**The Effects of Mycotoxins on Human and Animal Health**

## **A Special Focus on the Cellular and Molecular Mechanisms Responsible for Mycotoxin Toxicity**

Editors

**Daniela Eliza Marin Ionelia Taranu**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editors* Daniela Eliza Marin National Institut for Research and Development for Biology and Animal Nutrition Romania

Ionelia Taranu National Institut for Research and Development for Biology and Animal Nutrition Romania

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Toxins* (ISSN 2072-6651) (available at: https://www.mdpi.com/journal/toxins/special issues/Mycotoxins Cellular Molecular).

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### **Contents**

### **About the Editors**

**Daniela Eliza Marin** is Senior Researcher in the Laboratory of Animal Biology and Scientific Secretary of the National Institute for Research and Development in Biology and Animal Nutrition, Balotes,ti, Romania. For more than 20 years, she has acquired expertise in mycotoxins and their effects on the animal health. She has coordinated two international projects and five national projects in toxicology and she was involved in more than 30 national and international projects as team member. She is a reviewer for more than 20 international journals and Academic Editor for the journals *Toxins* and *Romanian Biotechnology Letters*. Out of 120 published papers, more than 50 were published as the main author or co-author in peer-reviewed journals, mainly in Toxicology. She was Evaluator or Scientific Officer for different research agencies (UEFISCDI—Romania, National Science Centre—Polonia, Agence Universitaire de la Francophonie etc.).

**Ionelia Taranu** is a senior researcher at the National Research and Development Institute for Biology and Animal Nutrition (INCDBNA-IBNA), Balotesti, Romania. She is the head of Animal Biology Laboratory, IBNA, in which she coordinates a team of seven scientists and four technicians working on nutrition and immunology field. The research work of Dr. Ionelia TARANU is substantiated in 152 scientific publications (of which 71 are ISI), mainly dealing with the impact of feed quality (optimization of dietary composition) and feed security (feed contamination) on the performance, gastro-intestinal barrier and health of farm animals (immune response modulation to various dietary factors: novel feed, contaminants: mycotoxins, pre- and probiotics: bioactive compounds, fibers, bacteria, etc.). She is an independent evaluator expert (ID: EX2002B070822) for the EU Commission and invited expert of EFSA and other research agencies (UEFISCDI—Romania, ANCSI—Romania, Dutch Technology Foundation, National Science Centre-Polonia, FWO, Suisse).

### **Preface to "The Effects of Mycotoxins on Human and Animal Health"**

Mycotoxins are secondary metabolites mainly synthesized by fungi belonging to the Aspergillus, Fusarium and Penicillium genera. The contamination of cereals and agricultural products by mycotoxins may lead to a variety of adverse health effects, ranging from acute to chronic toxicity, with serious health implications for both humans and animals. The toxic effects of mycotoxins are characterized by lesions in target organs such as the liver and kidneys and in the pulmonary, intestinal, immune and central nervous systems, which can vary according to the type of toxin, time of exposure, mycotoxin concentration, animal species, sex, etc. At the cellular level, many processes such as apoptosis, genotoxicity, oxidative stress and inflammation have been described as being responsible for the effects of mycotoxins.

Immune response is one of the most affected by the exposure to mycotoxins. Chronic or sub-chronic exposure to mycotoxins is responsible for impaired immunity, decreased resistance to disease, increase in the predisposition of animals to infections and, consequently, decreased productivity. However, the mode of action of mycotoxins on different effectors of the immune response and the molecular mechanisms involved need further research. Another subject of interest is related to the concomitant exposure of human and animals to mycotoxins and other food/feed contaminants such as viruses, bacteria, pesticides, heavy metals, etc., which can have additive or synergic toxic effects and thus can amplify the mycotoxins toxicity.

Mycotoxins represents an important threat for the global economy and there is a permanent need to find new solutions for the mitigation of the mycotoxin effects in human and animals. Among them, the nutritional solutions represent a more convenient approach due to their low toxicity and their high content in bioactive compounds that can improve the health of human and animals exposed to mycotoxins.

The present Special Issue aims to improve our knowledge concerning mycotoxin toxicity by investigating their mode of action and the molecular mechanisms involved at the level of organs involved in the metabolism and excretion (liver and kidney) as well as at the level of the immune response effectors. Additionally, the efficacy of some dietary byproduct antioxidants, such as mitigation agents, in improving the health of animals exposed to individual mycotoxins or combination of mycotoxins was investigated.

> **Daniela Eliza Marin, Ionelia Taranu** *Editors*

## **Grape Seed Waste Counteracts Aflatoxin B1 Toxicity in Piglet Mesenteric Lymph Nodes**

### **Daniela Eliza Marin \*, Cristina Valeria Bulgaru, Cristian Andrei Anghel, Gina Cecilia Pistol, Madalina Ioana Dore, Mihai Laurentiu Palade and Ionelia Taranu**

National Institute for Research and Development for Biology and Animal Nutrition, INCDBNA Balotesti, Calea Bucuresti nr 1, Balotesti, 077015 Ilfov, Romania<EMAIL_ADDRESS>(C.V.B<EMAIL_ADDRESS>(C.A.A<EMAIL_ADDRESS>(G.C.P<EMAIL_ADDRESS>(M.I.D<EMAIL_ADDRESS>(M.L.P<EMAIL_ADDRESS>(I.T.) **\*** Correspondence<EMAIL_ADDRESS>Tel.: +40-213512082

Received: 13 November 2020; Accepted: 13 December 2020; Published: 15 December 2020

**Abstract:** Aflatoxin B1 (AFB1) is a mycotoxin that frequently contaminates cereals and cereal byproducts. This study investigates the effect of AFB1 on the mesenteric lymph nodes (MLNs) of piglets and evaluates if a diet containing grape seed meal (GSM) can counteract the negative effect of AFB1 on inflammation and oxidative stress. Twenty-four weaned piglets were fed the following diets: Control, AFB1 group (320 μg AFB1/kg feed), GSM group (8% GSM), and AFB1 + GSM group (8% GSM + 320 μg AFB1/kg feed) for 30 days. AFB1 has an important antioxidative effect by decreasing the activity of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) and total antioxidant status. As a result of the exposure to AFB1, an increase of MAP kinases, metalloproteinases, and cytokines, as effectors of an inflammatory response, were observed in the MLNs of intoxicated piglets. GSM induced a reduction of AFB1-induced oxidative stress by increasing the activity of GPx and SOD and by decreasing lipid peroxidation. GSM decreased the inflammatory markers increased by AFB1. These results represent an important and promising way to valorize this waste, which is rich in bioactive compounds, for decreasing AFB1 toxic effects in mesenteric lymph nodes.

**Keywords:** aflatoxin B1; grape seed meal; mesenteric lymph nodes; piglets

**Key Contribution:** Aflatoxin B1 induces inflammation and oxidative stress in mesenteric lymph nodes. In the mesenteric lymph nodes of AFB1-intoxicated piglets, grape seed meal can improve some markers of inflammation and oxidative stress.

#### **1. Introduction**

The gut is constantly exposed to potentially harmful contaminants from food or feed, such as mycotoxins [1]. Aflatoxin B1 is a mycotoxin produced by different species of fungi, especially *Aspergillus flavus* and *A. parasiticus* [2]. AFB1 is a potent carcinogen in humans and animals [3] and, for this reason, was classified in Group 1 of human carcinogens on the basis of toxicological data. Aflatoxins can contaminate different commodities such as cereals, nuts, dried fruits, and spices [4].

AFB1 is absorbed in the proximal part of the gut at high rates regardless of the species [5]. As the passage of the toxin across the intestinal barrier is very high, AFB1 can compromise the intestinal epithelium even before the absorption in the upper part of the gut. Indeed, it was shown that AFB1 induced inhibition of epithelial cell growth [6], an increase of apoptosis [6], and an increase of apoptotic markers (Bax and caspase-3) [7]. Additionally, AFB1 induced dysregulation of intestinal microbiota [8] and affected intestinal barrier function [9,10]. After absorption in the intestinal villi, the lipophilic compounds, such as AFB1, are first transported via the mesenteric lymphatic duct and then through the

thoracic duct before entering the blood circulation [11]. Mesenteric lymph nodes (MSLs) are important for the proper functioning of the immune system of the gut, acting as filters for nutrients and microbial substances that enter through the lymph in the intestinal lamina propria [12]. MSLs are responsible for tolerance induction to food particles, but they also contribute to the prevention of the systemic inflammatory response that can result from the bypass of portal circulation [13].

AFB1 is a potent immunomodulator as the toxin has been shown to impair both the innate and acquired immune responses [14,15]. However, until now, little is known about the effect of AFB1 on gut immunity and, especially, mesenteric lymph nodes as local effectors of the immune response in the gut.

Grape seed meal (GSM) represents the residue left after grape seed oil extraction; this waste can be recycled in order to enrich the feed rations of farm animals [16,17]. GSM has a high content of bioactive compounds: polyunsaturated fatty acids (mainly linoleic acid, n-6), in addition to oleic acid (n-9), dietary fiber, phenolic acids, resveratrol, proanthocyanidins, and flavonoids [18,19]. In living organisms, the beneficial effects of bioactive plant compounds as counteracting agents for AFB1 toxicity have been investigated both in vivo and in vitro [20]. An in-vitro study performed on human hepatocytes has shown that an extract from palm kernel cake, rich in phenolics, has beneficial effects by upregulating the genes involved in the response to oxidative stress and downregulating the genes associated with inflammation and apoptosis in AFB1-exposed hepatocytes [21]. Additionally, in vivo studies have demonstrated that in rats, oxidized tea polyphenols can form a complex with AFB1 that leads to a decrease of AFB1 absorption and, implicitly, a reduction of its toxicity [22].

In particular, the bioactive compounds from grape seed were shown to have beneficial effects on intoxication with AFB1. In a recent study, the supplementation of an AFB1-contaminated diet with grape seed proanthocyanidin extract reduced AFB1 residue in the liver and significantly mitigated the negative effects caused by AFB1 in broiler chickens [23]. Through their alimentation, rich in cereals, especially maize, pigs are particularly exposed to mycotoxins. We have demonstrated that GSM addition into a diet contaminated with AFB1 ameliorates histological liver injuries, reduces the parameters associated with oxidative stress in piglets [24], and is able to change the microbiota composition already affected by AFB1 exposure [25]. The present study is a continuation of our previous studies and aims to investigate if AFB1 can affect mesenteric lymph nodes as the gate of toxic substances in intestinal lamina propria. Additionally, this study aims to evaluate if the administration of a diet containing grape seed meal can counteract the negative effects of AFB1 on inflammation and oxidative response.

#### **2. Results**

#### *2.1. E*ff*ect of Grape Seed Meal and Aflatoxin B1 on Oxidative Damage in Mesenteric Lymph Nodes*

AFB1 induces a significative decrease in the activity of the enzymes involved in the response to oxidative stress, with 27% for catalase (CAT; *p* = 0.0248), 22% for glutathione peroxidase (GPx; *p* = 0.0034), and 10.2% for superoxide dismutase (SOD; *p* < 0.0001), as compared with the control piglets (Figure 1). Additionally, exposure of piglets to 320 μg AFB1/kg feed, significantly decreased the total antioxidant status (−20.5%; *p* = 0.0001) associated with an increase of lipid peroxidation (+52.3%; *p* = 0.0001), as assessed by the TBARS method. When piglet diet included 8% of grape seed meal, no effect was observed on the activity of the enzymes involved in antioxidative defense, total antioxidant capacity (TAC), or lipid peroxidation. However, the concomitant administration of both grape seed meal and toxin resulted in a significant increase of GPx (by 119.3%; *p* = 0.0048), SOD (by 105.9%; *p* = 0.0046), and TAC (by 112%; *p* = 0.0180), as compared with the AFB1 group, while decreasing lipid peroxidation (by 12.3%; *p* = 0.018). GSM was not able to significantly increase CAT activity (*p* = 0.331, as compared with the AFB1 group).

**Figure 1.** Effect of grape seed meal and AFB1 on oxidative damage in mesenteric lymph nodes. Data represent mean ± SD, *n* = 6. One-way ANOVA tests, followed by a Fisher's PSLD test, were used for statistical data analyses. a–c indicate statistically significant differences between treatments (*p* < 0.05).

Piglets' exposure to AFB1 contaminated diet was responsible for a a significant increase in the synthesis of IL-1β (*p* = 0.0068), IL-8 (*p* = 0.0010), and for a tendency of increase in the synthesis of IL-6, IFN-γ, and TNF-α (*p* > 0.05; Figure 2).

**Figure 2.** Effect of grape seed meal and AFB1 on inflammatory cytokine synthesis in mesenteric lymph nodes. Data represent means ± SD, *n* = 6. One-way ANOVA tests, followed by Fisher's PSLD test, were used for statistical data analyses. a–c indicate statistically significant differences between treatments (*p* < 0.05).

GSM diet did not affect the synthesis of inflammatory cytokines, the concentration of analyzed cytokines being very similar to the concentration measured in the mesenteric lymph nodes of the control group.

GSM administration to the piglets exposed to AFB1 (AFB1 + GSM diet) had an important anti-inflammatory effect, decreasing inflammatory cytokine synthesis towards the control level. Inclusion of GSM into the AFB1-contaminated diet (AFB1 + GSM group) was responsible for a decrease in the synthesis of IL-1β (8.57 ± 2.97 to 5.16 ± 1.75 μg/g tissue), IL-8 (34.7 ± 2.07 to 27.59 ± 3.07 μg/g tissue), IL-6 (81.9 ± 14.9 to 57.97 ± 9.88), and IFN-γ (8.13 ± 2.54 to 6.72 ± 1.47 μg/g tissue) towards the levels of the control group.

This decrease was significant for IL-1β (*p* = 0.0009), IL-6 (*p* = 0.044), and IL-8 (*p* = 0.0018) and not for IFN-γ (*p* = 0.371). GSM was not able to counteract the increase of TNF-α induced by the toxin (*p* = 0.389).

