**1. Introduction**

Mycotoxins, secondary fungal metabolites, are frequent contaminants of cereals and cereal products. Delivered via these plants and products, they pose a serious health threat to humans and animals [1–3]. Whereas mycotoxins are sometimes regarded as stressors [4,5], they have a target-specific mode-of-action and so are true toxins rather than stressors per se [6]. The most common and important mycotoxins in Europe, produced by fungi of the *Fusarium* family, are deoxynivalenol (DON), and zearalenone (ZEN). Pigs are particularly sensitive to DON [7], but they also show high sensitivity to ZEN [8].

In pigs, high doses of DON cause reduced appetite, complete anorexia, vomiting [9], and reproductive disorders [10]. Absorption of DON is rapid, and the toxin reaches the peak

plasma concentrations within 30 min of oral administration [11]. The majority of ingested DON is absorbed in the proximal part of the small intestine [12]. In the liver, DON is metabolized into de-epoxy-DON [13]. An evidence showed that chronic ingestion of DON at low doses, which is clinically asymptomatic, alters the mucosal epithelial cells and villi of the small intestine [14–17] and affects the defense mechanisms of the large intestine [18].

ZEN and its metabolites are agonists of estrogen receptors, and they compete with endogenous hormones for the binding sites of estrogen receptors [2,19]. Treatment with ZEN leads to precocious puberty, reproductive disorders, and hyperestrogenism [20]. According to previous studies, domestic pigs are particularly sensitive to the estrogenic effect of ZEN owing to the very rapid and large (approximately 80–85%) absorption of the toxin in their digestive system [21]. In the liver, ZEN is metabolized into α- and β-zearalenol, which are considered more toxic than ZEN. The ratio of these two metabolites to each other is species-specific. Most studies showed that α-zearalenol predominates in pigs [13,21–25]. This form is more active than β-zearalenol, which provides another explanation for the high sensitivity of pigs to ZEN content in feed [21,24]. ZEN at low doses has deleterious effects on the morphology of the small intestine [16,17] and affects the defense mechanisms of the liver [26] and the large intestine [18].

During exposure to environmental mycotoxins, animals often encounter mixtures of toxins, rather than single toxins. Therefore, the toxicity of mycotoxins needs to be addressed in the context of their mixtures to assess their health risk [27–29]. However, studies on the effects of mycotoxin combinations are relatively rare, and their results are very ambiguous [27,28,30,31].

The liver and gastrointestinal parts of the digestive system are major sites of mycotoxin metabolism [32]. However, there is little molecular, metabolic, and histological research on the effects of mycotoxins on the liver. In addition, the outcomes of these studies are not conclusive [15,28]. In pigs, some histological changes have been observed in the liver as a result of the individual or combined effects of DON and ZEN [15,33]. Contrastingly, Renner and coauthors [34] showed that chronic dietary DON exposure (4 weeks, 4.59 μg/kg BW) did not affect the histology of pig liver.

The purpose of this study was to determine the effects of single and combined administrations of DON (12 μg/kg BW) and ZEN (40 μg/kg BW) to pigs for 1, 3, or 6 weeks on the histology and ultrastructure of the liver. The selection of the doses used has been widely discussed in our previously published papers [16–18].

### **2. Results and Discussion**

### *2.1. Light Microscopy Study*

### 2.1.1. Architecture of the Liver

The livers of control pigs had the typical structural characteristic for this species (Figures 1A and 2A). The hepatic lobules were neatly outlined by an envelope of fibrous connective tissue, which interconnected the portal areas. Within each lobule, hepatocytes were arranged in linear cords radiating from the central vein and separated by sinusoids, which had a uniform diameter over their entire length. The limiting plate of hepatocytes bordered the lobule interior from the connective tissue.

Prominent qualitative differences in the liver architecture were observed between the control group and the DON and DON + ZEN groups. The perilobular connective tissue widened and contained greater amounts of collagen fibers starting from the first week of treatment (Figures 1B and 2B, Table 1). In addition, connective tissue penetrated into the lobule and caused disruption of the limiting plate (Figure 1C). After 6 weeks of treatment, the presence of small scars was pronounced (Figure 1D). The disorganization of hepatic cords was observed in the livers of pigs fed diets contaminated with DON and DON + ZEN for 3 and 6 weeks.

