uterine section 0 0.50 ± 0.28 1.65 ± 0.18

D2 1.1 101.01/196.9/298.2 Ovary 1.04 ± 0.10 xx yy 2.65 ± 0.55 aa 3.00 ± 0.04 a

Uterine horn,

D1 0.8 80.5/161.9/242.7

(see Figure 2).

Middle part of the cervix 0 4.03 ± 0.16 1.77 ± 1.46 Hypothalamus 0 0.30 ± 0.20 0.94 ± 0.04 Pituitary gland 0 0 0 **Figure 2.** The CF of α-ZEL in the reproductive system tissues, hypothalamus, and pituitary glands of sexually immature gilts exposed to various ZEN doses. Key: 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. **Figure 2.** The CF of α-ZEL in the reproductive system tissues, hypothalamus, and pituitary glands of sexually immature gilts exposed to various ZEN doses. Key: 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.

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

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

In group ZEN5, an absence of significant differences in β-ZEL concentrations was noted between tissues on different exposure days (see Table 3). In group ZEN10, clear differences in the studied parameter were found in the ovarian section of the uterine horn and in the middle part of the cervix between samples taken D1 and those taken on D2 and D3. In group ZEN15, differences in β-ZEL levels were noted between the ovaries and the

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 accu-

mulation was inversely proportional to both the dose and exposure date.

uterine section of the uterine horn, but only on D3.

**Figure 3.** The CF of β-ZEL in the reproductive system tissues, hypothalamus, and pituitary glands of sexually immature gilts exposed to various ZEN doses. Key: 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. **Figure 3.** The CF of β-ZEL in the reproductive system tissues, hypothalamus, and pituitary glands of sexually immature gilts exposed to various ZEN doses. Key: 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.

The graphic presentation of the CF values of β-ZEL (see Figure 3) clearly indicates that the concentrations of this metabolite in the examined tissues were very low. Higher CF values that were proportional to the administered dose were noted only on D1 in groups ZEN10 and ZEN15. The ovaries were the only exception, where saturation with β-ZEL was higher than in the remaining tissues and not always proportional to the admin-In group ZEN5, an absence of significant differences in β-ZEL concentrations was noted between tissues on different exposure days (see Table 3). In group ZEN10, clear differences in the studied parameter were found in the ovarian section of the uterine horn and in the middle part of the cervix between samples taken D1 and those taken on D2 and D3. In group ZEN15, differences in β-ZEL levels were noted between the ovaries and the uterine section of the uterine horn, but only on D3.

istered dose. In the pituitary gland, the CF values of β-ZEL were also higher than in the remaining tissues (excluding on D1), with the exception of the ovaries. In group ZEN5, β-ZEL was not detected on D1 (see Figure S3) because the values determined in all tissues as below the sensitivity of the method (see Table 3 and Figure 3). In all groups, β-ZEL was not identified in the hypothalamus or pituitary gland on D1. On D2 and D3, the mathematical (see Table 3 and Figure 3) and hierarchical (see Figures S1- No significant differences in β-ZEL concentrations were observed between groups on D1 and D2 (see Table 3). On D3, the CF (see Figure 3) values of β-ZEL in the ovaries, the ovarian section of the uterine horn, and the middle part of the cervix were lower in group ZEN5 than in groups ZEN10 and ZEN15. On D3, considerable differences in β-ZEL levels in the hypothalamus were also found between group ZEN5 and group ZEN15. The concentrations of β-ZEL in the middle part of the cervix and the hypothalamus differed significantly between group ZEN10 and group ZEN15.

S3) values of β-ZEL were generally low and similar. The CF values of β-ZEL were much higher only in the ovaries and the pituitary gland. The hierarchical order of data was not maintained between group ZEN10 and group ZEN15, but it was maintained between exposure dates (D1, D2, and D3). The parent compound (ZEN) and its two metabolites (α-ZEL and β-ZEL) were deposited in reproductive system tissues, the hypothalamus, and the pituitary gland, even The graphic presentation of the CF values of β-ZEL (see Figure 3) clearly indicates that the concentrations of this metabolite in the examined tissues were very low. Higher CF values that were proportional to the administered dose were noted only on D1 in groups ZEN10 and ZEN15. The ovaries were the only exception, where saturation with β-ZEL was higher than in the remaining tissues and not always proportional to the administered dose. In the pituitary gland, the CF values of β-ZEL were also higher than in the remaining tissues (excluding on D1), with the exception of the ovaries.

