*2.7. Correlation Coefficients*

Linear correlation coefficients (Pearson's *r*) were calculated to assess the relationships between ZEN concentrations in the bone marrow microenvironment and the values of blood morphotic components (RBC, WBC, and PLT) on different exposure dates and in different experimental groups (see Table 6). These blood components were selected because erythropoiesis, leukopoiesis, and thrombopoiesis take place during the last processes of hemopoiesis in the bone marrow environment. The strength of the examined correlations can be evaluated based on the values presented in Table 6 [36]. A correlation is negative when *r* < 0. The strength of correlations is evaluated on the following scale: *r* < 0 to −0.2, no negative linear correlation; *r* = −0.21 to −0.39, weak negative correlation; *r* = −0.4 to −0.69, moderate negative correlation; *r* = −0.7 to −0.9, relatively strong negative correlation; *r* > −0.9, very strong negative correlation. The correlation coefficient denotes a statistical relationship, and it does not imply a cause-effect relationship.

In a negative correlation, an increase in the value of one variable is accompanied by a decrease in the value of another variable, which was observed in this study. A negative correlation coefficient must be lower than −0.710 (relatively strong negative correlation) to explain more than 50% of the variance in the examined values. A relatively strong negative correlation was noted in PLT values on D1 in group ZEN5 and on D3 in groups ZEN5 and ZEN10 (see Table 6), in WBC values on D2 in groups ZEN5 and ZEN15, and in RBC values on D1 in group ZEN15, on D2 in group ZEN10, and on D3 in groups ZEN5 and ZEN15. A moderate positive correlation was observed in PLT values on D1 in group ZEN10, which could be attributed to the lowest ZEN concentration at a relatively low value of CF. These findings indicate that ZEN dose and time of exposure exert a negative effect on the haematopoietic activity of the evaluated blood morphotic components in the bone marrow microenvironment.

*Toxins* **2022**, *14*, 105


**Table 3.** Selected white blood cell markers in group ZEN10 on different analytical dates (*x*, SD).

HCT, Haematocrit; MCV, Mean Corpuscular Volume; MCHC, Mean Corpuscular Haemoglobin Concentrations; HDW, Haemoglobin Distribution Width. Statistical symbols: a , relative to date 7; b , relative to date 8; c , relative to date 9; d , relative to date 10. Statistically significant differences: a,b,c and d at *p* ≤ 0.05; aa,bb and cc at *p* ≤ 0.01.


**Table 4.** Selected haematological parameters in group ZEN15 on different analytical dates (*x*, SD).

Key: Group ZEN15, 15 µg ZEN/kg BW; WBC, White Blood Cells; NEUT, Neutrophils; LYMPH, Lymphocytes; EOS, Eosinophils; MPV, Mean Platelet Volume. Statistical symbols: <sup>a</sup> , relative to date 1; <sup>b</sup> , relative to date 3; c , relative to date 4; <sup>d</sup> , relative to date 5; <sup>e</sup> , relative to date 7; <sup>f</sup> , relative to date 10. Statistically significant differences: a,b,c,d,e and <sup>f</sup> at *<sup>p</sup>* <sup>≤</sup> 0.05; aa,cc,ee and ff at *<sup>p</sup>* <sup>≤</sup> 0.01.

**Table 5.** Platelet count in the experimental groups on different analytical dates (*x*, SD).


Key: PLT count in groups 1, 2, and 3; PLT clumps in groups 2 and 3. Statistical symbols: <sup>a</sup> , relative to date 1; b , relative to date 3; <sup>c</sup> , relative to date 7; <sup>d</sup> , relative to date 9; <sup>e</sup> , relative to date 10. Statistically significant differences: a,b,c,d and <sup>e</sup> at *<sup>p</sup>* <sup>≤</sup> 0.05; bb,dd and ee at *<sup>p</sup>* <sup>≤</sup> 0.01.

**Table 6.** Correlation coefficients (Pearson's *r*).


Key: Ratio of ZEN concentrations in the bone marrow microenvironment and blood morphotic components (RBC, WBC, and PLT) on different exposure dates (D1, D2, and D3: exposure days 7, 21, and 42, respectively). The mycotoxin was administered once daily before morning feeding (group ZEN5, 5 µg ZEN/kg BW; group ZEN10, 10 µg ZEN/kg BW; group ZEN15, 15 µg ZEN/kg BW). Samples were collected from pre-pubertal gilts immediately before slaughter.