#### *2.3. E*ff*ect of Grape Seed Meal and Aflatoxin B1 on Cell Signaling Pathways in Mesenteric Lymph Nodes*

In order to validate the qPCR analysis and to demonstrate that GSM can restore the negative effects, such as inflammation and the oxidative response triggered by AFB1, we assessed by Western blot the expression of three proteins involved in NF-kB and Nrf2 signaling pathways: phospho-p38 MAPK, phospho-NF-kBp65, and Nrf2 (Figure 3A–C).

**Figure 3.** Effect of grape seed meal and aflatoxin B1 on cell signaling pathways in mesenteric lymph nodes: (**A**) phosphor-NF-kB1; (**B**) phosphor-p38 MAPK; (**C**) Nrf2. Data represent mean ± SD, *n* = 6. One-way ANOVA tests, followed by Fisher's PSLD test, were used for statistical data analyses. a–c indicate statistically significant differences between treatments (*p* < 0.05).

Feeding piglets with the diet contaminated with 320 μg AFB1/kg feed resulted in a significant nuclear increase in protein expression of both phosphorylated forms of NF-kBp65 (*p* = 0.049, Figure 3A) and p38 MAPK (*p* = 0.028, Figure 3B), while Nrf2 expression was not affected (*p* = 0.55, Figure 3C) in the AFB1 nuclear fraction when compared with control.

No significant alteration of the expression of these three proteins was noticed in the cytoplasm, although a slight increase of NF-kB expression in the AFB1 group was observed. As compared with the control, concomitant administration of both GSM and AFB1 resulted in a decrease of the expression of both phospho-p38 MAPK (4.86 ± 0.54 vs. 5.86 ± 0.59 A.U.) and phospho-NF-kBp65 (2.24 ± 0.28 vs. 2.19 ± 0.14 A.U.).

Additionally, the expression of Nrf2 increased toward the control level in the AFB1 + GSM group as compared with the control in both nucleus (1.64 ± 0.19 vs. 1.67 ± 0.69 A.U.) and cytoplasm (1.06 ± 0.11 vs. 1.17 ± 0.28 A.U.). The expression of the proteins involved in the inflammation signaling pathway decreased significantly in the AFB1 + GSM group as compared with the AFB1 group: −35% for phospho-p38 MAPK (*p* = 0.0017) and −27% for phospho-NF-kBp65 (*p* = 0.05), respectively.

#### *2.4. E*ff*ect of Grape Seed Meal and Aflatoxin B1 on mRNA Gene Expression Involved in Inflammation*

Exposure to AFB1-contaminated diet was responsible for a significant increase in the expression of the majority of the investigated genes. AFB1 contamination led to a highly significant increase in the expression of several proinflammatory markers: IL-8 (*p* = 0.001), IL-1β (*p* = 0.023), and IL-18 (0.07) (Table 1).

Looking at the gene expression encoding for signaling molecules with a key role in the inflammation pathway, a similar increase was observed for (i) transcription factors: c-Jun (*p* = 0.013), NF-kB p65 (*p* = 0.004), STAT3 (*p* = 0.011); (ii) MAP kinases: extracellular signal-regulated kinase ERK 1 (*p* = 0.039); (iii) metalloproteinase MMP2 (*p* = 0.001). When compared with the control diet, GSM alone had no effect on inflammatory markers or signaling molecules, with the exception of some genes, but resulted in a decrease in inflammatory response of the mesenteric lymph nodes, compared with the AFB1 group. In the AFB + GSM group, GSM was responsible for a significant decrease of the expression of ERK-1 (*p* = 0.006), c-Jun (*p* = 0.028), IL-18 (*p* = 0.002), and IFN-γ (*p* = 0.002), while the AFB1 × GSM effect for the other gene expressions was not significant (*p* > 0.05).

**Table 1.** Effect of the exposure of piglets to aflatoxin B1 and/or aflatoxin B1 + grape seed meal on selected gene expression in mesenteric lymph nodes.

*2.5. Correlations between Gene Expressions of Transcription Factors, MAP Kinases, Metalloproteinases, and Cytokines in Pigs Fed AFB1 and AFB1* + *GSM Diets*

In order to confirm the inflammatory effect of AFB1 and to better understand the mechanism involved in GSM action in counteracting the AFB1 effect, mathematical correlations were established between the expressions of transcription factors, MAP kinases, metalloproteinases, and cytokines in mesenteric lymph node samples derived from pigs fed AFB1 or AFB1 + GSM diets. As expected, the expression of all the inflammatory markers analyzed in the present paper was positively correlated (Figure 4).

**Figure 4.** Gene expression correlations in lymph nodes of piglets exposed to AFB1 versus control.

Highly significant correlation was obtained between the expression of NF-kB, the key signaling molecule of the inflammation pathway, and the expression of (i) transcription factor c-Jun (*R*<sup>2</sup> = 0.95); (ii) MAPK signaling molecules: ERK1 (*R*<sup>2</sup> = 0.927), JNK1 (*R*<sup>2</sup> = 0.808), JNK2 (*R*<sup>2</sup> = 0.792); (iii) metalloproteinase MMP2 (*R*<sup>2</sup> = 0.958); (iv) inflammatory cytokine IL-18 (*R*<sup>2</sup> = 0.75). When mathematical correlations were performed between inflammatory gene expressions of lymph nodes derived from the AFB1 + GSM group and those of the AFB1 group (Figure 5), a highly significant correlation were observed between the expression of NF-kB and the expression of c-JUN (*R*<sup>2</sup> = 0.965), p38 (*R*<sup>2</sup> = 0.924), JNK 1 (*R*<sup>2</sup> = 0.828), JNK 2 (*R*<sup>2</sup> = 0.986), STAT 3 (*R*<sup>2</sup> = 0.982), MMP 2 (*R*<sup>2</sup> = 0.952), TNF-α (*R*<sup>2</sup> = 0.817), IL-8 (*R*<sup>2</sup> = 0.822), IL-18 (*R*<sup>2</sup> = 0.872), and IFN-γ (*R*<sup>2</sup> = 0.875), which clearly demonstrate the anti-inflammatory effect of GSM on the mesenteric lymph nodes of AFB1-intoxicated piglets.

**Figure 5.** Gene expression correlations in lymph nodes of piglets exposed to AFB1+ GSM versus AFB1-exposed piglets.

#### **3. Discussion**

Mycotoxins are responsible for important decreases in productivity and health in the animal sector, and finding new solutions for the reduction of their toxic effects represent a continuous challenge for scientists. For some mycotoxins, such as aflatoxins, zearalenone, and ochratoxin, the use of high-affinity binders represents a promising solution as they can form stable complexes that cannot be adsorbed in the gut and are eliminated through feces, thus reducing the toxins' systemic absorption [26,27].

According to Commission Regulation (EC) no 386/2009, mycotoxin adsorbents, which are able to mitigate the toxins' effects through the reduction of intestinal absorption, were classified as a new functional group of feed additives. As stipulated in this regulation, the mycotoxin adsorbents should suppress or reduce the absorption, promote the excretion of mycotoxins and, at the same time, increase the quality of the feed, assuring the protection of public and animal health. At present, the use of mycotoxin adsorbents represents the best method for protecting animals against the harmful effects of contaminated feed [28,29].

From the mycotoxin adsorbents group, aluminosilicates are the most tested, but their use is limited due to their capacity to bind other molecules besides mycotoxins, such as vitamins and minerals that cannot be further absorbed in the gut [30]. Additionally, many binders have proven a high efficacy for mycotoxin adsorption using in-vitro abiotic systems, but these qualities were not confirmed by the in vivo studies [31].

Grape residues, as agroindustrial waste, are frequently discarded in open areas, representing a problem for the environment [32]. In order to avoid this, investigations have been done for their use for other purposes. Highlighting their chemical composition, rich in bioactive compounds, has led to their use as food/feed additives and pharmaceuticals [33]. For example, grape seed waste has been used in animal farm production in order to get functional foods, such as eggs or meat enriched in PUFAs or polyphenols [16,34].

The use of grape waste as binders of mycotoxins is of recent interest. Thus, the bioactive compounds from grape byproducts (grape pomace) were shown to efficiently adsorb AFB1 in abiotic systems [35]. Grape pomace was also efficient in reducing the urinary mycotoxin biomarker of AFB1 and zearalenone in an in-vivo trial, where piglets were fed a bolus contaminated with a mixture of mycotoxins (fumonisin B1, deoxynivalenol, zearalenone, AFB1, and ochratoxin A), through the reduction of gastrointestinal mycotoxin absorption [36].

As the information related to the effect of AFB1 on the lymphatic system and, in particular, the lymph nodes are scarce, the present study investigated the effect of AFB1 on the mesenteric lymph nodes of piglets after weaning. Additionally, we have investigated if a diet containing grape seed meal can counteract the negative effect of AFB1 on inflammation and oxidative response in piglets' MSL.

Many studies have shown that oxidative stress represents one of the causes for AFB1-induced toxicity, which leads to the generation of reactive oxygen species (ROS), resulting in lipid, protein, and DNA damage and, consequently, cell injury [37].

Oxidative stress plays a negative role in chronic inflammatory diseases [38], and our previous data indicate that oxidative stress and inflammation are tightly correlated in animals exposed to mycotoxins [39,40].

In the present study, our results have shown that AFB1 has an important antioxidative effect by decreasing the activity of principal enzymes involved in the response to oxidative stress (CAT, GPx, SOD) and total antioxidant status while increasing lipid peroxidation, as compared to oxidative stress parameters in the MSL of control piglets. As a result of the exposure to AFB1, an increase of transcription factors, MAP kinases as signaling molecules, and metalloproteinases and cytokines as effectors of inflammatory response were observed in the mesenteric lymph nodes of the intoxicated piglets. According to the most recent data, AFB1 has a rather biphasic effect on the immune response, with a stimulatory effect in the first phase, followed by a suppressive action in the second phase [41]. Immuno-stimulation induced by AFB1 increases the synthesis of inflammatory markers and free radicals, which lead to chronic inflammation and cancer [42,43]. Anti-inflammatory activities of plant bioactive compounds have been reported in acute and chronic inflammation in animal models [44,45], and recent data have shown that they can be used for counteracting the toxic effect of AFB1 [24,46,47]. Our previous results have shown that GSM addition into AFB1-contaminated diet induced the amelioration of liver injuries and decreased inflammation and oxidative stress in the liver of intoxicated piglets by decreasing the MAPKs and NF-κB signaling pathways overexpressed by the AFB1 diet [24].

Although the immune system represents one of AFB1's targets, few studies have investigated the potential beneficial role of bioactive compounds on the immune organs. For example, curcumin, a powerful plant antioxidant, decreases the weight of spleen and bursa of Fabricius, as well as the ratio of spleen/bursa, in broilers fed AFB1, towards similar values to the control group [48]. Additionally, in AFB1-intoxicated mice, lycopene, another plant bioactive compound, can alleviate AFB1-induced immunosuppression by increasing spleen weight, spleen coefficient, T-lymphocyte subsets, and IL-2, IFN-γ, and TNF-α gene expression in spleen [49]. Similarly, our results have shown that GSM was able to decrease proinflammatory cytokine gene expression and protein synthesis (IL-1β, IL-6, IL-8, IL-18, and IFN-γ), as well as the gene and protein expressions of important markers of signaling inflammation—ERK-1, c-Jun, phospho-p38 MAPK, phospho-NF-kBp65—as molecules involved in the NF-kB signaling pathway.

It was shown that phytocompounds can alleviate the immunosuppression induced by AFB1 in the spleen of mice through the inhibition of oxidative stress and mitochondria-mediated apoptosis [49]. Indeed, our results have shown that GSM induced a reduction of AFB1-induced oxidative stress by increasing the activity of enzymes involved in the response to oxidative stress (GPx and SOD) and decreasing lipid peroxidation.

The mechanism responsible for the beneficial effect of GSM in counteracting AFB1 toxicity is not clear. Recent studies have shown that grape pomace was able to efficiently bind AFB1, decreasing in this way the toxin's absorption and increasing its excretion [35,36]. Likewise, our in vitro studies, performed on a panel of eight agroindustrial wastes, have shown that GSM was the most efficient binder of AFB1; these results suggest that this waste has a high capacity to bind AFB1 (Palade et al., submitted). The decrease of the toxic effects of AFB1 in the MSL of the AFB1 + GSM group, observed in our study, can be related to the capacity of GSM to adsorb AFB1 and increase its elimination.

Some hypotheses concerning the GSM way of action can be formulated based on previous literature studies. The first one, related to the high cellulose content (37.8%) of GSM [24], has a great potential to adsorb AFB1 by electrostatic attractions and hydrogen bonding, resulting in the formation of a mycotoxin monolayer on its surface [48]. The second one is based on the ability of polyphenols to form a complex with AFB1 mycotoxins. In a recent study, Lu et al. (2017) demonstrated that polyphenols from fermented tea can reduce AFB1-induced liver injury as they bind the toxin in a complex (C-AFB1 complex) and, consequently, inhibit AFB1 absorption and increase toxin elimination through feces [22]. Another interesting hypothesis is that of Ali Rajput et al., who considered that the protective effects of the bioactive compound proanthocyanidin, from grape seed, may be due to AFB1 biotransformation in the gut, which leads to the reduction of AFB1 absorption [23]. However, more studies are necessary to elucidate the interaction between grape waste and AFB1.

#### **4. Conclusions**

In conclusion, our study demonstrates that before being transported via the mesenteric lymphatic duct and entering the blood circulation, AFB1 exerts proinflammatory and pro-oxidative effects in the mesenteric lymph nodes of intoxicated piglets. Grape seed meal, a waste product generated after oil extraction, has been shown to have the capacity to reduce the inflammation and oxidative stress triggered by AFB1. These results represent an important and promising way to valorize this waste, rich in bioactive compounds, for decreasing AFB1 toxic effects on mesenteric lymph nodes.

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

#### *5.1. Animals and Dietary Treatments*

Twenty-four crossbred weaned piglets (TOPIGS-40), 4 weeks old, were randomly assigned to the experimental groups and fed the following treatments for 30 days: Control (fed a maize-soybean diet), AFB1 group (diet contaminated with 320 μg AFB1/kg feed), GSM group (diet with 8% grape seed meal included), and AFB1 + GSM group (basal diet with 8% GSM and 320 μg AFB1/kg feed), as previously described by Taranu et al. [50]. Each experimental group was represented by 6 piglets (2 pens/treatment and 3 pigs/pen). Piglets had access ad libitum to water and feed during the experimental period.