**Figure 1.** (**A**,**B**) Architecture of the liver in a control pig, 6th week of experiment (**A**) and in a pig receiving deoxynivalenol (DON) for 6 weeks (**B**). Note the thickening of the interlobular septa. (**C**) A strip of connective tissue in the liver lobule. A pig was treated with DON for 3 weeks. (**D**) Focal fibrosis inside the liver lobule of a pig treated with DON for 6 weeks. Note that the central vein is surrounded by connective tissue. Figures (**A**–**D**) show hematoxylin and eosin stained sections.

**Figure 2.** (**A**,**B**) Trichrome staining of the liver in a control pig, 6th week of experiment (**A**) and in a pig receiving DON for 6 weeks (**B**). Pay attention to the thickness of the interlobular septa and the content of collagen fibers in them.


**Table 1.** Semiquantitative analysis of fibrosis.

Description of the score used: 0, no fibrosis, perilobular tissue septa with small number of collagen, characteristic for the pig liver; 1, mild enlargement of portal areas and perilobular tissue septa; 2, large enlargement of portal areas and perilobular tissue septa, limiting plate fibrosis; 3, partial cirrhosis of some lobules. The values presented are means.

The administration of DON and DON + ZEN significantly increased the thickness of perilobular connective tissue in the liver after 3 and 6 weeks of treatment. No significant differences were observed in the thickness of septa after ZEN treatment (Figure 3A). The cross-sectional area of the lobules did not differ between the control group and the groups treated with mycotoxins (Figure 3B).

The data show that DON and DON + ZEN, but not ZEN, affected the qualitative and quantitative characteristics of the liver architecture. The administration of DON and DON + ZEN resulted in an excessive accumulation of extracellular matrix, including collagen, in perilobular connective tissue, starting from the first week of treatment. Moreover, treatment with DON + ZEN for 3 and 6 weeks caused a progressive accumulation of extracellular matrix in the liver parenchyma. We interpreted the intra-lobular presence of mild scars as early fibrosis. The process of liver fibrosis is most often a consequence of chronic diseases, but it can also be caused by many factors that induce acute damage [35,36]. In experimental studies, administration of xenobiotics induced parenchymal fibrosis similar to cirrhosis in rodent liver [36]. The intensity of the fibrosis process caused by external factors

shows large differences [37]. Previous studies have shown that mycotoxins affect the histology of pig liver [15,34,38]; however, the authors did not report the process of liver fibrosis.

**Figure 3.** (**A**) Thickness of the connective tissue septa. (**B**) Cross-sectional area of the liver lobules. C—control group, Z—group treated with zearalenone (ZEN), D—group treated with deoxynivalenol (DON), and M—group treated with DON and ZEN. The values are presented as mean ± standard deviation. Bars labeled with different small lower-case letters differ significantly at *p* ≤ 0.05.

Disorganization of hepatic cords as one of the main lesions in piglets after chronic exposure to DON was observed by Gerez et al. [15]. In our study, similar changes were found after 6 weeks of DON and DON + ZEN ingestion.

#### 2.1.2. Microscopic Liver Scoring

The analysis using modified microscopic liver scoring (histology activity index (HAI)) comprised six histological criteria. Exemplary microphotographs of changes included in the individual criteria are presented in Figure S1 (Supplementary Material). The cumulative score and the contribution of each criterion are presented in Figure 4. The largest histopathological lesions of the liver were observed in pigs treated with DON for 1 week or DON + ZEN for 1, 3, 6 weeks. The total HAI score for DON resulted mainly from increased portal inflammation and focal lytic necrosis, whereas that for DON +

ZEN resulted from increased portal and periportal inflammation, confluent necrosis, and especially focal lytic necrosis, compared with control pigs. The total HAI scores in pigs receiving DON and DON + ZEN for 1, 3, and 6 weeks were significantly higher than in that in the control group.

**Figure 4.** Histopathological score of the examined livers according to the histology activity index (HAI) modified by Stanek et al. [38]. Six histopathological parameters were scored in hematoxylin and eosin (HE)-stained tissues (see Figure S1 in Supplementary material), and the cumulative HAI score was calculated for each experimental group. The mean value of the cumulative HAI scores in each group is represented by the total height of the bar. C—control group, Z—group treated with zearalenone (ZEN), D—group treated with deoxynivalenol (DON), and M—group treated with DON and ZEN. The values of the cumulative HAI score labeled with different lower-case letters above the bars differ significantly at *p* ≤ 0.05.

The foci of hepatocyte necrosis with lymphocytic infiltrates, portal, periportal, and acinar inflammation occur in the liver as a result of drug usage, intoxication, or viral and bacterial infections [39]. The occurrence of necrosis is also associated with the activation of immune mechanisms in response to the adverse effects of toxic substances [36,40]. These changes are generally associated with pathological symptoms; however, they also occur in seemingly healthy animals, and, in this case, they are considered as preclinical conditions [41].