#### **3. Discussion**

In this study and in our previous research, attempts were made to fill the gap in knowledge about the in vivo effects of low and very low doses of ZEN and its metabolites on mammals [7,16,28,29]. The present study was undertaken to address the general scarcity of published data on the influence of feed contamination with ZEN and its metabolites on haematopoietic activity and the bone marrow microenvironment in pre-pubertal gilts. In this study, selective ZEN doses, in each range of their values, influenced ZEN concentrations in the bone marrow microenvironment on different dates of exposure.

#### *3.1. Zearalenone and Its Metabolites in the Bone Marrow Microenvironment*

The first exposure date (D1) marks the end of stimulatory processes after seven days of exposure to an undesirable substance, such as ZEN. The final effects of adaptive processes [37], accompanied by decreased haematopoiesis in the bone marrow microenvironment, are manifested on D2 [28]. These processes can be accompanied by increased Ca2+ levels, in particular in the mitochondria [38], and changes in the activity of selected enzymes, such as hydroxysteroid dehydrogenases [39], thus disrupting steroidogenesis [13]. Excess ZEN (which was not biotransformed or was recovered after enterohepatic recirculation) probably led to hyperestrogenism on D3. The resulting "free ZEN" can affect steroidogenesis [28].

The present findings demonstrate that the process of ZEN absorption in the bone marrow microenvironment was highly individualised, due to considerable differences in standard deviation values and specifically due to the absence of ZEN metabolites in the analysed tissue in pre-pubertal gilts. Low ZEN concentrations and the absence of ZEN metabolites in the bone marrow microenvironment on all exposure dates confirm the previous suggestion that the physiological demand for exogenous oestrogen-like compounds [28] in pre-pubertal gilts is high due to supraphysiological hormone levels [40]. However, the values noted in this study were higher than those reported in the blood serum [28] or the heart muscle [19] of the same pre-pubertal gilts. This observation could be attributed to a much higher number of oestrogen receptors in the bone marrow microenvironment than in other tissues or organs [41,42]. In menopausal females, the reproductive system, followed by the bone marrow microenvironment, are most sensitive to the presence of gonadal hormones (long-range signals) [43]. This order is reversed in maturing females (the signals are initiated by oestradiol–oestrogen receptors [44]), which could explain higher ZEN concentrations in the bone marrow environment of pre-pubertal gilts. Exposure to higher ZEN doses leads to the production of "free ZEN", which plays different and not always positive roles. Oestradiol and "free ZEN" levels increase proportionally to the ZEN dose. These compounds decrease the concentrations of progesterone and testosterone [28], but they also play important protective roles in skeletal homeostasis [42], which is an important consideration in pre-pubertal gilts.

The above observations are confirmed by ZEN concentrations on D1 in all experimental groups. According to most researchers, metabolites are produced during the biotransformation of ZEN in pigs [45]. Therefore, the absence of metabolites in the bone marrow microenvironment could be regarded as a consequence of biotransformation processes in pre-pubertal gilts, where ZEN does not induce hyperestrogenism, but compensates for the physiological deficiency of endogenous oestrogens [40]. The above situation could result from a higher demand of the host organism for compounds with high estrogenic activity, such as ZEN and its metabolites (whose concentrations were below the sensitivity of the method) [46], or perhaps these were the physiological values that are required for the body to function [5]. This implies that it was a satisfactory effect of exposure to very low doses of ZEN during the ongoing detoxification processes. Other explanations are also possible: (i) the seventh day of exposure to an undesirable substance such as ZEN marks the end of adaptive processes [37], or (ii) ZEN is utilized as a substrate that regulates the expression of genes encoding HSDs, which act as molecular switches for the modulation of steroid hormone pre-receptors [28], or (iii) undesirable substances undergo enterohepatic circulation before they are completely eliminated from the body [47], and/or (iv) the gut microbiome exhibits specific behaviour during exposure to ZEN [29]. The above factors, alone or in combination, could affect the concentrations of ZEN in the bone marrow microenvironment, because ZEN is a promiscuous compound that can decrease the values of hematopoietic parameters [48].