The experimental protocol was approved on 5 February 2020 by the Ethical Committee (no. 52/2014 of INCDBNA Balotesti) and the animal handling was done in accordance with EU Council Directive 98/58/EC and Romanian Law 206/2004.

Animals were slaughtered at the end of the experiment by exsanguination, and samples of the mesenteric lymph nodes were taken on ice and stored at −80 ◦C until the assessment of the immune and stress oxidative parameters.

#### *5.2. Composition of the Grape Seed Meal*

Dried grape seed meal (GSM), resulted after oil extraction, was provided by S.C. OLEOMET-S.R.L., Bucharest, Romania. Analyses consisting of chemical composition (fat, protein, ash, fibers), total and specific polyphenol content, as well as polyunsaturated fatty acids (PUFA) and antioxidant capacity, were performed for GSM characterization, as already described by Taranu et al. [51].

#### *5.3. Toxin*

Pure AFB1 (FERMENTEC, Jerusalem, Israel) was used to contaminate both AFB1 and GSM + AFB1 diets. Briefly, 50 mg toxin was dissolved in dimethyl sulfoxide and mixed with the control diet in order to achieve a final AFB1 concentration of 320 μg/kg diet. The final AFB1 concentration was confirmed by ELISA and UPLC analysis (320 ± 10.9 μg AFB1/kg diet). In the control diet, the AFB1 concentration was 2.4 ± 0.15 μg/kg diet. The diets were analyzed for contamination by other mycotoxins (ochratoxin A, fumonisins, deoxynivalenol, zearalenone); the levels found were under the EU limits for pigs.

#### *5.4. Determination of Total Antioxidant Status*

A total antioxidant capacity (TAC) assay has already been described by Marin et al. [39]. The method consists of the measurement of the absorption of 2,20-azinobis-[3-ethylbenzothiazoline-6-sulfonic acid cation (ABTS+)] in samples of mesenteric lymph nodes, and the results are expressed as mmol TEAC (trolox equivalent antioxidant capacity)/g tissue.

#### *5.5. TBARS Assessment*

Thiobarbituric acid reactive substances (TBARS) were measured in samples of frozen mesenteric lymph nodes, as already described [39]. The results are expressed as nmol/mg protein.

#### *5.6. Enzyme Activity Assessment*

The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were measured using Cayman kits (Cayman Chemical, Ann Arbor, MI, USA), according to the instructions provided by the manufacturer [52]. A Tecan microplate reader (SunRise, Vienna, Austria) was used for the measurement of the absorbance.

#### *5.7. Cytokine Measurement*

Cytokine concentration was assessed in mesenteric lymph nodes, as already described by Marin et al. [39]. Briefly, the homogenates from 1 g of frozen sample for each animal, in buffer with complete protease inhibitor cocktail, were used for the analyses of cytokine content by ELISA. Bradford assay was used for the analyses of total protein content. Monoclonal antiporcine antibodies for IL-1beta, IL-6, IL-8, and TNF alpha were used as capture antibodies, and biotinylated antiporcine IL-1beta, IL-6, IL-8, and TNF alpha were used as secondary antibodies (R&D Systems; Minneapolis, MN, USA). Results were presented as micrograms of cytokine/g tissue.

#### *5.8. Extraction of Total RNA and cDNA Synthesis*

RNA extraction followed by cDNA synthesis was carried out as described by Pistol et al. [53]. Briefly, a Qiagen RNeasy midi kit (QIAGEN GmbH, Hilden, Germany) was used for the extraction of total RNA from frozen mesenteric lymph node samples and then treated with ribonuclease inhibitors and purified on columns of silica gel. Concentration and quality were analyzed using a Nanodrop ND-1000 (Thermo Fischer Scientific, Waltham, MA, USA), and integrity was analyzed using agarose gel electrophoresis. Then, 100 ng of total RNA samples were used for the generation of cDNA using an M-MuLV Reverse Transcriptase Kit (Thermo Fischer Scientific, Waltham, MA, USA). A GeneQuerry™ Pig cDNA Evaluation Kit (ScienCell, Carlsbad, CA, USA) was used for the assessment of the absence of contamination with genomic DNA and the successful reverse transcription of tRNA to cDNA and cDNA quality.

#### *5.9. Detection of Inflammatory and Signalling Gene Expression by qPCR Array*

Real-time PCR was used to evaluate the expression of transcription factors: c-Jun, nuclear factor NF-kappa-B p65 (NF-kB p65), and signal transducer and activator of transcription 3 (STAT3); MAP kinases: extracellular signal-regulated kinase (ERK 1), c-Jun N-terminal kinase (JNK 1, JNK2) and p38, and metalloproteinase MMP2; cytokines: tumor necrosis factor (TNF alpha), interleukin beta (IL-1 beta), interferon gamma (IFN gamma), interleukin 8 (IL-8), and interleukin 18 (IL-18), as already described by Marin et al. [40]. The sequence of gene-specific primer pairs and the conditions used for the reactions have already been published in our previous papers [54–56]. Duplicates were performed for each gene, and melting curves were used for the confirmation of the formation of single PCR products. For all primers, negative controls were used, consisting of qPCR mix except for cDNA. Two reference genes—beta-actin (ACTB) and hypoxanthine-guanine phosphoribosyl-transferase (HGPRT)—were used for the relative quantification of gene expression changes, and the results were expressed as fold change, as compared with the control group (Fc), using the 2(−ΔΔCT) method [57].

#### *5.10. Western Blot Analysis*

The protein expression level of three proteins involved in cell signaling—MAPK-p38, NF-kB phosphorylated form and Nrf2 (Nuclear factor erythroid 2-related factor 2)—were measured by Western blot. Cytoplasmic and nuclear fractions of tissue lysates were obtained using the protocol recommended by Thermo Fisher Scientific (Rockford, IL, USA NE-PER) for Nuclear and Cytoplasmic Extraction Reagent Kits, as described by Pistol et al. [53]. After assessment of protein concentration (Pierce BCA Protein Assay Kit, Thermo Fischer Scientific, Waltham, MA, USA), lymph nodes lysates, undiluted (cytoplasmic lysates) or <sup>1</sup> <sup>2</sup> diluted (nuclear lysates), were separated on a 10% SDS-PAGE and transferred onto a 0.45-μm nitrocellulose membrane. After being blocked overnight with 5% nonfat dry milk, the membrane was incubated after washing with primary antibodies from Cell Signaling Technology (Beverly, MA, USA) for phospho-MAPK-p38 (rabbit antiporcine phospho-MAPK-p38), phospho-NF-kB/p65 (rabbit antiporcine phospho-NF-kB p65), β-actin (rabbit antiporcine β-actin), and Nrf2 (rabbit polyclonal antibody; Abbexa, Cambridge, UK) for 2 h at room temperature. Then, the membranes were incubated with a horseradish-peroxidase-conjugated antirabbit IgG antibody for 1 h (Cell Signaling Technology, Danvers, MA, USA). The immunoreactivity was assessed by using Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA). A MicroChemi Imager (DNR Bio-Imaging Systems LTD, Neve Yamin, Israel) was used for developing immunoblotting images, and GelQuant software (DNR Bio-Imaging Systems LTD, Neve Yamin, Israel) was used for the evaluation of the level of protein expression. The results represent the ratio between the expression level of the protein of interest (p38 MAPK, NF-kB/p65, Nrf2) and β-actin.

#### *5.11. Statistical Analyses*

The differences for all the investigated parameters were analyzed using one-way ANOVA tests, followed by Fisher's PSLD test.

**Author Contributions:** Conceptualization, D.E.M. and I.T.; assessment of oxidative stress parameters, M.L.P. and M.I.D., Western blot analyses, C.A.A., C.V.B. and G.C.P.; qPCR analyses C.V.B. and G.C.P., data analysis and statistics, D.E.M.; writing—original draft preparation, D.E.M.; writing—review and editing, I.T.; funding acquisition, D.E.M. and I.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by National Research Projects PN III—8PCCDI, granted by the Romanian Ministry of Education and Research.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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### *Article* **The Effectiveness of Dietary Byproduct Antioxidants on Induced CYP Genes Expression and Histological Alteration in Piglets Liver and Kidney Fed with Aflatoxin B1 and Ochratoxin A**

**Roua Gabriela Popescu 1, Cristina Bulgaru 2, Arabela Untea 2, Mihaela Vlassa 3, Miuta Filip 3, Anca Hermenean 4, Daniela Marin 2, Ionelia T, ăranu 2,\*, Sergiu Emil Georgescu 1,\* and Anca Dinischiotu <sup>1</sup>**

**Abstract:** The purpose of this study was to investigate the potential of a byproduct mixture derived from grapeseed and sea buckthorn oil industry to mitigate the harmful damage produced by ochratoxin A and aflatoxin B1 at hepatic and renal level in piglets after weaning. Forty cross-bred TOPIGS-40 hybrid piglets after weaning were assigned to three experimental groups (E1, E2, E3) and one control group (C), and fed with experimental diets for 30 days. The basal diet was served as a control and contained normal compound feed for starter piglets without mycotoxins. The experimental groups were fed as follows: E1—basal diet plus a mixture (1:1) of two byproducts (grapeseed and sea buckthorn meal); E2—the basal diet experimentally contaminated with mycotoxins (479 ppb OTA and 62ppb AFB1); and E3—basal diet containing 5% of the mixture (1:1) of grapeseed and sea buckthorn meal and contaminated with the mix of OTA and AFB1. After 4 weeks, the animals were slaughtered, and tissue samples were taken from liver and kidney in order to perform gene expression and histological analysis. The gene expression analysis showed that when weaned piglets were fed with contaminated diet, the expression of most analyzed genes was downregulated. Among the CYP450 family, *CYP1A2* was the gene with the highest downregulation. According to these results, in liver, we found that mycotoxins induced histomorphological alterations in liver and kidney and had an effect on the expression level of *CYP1A2*, *CYP2A19*, *CYP2E1*, and *CYP3A29*, but we did not detect important changes in the expression level of *CY4A24*, *MRP2* and *GSTA1* genes.

**Keywords:** piglets; antioxidant effect; feed additives; mycotoxins; CYPs gene expression

**Key Contribution:** The addition of some plant-derived antioxidants in feed could be a better solution to diminish the deleterious effects of mycotoxins on animal health.

#### **1. Introduction**

Mycotoxins are secondary toxic metabolites produced by certain strains of filamentous fungi. These low molecular weight compounds (up to 500 Da) can contaminate a variety of raw materials and cause an increased risk to human and animal health [1]. The number of mycotoxins characterized and with well-known effects is relatively small due to the

**Citation:** Popescu, R.G.; Bulgaru, C.; Untea, A.; Vlassa, M.; Filip, M.; Hermenean, A.; Marin, D.; T, aranu, I.; ˘ Georgescu, S.E.; Dinischiotu, A. The Effectiveness of Dietary Byproduct Antioxidants on Induced CYP Genes Expression and Histological Alteration in Piglets Liver and Kidney Fed with Aflatoxin B1 and Ochratoxin A. *Toxins* **2021**, *13*, 148. https://doi.org/ 10.3390/toxins13020148

Received: 11 January 2021 Accepted: 13 February 2021 Published: 15 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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/).

multitude of metabolites with toxic potential generated by fungi [2–4]. They are classified into five groups, with specific chemical structures that occur frequently in feed and food: trichothecenes, zearalenone, ochratoxins, fumonisins, and aflatoxins. The mycotoxins producing fungi found in food and feed are divided into two groups: those that invade before grain harvesting, called field fungi, and those that grow only after harvesting, called storage fungi [5]. At the European level, there are regulations and recommendations regarding the maximum accepted level for six types of mycotoxins commonly found in pigs' feed: aflatoxins, fumonisins, ochratoxins, deoxynivalenol, T2 toxin, and zearalenone [6–8].

Among the farm animal species, pigs are very sensitive to mycotoxins due to their exposure to cereal-based fodders [9]. Swine metabolism is not effective in detoxifying and excreting mycotoxins, which increases the risk of mycotoxicosis. This susceptibility also varies with age, concentration of mycotoxins in feed, and duration of exposure. Liver is organ most affected by the ingestion of these toxins [10]. Furthermore, these toxins increase the permeability of the intestinal epithelial barrier in swine and poultry, which could generate predisposition for necrotic enteritis [11] and the decrease of innate immunity.

Aflatoxins represent the most abundant mycotoxins found in foodstuffs, oilseeds, cereals, milk, soils, animals, and humans. All types of aflatoxins are derived from fungal species belonging to the genus *Aspergillus* and are considered among the most harmful mycotoxins for animals and humans [4,10–17]. As mentioned above, in suckling piglets and growing, finished, and breeding pigs, the main biological effects of aflatoxins are carcinogenicity, immunosuppression, mutagenicity, teratogenicity, decreased feed efficiency and poor weight gain, impaired liver, and altered serum biochemical parameters [18,19]. Severe effects in swine can lead to acute hepatitis, systemic hemorrhages, nephrosis, and death [20], as well as decreased resistance to stress [21]. Some authors have also shown that swine fed with low levels of aflatoxins presented signs of pulmonary edema, reduced feed consumption and body weight gain, and a decrease in the enzymatic activities implicated in oxidative decarboxylation, as well as total serum protein, blood pressure, and total leukocyte count [18,22–24]. In this context, according to the European Commission Directive 2003/100/EC, the maximum aflatoxin B1 (AFB1) accepted level for pigs is set at 0.02 mg/kg.

Ochratoxins are secondary metabolites produced by fungal species belonging to the genus *Aspergillus* and *Penicillium*. Divergent opinions regarding the genotoxic or nongenotoxic mechanisms of ochratoxins toxicity have been published [25,26]. In vitro and in vivo studies revealed that guanine-OTA-specific DNA adducts persisted for more than 16 days at renal level, whereas in liver and spleen, they were removed after 5 days [27]. Due to this, their main toxic and carcinogenic effects were exerted in kidney [28].