The results of our HAI analysis showed that the liver, being the first metabolic station, is affected to varying degrees by the administered toxins. Treatment with ZEN had no significant effect on liver histopathology. However, DON and DON + ZEN significantly affected pig liver regardless of the administration period of toxins. The highest intensity of necrosis foci was observed in the livers of animals receiving both mycotoxins. The results obtained may be interpreted as an effect of the toxic effects of DON and the synergistic toxic effect of the combined toxins. In addition, they may be caused by the stimulation of inflammatory processes in response to the administered mycotoxins. This is due to the pro-inflammatory actions of DON and ZEN [42].

The results of previous studies on the effect of mycotoxins on the histology of the liver parenchyma are diverse. Histological changes, including the disorganization of hepatic cords, the cytoplasmic vacuolization of hepatocytes, megalocytosis, and focal necrosis, were reported in pigs subjected to 28 days of diet contaminated with DON (3 mg/kg) or DON (3 mg/kg) + NIV (1,5 mg/kg) + ZEN (1,5 mg/kg) [15]. Acute exposure to DON at a dose of 1 mg/kg BW for 6 and 24 h led to apoptosis of hepatocytes [33]. On the other hand, exposure to DON at a concentration of 4.59 mg/kg feed for 28 days [34] or 3.1 mg/kg feed for 37 days did not affect liver HAI [38].

Megalocytosis and cytoplasmic vacuolization were the main histological lesions reported in piglets subjected to chronic exposure to DON [15]. Magalocytosis potentially indicates irreversible hepatocyte injury [43]. However, in our study, this phenomenon concerned only a few hepatocytes, and there were no differences in their presence between control and experimental pigs.

### 2.1.3. Hepatic Sinusoids

The livers of pigs from all experimental groups were characterized by significant dilatation of the hepatic sinusoids in Zone III (drainage) of acinus compared with those of control animals (Table 2; Supplementary material, Figure S2). The largest sinusoidal dilatation was observed in pigs receiving DON + ZEN. In these animals, significant sinusoidal dilatation occurred after 1, 3, and 6 weeks of mycotoxin administration. In the groups treated with ZEN alone and DON alone, sinusoidal dilatation decreased after 3 and 6 weeks (Table 2).


**Table 2.** Semiquantitative analysis of sinusoidal dilatation.

Description of the score used in estimating changes: 0, absence; 1, sporadic presence; 2, few presence; 3, middle presence; and 4, numerous presence.

Under physiological conditions, the liver sinusoids are characterized by a constant, uniform diameter. Sinusoidal dilatation in Zone I has been observed in pregnancy, long-term steroid administration, and hepatomegaly [44]. This phenomenon also occurs after exposure to vinyl chloride and arsenic [40], and may also be the result of the administration of certain chemotherapy drugs [45]. Sinusoidal dilatation in Zone III was observed in the case of local disorders of blood flow in the portal veins or hepatic veins [40]. In our study, pronounced sinusoidal dilatation in Zone III was observed as a result of DON and ZEN administration, which suggested a disruptive effect of mycotoxins on blood flow in the liver. However, the histological findings did not provide any suggestions about the possible cause of this disturbance.

### 2.1.4. Glycogen Storage

We used periodic acid-Schiff (PAS) staining to evaluate changes in glycogen content. An increase in glycogen deposits was observed in the groups treated with ZEN, DON and DON + ZEN compared with those in the control group after the first week of experiment (Figure 5, Table 3). The staining was particularly intense in Zone I. After 3 and 6 weeks of treatment, there were no differences in glycogen content between the control and experimental groups.


**Table 3.** Semiquantitative analysis of glycogen content in the central and peripheral parts of liver lobules.

For a description of the score, see Table 2.

**Figure 5.** (**A**,**B**) Distribution of glycogen in hepatocytes in a control pig (**A**) and a pig treated with ZEN + DON (**B**). The first week of experiment. Periodic acid-Schiff reaction. Note the increase in the amount of glycogen in a pig treated with mycotoxins.

The liver is a major site of glycogen accumulation, and the main role of glycogen in the liver is to store glucose for release during fasting. The amount of stored glycogen depends on nutritional and living factors and shows large individual fluctuations [36,40]. In previous studies, no effects of mycotoxins on glycogen storage were observed in pig liver [7]. The results of our current studies showed that the effect of DON and ZEN on liver glycogen was temporary.
