**1. Introduction**

Zearalenone is an undesirable substance that is widely encountered in cereal kernels and cereal products [1]. It is produced mainly by *Fusarium graminearum* as a metabolite with estrogenic properties. Pure ZEN is a white crystalline compound with the chemical formula C18H22O5, a molecular mass of 318.36 g/mol, and a melting temperature range of 161–163 ◦C. Zearalenone is not soluble in water, but it dissolves easily in alkaline solutions, ether, benzene, methanol, and ethanol. This mycotoxin can be metabolized by various organisms during phase I and II biotransformation processes [2]. Feeds contain modified forms of ZEN (ZELs), including phase I biotransformation products such as α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol, and zearalanon, as well as phase II biotransformation products conjugated with glucose, sulfate, and glucuronic acid. The

**Citation:** Mróz, M.; Gaj˛ecka, M.; Brzuzan, P.; Lisieska-Zołnierczyk, S.; ˙ Leski, D.; Zielonka, Ł.; Gaj˛ecki, M.T. Carry-Over of Zearalenone and Its Metabolites to Intestinal Tissues and the Expression of CYP1A1 and GSTπ1 in the Colon of Gilts before Puberty. *Toxins* **2022**, *14*, 354. https://doi.org/10.3390/ toxins14050354

Received: 20 April 2022 Accepted: 17 May 2022 Published: 18 May 2022

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**Copyright:** © 2022 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/).

presence of ZEN in the food chain can pose a health threat to humans and livestock [3]. This promiscuous compound induces the expression of estrogen receptors [4], contributes to reproductive disorders, and can even lead to changes in reproductive organs. Zearalenone also disrupts the hormonal balance [2,5,6], metabolic profile [7], and gut microbiota [8]. It exerts hepatotoxic [9,10], immunotoxic [11], hematotoxic [12], and genotoxic effects [13].

In humans and animals, ingested ZEN initially interacts with the gastrointestinal system [14]. Two research problems can be formulated in this stage. Firstly, ZEN's influence on intestinal health [15,16] and its carry-over from the intestinal digesta to intestinal tissues [16] has attracted considerable research interest in recent years. Secondly, in line with the hormesis paradigm [17], efforts are being made to evaluate the macroorganism's response to low mycotoxin doses that are frequently encountered in feed materials. Researchers have also attempted to determine tissue and cell dysfunctions in mammals exposed to the pure parent compound without metabolites or modified mycotoxins [4,18]. In view of our previous findings [5,7,8,13], potential clinical symptoms of ZEN mycotoxicosis of varying severity were taken into account. Therefore, three ZEN doses were considered in the interpretation of our previous results and the findings of other authors: the lowest adverse effect observed level (LOAEL, >10 µg ZEN/kg BW) [19–21] that causes clinical symptoms [22]; (ii) the no adverse effect observed level (NOAEL, 10 µg ZEN/kg BW) as the highest dose that does not cause clinical symptoms (sub-clinical states) [23]; and (iii) the minimal anticipated biological effect level (MABEL, <10 ZEN/kg BW) as the lowest dose that can be measured in tissues, entering into positive interactions with the host at various stages in life [24,25].

The observation that mycotoxins modulate the expression of enzymes that participate in phase I and II biotransformation processes [26] was taken into consideration in the current experiment. The biotransformation of undesirable substances, including ZEN and its metabolites, is affected by two types of enzymes in cells. During phase I biotransformation, enzymes such as CYP P450 trigger chemical reactions (hydroxylation) to remove undesirable substances that can act as enzyme substrates [27]. These enzymes exhibit maximum activity in the liver and the small intestine, which are responsible for detoxication. They are also involved in the biosynthesis of steroid hormones [28] and fatty acid metabolism, as well as the activation/inactivation of drugs and other exogenous compounds in the body [29,30]. During phase II biotransformation, metabolic rates can be measured based on the activity of glutathione S-transferase (GST). The rate of biotransformation and the rate at which undesirable feed-borne substances are excreted from the body affect the response of the intestinal mucosal immune system to toxins. The π isoform of glutathione transferase (GSTπ1) codes for various proteins involved in these processes. Glutathione S-transferase reduces the biological activity of various (not only exogenous) substances by creating conjugates of glutathione with electrophilic drugs and other exogenous toxins. These conjugates protect the body against the adverse effects of oxidative stress and prevent damage to lipids and nucleic acids. It is worth noting that conjugates can break down again in the lumen of the gut, thus increasing the concentration of mycotoxins and theoretically increasing the activity of enzymes such as CYP1A1 and GSTπ1 [26,30].