Most metabolites of ochratoxins from Phase I and Phase II detoxification have low toxicity. In the stomach, a part of ochratoxins is hydrolyzed to ochratoxin α by proteolytic enzymes. Another possibility for their hydrolysis is the opening of the lactone ring under alkaline conditions of intestine, thus resulting in a compound with high toxicity. Due to the strong binding to albumin, the elimination of ochratoxins by glomerular filtration is negligible, with the excretion being mainly through tubular secretion. The tubular resorption is considered partially responsible for the intracellular accumulation of ochratoxins [29,30].

Generally, in farm animals, ochratoxins are rapidly absorbed after ingestion through the gastrointestinal tract (stomach and proximal portion of the jejunum) in a passive manner, which is favored by the high affinity of binding of ochratoxins to plasma proteins, and in a nonionized form, which explains their persistence in the body. In porcine serum, ochratoxins bind more specifically to proteins with a molecular mass less than 20 kDa, allowing them to pass through the glomerular basement membrane and exert nephrotoxic effects. Ochratoxins also accumulate in liver and muscles. However, kidneys are the main site of ochratoxins storage, with their reabsorption at the proximal and distal tubules contributing to the body persistence and increased nephrotoxicity [27,31].

On the other hand, once AFB1 is absorbed at the intestinal level, it reaches liver where it is transformed by Phase I metabolizing enzymes by hydroxylation, hydration,

demethylation, and epoxidation. The first three reactions generate nontoxic metabolites, whereas the fourth produces AFB1-8,9 epoxide that forms adducts with DNA at the N7 site of guanine [32]. Also, AFB1 can be conjugated with reduced glutathione in a reaction catalyzed by glutathione-S-transferases [33] and glucuronic acid [34]. Excretion of AFB1 occurs primarily through the biliary pathways, followed by the urinary pathway [35].

One of the main difficulties encountered in controlling mycotoxins is that more than one type of mycotoxin is present in a batch of fodder or cereal at the same time. Thus, feeding of piglets and pigs with contaminated feed with several types of mycotoxins, even if they are in minimum concentrations, can cause numerous negative consequences due to their synergistic effect [36–40]. In this context, diminishing and eliminating the negative effects of mycotoxins found in swine feed could decrease production cost and loss in the pig industry.

To date, numerous strategies have been developed to prevent, reduce, or even eliminate mycotoxin contamination from animal feed by biological, chemical, and physical detoxification methods. These methods allow the degradation of mycotoxins and their corresponding metabolites and maintain the nutritional value of the food without introducing other substances with toxic potential into the biological systems [6,14,41].

Biological decontamination of mycotoxins using competitive inhibition by other fungi strains or addition of antioxidant compounds in animal feed in order to reduce the toxic effects of mycotoxins and/or to inhibit the growth of mycotoxin-producing fungus species represents a good solution. The most used method to counteract the negative impact of mycotoxins on farm animals is adding "mycotoxin binders" or "mycotoxin modifiers," which are aluminosilicates with a porous structure that are able to adsorb and trap mycotoxins [42–44]. They are very effective for aflatoxins and have limited activity against other types of mycotoxin. However, being nonspecific, they also bind vitamins and trace elements, generating deficiencies [45–47]. Adding some plant-derived antioxidants in feed could be a better solution [48] to diminish the deleterious effects of mycotoxins on animal health.

P450 cytochromes enzymes, mainly present in liver, intestinal tract, and kidney, play an important role in phase I biotransformation of xenobiotics, especially those belonging to the families 1 and 3 [49]. Mycotoxins can be substrates, inhibitors, or inducers of these metabolizing enzymes. Changes in the specific activity and inducibility of cytochromes P450 will ultimately determine the relative change in the metabolism of a xenobiotic. Mycotoxins may alter the gene expression of these proteins, leading to an altered absorption and biotransformation of nutrients and other substrate drugs from feed. Due to this, the aim of the present study was to investigate the potential of a byproduct mixture derived from *Vitis vinifera* (grapeseed) and *Hippophae rhamnoides* (sea buckthorn) oil industry to mitigate the harmful damage produced by the concomitant presence ochratoxin A (OTA) and aflatoxin B1 (AFB1) in feed at the hepatic and renal level in piglets after weaning.

#### **2. Results**

#### *2.1. Diet Composition*

The chemical composition of byproducts meal showed that sea buckthorn meal is richer in protein (+38.4%), fat (+66.6%), and carbohydrates and lower in ash than grapeseed meal (Table 1).

**Table 1.** Chemical composition of grapeseed and sea buckthorn.

DM = Dry matter; CP = Crude protein; EE = Fat (ethyl esters).

The chemical analysis also showed a different profile of the two byproducts in fatty acids, flavonoids, phenolic acids, and minerals. Thus, the sea buckthorn meal has a higher content of saturated fatty acids (palmitic and palmitoleic), omega-9 acids (cis oleic acid), and omega-3 acids (α-linolenic acid) than the grapeseed meal. In contrast, the grapeseed meal has a very high omega-6 acids (linoleic acid) content (67.35% compared to 18.59% in sea buckthorn meal) (Table 2).

**Table 2.** Fatty acid composition of grapeseed and sea buckthorn (g FAME/100 gTotal FAME).

FAME = Fatty Acid Methyl Esters; SFA = Saturated fatty acids; UFA = Unsaturated fatty acids; MUFA = Monounsaturated fatty acids; PUFA = Polyunsaturated fatty acids.

> Both byproducts contain flavonoids and phenolic acids, bioactive compounds known for their antioxidant, anti-inflammatory and immunomodulatory properties [50,51]. Thus, the total concentration of polyphenols was 74.8% higher in grapeseed meal (133.84 mg GAE/L) than in sea buckthorn (76.57 mg GAE/L). Concerning the different classes of polyphenols, grapeseed meal contains higher concentration of catechin and vanillic acid than sea buckthorn, while sea buckthorn is richer in rutin, quercitrin, luteolin, p-coumaric acid, and ferulic acid (Table 3).

> Regarding the mineral composition, sea buckthorn meal shoeds a higher content of K, Mg, Fe, Mn, and Zn than grapeseed meal. In contrast, grapeseed meal contained twice as much copper as sea buckthorn meal. Of note is the high concentration of iron from sea buckthorn meal (Table 4).

**Table 3.** Flavonoids and phenolic acids composition of byproducts.

#### *2.2. Animal Performance*

Exposure of piglets from E2 group to ochratoxin plus aflatoxin B1 mixture had no adverse effects on body weight, weight gain, and feed intake, as the differences were not significant compared to the control. In contrast, the administration of the diet containing the byproducts mixture alone (E1) increased significantly the body weight of piglets fed this diet when compared to control (32.14 ± 1.63 vs. 27.09 ± 1.31) and to group E2, which was fed the contaminated diet (32.14 ± 1.63 vs. 28.72 ± 1.07). It should be noted that the group of piglets receiving contaminated feed and the mixture of byproducts had a tendency to gain weight compared to the group of mycotoxin-intoxicated piglets, although the difference was not significant. Biochemical parameters analysis, which characterizes the general state of animal health and the functionality of liver and kidneys, registered normal values for the age and weight category of weaned piglets. No significant differences were identified between groups for most of them (Table 5). However, the mycotoxin mixture increased ALP and gamma GT activity compared to control and decreased activity in the control level in group E3 receiving the byproduct mixture.

**Table 5.** Biomarkers of liver and kidney function in plasma.

ALAT = alanine transaminase; ASAT = aspartate transaminase; ALK = alkaline phosphatase; GGT = gamma glutamyl transferase; SEM = standard error of mean; a,b,c Mean values within a row with unlike superscript letters were significantly different (*p* < 0.05).

#### *2.3. Histology of Liver and Kidney*

Light microscopic analysis of the livers from E2 group, fed with a basal diet contaminated with a mixture of OTA and AFB1, showed focal areas of necrosis, dilatation of sinusoid, and inflammatory parenchymal infiltration. The portal areas revealed mononu-

clear cellular infiltration and periportal fibrosis. The fibrotic perilobular fibrotic septa were also noticed (Figure 1).

**Figure 1.** Histopathological changes in liver of weaned piglets subjected to experimental diets. The Control group (C) showed the normal aspect of hepatocytes and sinusoids (**a**,**b**) in the H&E stain and the normal aspect of thin perilobular (**c**) and priportal (**d**) fibrous spikes in Gomori's trichrome stain. The E1 group showed the normal aspect of the liver in the H&E stain (**a**,**b**) and Gomori's trichrome stain (**c**,**d**). The E2 group showed dilated sinusoids and inflammatory infiltrates (arrows) and necrotic hepatocytes (\*) in the H&E stain (**a**–**c**,**e**–**g**), and perilobular (**d**) and priportal (**h**) fibrosis (arrowhead) in Gomori's trichrome stain. The E3 group displayed marked improvement of the histological aspect of the liver, which is comparable to that of the control group, in the H&E (**a**,**b**) and Gomori's trichrome stains (**c**,**d**). Scale bar = 50 μm.

> Mycotoxin administration caused structural changes in kidneys that affected both the cortex and medulla. Atrophy of the glomerular tufts and alteration of the Bowmann's capsule were noticed (Figure 2). The tubules showed necrosis of lining epithelial cells with inflammatory cells infiltration in between. Focal aggregates of inflammatory cells were observed in between the glomeruli and tubules in association with the focal areas of congestion in blood vessels, especially in the medullary region. Apparently, the collagen proliferation was mainly observed in areas of tubular injury. Furthermore, kidney sections from the E3 groups, the group fed with a basal diet containing a mixture of grapeseed and sea buckthorn meal and contaminated with the mix of OTA and AFB1, revealed minor pathomorphological changes, almost similar to control.

**Figure 2.** Histopathological changes in kidney of weaned piglets subjected to experimental diets. The Control group (C) showed the normal aspect of kidney cortex (**a**) and medulla (**b**) in the H&E stain and few collagen fibers surrounding glomeruli and tubules in cortex (**c**) and medulla (**d**) in Gomori's trichrome stain. The E1 group showed the normal aspect of kidney cortex (**a**) and medulla (**b**) in the H&E stain and few collagen fibers surrounding glomeruli and tubules in the cortex (**c**) and medulla (**d**) in Gomori's trichrome stain. The E2 group (**a**–**d**) kidney cortex showed glomerular atrophy (\*), Bowmann's capsule injury (arrow), inflammatory cell infiltrates (arrowhead) (**a**,**b**), or glomerular degeneration (\*) in the H&E stain (**a**–**c**) and slight proliferation of peritubular collagen in Gomori's trichrome stain (arrow) (**d**). (**e**–**h**) The kidney medulla showed altered tubuli (arrow), inflammatory infiltrates (arrowhead), and congestion in blood vessels in the H&E stain (**e**–**g**) and the proliferation of peritubular collagen in Gomori's trichrome stain (arrow) (**h**). The E3 group displayed marked improvement of the renal histological aspect, which is comparable to that of the control group, in the kidney cortex (**a**) and medulla (**b**) in the H&E stain and in the cortex (**c**) and medulla (**d**) in Gomori's trichrome stain. Scale bar = 50 μm.

Moreover, the morphometric analysis of the structural injuries in liver and kidney of experimental groups was evaluated (Table 6).

**Table 6.** The morphometric analysis of the structural injuries in liver and kidney of experimental groups.

One-Way ANOVA test. \* (All groups vs. Control group; \*\*\* *p* < 0.001). ˆ (E3 group vs. E2 group; ˆ *p* < 0.05; ˆˆ *p* < 0.01). MAV = Mean assessment value.

#### *2.4. The Level of Gene Expression*

We found that modifying the piglets' diet caused significant liver changes to the *CYP2E1* and *GSTA1* genes in the E1 group fed with a basal diet supplemented with a mixture of grapeseed and sea buckthorn meal, and to the *CYP4A24*, *MRP2*, and *GSTA1* genes in the E2 group fed with a basal diet contaminated with a mixture of AFB1 and OTA. The modifications caused insignificant changes to all the other target genes (Figure 3).

**Figure 3.** Gene expression level in the liver for *CYP1A2*, *CYP2A19*, *CYP2E1*, *CYP3A29*, *CYP4A24*, *MRP2*, and *GSTA1* of weaned piglets subjected to experimental diets. The data are illustrated as average values of the groups (n = 4) ± standard deviation of the mean (STDEV). Statistical significance: \* *p* < 0.05; \*\* *p* < 0.01. The statistical significance of the changes is related to the control group level.

In liver, the gene expression for *CYP1A2* decreased by 18% for E2 and 44% for E3, respectively, compared to the E1 group. The *CYP2A19* gene expression was unmodified in groups E1 and E2, whereas in group E3, it decreased by almost 62%. A significant increase by 29% was observed in *CYP2E1* gene expression in the E1 group fed with a basal diet supplemented with a mixture of grapeseed and sea buckthorn meal compared to the E2 group. In contrast, the administration of basal diet enriched with a mixture of grapeseed and sea buckthorn meal (E1 group) downregulated the *CYP3A29* gene expression by 24% compared to the E2 group level. Another contrast was observed in the *CYP4A24* gene expression, with a 33% decrease for the E1 group and 24% decrease for the E3 group, and a significant 41% increase in the E2 group fed with a basal diet supplemented with a mixture of AFB1 and OTA, compared to the control level. In the case of *MRP2*, the gene expression pattern was similar to that of the *CYP4A24* gene, with an insignificant 35% decrease for the E1 group and 24% decrease for the E3 group, and a significant 28% increase in the E2 group, compared to the control level. Similarly, to the *CYP4A24* gene expression, the *GSTA1* gene expression showed a significant 14% increase in the E2 group, a 9% increase in the E1 group, and a 30% decrease for the E3 group. Obviously, the concomitant administration of

the mixture of grapeseed and sea buckthorn meal and OTA and AFB1 generated a decrease of all analyzed genes expressions in liver compared to control.

Regarding the expression level of these genes in kidneys, compared to liver samples, no statistically significant changes were observed (Figure 4). However, changes in the regulation of gene expression level could be observed.

**Figure 4.** Gene expression level in the kidney for *CYP1A2*, *CYP2A19*, *CYP2E1*, *CYP3A29*, *CYP4A24*, *MRP2*, and *GSTA1* of weaned piglets subjected to experimental diets. The data are illustrated as average values of the groups (n = 4) ± standard deviation of the mean (STDEV).

