*2.3. The Prognostic Value of the ERs Expression Profile*

A total of 432 samples were analysed to determine the ER expression indicator (P-ERs). In many of the analysed samples, there were no significant differences in ER expression. The mean values of P-ERs were 42 ± 27 for ERα and 38 ± 26 for ERβ. P-ERs values were not normally distributed (Table 1).


**Table 1.** ERα and ERß expression at various absorption levels in the analysed sections of the GI tract in pre-pubertal gilts.


**Table 1.** *Cont.*

Abbreviation: In group E, the value of P-ERα was 35, reaching 8 in the lower quartile and 62 in the upper quartile. The analysed expression values were divided into four subgroups based on the values of the median, and the upper and lower quartiles: A—very low P-ERα (P-Erα ≤ 8), B—low P-ERα (8 ≤ P-Erα < 35), C—high P-ERα (35 ≤ P-Erα < 62), and D—very high P-ERα (P-Erα ≥ 62) (Table 1). In group E, very low (A), low (B), high (C), and very high (D) values of P-ERα were noted in six (25%), 12 (50%), five (21%), and one (4%) cases, accordingly. The statistical analysis was carried out for different mean, median, upper and lower quartile cut-off points, but no meaningful differences were noted.

#### 2.3.1. P-ER Values for ERα

In group C, the P-ERα value was 42, reaching 15 in the lower quartile and 69 in the upper quartile. An analysis of the median and the upper and lower quartiles revealed that the expression values could be divided into four subgroups: A—very low P-ERα (P-ERα <15), B—low P-ERα (15 ≤ P -ERα < 42), C—high P-ERα (42 ≤ P-ERα < 69) and D—very high P-ERα (P-Erα ≥69) (Table 1). In group C, very low (A), low (B), high (C), and very high (D) P-ERα values were found in 11 (46%), seven (29%), five (21%), and one (4%) cases, respectively. The statistical analysis was conducted for different means, medians, upper and lower quartiles of the separation points, but no meaningful differences were observed.

The results of the analyses involving the uptake of only Erα or ERβ are difficult to interpret. The values of P-ERs (Table 1) provide new information on the presence of a low ZEN dose in the diet. These were very similar in both groups, but at absorption level 3, an increase in P-ERs was observed in group E, resulting in a shift from quartile A to quartile B from the jejunum directly to the descending colon. The results described above and previous research findings suggest that ZEN may compensate for E<sup>2</sup> deficiency by triggering ERα [27].

#### 2.3.2. P-ER Values for ERβ

In group C, the P-ERβ worth was 35, reaching 9 in the lower quartile and 61 in the upper quartile. Based on the average value of the median, and the upper and lower quartiles, expression values were divided into four subgroups: A—very low P-ERβ (P— ERβ ≤ 9), B—low P-ERβ (9 ≤ P-ERβ < 35), C—high P-ERβ (35 ≤ P-ERβ < 61), and D—very high P-ERβ (P-Erβ ≥ 61) (Table 1). In group C, very low (A), low (B), high (C), and very high (D) levels of P-ERβ were found in six (25%), 11 (46%), 6 (25%), and one (4%) cases, respectively. The statistical analysis was carried out for different means, medians, upper and lower quartiles, but no meaningful differences were found.

In group E, the P-ERβ value was 38, reaching 12 in the lower quartile and 64 in the upper quartile. Based on the values of the median, the upper and lower quartiles and expression values were divided into four subgroups: A—very low P-ERβ (P-Rβ < 12), B—low P-ERβ (12 ≤ P-ERβ < 38), C—high P-ERβ (38 ≤ P-ERβ < 64) and D—very high P-ERβ (P-Erβ ≥ 64) (Table 1). In the experimental group, very low (A), low (B), high (C), and very high (D) P-ERβ values were known in eight (33%), 10 (42%), three (12%), and three (12%) cases, respectively. The statistical analysis was carried out for different means, medians, upper and lower quartiles, but no meaningful differences were found.

The values of P-ERβ (Table 1) shifted to the right from quartile C to quartile D at absorption level 3 in the caecum and the ascending colon. An analysis of the expression of both receptors demonstrated that the P-ERα levels shifted significantly to the lower quartiles (to the left) in animals exposed to low ZEN doses.