On the other two exposure dates, similar correlations were noted in the average concentrations of ZEN in the bone marrow microenvironment. On these dates, ZEN concentrations were higher, but still low or very low, and ZEN metabolites were not detected. These observations could be attributed to the accumulation of ZEN (due to the saturation of active oestrogen receptors [48] and other factors that determine the concentrations of steroid hormones [45]) during prolonged exposure. However, due to the general scarcity of studies investigating the influence of low ZEN doses on the bone marrow environment and haematopoiesis, our results cannot be compared with the findings of other authors. According to the hormesis principle, exposure to very low ZEN doses affects the synthesis and secretion of sex steroid hormones. Therefore, very low ZEN doses were biotransformed in an identical manner, but the parent compound (ZEN) and, possibly, its metabolites were utilized more efficiently or completely by the macroorganism and, in particular, the bone marrow microenvironment. The interactions between endogenous and environmental (exogenous) steroids could also be influenced by other endogenous factors [49].

#### *3.2. White Blood Cells*

Haematological parameters somewhat exceeded the upper reference limit, in particular the percentage of lymphocytes, which increased in groups ZEN5 and ZEN10 on D1-relative lymphocytosis [50]. The increase in the percentage of lymphocytes was accompanied by a decrease in the percentage of neutrocytes. Contrary results were reported by [51], where very high ZEN concentrations exerted apoptotic effects on lymphocytes in vitro. In the present study, apoptotic effects were observed only on blood sampling date 10.

Based on the observations made by Etim et al. [52], the fact that haematological parameters were within the reference ranges indicates that feed composition had no negative effect on their values throughout the experiment. However, in view of the results of another study by Etim et al. [31], the fact that the values of WBC, NEUT, and LYMPH were within the reference ranges, but were lower than in group C, suggests that feed components did not stimulate the immune system. The neutrophil-to-lymphocyte ratio must be analysed (see Figure 3) to validate the hypothesis that the immune system was more stimulated by nutritional factors (including ZEN) and dietary stress than by functional stress [32,53]. The sizeable increase in the percentage of EOS in total WBC counts in group ZEN5, relative to group C during the entire experiment, should also be considered. In the remaining experimental groups, the percentage of EOS was lower than in group C. This observation suggests that ZEN can exert allergizing effects, which was confirmed in our previous study [54].

The observed decrease in WBC counts in all groups during the experiment points to probable immune suppression (as a consequence of gilt maturation), but this trend was least pronounced in group ZEN5. It should also be noted that during ZEN exposure, lymphocytes migrate to tissues with a physiologically higher number of oestrogen receptors, which intensifies proliferation processes and stimulates vascular filling [55]. Similar observations were made by Przybylska-Gornowicz et al. [56].

The decrease in WBC counts in the experimental groups suggests that ZEN affects adaptive processes by inducing reversible changes in the morphological and functional organization of the local immune system [50,55].

#### *3.3. Red Blood Cells*

Red blood cell counts remained stable throughout the experiment. Considerable differences were noted on blood sampling dates 4 and 10, with a rising trend in group C. In the experimental groups, a decrease in Fe levels [7] had no effect on RBC counts. Haemoglobin distribution width values were stable, but very low, and they were accompanied by a drop in glucose levels [7]. The above values are characteristic of pre-pubertal animals [57].

*3.2. White Blood Cells* 

previous study [54].

10.

**Figure 3.** Selected haematological parameters in groups **(** *x* **).** Key: C, control group; Group ZEN5, 5 **Figure 3.** Selected haematological parameters in groups (*x*). Key: C, control group; Group ZEN5,

μg ZEN/kg BW; Group ZEN10, 10 μg ZEN/kg BW; Group ZEN15, 15 μg ZEN/kg BW.

5 µg ZEN/kg BW; Group ZEN10, 10 µg ZEN/kg BW; Group ZEN15, 15 µg ZEN/kg BW.

**Neutrocytes/Lymphocytes Eosinophils %**

of active oestrogen receptors [48] and other factors that determine the concentrations of steroid hormones [45]) during prolonged exposure. However, due to the general scarcity of studies investigating the influence of low ZEN doses on the bone marrow environment and haematopoiesis, our results cannot be compared with the findings of other authors. According to the hormesis principle, exposure to very low ZEN doses affects the synthesis and secretion of sex steroid hormones. Therefore, very low ZEN doses were biotransformed in an identical manner, but the parent compound (ZEN) and, possibly, its metabolites were utilized more efficiently or completely by the macroorganism and, in particular, the bone marrow microenvironment. The interactions between endogenous and environmental (exogenous) steroids could also be influenced by other endogenous factors [49].