Analyzing Figure 4, it could be noticed that the mixture of grapeseed and sea buckthorn meal downregulated the *CYP1A2* gene expression and upregulated the *CYP2A19, CYP2E1, CYP3A29*, and *CYP4A24* gene expression in an insignificant way, whereas *MRP2* and *GSTA1* gene expression remained unmodified. Also, the presence of OTA and AFB1 in piglets feed downregulated *CYP1A2* and *CYP2A19* gene expression in an insignificant way, whereas *MRP2* and *GSTA1* were unmodified. The concomitant administration of the mixture of grapeseed and sea buckthorn meal and OTA and AFB1 determined the return of all genes expression levels to control levels with the exception of *GSTA1*, which presented an important increase compared to E1 group.

#### **3. Discussion**

Mycotoxins such as AFB1 and OTA are natural toxins contaminating a large variety of plant products. As a consequence, AFB1, OTA, and their metabolites are present in food and feed, as well as in the products of animal origin [52]. Most of the toxicological studies regarding the effects of mycotoxins have considered the exposure to a single type of mycotoxin without considering the combination and the interaction between them, respectively, the synergistic or antagonistic effects which often occur in nature. Data regarding the toxic effects of mycotoxin combinations are limited, so the risks of exposure to several types of toxins are still unknown.

The occurrence of mycotoxins such as AFB1, DON, ZEA, OTA, FB1, and FB2 in cereal, cereal products, and complementary and complete feeding stuffs for pigs [16] is related to the geographical location and climate change, which increases the risk associated with mycotoxin contamination during the storage and processing of feed products for pigs [53]. The co-contamination of cereals and other raw materials occurs more frequently in real life than single mycotoxin contamination [7]. For example, the co-occurrence of aflatoxin B1 and ochratoxin A has been found in different food or feed ingredients, such as wheat [54], barley [55], cereal flours [56], spice [57], etc. The proportion between AFB1 and OTA in feed was found to be about 1 to 6 [37]. Also, the global feed content in AFB1 and OTA ranged between not determined and 100 ppb and not determined and 211 ppb, respectively [58]. In this context, in order to mimic the field conditions, we studied the effects of these mycotoxins together and to assess the effectiveness of the by-product mix in counteracting the effects of mycotoxins. The natural additives (grapeseed and sea buckthorn byproducts) were selected based on their ability to ameliorate mycotoxicosis upon dietary supplementation [59,60].

In the present study, the exposure of piglets (E2 group) to mycotoxins mixture did not influence the performance of animals (27.83 ± 1.1 vs. 27.09 ± 1.3 for body weight and 1.48 ± 0.9 vs. 1.40 ± 0.8 for feed intake) and biochemical parameters when compared to control. Similarly, Balogh et al. [61] reported that piglets fed with approximately 0.4 mg/kg of OTA during the starter (0–28 days) and grower (29–49 days) period did not register significantly changes in the production traits and clinical signs of toxicity in the grower phase. In contrast, a significant decrease of body weight gain was observed during the starter period when the animals were more sensitive. In this study, the dietary inclusion of the byproduct mixture alone had a significant influence on animal performance (group E1) and tended to increase piglets' weight when the mixture was associated with contaminated food (group E3).

From a toxicological point of view, OTA is classified by IARC (International Agency for Research on Cancer) in the same group (2B) of carcinogenic substances for humans, having a similar toxicity with AFB1 [62]. Toxicokinetic patterns of absorption, distribution, and elimination for these mycotoxins are, for the most part, entirely elucidated. In contrast, despite recent progress, our knowledge of the toxicokinetic biotransformation steps is not elucidated in detail. A number of studies have shown that AFB1 and OTA are metabolized by liver microsomes from humans, pigs, and rats into several epimers [63]. Changes in the specific activity and inducibility of cytochromes P450 ultimately determine the relative change in the metabolism of any xenobiotic.

It has been found that exposure to AFB1 and OTA decreased the gene expression of *CYP1A2*, *CYP2E1*, *CYP3A29,* and *MRP2* genes in pig's liver and resulted in several changes in liver histology and ultrastructure, including focal areas of necrosis, dilatation of sinusoid, inflammatory parenchymal infiltration, and periportal fibrosis. Regarding the gene expression level of CYP450 isoforms in pig's kidney, no data were available in the scientific literature.

The *CYP1A2*, *CYP2A19*, *CYP2E1*, *CYP3A29*, *CYP4A24*, *MRP2*, and *GSTA1* genes were chosen for this study because they encode proteins with enzymatic activity or transporter function that are involved in Phase I and Phase II of biotransformation and detoxification of xenobiotics to form electrophilic reactive metabolites [64].

According to these results, it appears that the by-product administration determined a decrease in *CYP1A2* gene expression and an increase in *GSTA1* gene expression. Similar results were noticed in HT-29 human colon cancer cells treated with *Salicornia freitagii* extract, known for its antioxidant and anti-inflammatory activity. In this case, due to its content in bioactive phenols, a downregulation of *CYP1A2* mRNA and an upregulation of GSTA1 mRNA occurred [65]. In contrast to our results, mRNA and protein expression of *CYP1A2* were increased in liver of chicory fed pigs [66]. These different results were probably caused by the different natural compounds present in chicory compared to the byproducts used in the present study, mainly chlorogenic, caffeic, and p-coumaric acids [67].

On the other hand, OTA and AFB1 probably interacted with and activated the aromatic hydrocarbon receptor, leading to its nuclear translocation. After the heterodimerization, OTA and AFB1 probably interacted with hydrocarbon receptor nuclear translocator, the heterodimer, bound to xenobiotic-responsive elements and transactivated genes such as *CYP1A1*, *CYP1A2*, and *GST* [68]. This xenobiotic-responsive element is shared between *CYP1A1* and *CYP1A2* genes [69], and the two enzymes codified by them present overlapping substrate specificity [70]. In pig liver, only *CYP1A2* activity is present, and its

relative amount of total detected CYP450 is 4% [71]. In the human liver, AFB1 and OTA are inducers for CYP1A1, 1A2, 2B6, 2C9, 3A4, and 3A5 [72]. AFB1, as well as OTA exposure, generate mitochondrial dysfunction characterized by an increase in ROS production [14] that could increase TGF-β1 expression or activate latent TGF-β1 [73]. Taking into consideration, the previous evidence that TGF-β1 decreased *CYP1* expression in humans and rats, it is possible that the same mechanism [74] occurred under our conditions. The effects of the concomitant exposure to both mycotoxins and grapeseed and sea buckthorn by-products were probably synergistical, and the expression of *CYP1A2* was lower in E3 compared to E1, E2, and the control group. *CYP1A2* is expressed in lower levels in extrahepatic tissues [75].

The kidney is an organ that receives about 25% of cardiac output and purifies metabolic residue and xenobiotics from the circulatory system. During this discharging process, toxic substances are concentrated in the kidney [76]. In piglet kidneys, the variation of *CYP1A2* gene expression was similar with the expression levels in liver for E1 and E2. Interestingly, in the E3 group, the expression of this gene was at a higher level than the control group. This could possibly be due to the activation of noncanonical signaling pathway for AhR transcription in the kidney cells [77].

In pig liver, the relative amounts of *CYP2A19* and *CYP2E1* represent 31% respectively 13% of total CYP450 [71]. Porcine *CYP2A19* and *CYP2E1* genes are responsible for the biotransformation for endogenous compounds (skatole, sex hormones) as well as exogenous compounds (food components). Both types of compounds are highly expressed in the liver and less in the kidney and adipose tissue. *CYP2A19* transcription is controlled by the CAR transcription factor [78]. Its human orthologue, *CYP2A6* is controlled by CAR, PXR, glucocorticoid receptor (GR), estrogen receptor *α*, HNF4 *α*, and PGC-1*α* [79]. Also, the constitutive hepatic expression of *CYP2A6* in mice is governed by an interplay between HNF4 *α*, CCAAT-box/enhancer binding protein (C/EBP *α*, C/EBP *β*) and octamer transcription factor-1 (Oct-1) [80]. Previously, a positive correlation between mRNA and protein levels for *CYP2A19* gene was observed [81]. Unlike other CYP 450 genes, *CYP2A19* plays a less important role in the xenobiotics' metabolism but is involved in the reaction of cells to stress, Nrf-2, being also involved in *CYP2A19* transcription [82]. The *CYP2A19* gene is probably highly polymorphic compared to the *CYP2A6* gene [83], and an extensive interindividual variation of its product could occur. Previous studies revealed that duck P450 orthologues of the mammalian *CYP2A6* and CYP3A4 are involved in AFB1 bioactivation into its epoxide form [84]. Unlike these results, in the present study, no significant changes of *CYP2A19* gene expression were noticed in the E1 and E2 groups, probably due to the high level of expression of this gene in piglet liver. For now, it is difficult to explain why the co-exposure of both mycotoxins and the mixture of grapeseed and sea buckthorn meal decreased the expression of *CYP2A19.* However, this decrease of expression diminished the risk of generation of toxic metabolites.

In pig kidney, the expression of *CYP2A19* is lower compared to that found in liver [79]. Probably due to this lower expression, animal exposure to the mixture of grapeseed and sea buckthorn meal generated an upregulation of Nrf-2 induced *CYP2A19* gene expression due to the luteolin [85] and ferulic acid [86] content.

On the other hand, there is evidence that only two transcription factors, i.e., chick ovalbumin upstream promoter transcription factor (COUP-TF1) and hepatocyte nuclear factor (HNF-1), are involved in the regulation of *CYP2E1* transcription in pigs [87]. *CYP2E1*, like other xenobiotic-metabolizing P450s, is mainly located in the membrane of the endoplasmic reticulum (ER) and can be induced under a variety of metabolic or nutritional conditions. ER stress can be induced by metabolic stress, which is caused by overload of protein/lipid biosynthesis, and oxidative stress, which could trigger the evolutionarily conserved complex homeostatic signaling pathway known as the unfolded protein response (UPR) [88].

It is likely that the level of *CYP2E1* mRNA was approximately the same in the E1 and control groups due to the antagonistic actions of palmitic acid [89], linoleic, and *α*-linolenic acids [90] that increased this gene transcription and the actions vanillic and p-coumaric acids which decreased it [65].

Recently, it was proved that OTA-containing feed altered the intestinal microbiota in ducks, affecting the cecum microbiota diversity and composition as well as the intestinal barrier. As a result, Gram-negative bacterial-derived lipopolysaccharides entered the blood and liver, causing liver inflammation [91]. In the case of immune-mediated liver injury, the expression of *CYP2E1* was decreased [92]. This situation could occur in the E2 group. It is likely that the cumulative effects of the two mycotoxins and dietary by-products decreased the expression of *CYP2E1* in liver of the E3 group.

In the kidney, free fatty acids, such as palmitate, oleate, and linoleate, are stored in the nephron [93], and these acids probably increased the expression of the *CYP2E1* gene in the kidneys of the E1 group compared to the control level. According to Pfohl-Leszkowicz and Manderville [25], OTA forms adduct with DNA, generating renal genotoxicity and carcinogenesis. It is likely that high levels of OTA stimulated *CYP2E1* gene expression in the kidneys of the E2 group compared to the control level. In the E3 group, it appears that the coadministration of the two mycotoxins and dietary byproducts had antagonistic effects, with the expression of *CYP2E1* gene returning to the control level. Moreover, histological evaluation for the E3 group showed that the byproduct mixture derived from grapeseed and sea buckthorn oil mitigated the harmful damage produced by aflatoxin B1 and ochratoxin A at the hepatic and renal level in piglets after weaning. *CYP2E1*, like other xenobiotic-metabolizing P450s, is mainly located in the membrane of the ER and can be induced under a variety of metabolic or nutritional conditions [89]. The regulation of the *CYP2E1* gene in the E1 group was probably due to the hydroxylation of coumarin-derived compounds that were catalyzed by CYP2A enzymes, which are considered to be specific indicators for the presence of CYP2 enzymes [94], with the p-coumaric acid being present in grapeseed and sea buckthorn byproducts.

In the case of pigs, very little is known about the presence of CYP3As enzymes in the renal tissue, and nothing is known about their inducibility [95]. Several genes have been identified in the CYP3A subfamily of mammals (for example, five in rat and four in human), but the expression of these genes in renal tissues has been poorly investigated [96]. In terms of gene expression, Ayed-Boussema et al. (2012) [63] and Gonzalez-Arias et al. [97] described an increase of expression levels in all cytochromes assayed (CYP3A4, 2B6, 3A5, and 2C9) in a primary human hepatocyte culture. Previous studies have reported various results regarding the effects of AFB1 and OTA in primary cultured human hepatocytes in which increasing concentrations of these mycotoxins clearly induced CYP3A4 and CYP2B6 mRNA levels in a dose-dependent manner [63].

In contrast, it has been found that in the presence of OTA and AFB1 in liver (Figure 3, group E2), the *CYP3A29* expression level is decreased compared to the control level, perhaps due to activation of the AhR [98]. These data differ from those of Zepnik et al. [99], who reported an increase of OTA hydrolysis by microsomal enzymes from rat liver, specifically for P450 3A1/2 and 3A4, suggesting that this gene expression is modulated in a speciesdependent manner.

In some cases, the inhibition of P450 enzymes by polyphenols may have a chemopreventive effect due to the potential activation of carcinogens by P450 enzymes within the course of their natural metabolic activity. The inhibition of xenobiotic-metabolizing Phase I enzymes could be one target of the chemo-preventive effects of naturally occurring polyphenols.

The increase of *CYP4A24* observed in liver could be a physiological response in the unusual context of aberrant lipid accumulation and absence of *CYP2E1* activity, due to the fact that *CYP2E1* and CYP4A are inducible hepatic microsomal cytochromes *P*-450 involved in hydroxylation of fatty acids, and both can initiate the auto-propagative process of lipid peroxidation. They might be complementary, leading to interactions in the regulation of the individual enzymes [100]. It is therefore clear that CYP4A proteins are key intermediaries in an adaptive response to perturbation of hepatic lipid metabolism [101]. The decreased *CYP4A24* level in the kidney probably leads to the toxic effects generated in liver due to the mycotoxin-contaminated diet, which means that *CYP4A24* regulates hepatic ER stress [102,103].