#### **3. Discussion**

This study confirmed our recent observations that low ZEN doses improve somatic [36] and reproductive health (our previous mechanistic studies) [2,19,37]. On the first day of exposure, ZEN exerted a stimulatory effect on the body, with the exception of the reproductive system [18,38]. This effect was minimised after the second or third day of exposure, probably due to: (i) the negative effects of extragonadal compensation for oestrogen synthesis [39,40] by androgen conversion or the acquisition of exogenous oestrogens or oestrogen-like substances [2,9,41]; (ii) adaptive mechanisms [37]; (iii) higher energy and protein utilisation, indicating more efficient feed conversion (productivity in group E) [41–43]; or (iv) detoxification processes (biotransformation) [3]. The last argument is difficult to confirm since an analysis of the carry-over factor in the GI tract of the same animals did not reveal the inherence of α- ZEL or β- ZEL (ZEN metabolites) in the intestinal walls or that the registered levels were below the detection limit [20,25]. According to López-Calderero et al. [44], a higher ERα/ERβ ratio indicates that proliferative processes are stimulated or silenced, and it is unrelated to apoptosis [38]. Similar observations were made by Cleveland et al. [45] and Williams et al. [46]. These results suggest that low levels of ZEN in the diet stimulate proliferative processes in the gastrointestinal tract of prepubertal gilts, especially in the colon. In sexually mature animals, this is a good predictor of weight gain or the time needed to reach slaughter weight [41], and it suggests that the gastrointestinal tract regulates somatic health [9,38]. Thus, the digestive system acts as a "second brain" [47] as it performs numerous functions including a modulatory role between the intestinal contents and tissues vis. the central nervous system [48]. These findings also suggest that ZEN and endogenous oestrogens control growth, differentiation and other important functions in tissues including in the gastrointestinal tract [2] of prepubertal gilts with supraphysiological oestrogen levels [18]. The above also suggests that oestrogen signalling (e.g., ZEN and its metabolites), regardless of its origin, is the major regulator of genomic mechanisms. Oestrogen receptors play a special role: (i) they are activated by ligand-dependent and ligand-independent pathways; (ii) they act as transcription factors that activate and trigger the expression of all sensitive genes; and (iii) the feedback loop regulated by oestrogens contributes to the maintenance or modification of all genomic processes.

#### *3.1. Oestrogen Receptors*

The biological effects of oestrogens are determined by the type of ERs including the classical nuclear ERα and ERβ as well as the G-protein-coupled ERs (GPER; its expression has not been analysed). Therefore, the levels of different ERs determine the effects of endogenous and exogenous oestrogens on cells (tissues).

#### 3.1.1. Oestrogen Receptor Alpha

The expression of ERα in the control group could be attributed to the physiological deficiency of E<sup>2</sup> in the gilts before puberty [4,24,49], which could point to supraphysiological hormone levels rather than hypoestrogenism [18,50]. Zearalenone mycotoxicosis contributes to an increase in steroid levels (endogenous steroids such as E2, progesterone, and testosterone as well as exogenous steroids such as ZEN), which may restore or enhance ER signalling in cells [18,51], but only in relation to hormone-dependent ERs [27]. As a result, ERα expression is not stimulated but deregulated [51]. Most importantly, circulating steroid hormones are bioavailable (not bound to carrier proteins) and their cellular effects are observed at very low concentrations of approximately 0.1–9 pg/mL E<sup>2</sup> [49]. The concentrations of active hormones are determined by the age and health status of animals [2,8,18,24,52].

Various conclusions can be drawn from the observations of the role of ERα in mammals and the results of the experimentally induced ZEN mycotoxicosis. According to Suba [38], both high and low levels of E<sup>2</sup> stimulate the expression and transcriptional activity of ERs to restore or enhance ER signalling in cells, which was not observed in the current study. However, the IE of ERα was suppressed to a greater extent. Low ZEN doses in the diet decrease the IE of ERα, which directly affects the somatic (higher weight gain) [41] and reproductive health (delayed sexual maturity [53]) of animals. It should also be noted that low serum E<sup>2</sup> levels may induce compensatory effects to increase the expression and transcriptional activity of ERs, while increased synthesis of endogenous E<sup>2</sup> may compensate for low ER signalling [54]. However, it remains uncertain as to whether low ZEN doses are sufficient to meet the requirements of sexually immature gilts. The present findings suggest that this may be the case, with positive implications for pig farmers.