Haematological parameters somewhat exceeded the upper reference limit, in particular the percentage of lymphocytes, which increased in groups ZEN5 and ZEN10 on D1 relative lymphocytosis [50]. The increase in the percentage of lymphocytes was accompanied by a decrease in the percentage of neutrocytes. Contrary results were reported by [51], where very high ZEN concentrations exerted apoptotic effects on lymphocytes in vitro. In the present study, apoptotic effects were observed only on blood sampling date

Based on the observations made by Etim et al. [52], the fact that haematological parameters were within the reference ranges indicates that feed composition had no negative effect on their values throughout the experiment. However, in view of the results of another study by Etim et al. [31], the fact that the values of WBC, NEUT, and LYMPH were within the reference ranges, but were lower than in group C, suggests that feed components did not stimulate the immune system. The neutrophil-to-lymphocyte ratio must be analysed (see Figure 3) to validate the hypothesis that the immune system was more stimulated by nutritional factors (including ZEN) and dietary stress than by functional stress [32,53]. The sizeable increase in the percentage of EOS in total WBC counts in group ZEN5, relative to group C during the entire experiment, should also be considered. In the remaining experimental groups, the percentage of EOS was lower than in group C. This observation suggests that ZEN can exert allergizing effects, which was confirmed in our

Erythrocytes, the main morphotic components of the blood, are highly sensitive to endogenous factors such as oxidation. Red blood cells have effective antioxidant mechanisms that remove reactive oxygen species that are produced outside or inside erythrocytes [58]. According to Tatay et al. [59], ZEN is a powerful antioxidant, and one of the side effects of ZEN exposure is a decrease in RBC counts, which could be attributed to "antioxidant competition". Similar suggestions were made by other authors who reported that ZEN induced eryptosis in mature human erythrocytes [60]. According to the cited authors, eryptosis can take place even at low concentrations of ZEN.

Eryptosis can also be induced by a mechanistic increase in intracellular Ca2+ levels [61,62], oxidative stress, energy depletion [63], and exposure to undesirable substances such as ZEN [56]. However, the loss of extracellular Ca2+ can also inhibit the eryptotic effect [64] due to anaemia and microcirculatory disorders. Microcirculatory disorders are observed in various tissues during exposure to ZEN, and they lead to numerous extravasations and vasodilation [65,66] in pre-pubertal female mammals, due to the relaxation of smooth muscle cells in vessel walls, in particular in large veins, large arteries, and smaller arterioles. An analysis of RBC values clearly indicates that all of the tested ZEN doses decreased erythrocyte counts and induced eryptosis [41].

#### *3.4. Platelets*

Platelet counts were above the norm in all groups, which is indicative of thrombocytosis [67]. The percentage of PLT Clumps was also elevated (pseudothrombocytopenia). These results could be explained by inadequate analytical standards or blood artefacts [68]. It should also be noted that platelets participate in the early stages of liver regeneration, but the underlying mechanisms of action have not yet been fully elucidated [69]. The liver is the main organ responsible for the biotransformation (detoxification) of undesirable substances such as ZEN [13]. The observed differences in values can be attributed to: (i) intensified proliferation in group ZEN5, and (ii) the influence of substances that stimulate proliferation in maturing animals [7,67,70].

In an analysis of PLT values, a positive correlation coefficient was noted only once. On the remaining exposure dates, the correlation coefficient assumed negative values, and it revealed the presence of a relatively strong negative correlation on three occasions. The strongest negative correlations were noted on D1 (during the highest exposure to ZEN) and D3 (during prolonged hyperestrogenism resulting from the accumulation of ZEN).

#### *3.5. Summary and Conclusions*

The exposure to very low doses of ZEN, administered *per os* to pre-pubertal gilts over a period of 42 days, induced a specific response in the bone marrow microenvironment. Zearalenone metabolites were not detected, and the concentration of the parent compound was higher in the bone marrow microenvironment than in other tissues. Low doses of ZEN affected haematopoiesis and induced completely different responses in selected haematological parameters than high doses (LOAEL). Zearalenone's multidirectional effects on blood morphotic components at the beginning and end of the experiment were probably modified by compensatory and adaptive mechanisms. Zearalenone also induced reversible changes in the morphological and functional organization of the local immune system. The values of the analysed haematological parameters pointed to eryptosis and the stimulation of detoxifying organs such as the liver, where the intensity of biotransformation processes decreases under exposure to low doses of the studied mycotoxin. Further research is needed to elucidate the mechanisms responsible for changes in the haematological parameters of pre-pubertal gilts exposed to low doses of ZEN.