In the present study, the addition of a mixture of grapeseed and sea buckthorn meal by-products increased expression levels in the kidney, which would be expected to favor the elimination processes and maintenance of the balance of intracellular substances [104]. Moreover, OTA was absorbed in the intestine where the multidrug resistance protein 2 (*MRP2* gene) plays an important role, acting as a xenobiotic outward transporter to reduce the oral bioavailability and the toxin load to organs and, thereby, OTA toxicity. Once OTA reaches the bloodstream, it can reach other organs such as liver, and the MRP2 transporter is again a key primary active transporter involved in anionic conjugate and xenobiotic extrusion into the extracellular space which contributes to bile formation and the subsequent elimination of the toxin [97,105]. Also, the MRP2 transporter is present in the apical membranes of enterocytes, kidney-proximal tubules, and other cells [105]. OTA toxicity has been attributed to its isocoumarin moiety, and it is well known that OTA is inactivated or bioactivated by cytochrome P450 enzymes [29]. Previously, the presence of OTA in feed was linked to the development of nephrotoxicity, which, in rats, has been associated with renal adenomas and kidney tumors [97]. In the present study, a decrease of *MRP2* expression in the liver was found, indicating an impairment of the secretion of mycotoxins in the E2 group.

In rats, OTA was observed to be excreted 15% less in the proximal tubules of the kidney, while the proximal tubular transport of amino acids was not impaired [97,106]. Therefore, the decrease of *MRP2* in liver found in this study could be the mechanism through which mycotoxins reach high percentages of bioavailability in vivo. In this way, the AFB1 and OTA exposure of piglets would be magnified, contributing to the hepatotoxicity.

Considering the nephrotoxic potential of OTA and AFB1, the decrease of the *MRP2* gene product may also have a major impact on the proximal tubule, leading to a decreased capacity to eliminate OTA [97]. However, further studies are needed on the AFB1 and OTA transporter mechanism to support this hypothesis.

In Phase II of metabolic detoxification, the original xenobiotic compound or the intermediate metabolites modified during Phase I are conjugated in order to be suitable for excretion. Glutathione S transferases (GSTs) and UDP glycurosyltranferases (UGTs) contribute to Phase II processing [107].

In the presence of a mixture of grapeseed and sea buckthorn meal byproducts in pigs feed, the *GSTA1* expression level in liver is significantly increased, possibly by an antioxidant-responsive element (ARE) and β-NF-responsive element (β-NF-RE), respectively, which, in the presence of phenolic antioxidants, activate the GST isoforms without the need for aryl hydrocarbon (Ah) receptors [108]. Surprisingly, in the study of Ghadiri et al. (2019) [109], the AFB1-mediated mRNA downregulation of *GSTA1* was observed in the cow's liver in the presence of an antioxidant.

Previous studies [110] showed that OTA and AFB1 compete for the same CYP450 enzymes which represent the bioactivation route of AFB1, with less AFB1-DNA adducts being produced. Due to this competition, AFB1 could probably be conjugated with reduced glutathione in a reaction catalyzed by GST enzymes, with their codding genes being upregulated. AFB1 could be involved in other types of Phase II reactions, i.e., glucuronidation and sulfatation, whereas OTA is mainly conjugated with reduced glutathione [72]. Moreover, in response to concomitant administration in the pigs, the feed of two mycotoxins (AFB1 and OTA) increased the generation of the oxidative stress biomarkers. Therefore, defense mechanisms were activated, promoting adaptation and survival in response to oxidative stress [111]. For example, ROS and oxidants could activate the transcription of GST isoforms through ARE [108], as observed in both the liver and kidneys through an increase in the expression level of the *GSTA1* gene.

#### **4. Conclusions**

Our data revealed the existence of differences between piglet's kidney and liver regarding the reaction against both mycotoxins and by-products used in this study. Generally, the by-products with antioxidant action decreased the expression of the analyzed CYPs mRNA in liver and increased them in kidney. Also, in both organs, the co-exposure of piglets to OTA and AFB1 generated an increase or a decrease of gene expression dependent on the gene type. The inclusion of grapeseed and sea buckthorn meal in the diet of OTA and AFB1-intoxicated pigs decreased the CYP P450 gene expression, suggesting the decrease of bioactivation of these mycotoxins, probably resulting in a diminished toxicity in both organs, as the histological studies have revealed.

These findings suggest that grapeseed and sea buckthorn meal waste represent a promising source in counteracting the harmful effect of ochratoxin A and aflatoxin B1. Although additional work is needed to unravel the mechanisms by which grapeseed and sea buckthorn byproducts affects AFB1 and OTA biotransformation, and hence the generation of toxic metabolites, the protective effects seem to be at least partly mediated by the enhancement of the antioxidant defense at the liver and kidney level.

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

#### *5.1. Experimental Design and Samples Collection*

Forty cross-bred TOPIGS-40 hybrid (♀Large White × Hybrid (Large White × Pietrain) × ♂Talent, mainly Duroc) piglets after weaning with an average body weight of 9.11 ± 0.03 kg were assigned to three experimental groups (E1, E2, E3) and one control group (C), housed in pens (two replicates of five pigs per pen per treatment) and fed with experimental diets for 30 days. Feed and water were offered ad libitum during the experiment. The basal diet was served as a control and contained normal compound feed for starter piglets without mycotoxin (corn 68.46%, soya meal 19%, corn gluten 4%, milk replacer 5%, L-lysine 0.3%, DL-methionine 0.1%, limestone 1.57%, monocalcium phosphate 0.35%, salt 0.1%, choline premixes 0.1%, and 1% vitamin-mineral premixes). The experimental groups were fed as follows: E1—basal diet plus a mixture (1:1) of two byproducts (grapeseed and sea buckthorn meal) in a percentage of 5% by replacing corn and soya bean meal; E2—the basal diet artificially contaminated with mycotoxins (a mixture of 62 ppb aflatoxin B1- AFB1 and 479 ppb ochratoxin A-OTA); and E3—basal diet containing 5% of the mixture (1:1) of grapeseed and sea buckthorn meal and contaminated with the mix of AFB1 and OTA. The mixture of OTA and AFB1 mycotoxins was kindly provided by Dr. Boudra and Dr. Morgavi from I. N. R. A, Centre of Clermont Ferrand, and was produced by the cultivation of *Aspergillus flavus* and *Aspergillus ochraceous* on wheat as already described by Boudra et al. [112]. The contaminated material obtained was incorporated into the diets for the E2 and E3 groups, resulting in a final concentration of 479 ppb OTA and 62 ppb AFB1. Animals from all experimental groups had free access to the treatment feed and water every day of the experimental period (30 days). The grapeseed meal and sea buckthorn meal were provided by two local commercials, S.C. OLEOMET-S.R.L. and BIOCATINA, Bucharest, Romania. After 4 weeks, the animals were slaughtered with the approval of the Ethical Committee of the National Research-Development Institute for Animal Nutrition and Biology, Balotes,ti, Romania (Ethical Committee no. 118/02.12.2019) and in accordance with the Romanian Law 206/2004 and the EU Council Directive 98/58/EC for handling and protection of animals used for experimental purposes. At the end of the experimental period of this study, the productive parameters, weight, and feed consumption were measured. Liver and kidney samples were collected from four animals per group and perfused with ice-cold saline solution to remove blood. Fragments of ~50 mg from the right liver lobe and renal cortex (three from each) were collected in RNAlater Stabilization Reagent (Qiagen, Germantown, Maryland) and then stored at −80 ◦C until RNA isolation step.

Due to ethical reasons, maximizing the use of each animal, minimizing the loss of animals, and statistical analysis, the number of individuals was reduced as much as scientifically possible. Good science and good experimental design help to reduce the number of animals used in any research study, allowing scientists to gather data using the minimum number of animals required [113].

#### *5.2. Feed Characterization*

Feed diets were analyzed for basal chemical composition (dry matter, crude protein, crude fat, crude fiber, and ash) according to the International Standard Organization methods (SR ISO 6496/2001, Standardized Bulletin (2010). http://www.asro.ro (accessed on 13 February 2021)). Bioactive compounds from byproducts meals, such as polyphenols, polyunsaturated fatty acids (PUFA), and minerals, were determined by Folin-Ciocalteu reaction, HPLC-UV-Vis, and gas chromatography as described by the authors of [113,114]. Antioxidant activity was determined by the DPPH method as described previously by the authors of [115].

#### *5.3. Plasma Biomarkers Analysis*

On day 30, blood samples were aseptically collected from fasted piglets. Markers that reflect the functionality of liver (aspartate transaminase-AST, alanine transaminase-ALT, gamma glutamyl transferase-GGT, total protein, alkaline phosphatase-AKL), and kidneys (albumin, creatinine) were determined after blood centrifugation using a Clinical Chemistry benchtop analyser Horiba Medical—ABX Pentra 400, (Irvine, CA, USA).

#### *5.4. Light Microscopy Examination*

Liver and kidney biopsies were fixed in 4% phosphate-buffered formaldehyde solution, dehydrated, clarified, and included in paraffin blocks. The 5 μm sections were processed routinely for hematoxylin-eosin and Gomori trichrome (Leica Biosystems, 38016SS1, Nussloch, Germany) staining, respectively, according to Leica's protocol. Microscopic sections were analyzed with an Olympus BX43 microscope equipped with a digital camera Olympus XC30. The histopathological alterations of liver and kidney were graded by the severity of lesions as belonging to grades 1–4, as previously described [116]. For liver, grade 1: Normal aspect; grade 2: Normal hepatocytes, slight dilated sinusoids and congestion; grade 3: Vacuolated hepatocytes, dilated sinusoids and congestion; moderate collagen proliferation; grade 4: Necrosis, inflammatory infiltrates, collagen proliferation. For kidney, grade 1: Normal aspect; grade 2: Slight tubular/glomerular injuries, inflammation, and collagen proliferation; grade 3: Mild tubular/glomerular injuries, inflammation, and collagen proliferation; grade 4: Marked tubular/glomerular injuries, inflammation, and collagen proliferation. A "mean assessment value" (MAV) was calculated as a mean of all data per experimental group.

#### *5.5. RNA Isolation*

The isolation of total RNA was performed from 10 mg of tissue using the RNeasy Plus Universal Mini Kit (Qiagen) following the manufacturer's protocol. Moreover, it included the On-column DNase digestion step. After RNA isolation, aliquots were made in order to prevent degradation induced by freeze-thaw cycles. The concentration and purity of total RNA were determined using NanoDrop 8000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA).

#### *5.6. RNA Integrity Number (RIN)*

RIN values of the RNA samples were determined using the Agilent RNA 6000 Nano Kit (Agilent, Santa Clara, CA, USA) and Agilent 2100 Bioanalyzer using the manufacturer's protocol. Samples with RIN values smaller than 8 were not included in further analysis, and the isolation steps were repeated.

#### *5.7. Reverse Transcription*

For cDNA synthesis, 1000 ng of total RNA was subjected to reverse transcription using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). A 4 μL reaction mix and 1 μL reverse transcriptase were mixed with 1 μL RNA samples and completed with RNase free water to a total volume of 20 μL. The final concentration of RNA was 1000 ng per reaction. The reaction was performed using a Veriti 96-Well thermal cycler (Applied Biosystems, Foster City, CA, USA) with the following program: One cycle of 25 ◦C for 5 min, one cycle of 42 ◦C for 30 min and one cycle of 85 ◦C for 5 min. The concentration and purity of the cDNA samples was determined using NanoDrop 8000 spectrophotometer (Thermo Scientific).

#### *5.8. Primer Design*

Because of the lack of data regarding genes involved in the hepato-nephrotoxicity in the mycotoxin exposure of weaned pigs, primer sequences (Table 7) were designed in silico using Primer3Plus [59] and verified by BLAST program [117]. Those with the highest specificity for the target sequence were selected in order to amplify the *CYP1A2*, *CYP2A19*, *CYP2E1*, *CYP3A29*, *CYP4A24*, *MRP2*, and *GSTA1* genes and three reference genes encoding for TATA-box binding protein, ribosomal protein L4, and beta-2-microglobulin in *Sus scrofa*. The annealing temperatures of the primers were determined by temperature gradient PCR.

#### *5.9. Real-Time PCR*

The Real-Time PCR reaction was carried out on the iCycler iQ Real-Time PCR Detection System (Bio-Rad) using iQ SYBR Green SuperMix (Bio-Rad). In a 96-well plate, 1 μL of 100 ng/μL cDNA, 12.5 μL iQ SYBR Green SuperMix (Bio-Rad), 0.5 μL of 20 pmol/μL forward primer, 0.5 μL of 20 pmol/μL reverse primer, and 10.5 μL of MilliQ water were added. The total volume was 25 μL. The amplification program was comprised of 1 cycle of 95 ◦C for 5 min, 45 cycles of 95 ◦C for 30 s 55/56 ◦C for 30 s, 72 ◦C for 45 s, and 85 cycles of 55 ◦C, with an increase of set point temperature by 0.5 ◦C per cycle for 10 s. The samples were run, and the threshold cycles (Ct) values were recorded. Melting curves were also performed.

#### *5.10. Data Analysis*

The Ct values were processed as stated in "The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments" [118] using OpenOffice

Calc according to the 2-ΔΔCt method described by Livak and Schmittgen (2001) [119]. The reference genes (TBP, RPL4, and B2M) were chosen in order to be stably expressed across different tissue types and treatments on swine specimens [120,121]. The relative expression value (2−ΔΔCt) was obtained by normalization, subtracting the arithmetic mean of the reference genes from each gene of interest. Technical replicates were averaged before statistical analysis. The data are illustrated as average values of the groups (n = 4) ± standard error deviation of the mean (STDEV). All data were statistically analyzed using a one-way ANOVA method performed with GraphPad Prism 3.03 software (GraphPad Software, La Jolla, CA, USA). Post-hoc comparisons between all groups were run using the Bonferroni test. The statistical significance (*p* value) was presented for all groups in contrast to the Control group (C).