in gilts exposed to very low doses of ZEN (MABEL, NOAEL, and LOAEL). In the analyzed tissues, ZEN accumulation decreased proportionally on successive dates of exposure, and it decreased by ± 50% between D1 and D2, and between D2 and D3. 2.2.2. Blood Concentrations of estradiol and progesterone Estradiol (E2) levels were higher in all experimental groups than in group C on all In group ZEN5, β-ZEL was not detected on D1 (see Figure S3) because the values determined in all tissues as below the sensitivity of the method (see Table 3 and Figure 3). In all groups, β-ZEL was not identified in the hypothalamus or pituitary gland on D1. On D2 and D3, the mathematical (see Table 3 and Figure 3) and hierarchical (see Figures S1–S3) values of β-ZEL were generally low and similar. The CF values of β-ZEL were much higher only in the ovaries and the pituitary gland. The hierarchical order of data was

analytical dates (see Figure 4). However, significant differences were noted only on D3 and on successive exposure dates. On these dates, E2 concentrations were highest in group not maintained between group ZEN10 and group ZEN15, but it was maintained between exposure dates (D1, D2, and D3).

The parent compound (ZEN) and its two metabolites (α-ZEL and β-ZEL) were deposited in reproductive system tissues, the hypothalamus, and the pituitary gland, even in gilts exposed to very low doses of ZEN (MABEL, NOAEL, and LOAEL). In the analyzed tissues, ZEN accumulation decreased proportionally on successive dates of exposure, and it decreased by ± 50% between D1 and D2, and between D2 and D3. *Toxins* **2022**, *14*, 790 10 of 19

#### 2.2.2. Blood Concentrations of estradiol and progesterone

Estradiol (E2) levels were higher in all experimental groups than in group C on all analytical dates (see Figure 4). However, significant differences were noted only on D3 and on successive exposure dates. On these dates, E<sup>2</sup> concentrations were highest in group ZEN5 and lowest in group C (the differences were determined at 6.29 pg/mL on D3, 7.52 pg/mL on D4, 4.53 pg/mL on D5, and 4.96 pg/mL on D6). Estradiol levels were also higher in groups ZEN10 and ZEN15 than in group C on all exposure days, but the observed differences were not significant. ZEN5 and lowest in group C (the differences were determined at 6.29 pg/mL on D3, 7.52 pg/mL on D4, 4.53 pg/mL on D5, and 4.96 pg/mL on D6). Estradiol levels were also higher in groups ZEN10 and ZEN15 than in group C on all exposure days, but the observed differences were not significant.

The distribution of P4 (progesterone) concentrations (see Figure 5) differed throughout the entire experiment. On the first four exposure dates, P4 levels were highest in group C. On D5 and D6, the analyzed parameter peaked in group ZEN10. Significant differences were observed between group ZEN10 and groups ZEN5 and C (on D5 0.21 ng/mL and D6 The distribution of P<sup>4</sup> (progesterone) concentrations (see Figure 5) differed throughout the entire experiment. On the first four exposure dates, P<sup>4</sup> levels were highest in group C. On D5 and D6, the analyzed parameter peaked in group ZEN10. Significant differences were observed between group ZEN10 and groups ZEN5 and C (on D5 0.21 ng/mL and D6 0.12 ng/mL).

0.12 ng/mL).

**Figure 5.** The effect of ZEN on blood P4 concentrations in pre-pubertal gilts: arithmetic means ( *x* ) of five samples collected on each analytical date (D1–D6) in every group (control [C], ZEN5, ZEN10 and ZEN15). Statistics significant differences were determined at \**p* ≤ 0.05 and \*\**p* ≤ 0.01. **Figure 5.** The effect of ZEN on blood P<sup>4</sup> concentrations in pre-pubertal gilts: arithmetic means (*x*) of five samples collected on each analytical date (D1–D6) in every group (control [C], ZEN5, ZEN10 and ZEN15). Statistics significant differences were determined at \* *p* ≤ 0.05 and \*\* *p* ≤ 0.01.