The results of these observations should play an important role in clinical veterinary practice, where ZEN could become a biomarker of the subclinical state of pre-pubertal gilts.

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

### *4.1. General Information*

All experimental procedures involving animals were carried out in compliance with Polish regulations setting forth the terms and conditions of animal experimentation (Opinions No. 12/2016 and 45/2016/DLZ of the Local Ethics Committee for Animal Experimentation of 27 April 2016 and 30 November 2016).

#### *4.2. Experimental Feed*

Analytical samples of ZEN were dissolved in 96 µL of 96% ethanol (SWW 2442-90, Polskie Odczynniki SA, Katowice, Poland) in doses appropriate for different BW. Feed was placed in gel capsules saturated with the solution and kept at room temperature to evaporate the alcohol. All groups of gilts received the same feed throughout the experiment. The animals were weighed at weekly intervals, and the results were used to adjust individual mycotoxin doses [12,16,29].

The feed administered to all experimental animals was supplied by the same producer. Friable feed was provided *ad libitum* twice daily, at 8:00 a.m. and 5:00 p.m., throughout the experiment. The composition of the complete diet, as declared by the manufacturer, is presented in Table 7 [12,16,29].

**Table 7.** Declared composition of the complete diet.


<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 proximate chemical composition of the diets fed to pigs in groups C, ZEN5, ZEN10, and ZEN15 was determined using the NIRS™ DS2500 F feed analyser (FOSS, Hillerød, Denmark), a monochromator-based NIR reflectance and transflectance analyser with a scanning range of 850–2500 nm [12,16,29].

#### Toxicological Analysis in Feed

Feed was analysed for the presence of mycotoxins and their metabolites: ZEN, α-ZEL, and deoxynivalenol (DON). Mycotoxin concentrations in feed were determined by separation in immunoaffinity columns (Zearala-TestTM Zearalenone Testing System, G1012, VICAM, Watertown, MA, USA; DON-TestTM DON Testing System, VICAM, Watertown, MA, USA). The feed samples were ground in a laboratory mill. 25 g of the ground sample was taken up in 150 mL of acetonitrile (90%) to extract the mycotoxins. 10 mL of the resulting solution was withdrawn and diluted with 40 mL of water. Again, 10 mL of the next solution was taken and passed through the immunoaffinity column (VICAM). The immunoaffinity bed in the column was subsequently washed with demineralized water (Millipore Water Purification System, Millipore S.A., Molsheim, France). The column was eluted with 99.8% methanol (LIChrosolvTM, No. 1.06 007, Merck-Hitachi, München, Germany) to wash away the bound mycotoxin. The obtained solutions were analyzed by high-performance liquid chromatography (HPLC system, Hewlett Packard type 1050 and 1100, Beeston Court, United Kingdom) coupled with a diode array detector (DAD) and a fluorescence detector (FLD) and chromatography columns (Atlantis T3, 3 µm 3.0 150 mm Column No. 186003723, Waters, AN Etten-Leur, Ireland). Mycotoxins were separated using a mobile phase of acetonitrile:water:methanol (46:46:8, *v*/*v*/*v*). The flow rate was 0.4 mL/min. The limit of detection was set at 5 µg/kg of feed for DON and 2 µg/kg of feed for ZEN, based on validation of chromatographic methods for the determination of ZEN and DON levels in feed materials and feeds. Chromatographic methods were validated at the Department of Veterinary Prevention and Feed Hygiene, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Poland [11].

#### *4.3. Experimental Animals*

The experiment was performed at the Department of Veterinary Prevention and Feed Hygiene of the Faculty of Veterinary Medicine at the University of Warmia and Mazury in Olsztyn Poland on 60 clinically healthy pre-pubertal gilts with initial BW of 14.5 ± 2 kg. The animals were housed in the same building, in adjoining pens with latticework walls, and had free access to water. They were randomly assigned to three experimental groups (group ZEN5, group ZEN10, and group ZEN15; *n* = 15) and a control group (group C; *n* = 15) [71,72]. Group ZEN5 gilts were orally administered ZEN at 5 µg/kg BW, Group ZEN10, 10 µg/kg BW, and Group ZEN15, 15 µg/kg BW. Group C animals were orally administered a placebo. Zearalenone was administered daily in gel capsules before morning feeding. Feed was the carrier, and group C pigs were administered the same gel capsules, but without mycotoxins [12,16,29].