**Author Contributions:** Conceptualization, A.D. and S.E.G.; methodology, R.G.P., C.B., A.U., M.V., A.H., D.M. and M.F.; software, R.G.P.; validation, R.G.P. and S.E.G.; formal analysis, R.G.P. and S.E.G.; investigation, R.G.P., C.B., A.U., M.V., A.H. and M.F.; writing—original draft preparation, R.G.P., C.B., A.H. and M.V.; writing—review and editing, A.D., I.T, . and S.E.G.; supervision, A.D. and S.E.G.; project administration I.T, . and S.E.G.; funding acquisition, R.G.P. and S.E.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by a grant of the Romanian Ministry of Research and Innovation, CCCDI—UEFISCDI, project number PN-III-P1-1.2-PCCDI-2017-0473/"From classical animal nutrition to precision animal nutrition, scientific foundation for food security", within PNCDI I.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethical Committee of the National Research-Development Institute for Animal Nutrition and Biology, Balotes,ti, Romania (Ethical Committee no. 118/02.12.2019) and in accordance with the Romanian Law 206/2004 and the EU Council Directive 98/58/EC for handling and protection of animals used for experimental purposes.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy reason.

**Acknowledgments:** This work was supported by a grant of the Romanian Ministry of Research and Innovation, CCCDI—UEFISCDI, project number PN-III-P1-1.2-PCCDI-2017-0473/"From classical animal nutrition to precision animal nutrition, scientific foundation for food security", within PNCDI I "Use of agro-food residues by feeding solutions which to control the feed contaminants".

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

### *Article* **Rat Tumour Histopathology Associated with Experimental Chronic Dietary Exposure to Ochratoxin A in Prediction of the Mycotoxin's Risk for Human Cancers**

**Diana Herman <sup>1</sup> and Peter Mantle 2,\***

**Abstract:** Mammalian animal toxicity of ochratoxin A (OTA) has focused largely in the past halfcentury on pigs because of initial recognition of it as a principal cause of intermittent growth suppression and renal disease caused by mouldy feed. Subsequent classical toxicology has used laboratory rodents because renal pathology in pigs raised questions concerning possible involvement in the human idiopathic bilateral renal atrophy of Balkan endemic nephropathy for which OTA was a focus of attention for human nephropathy through 1980s and into 2000s. Emphasis on human nephropathy has more recently concerned the plant metabolite aristolochic acid. Recognition that agricultural management can often minimise food and feed-stuff spoilage by OTA-producing Aspergilli and Penicillia has moderated some of the risks for animals. Legislation for human food safety combined with sophisticated analysis generally provides safety in the developed world. Chronic experimental exposure of male rats, in the absence of clinical dis-ease, specifically causes renal cancer. The possibility of this as a unique model for the human has generated considerable experimental evidence which may be more directly relevant for carcinogenesis in the complex kidney than that obtained from biochemical toxicities in vitro. Nevertheless, there does not appear to be any case of human renal or urinary tract cancer for which there is verified etiological proof for causation by OTA, contrary to much claim in the literature. To contribute to such debate, histopathology review of OTA/rat renal cancers, augmented where appropriate by immune profiles, has been completed for all remaining tumours in our research archive. Overall consistency of positivity for vimentin, is matched with occasional positives either for CD10 or the cytokeratin MNF 116. The current situation is discussed. Suggestion that OTA could cause human testicular cancer has also been challenged as unsupported by any experimental findings in rats, where the Leydig cell tumour immune profile does not match that of human germ cell neoplasms.

**Keywords:** vimentin; CD10; MNF 116; renal cell cancer; urothelial cancer; testicular cancer; immunohistochemistry

**Key Contribution:** Immunohistochemical confirmation of the cytokeratin clone MNF 116 in some ochratoxin/rat kidney cancer contrasts with the protein's apparent absence from human kidney cancer. Experimental animal findings for ochratoxin A toxicity are discussed concerning application to carcinogenesis in humans.

#### **1. Introduction**

Ochratoxin A (OTA) was discovered in South Africa in the early 1960s to explain the general toxicity of cultured *Aspergillus ochraceus* as a dietary additive for experimental rats. Concurrently, the term mycotoxins became used to encompass toxic metabolites of other food-spoilage moulds such, as the aflatoxins of *A. flavus,* found to have widespread occurrence. Focus for OTA soon shifted to the spasmodic occurrence of an idiopathic porcine nephropathy in the Danish bacon industry, economically linked to home-grown

**Citation:** Herman, D.; Mantle, P. Rat Tumour Histopathology Associated with Experimental Chronic Dietary Exposure to Ochratoxin A in Prediction of the Mycotoxin's Risk for Human Cancers. *Toxins* **2021**, *13*, 205. https://doi.org/10.3390/ toxins13030205

Received: 4 January 2021 Accepted: 10 March 2021 Published: 12 March 2021

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**Copyright:** © 2021 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/).

barley; in some high rainfall years this had been insufficiently dried before storage. Seasonal opportunist moulding by a common *Penicillium*, and chromatographic recognition of OTA partly by its fluorescence under UV254 light, revealed another biosynthetic source of the mycotoxin in amounts subsequently demonstrated experimentally as a major cause of the disease in pigs. This had been expressed as reduced growth rate, low carcass weight and mottled and disproportionally enlarged kidneys, readily recognised at meat inspection and the carcass rejected [1].

Several *Aspergillus* and *Penicillium* moulds across both tropical and temperate latitudes were subsequently found to elaborate OTA during spoilage of major staple agricultural products such as cereals, and in crops for high-end commodities such as coffee, cocoa and red wines. Concern that traces of OTA in major cereal products such as pasta might pose a human health risk, and of potential economic threat to commercial images of the high-end commodities, stimulated extensive research into OTA toxicology. Concurrently, worldwide food safety authorities (e.g., for Europe, Joint expert committee on food additives, International agency for research on cancer, European food standards agency) addressed potential health risks and formulated documents to guide regulatory legislations.

Pigs being important in the human food chain in many economies prompted a major US experimental toxicology study in the 1980s with lifetime chronic exposure in rats [2]. The striking finding of renal tumours, expressed late in life mainly in males in the absence of overt toxicity, was the more notable as designating OTA as the most potent chemical for renal carcinoma in rats, although the extensive technical report did not raise any specific concerns for humans. The kidney cancer incidence findings demonstrated across the three gavage dose rates chosen for the NTP study later demonstrated OTA as a model for classic log dose/response for chemical carcinogens [3].

Subsequent developments in sensitive and definitive analytical methodology have shown widespread occurrence of relevant moulds and their mycotoxins, although general occurrence in well-managed agriculture is generally low. For human health, there has been little verified etiological evidence of general toxicities attributable other than to spasmodic episodes. The initial cautious classification of possible carcinogenetic risk [4] seems never subsequently to have had evidence that could stand classical epidemiological scrutiny. Particularly, the relevance of rat and mouse as pathological models for the human has become insecure because of the wide quantitative gulf (>104-fold) between necessary daily carcinogenic exposure for rodents (>30 μg OTA/kg body weight daily for at least half a rat lifetime, and much more for a mouse) and surveyed average natural daily human intake data (UK, 0.26–3.5 ng/kg b. wt.) [5].

The classical US toxicity study in rats [2] followed strict toxicology protocols involving gavage administration of OTA five days per week for up to 2 years. The more recent London studies, part of a European Commission project on OTA toxicity (2001-4), provided daily dietary ochratoxins as elaborated during moulding of shredded wheat breakfast cereal by *Aspergillus ochraceus* in a shaken solid substrate fermentation. The product, analysed for OTA, was homogenised (c.103-fold) into powdered rat diet for consumption during natural diurnal habit for Fischer male rats. Although blood OTA analysis had not been available for the US study, values in London followed a gradual rise to 8–10 μg/mL during the first month. For the highest daily OTA intake employed (300 μg/kg b wt) at least 9 months exposure seemed necessary to initiate kidney cancer, during which the common prevalence of monocytic leukaemia in ageing Fischer males was apparently repressed [6]. The tumour-free outcome for the US study lowest dose (21 μg/kg b wt) was raised to c. 30 μg/kg b wt in a London study in Dark Agouti male rats [7]. The dose/response criteria adopted for OTA's recognition as the model for log dose/response for chemical carcinogens [3] was thus reinforced by all subsequent findings in London [7] describing OTA/rat renal carcinogenicity as thresholded. Notably, this was not considered later in a major analysis of human risk assessment for OTA in Canada [8] in which a curious distorted non-thresholded graphical illustration is presented.

Critical histology review of some OTA/rat renal tumours has been described [9], including exploratory application for the first time of clinical immunohistochemistry where most tumours showed a range of positive responses to the clinical immuno-stains. Although designed for human histopathological diagnosis some immuno-stains helpfully showed cross-reactivity to rats. The primary experimental objective here is to consolidate those findings by completing histology review of all remaining archived cases in our archive.

#### **2. Results**

In confirming a renal cell origin for rat/OTA tumours, all four cases were diffusely and intensely positive for vimentin (Figure 1A, Case 1, tumour dimensions 10 × 20 mm)

(**A**)

**Figure 1.** *Cont.*

(**B**)

**Figure 1.** (**A**) Case 1, Vimentin (100×). (**B**) Case 1, CD10 (100×).

For case 1, CD10 is diffusely and intensely positive, as illustrated on approximately the same area as vimentin, in Figure 1B.

For Case 2, tumour diameter 20 mm, CD10 is negative but cytokeratin clone MNF 116 is diffusely positive, with heterogeneous pattern and variable intensity (Figure 2A, with adjacent Figure 2B as positive control illustrating staining in human tonsil and validating immunostaining cross reactivity in rat).

(**A**)

**Figure 2.** *Cont.*

(**B**)

**Figure 2.** (**A**) Case 2, CK MNF 116 (200×). (**B**) Control (human tonsil), CK MNF 116 (200×).

Case 3 tumour (5 mm) is not immuno-positive other than for vimentin (Figure 3); the diagnosis of renal cell carcinoma is being made on the histological aspect on H&E.

**Figure 3.** Case 3, Vimentin (200×).

Case 4 tumour (5 mm) is additionally positive for CD10. (not illustrated).

#### **3. Discussion**

Histopathology and immune profiles of the present four rat renal tumours caused by chronic exposure to dietary OTA are complementary to our other similar tumours to which immunohistochemical profiles have recently been ascribed [9]. The combined profiles in ten cases dis-associate the rat tumours from implying a model for an etiological role of OTA for the renal pelvic tumours sometimes associated with the Balkan endemic nephropathy. The latter have also recently been shown to have an immune profile indistinguishable from urothelial tumours studied in Slovakia where the Balkan nephropathy has not been reported, thereby dis-associating OTA from urothelial tumours sometimes occurring in Balkan nephropathy cases. A putative model role for human renal cell cancer might still persist if the vital factor of extraordinary dietary exposure to OTA could be established at a plausibly indicative value. Suggestion of OTA involvement in human testicular cancer has also been discounted experimentally in rats.

Immunostaining for vimentin was invariably extensive as is typical for human tissues of mesenchymal origin. The accompanying positivity for CD10 in three of the 10 rat tumours could fit for humans [10].  Immunoprofiles for these renal tumours do not fit easily with the OTA/rat renal expression assisting in predicting OTA as a human carcinogen. Notably, a recent review, co-authored from IARC [11], concludes that defining DNA adducts, oxidative stress and epigenetic factors that operate in humans could lead to reclassification of OTA as a carcinogen. It is not clear whether this indicates official IARC policy. It is unfortunate that authors mis-cite that 'DNA diploidy in OTA-induced rat tumours is associated to genetic change' [12]. It is hoped that any further consideration by IARC would consider that much or most of the in vitro toxicology literature avoids relating the OTA concentration, used to obtain a measured result, to how this relates to kidney parenchyma in vivo during carcinogenesis. It is also important to avoid some literature's assumption of accumulation of OTA in kidney [13].

Gender specificity in rodents is a neglected problem for matching a potential model with the human for whom there seems none for renal cell carcinoma. For the mouse, OTA renal cancer seems specifically male [14] and for the rat is nearly so [2]. A possible explanation for the rat [15] concerns ability of OTA to bind not only to plasma proteins but also to small male-specific urinary peptides in blood. These are subsequently transported through glomerular filtration to pass down nephrons. They follow the usual fate of at least partial salvage absorption in kidney cortex, thereby potentially augmenting delivery of OTA to cortical epithelia. It is hypothesised that the phenylalanine moiety of free OTA predisposes some direct cortical salvage for that essential amino acid in both genders, but that binding to male urinary peptides boosts the overall toxic impact in the potential tissue target for tumorigenesis. Further experimental study could readily detect by analytical gel electrophoresis the extent to which OTA binds to particular urinary proteins, traces of which must escape cortical salvage to appear in urine as pheromones. Such escape is essential in mouse biology, providing the olfactory language of gender and sexuality in the dark. Similarly, in the rat, verification of association of OTA with urinary protein [15] and extension, possibly by use of OTA radiolabelled to high specific activity, could help to clarify some mystery about OTA circulation in blood. OTA binds to serum albumin rather strongly in humans, but the competition in mice and rats between albumin and the smaller proteins that pass though glomeruli is unclear. Some preliminary exploration across puberty in male rats has occurred [16] but there is room for much more in connection with the pharmacokinetics of OTA. Extension of the previous findings [14] was only prevented by the tragic death of the key scientist.

Another way of gaining understanding of the relative dynamics of OTA in blood of male and female rats, to focus on gender differential in renal tumorigenesis, would be by first establishing and quantifying stable circulating OTA in male and female rats during continuous dietary exposure; then to castrate some of the males while continuing the OTA. Predictably, females would have initially attained a higher stable concentration of OTA in blood than males, which were actively excreting more bound to small urinary proteins. Post-castration OTA concentration in male blood could be expected to rise as testosterone-regulated urinary protein synthesis in liver declines. This could be a step towards evaluating the role of urinary proteins in the male rat tendency to develop renal tumours in response to chronic exposure to dietary OTA.

An experimental challenge to explore could be the use of an established technique of rat renal transplantation [17] to study male kidney neoplasia in a female body exposed to dietary OTA for 9–10 months, and vice versa. The graft would have to remain functional for well over a year. However, it is a curious thought that the global concern for OTA as a risk to human health, and the vast expense of analysis and legislation, could be an illusion created experimentally before discovery that small urinary proteins are sex pheromones in mice and rats, where they may transport OTA into kidney.

The above histopathology review [9] also notes that the very small group of female renal tumours in the 1989 NTP study had rather more variable histopathology than males, but the largest example was found to conform to the same immune profile as for males. Qualitative rat renal tumour pathology in response to OTA has thus been found to have a consistent characteristic pattern across genders.