#### **3. Discussion**

**3. Discussion** The results of this quantitative analysis of ZEN, α-ZEL, and β-ZEL levels in selected tissues of the reproductive tract and the HPG axis, and of E2 and P4 concentrations in the blood of pre-pubertal gilts may be difficult to interpret because very few studies have The results of this quantitative analysis of ZEN, α-ZEL, and β-ZEL levels in selected tissues of the reproductive tract and the HPG axis, and of E<sup>2</sup> and P<sup>4</sup> concentrations in the blood of pre-pubertal gilts may be difficult to interpret because very few studies have addressed this issue.

addressed this issue. On exposure date D1, the levels of ZEN and its two metabolites (see Table 1) in the examined tissues can be attributed to ongoing transformation processes in maturing gilts, which do not lead to hyperestrogenism, but merely cater to the physiological demand for endogenous steroids (see Figures 4 and 5) in these animals [37,38]. This observation is problematic because physiologically maturing gilts (during puberty) are naturally deficient in endogenous steroids, and exposure to feed-borne ZEN leads to an increase in steroid levels. The question that arises is: how does this happen? One of the possible explanations is that pre-pubertal gilts adapt to frequent or prolonged exposure to low doses of exogenous steroid-like substances. The results noted for group ZEN5 (MABEL dose) on On exposure date D1, the levels of ZEN and its two metabolites (see Table 1) in the examined tissues can be attributed to ongoing transformation processes in maturing gilts, which do not lead to hyperestrogenism, but merely cater to the physiological demand for endogenous steroids (see Figures 4 and 5) in these animals [37,38]. This observation is problematic because physiologically maturing gilts (during puberty) are naturally deficient in endogenous steroids, and exposure to feed-borne ZEN leads to an increase in steroid levels. The question that arises is: how does this happen? One of the possible explanations is that pre-pubertal gilts adapt to frequent or prolonged exposure to low doses of exogenous steroid-like substances. The results noted for group ZEN5 (MABEL dose) on D1 seem to validate this hypothesis.

D1 seem to validate this hypothesis. We found a lack of both ZEN metabolites in the ZEN5 group at the D1 date (see Tables 2 and 3), probably due to the low supply of endogenous steroid hormones and very low concentrations of exogenous ("free") ZEN [37]. Exogenous ZEN was accumulated in large quantities in the studied tissues, including in the hypothalamus and pituitary gland (see Figure 1). The above could suggest that ZEN biotransformation in the intestines and blood vessels proceeded at a "slower" rate than ZEN binding to steroid hormone receptors, possibly to cater to a very high demand for steroids. This observation is supported by the CF values (see Figure 1) and the hierarchical order of data in the tree maps (see Figure S1), which suggest that ZEN was accumulated mostly in the ovaries and the cervix, followed by the pituitary gland, on D1 in group ZEN5 (MABEL dose) (see Figure S1). This process was accompanied by a marked increase in E2 concentrations (see Figure 4) in all experimental groups relative to the control group. On D3, E2 levels differed significantly We found a lack of both ZEN metabolites in the ZEN5 group at the D1 date (see Tables 2 and 3), probably due to the low supply of endogenous steroid hormones and very low concentrations of exogenous ("free") ZEN [37]. Exogenous ZEN was accumulated in large quantities in the studied tissues, including in the hypothalamus and pituitary gland (see Figure 1). The above could suggest that ZEN biotransformation in the intestines and blood vessels proceeded at a "slower" rate than ZEN binding to steroid hormone receptors, possibly to cater to a very high demand for steroids. This observation is supported by the CF values (see Figure 1) and the hierarchical order of data in the tree maps (see Figure S1), which suggest that ZEN was accumulated mostly in the ovaries and the cervix, followed by the pituitary gland, on D1 in group ZEN5 (MABEL dose) (see Figure S1). This process was accompanied by a marked increase in E<sup>2</sup> concentrations (see Figure 4) in all experimental groups relative to the control group. On D3, E<sup>2</sup> levels differed significantly between the experimental groups and the control group. At the same time, P<sup>4</sup> concentrations

between the experimental groups and the control group. At the same time, P4

(see Figure 5) were much lower in the experimental groups than in group C on all exposure days and remained at a similar level throughout the experiment.