#### 3.1.2. Oestrogen Receptor Beta

According to the literature, intense ERβ expression or a high level of absorption (3 points on a 4-point grading scale) contributes significantly to gut health, especially colon health, and intensifies metabolic processes [55,56]. In turn, ERβ silencing increases the risk of duodenal inflammation and enhances oncogenesis not only in the gastrointestinal tract, but also in the reproductive system [22,40,45,46,57]. Deletion processes suggest that ERβ has anti-inflammatory and anti-carcinogenic properties, and exerts chemopreventive effects in the colon [58], which was confirmed in a study of low-dose ZEN mycotoxicosis [59].

Apart from the previously published research on the effects of E<sup>2</sup> deficiency in prepubertal animals, another issue should be addressed. Williams et al. [46] and Gaj˛ecka et al. [59] reported that selected phytoestrogens (silymarin and silibinin) and mycoestrogens (ZEN) have a selective affinity for ERβ [60,61]. This is the result of the increased expression of the ERβ gene, suggesting that natural exogenous dietary oestrogens may have anti-inflammatory properties [35]. These oestrogens also exert chemopreventive effects [22], and they can reverse minor carcinogenic changes in the colon [62]. Calabrese et al. [63] found that a mixture of phytoestrogens and lignans reduced the size and number of duodenal polyps and exerted therapeutic effects in this segment of the gastrointestinal tract [64].

As stated in the research objective, this study was conducted to determine if low ZEN doses naturally occurring in feeds could produce similar effects, and the present results suggest that it is possible. This conclusion is also consistent with the results of previous studies conducted as part of the same research project [2,19,29,31–36,52].

#### 3.1.3. ER Expression Indicator

In animals exposed to ZEN, the P-ER levels differed between quartiles. In group E, the P-ERα values shifted from quartile A to quartile B, while the P-ERβ values shifted from quartiles B and C to quartiles A and D. The expression levels of ERα confirm that low ZEN doses can exert oestrogenic effects on the studied ERs.

The endogenous ligand that triggers ERβ [27] and the cells that are activated by specific receptors could not be identified based on the existing knowledge. For this reason, the influence of ZEN on ERβ is difficult to interpret. It seems that E<sup>2</sup> does not bind to ERα and ERβ with equal affinity, but it binds to oestrogen response elements. However, ERβ is a much weaker transcriptional activator than ERα. In turn, the oestrogen response element activator protein-1 is responsible for the proliferation processes induced by E2. Nevertheless, E<sup>2</sup> has no effect on ERβ, which may indicate that ERβ can modulate ERα activity in cells where both receptors are co-expressed. However, in many cells, ERβ is expressed in the absence of ERα, and in these cells, ERβ remains active independently of ERα [56]. This is the case in epithelial cells of the colon [65], where ERβ-driven enhanced metabolic processes occur [55].

Preclinical models have shown that ERα activity can be modulated by ERβ, which inhibits oestrogen-dependent proliferation and promotes apoptosis [66]. There is evidence that uncontrolled proliferation, progression, and/or failure to respond to treatment may disrupt oestrogen signalling. ERα may be associated with proliferative disorders, and it can be used to determine the efficacy of hormone therapy. In contrast, ERβ is present in healthy colonic mucosa and its expression is significantly delayed in colonic proliferative disorders [44,56].

#### 3.1.4. Summary

The observed silencing of ERs indicates that: (i) low monotonic doses of ZEN elicited the strongest responses on analytical dates III, IV, and VI, whereas on the last date, the prepubertal gilts developed tolerance to the analysed undesirable substance; (ii) ERα expression was increased in the duodenum and ERβ expression was increased in the descending colon; (iii) the opposite was observed in the caecum and the ascending colon; and (iv) the gastrointestinal tract of sexually immature gilts was adapted to the presence of ZEN in the feed after the first two exposure dates. Due to the very low concentrations of E2, ZEN was bound to ERs and triggered qualitative changes in ERs during the successive weeks of the experiment (activation?). Qualitative changes were manifested by a shift in the ER expression levels from absorption level 0 to 3, especially ERβ expression in the descending colon. The observed shift in ERβ expression suggests that zearalenone and its metabolites are involved in the control of proliferation and apoptosis in enterocytes.

#### **4. Materials and Methods**

#### *4.1. Experimental Animals*

The experiment was carried out at the Department of Veterinary Prevention and Feed Hygiene of the Faculty of Veterinary Medicine of the University of Warmia and Mazury in Olsztyn, Poland, on 36 clinically healthy gilts with an initial body weight (BW) of 25 ± 2 kg. Pre-puberty gilts were kept in groups and had ad lib access to water.