There had been no agreed conclusion in an EU project (European Commission 2001–2004) concerning putative genotoxic, epigenetic or oxidative stress mechanisms in male rat renal carcinogenesis caused experimentally by OTA. However, definitive structural evidence of DNA adduction was subsequently obtained [18] after MS analysis of a synthetic adduct following preparative isolation from photoreaction of OTA and DNA. A previous plan, for MS data of the principal adduct isolated from kidneys of rats given OTA, had failed only due to misunderstanding that all of the extremely small amount of sample would need to go on the MS probe. Unfortunately, this was not practically a repeatable enterprise.

A third lifetime rat study, in Hannover Germany in the 1990s using Dark Agouti rats with gavage exposure to OTA [19] similar to that of the NTP study, also revealed some renal tumours. However, these were noted mainly as indicators of carcinogenicity in six of 15 individuals whose kidneys were subsequently found to contain OTA/DNA adducts. OTA dose was similar in cumulative amount to that in the high dose of the NTP study [2]. Giving OTA in aqueous vehicle, instead of corn oil, would have caused daily surges in circulating toxin concentration with greater toxic impact. Nevertheless, finding consistent occurrence of adducts in elderly rats correlated with continuing exposure to OTA is several months too late to imply an epidemiological role in tumour initiation. The same applies in principle also to human kidney cancer.

Subsequent use also of male Dark Agouti rats [7] generally corroborated the tumour findings in Hannover. However, this was only by doubling the OTA dose through 9 months of dietary exposure in the first year of life, during which OTA was consumed normally and slowly mainly by rat habit during the 12 h human night period. Other gavage regimens have delivered immediately during human daytime. Predictably, no adducts could have been found a year later due to repair of any formed during OTA exposure. In any case, it would be necessary to perfuse-wash tumorous kidneys in situ to remove vascular blood, within which adducts also occur [20], to demonstrate OTA/DNA adducts within kidney or tumour parenchyma. A similar strategy has also been necessary to explain a misconception of accumulation of OTA in mammalian kidney [13].

Across the three main centres for lifetime experiments, there was a consistently lower incidence of renal neoplasms in females than in males for Fischer, Dark Agouti and Lewis rats. A putative mechanism for this differentiation has been proposed [15]. It requires further experimental verification but can not be applied to humans which do not have analogous urinary proteins. This gender focus for OTA also potentially diminishes rat carcinogenicity relevance to humans and is an open question for other mammals.

In recent years ethical considerations have limited some whole animal experimentation, in addition to the economic cost of experimental mycotoxins, and to the lifetime

maintenance cost for rodents and particularly for primates. Thus, the scientific literature has reports mainly on tissue culture experiments. Whereas this may provide model information applicable to some mammalian tissues, there is less confidence in application to kidney with its exceptionally complex internal dynamics for sequential stages in filtration, key metabolite recovery, water and ion regulation, metabolic waste excretion and the huge replication of separate nephrons. Particularly, relative roles of trans-membrane ion transfer of OTA, as a phenylalanine derivative, from capillaries to nephron epithelia versus classical understanding of glomerular filtration, are not clear. Very rare finding of very simple early nuclear proliferation in situ within a nephron is tantalising evidence of early neoplasm. We have not yet seen this for OTA, but are aware of a possible model illustrated in a rat in an experiment on chronic exposure to aristolochic acid [21]. In our experience, continuous dietary exposure of male rats to OTA for 6 months, during which nephron epithelia experience millions of OTA molecules, is insufficient to initiate any renal tumour. Nine or ten months is sufficient, but the subsequent point at which pre-cancerous neoplasms might be visible in serial-sectioned kidney is unknown. Is this largely a matter of statistical probability of causing a critical, highly focal, genetic lesion? More experimental understanding is needed for this topic. OTA exposure starting at one year has failed to cause cancers [22]. What factor(s) between 6 and 9 months of age is influential in OTA tumorigenesis?

Experimental evidence shows that the Fischer and other OTA/rat tumours which have generated concern for humans may simply mimic the mechanism operating spontaneously in the EKER strain [23]. The findings are reminiscent of constitutive changes in the rat tuberous sclerosis gene complex which in the EKER strain are correlated with renal neoplasms. Thus, rat renal carcinogenesis caused by OTA does not obviously mimic human urinary tract tumorigenesis.

Another tumorigenesis topic in the literature in the past decade concerns whether OTA is a cause of human testis cancer. This was reiterated prominently in a review abstract [24] with the assertion 'that OTA is a biologically plausible cause of testicular cancer in man'. This was unfortunately based partly on misreading of the literature [22]. There was also insistence that experimental creation of OTA/DNA adducts in testes of newborn mice, from the mother's intrauterine exposure to a quite large OTA insult (2.5 mg/kg b wt) about 4 days previously, without discounting OTA/DNA adducts in newborn blood. Application of immunohistochemistry to histology review of rat testis tumours has since showed [25] the distinctive difference between the natural rat Leydig cell tumours and the germinal cell preponderance of human testicular tumours.

#### **4. Conclusions**

Rat and mouse renal tumour response to long-term dietary OTA has cautioned possible analogous cancer risk for humans, but there is yet no verified case of disease. A recent EFSA report [26] expresses continued uncertainty about human risks for OTA contaminations in food. Tumorigenic mechanisms have been proposed from in vitro studies but are unconvincing for tumours of highly focal origin in rodent or human. Review of rat tumour histopathology, including immune profiles, makes the rat a poor model in humans for cancers in kidney and testis. However, several experimental findings for kidney point to OTA mimicking the natural spontaneous renal tumours of the EKER strain. Natural OTA exposure for humans is very much less than that often applied to experimental cells and whole animal carcinogenicity follows a classical log dose/response relationship. Focus on satisfying Bradford Hill criteria for epidemiology of OTA is encouraged to avoid biases.

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

Four renal tumours from Fischer male rats given protracted dietary OTA (300 μg/kg body weight daily) [6] were embedded in paraffin blocks. Animals were from the same lifetime experimental group [6] as those whose immunoprofiles were previously described [9]. Ethical review and approval were waived for this study because no new live animals were involved. Sections (3 μm) were mounted on charged slides (TOMO, Matsunami, Japan)

and processed for immunohistochemistry in the Cell Pathology Laboratory of South West London Pathology at St George's Hospital, Tooting, variously applying panels of antibodies in fully automated BenchMark ULTRA immunohistochemistry processing, as required to assist clinical diagnoses. Procedures followed exactly those previously described [9]. The following antibodies were used: CK MNF 116, clone MNF 116 (Dako); Vimentin, clone V9 (Dako, Novocastra); CD10, clone 56C6 (Dako). After applying DAB chromogen, nuclei were counterstained blue with haematoxylin. The brown immune reaction product of DAB chromogen is cytoplasmic and/or membranar for the listed antibodies. Haematoxylin and Eosin staining was also performed for preliminary standard tissue differentiation of nuclear (blue) and cytoplasmic components (red).

**Author Contributions:** Conceptualization, D.H. and P.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Ethical review and approval were waived for this study because no new live animals were involved.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

### *Article* **The Impact of the Nephrotoxin Ochratoxin A on Human Renal Cells Studied by a Novel Co-Culture Model Is Influenced by the Presence of Fibroblasts**

**Gerald Schwerdt \*, Michael Kopf and Michael Gekle**

Julius-Bernstein-Institut für Physiologie, 06112 Halle, Germany<EMAIL_ADDRESS>(M.K<EMAIL_ADDRESS>(M.G.)

**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** The kidney is threatened by a lot of potentially toxic substances. To study the influence of the nephrotoxin ochratoxin A (OTA) we established a cell co-culture model consisting of human renal proximal tubule cells and fibroblasts. We studied the effect of OTA on cell survival, the expression of genes and/or proteins related to cell death, extracellular matrix and energy homeostasis. OTAinduced necrosis was enhanced in both cell types in the presence of the respective other cell type, whereas OTA-induced apoptosis was independent therefrom. In fibroblasts, but not in tubule cells, a co-culture effect was visible concerning the expression of the cell-cycle-related protein p21. The expression of the epithelial-to-mesenchymal transition-indicating protein vimentin was independent from the culture-condition. The expression of the OTA-induced lncRNA WISP1-AS1 was enhanced in co-culture. OTA exposure led to alterations in the expression of genes related to energy metabolism with a glucose-mobilizing effect and a reduced expression of mitochondrial proteins. Together we demonstrate that the reaction of cells can be different in the presence of cells which naturally are close-by, thus enabling a cellular cross-talk. Therefore, to evaluate the toxicity of a substance, it would be an advantage to consider the use of co-cultures instead of mono-cultures.

**Keywords:** ochratoxin A; cell culture; energy metabolism; apoptosis-necrosis balance; mitochondria

**Key Contribution:** Co-culture of human renal tubule cells with human fibroblasts demonstrate that the impact of a toxic substance, here ochratoxin A (OTA), can be underestimated when only one cell type is used. Based on gene expression studies, OTA interferes with energy metabolism leading to disturbed mitochondrial function and enhanced glucose mobilization from glycogen stores.

### **1. Introduction**

Due to its excretory function, the kidney is threatened by a variety of harmful substances such as drugs or food contaminants, like mycotoxins leading to acute or—even worse—chronic kidney diseases with a prevalence of about 10% [1,2]. To understand the mechanisms of the nephrotoxic action it is helpful to find strategies to alleviate these detrimental scenarios and many studies have been performed to solve the question of why and how kidneys are endangered [3–5].

To study the influences of a substance on an organism, it is often difficult to use whole animals because of ethical concerns and organizational, costly and elaborate prerequisites. Furthermore, a transfer of knowledge to the human situation is associated with uncertainties. Therefore, cell culture models have been established and are used widely, and have the advantage that a specific cell type and its response to a substance or to a treatment can be studied under controlled conditions. Although many and important findings have been made using this approach, some disadvantages are inherent: cells of a cell line often have been immortalized by mutagenesis or other—sometimes drastic—methods [6]. This allows easy handling and long usage but with the hazard that results found in a specific model

**Citation:** Schwerdt, G.; Kopf, M.; Gekle, M. The Impact of the Nephrotoxin Ochratoxin A on Human Renal Cells Studied by a Novel Co-Culture Model Is Influenced by the Presence of Fibroblasts. *Toxins* **2021**, *13*, 219. https://doi.org/10.3390/ toxins13030219

Received: 25 February 2021 Accepted: 16 March 2021 Published: 18 March 2021

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**Copyright:** © 2021 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/).

system may not be transferable to the situation in the whole organ or organism. To overcome this disadvantage, instead of immortalized cell lines, primary cells can be used, but the generation of primary cells is often very difficult and requires advanced technical skills. Additionally, primary cells often do not survive for a long period of time and need special individual culture conditions. But primary cells are a step closer to natural conditions and at least sometimes it turned out that they are more sensitive to, e.g., toxic stimuli as cell lines are [7], meaning that cell lines might be more robust. Another disadvantage is the fact that cells are often kept in monoculture, i.e., without the influences of other cell types, which in their home organ usually are close-by. Therefore, it might be a step towards a more realistic situation to study the response of a cell type to a substance or treatment in the presence of those cells, which are in the native organ in close proximity.

In the kidney, proximal tubule cells are surrounded by fibroblasts and a—probably mutual—influence can be assumed. This is also the case in kidney damaging scenarios that in most cases lead to tubulo-interstitial inflammation and fibrosis [8,9], and are decisive for the decline of kidney function. Because of their transport and enzymatic capabilities, renal proximal tubule cells are endangered by a variety of potential toxic substances such as, e.g., drugs or their remnants or mycotoxins. One role of the surrounding fibroblasts is to furnish the extracellular matrix by release of collagens and other matrix components and therefore to participate in the integrity of the tissue [10]. But they are also—together with epithelial cells—involved in inflammatory processes or in fibrotic kidney diseases [11] with the risk of developing renal failure.

An intensively studied mycotoxin with relevance for human health is ochratoxin A (OTA) [12,13]. It can be found in a variety of foodstuffs [12,14] and to avoid its exposure and uptake is almost impossible [9,15]. This leads to the observation that OTA is detected frequently in human blood in low nanomolar concentrations [16]. In exposed animals, OTA leads to kidney failure and fibrotic changes [17,18]. OTA exposure is assumed to be involved in human kidney diseases [19]. In human primary proximal tubule cells, a toxic effect of OTA has been shown, which is also observable in human primary fibroblasts, although it is not as prominent as in proximal tubule cells [7]. The mechanisms behind the toxic action of OTA are still not completely understood and are subject of many ongoing studies. How far neighboring cells with different functions interfere and thereby modulate cell function is almost not known but is expectable. In a previous study using a co-culture model consisting of rat kidney proximal tubule and fibroblast cells, it turned out that a kind of crosstalk between both cell types takes place, leading to the observation that effects of OTA as epithelial-to-mesenchymal transition (EMT) occurred only under co-culture conditions [20]. Another conclusion drawable from that study and others was that rat cells are more robust concerning the tolerance to OTA as compared to human proximal tubule cells [7,20] and therefore a model system based on human cells is required to closer evaluate the human situation and the risk of OTA exposure.

Therefore, in the present study, we establish an advanced cell co-culture model consisting of human proximal tubule cells (HK2 cells) and human fibroblasts (CCD-1092SK cells) to study the effects of OTA on cell survival (apoptosis, necrosis) and expression of some exemplarily chosen genes related to cell cycle, cell death, extracellular matrix, and metabolism. Similar to previous studies using rat cells [20], the human proximal tubule cells were placed on filter devices and the filters were put above a layer of fibroblasts seeded on the bottom of a petri dish so that the basolateral side of the epithelial cells faces towards the fibroblasts, enabling a kind of conversation between both cell types.

#### **2. Results**

#### *2.1. Protein, Lactate Dehydrogenase Release and Caspase-3 Activity*

To obtain a first impression about possible effects of culture conditions itself as well as about effects on OTA-induced alterations, we compared caspase-3 activity as a measure for apoptosis of cells grown in monoculture with the activity of cells grown in co-culture incubated with or without 100 nM OTA for two points of time, 24 and 48 h. In addition,