In groups ZEN10 and ZEN15 (NOAEL and LOAEL doses, respectively), ZEN concentrations in the hypothalamus and pituitary gland were inversely proportional to the administered doses, and a shift to other levels in the hierarchy was observed (see Figures S2 and S3, respectively). On D2 (see Figure S2), nervous system tissues ranked second in the data hierarchy. On D3 (see Figure S3), nervous system tissues ranked third in the data hierarchy only in group ZEN5 (MABEL dose). These results could point to the interdependence between the HPG axis and the ovaries, which is very important because ZEN exerts a direct negative effect on the neuroendocrine coordination of reproductive competence [39]. Similar observations were made by Rykaczewska et al. [10,37].

According to other researchers, α-ZEL is the dominant ZEN metabolite in the peripheral blood of pigs [17,40], and the results noted for group ZEN5 (MABEL dose) corroborate this observation. β-ZEL was the dominant ZEN metabolite in only one study [37]. The above could be explained by the host organism's increased demand for compounds characterized by steroid (not only estrogenic) activity, such as α-ZEL and ZEN, but not β-ZEL [41]. These compounds are a source of endogenous steroid hormones which are essential for healthy functioning [42]. It should also be noted that ZEN and α-ZEL form highly stable complexes with albumins that prolong their half-life [7], which is a very important consideration in the interplay of steroid hormones. During exposure to ZEN, adaptive processes, in particular adaptive immunity, end on exposure date D1 [43]. Zearalenone is an endocrine-disrupting chemical (EDC) [44] and a substrate that regulates (inversely) the expression levels of genes encoding hydroxysteroid dehydrogenases (epigenetically [45]), which act as molecular switches and modulate steroid hormone pre-receptors [46]. Zearalenone also slows down proliferative processes in granulosa cells, proportionally to the administered dose, and provokes apoptosis in the ovaries [47]. These processes are accompanied by enterohepatic recirculation which slows down the elimination of mycotoxins [48]. Zearalenone was also found to affect gut microbiota [11] by increasing the log values of specific microbial counts in the distal segment of porcine intestines in the static and dynamic system.

Much like other EDCs [30,44], ZEN can cross the blood-brain barrier, modify hypothalamic and pituitary functions, and exert a negative influence on central and peripheral reproductive tissues. According to He et al. [49], Rykaczewska et al. [37], and Zheng et al. [40], ZEN disrupts biological functions during the release of neurotransmitters which control physiological homeostasis, including levels of the FSH [33]. The discussed mycotoxin also blocks neuroactive ligand-receptor interaction pathways [39] and calcium signaling pathways [50–52] in the mitochondria. The resulting negative feedback initially decreases steroid production [45] and increases steroid synthesis on final dates of exposure. The results of this study could be also explained by the fact that ZEN blocks the activity of the HPG axis, which increases P<sup>4</sup> levels, enhances endometrial receptivity, and induces specific morphometric changes in the reproductive system [37].

These observations suggest that the estrogen-sensitive tissues were saturated with ZEN (already on D1, proportionally to the administered ZEN dose), which leads to changes to the supraphysiological hormonal levels of pre-pubertal gilts, accompanied by an inverse correlation between steroid concentrations [37,38]. In pre-pubertal gilts exposed to ZEN, the strongest response was observed on D1, when hormonal receptors (not only estrogen receptors) were saturated, and neuroactive ligand-receptor interaction pathways were blocked. The accumulation of ZEN decreased considerably on successive dates of exposure, probably because the administered doses were very low, and the gilts' physiological status perpetuated the processes that accompany ZEN mycotoxicosis. Extrapolation of the current findings indicates that ZEN, evaluated quantitatively in the analyzed tissues, affects HPG activity in pre-pubertal gilts in all examined doses due to its promiscuous properties [25].

Analysis of the concentrations (see Tables 1–3) and CF values (see Figures 1–3 and Figures S1–S3) of α-ZEL and β-ZEL based on arithmetic means and hierarchal data revealed certain trends. The concentrations and CF values of ZEN metabolites were inversely proportional to the corresponding values of the parent compound, in all tissues except the ovaries. In the examined nervous system tissues, ZEN metabolites accumulated gradually at concentrations that were inversely proportional to the administered dose (experimental group) and exposure date. This observation indirectly demonstrates that ZEN and α-ZEL (the most prominent mycoestrogens) exerted complementary effects and blocked estrogen receptors in the analyzed reproductive system tissues. However, ZEN probably slowed down the synthesis and release of FSH in the porcine pituitary gland [49]. Inhibited FSH secretion decreased the production of sex steroids, including E2, P4, and testosterone [34,37,45,53], and probably enhanced the conversion of steroids into E2, which increased feed intake and promoted the storage of excess energy in fat cells [10]. Therefore, inhibited pulsatile HPG activation slowed down gonadal development, prolonged sexual maturation, and delayed reproductive maturity.

