues, hypothalamus, and pituitary glands of sexually immature gilts.

Abbreviations: D1—exposure day 7; D2—exposure day 21; D3—exposure day 42. Experimental groups: Group ZEN5—5 µg ZEN/kg BW; Group ZEN10—10 µg ZEN/kg BW; Group ZEN15—15 µg ZEN/kg BW. LOD > values below the limit of detection were expressed as 0. In the pituitary gland, α-ZEL concentrations were assayed in aggregate samples. The differences were regarded as statistically significant at <sup>a</sup> , <sup>x</sup> *<sup>p</sup>* <sup>≤</sup> 0.05 and aa , bb , xx , yy *<sup>p</sup>* <sup>≤</sup> 0.01; a , aa significant difference between exposure date D1 and exposure dates D2 and D3; <sup>b</sup> , bb significant difference between exposure date D2 and exposure date D3; <sup>x</sup> , xx significant difference between group ZEN15 and groups ZEN5 and ZEN10; yy significant difference between group ZEN5 and group ZEN10.

On D2 (see Table 2), significant differences in α-ZEL levels in the ovaries and the ovarian section of the uterine horn were observed between group ZEN5 and group ZEN15, and in the ovaries and the ovarian and uterine sections of the uterine horn between group ZEN5 and group ZEN10. On D3, significant differences in α-ZEL levels were noted only in the middle part of the cervix between group ZEN5 and groups ZEN10 and ZEN15.

A graphic presentation of the CF values of α-ZEL (see Figure 2) revealed a certain trend: all CF values were inversely proportional to the results presented in Figure 1. The CF values of α-ZEL in the ovaries of group ZEN5 gilts on D1 were the only exception (the results were below the sensitivity of the method, and these CF values were expressed as 0). An analysis of ZEN and α-ZEL concentrations (see Tables 1 and 2, respectively) on the remaining exposure dates in all groups revealed that α-ZEL levels were much lower than ZEN concentrations, and that the highest α-ZEL concentrations were noted in group ZEN5

on the last two exposure dates. The examined ZEN metabolite was present in nearly all hypothalamus samples. In the pituitary gland, the CF values of α-ZEL were very low in group ZEN15 on D2, and in groups ZEN10 and ZEN15 on D3 (see Figure 2), which indicates that α-ZEL was accumulated gradually in the pituitary gland and that its accumulation was inversely proportional to both the dose and exposure date.

**Table 3.** The CF and the mean (±) concentrations of β-ZEL (ng/g) in the reproductive system tissues, hypothalamus, and pituitary glands of sexually immature gilts.


Abbreviations: D1—exposure day 7; D2—exposure day 21; D3—exposure day 42. Experimental groups: Group ZEN5—5 µg ZEN/kg BW; Group ZEN10—10 µg ZEN/kg BW; Group ZEN15—15 µg ZEN/kg BW. LOD > values below the limit of detection were expressed as 0. In the pituitary gland, β-ZEL concentrations were assayed in aggregate samples. The differences were regarded as statistically significant at <sup>a</sup> , x , <sup>z</sup> *<sup>p</sup>* <sup>≤</sup> 0.05 and at aa , xx , **yy**, zz *<sup>p</sup>* <sup>≤</sup> 0.01; <sup>a</sup> , aa significant difference between exposure date D1 and exposure dates D2 and D3; <sup>x</sup> , xx significant difference between group ZEN5 and group ZEN10; yy significant difference between group ZEN5 and group ZEN15; <sup>z</sup> , zz significant difference between group ZEN10 and group ZEN15.

A hierarchical visualization of the CF values of α-ZEL in the examined tissues (see Figure S1) revealed several interesting findings. Firstly, α-ZEL was not detected in group ZEN5 on D1 (its levels were below the sensitivity threshold, see Table 2), and its concentrations peaked on D3. Secondly, α-ZEL concentrations in groups ZEN10 and ZEN15 were higher on D2 than on D1 and D3. Thirdly, the highest α-ZEL levels were noted in the middle part of the cervix, the ovaries, and both sections of the uterine horn, and its

accumulation was highest (mathematically and hierarchically) in group ZEN5 on D2 and D3 (see Figure 2).