A comparison of ZEN and α-ZEL concentrations (see Tables 1 and 2) and the CF values of both mycotoxins, presented graphically, leads to another question: why in group ZEN5 (MABEL dose) was ZEN the dominant mycotoxin on D1, whereas α-ZEL was the dominant compound on D3? It could be postulated that "free ZEN" is initially used due to supraphysiological hormonal levels in pre-pubertal gilts [37], whereas biotransformation processes are initiated later, and excess "free" ZEN is metabolically transformed into, for example, α-ZEL which is bound to free estrogen receptors or used to block HPG activity. Therefore, both the parent compound and its metabolite act as epigenetic switches that regulate sexual maturation [45]. In the first week of exposure, only ZEN acts as an epigenetic switch, and on successive dates of exposure, both ZEN and α-ZEL play this role. The role of β-ZEL remains unknown.

#### **4. Summary**

The presented quantitative analysis suggests that ZEN and its metabolites can be among the factors that inhibit the somatic development of reproductive system tissues by decreasing the activity of the HPG axis in pre-pubertal gilts [23,34], which exerts a direct negative effect on the neuroendocrine coordination of reproductive competence [37,39]. However, from the breeders' point of view, this is a positive phenomenon because low-dose ZEN mycotoxicosis enables maturing gilts to utilize nutrients for somatic development (most effectively in group ZEN5-MABEL dose) [10] rather than for reproductive development and performance.

#### **5. Materials and Methods**

#### *5.1. General Information*

This article is a continuation of a previously published study protocol [54].

### *5.2. Experimental Feed*

Analytical samples of ZEN were dissolved in 96 µl of 96% ethanol (SWW 2442-90, Polskie Odczynniki SA, Poland). Gilts were weighed at weekly intervals, and mycotoxin doses were calculated individually based on their BWs [6,11,12]. Gel capsules were saturated with the solution, feed was placed inside the capsules, and they were stored at room temperature to evaporate the alcohol. Throughout the trial, all gilts were given the same feed.

The feed given to all test animals was supplied by the same manufacturer. Friable feed was provided ad libitum twice everyday, at 8:00 a.m. and 5:00 p.m., during the experiment. The composition of the complete diet, as declared by the producer, is presented in Table 4 [6,11,12]. Pigs in the experimental groups received ZEN in gel capsules everyday before morning feeding. Feed was the carrier, and group C gilts received identical gel capsules without ZEN [10,11].


**Table 4.** Declared composition of the complete diets [37].

<sup>1</sup> Composition of the vitamin-mineral premix per kg: vitamin A—500,000 IU; iron—5000 mg; vitamin D3— 100,000 IU; zinc—5000 mg; vitamin E (alpha-tocopherol)—2000 mg; manganese—3000 mg; vitamin K—150 mg; copper (CuSO4·5H2O)—500 mg; vitamin B1—100 mg; cobalt—20 mg; vitamin B2 —300 mg; iodine—40 mg; vitamin B6—150 mg; selenium—15 mg; vitamin B12—1500 µg; L-lysine—9.4 g; niacin—1200 mg; DL-methionine + cystine—3.7 g; pantothenic acid—600 mg; L-threonine—2.3 g; folic acid—50 mg; tryptophan—1.1 g; biotin— 7500 µg; phytase + choline—10 g; ToyoCerin probiotic+calcium—250 g; antioxidant+mineral phosphorus and released phosphorus—60 g; magnesium—5 g; sodium and calcium—51 g.

The approximate chemical composition of the diets fed to pigs in groups C, ZEN5, ZEN10, and ZEN15 was determined using the NIRS™ DS2500 F feed analyzer (FOSS, Hillerød, Denmark), a monochromator-based NIR reflection and transflectance analyzer (FOSS, Hillerød, Denmark) with a scanning range of 850–2500 nm [54].