The objective of this study was to determine how in vivo or low doses of ZEN (MABEL (5 µg/kg BW), the greatest values of NOAEL (10 µg/kg BW), and LOAEL (15 µg/kg BW)) administered orally for 42 days influence the level of ZEN and selected ZELs in intestinal wall tissues on different dates of exposure and contribute to the expression of genes encoding selected enzymes (CYP1A1 and GSTπ1) in the colon in prepubertal gilts.

#### **2. Results**

The present results confirmed that ZEN is biotransformed in the initial stage of the carry-over process when this mycotoxin and its metabolites are transported from the lumen of the gut to the intestinal wall. Parent mycotoxin and its two most widely investigated metabolites (α-ZEL and β-ZEL) are accumulated in this stage.

The above observation was confirmed by the mean concentrations of ZEN and its metabolites in the intestinal wall on different exposure days (see Figure 1). The percentage share of each substance in the total pool was consistent with the anticipated values for pigs, but the carry-over factor (CF) was much lower, which cannot be attributed solely to low-dose ZEN mycotoxicosis. share of each substance in the total pool was consistent with the anticipated values for pigs, but the carry‐over factor (CF) was much lower, which cannot be attributed solely to low‐dose ZEN mycotoxicosis.

gated metabolites (α‐ZEL and β‐ZEL) are accumulated in this stage.

The present results confirmed that ZEN is biotransformed in the initial stage of the carry‐over process when this mycotoxin and its metabolites are transported from the lu‐ men of the gut to the intestinal wall. Parent mycotoxin and its two most widely investi‐

The above observation was confirmed by the mean concentrations of ZEN and its metabolites in the intestinal wall on different exposure days (see Figure 1). The percentage

*Toxins* **2022**, *14*, 354 3 of 22

**2. Results**

#### *2.1. Experimental Feed 2.1. Experimental Feed*

The analyzed feed did not contain mycotoxins, or its mycotoxin content was below the sensitivity of the method (VBS). The concentrations of masked mycotoxins were not analyzed. The analyzed feed did not contain mycotoxins, or its mycotoxin content was below the sensitivity of the method (VBS). The concentrations of masked mycotoxins were not analyzed.

#### *2.2. Clinical Observations 2.2. Clinical Observations*

The results presented in this paper were acquired during a large‐scale experiment where clinical signs of ZEN mycotoxicosis were not observed. However, changes in spe‐ cific tissues or cells were frequently noted in analyses of selected serum biochemical pa‐ rameters, hematological parameters, the heart muscle, the bone marrow microenviron‐ ment, coronary artery, the genotoxicity of cecal water, selected steroid concentrations, gut microbiota parameters, and weight gains. Samples for laboratory analyses were collected from the same prepubertal gilts. The results of these analyses were presented in our pre‐ vious studies [5,7,8,12,13,31,32]. The results presented in this paper were acquired during a large-scale experiment where clinical signs of ZEN mycotoxicosis were not observed. However, changes in specific tissues or cells were frequently noted in analyses of selected serum biochemical parameters, hematological parameters, the heart muscle, the bone marrow microenvironment, coronary artery, the genotoxicity of cecal water, selected steroid concentrations, gut microbiota parameters, and weight gains. Samples for laboratory analyses were collected from the same prepubertal gilts. The results of these analyses were presented in our previous studies [5,7,8,12,13,31,32].

#### *2.3. Concentrations of Zearalenone and Its Metabolites in the Intestinal Tract of Prepubertal Gilts*

Significant differences in ZEN levels between exposure date D1 and exposure dates D2 and D3 (see Table 1) were observed in the ascending colon centrifugal gyrus and the ascending colon centripetal gyrus in group ZEN5. In group ZEN10, significant differences were found in the transverse colon between D3 vs. D1 and D2. In group ZEN15, no significant differences were noted between exposure dates.

A comparison of ZEN concentrations in the examined segments of the intestinal tract (see Table 1) on the analyzed exposure dates in group ZEN5 revealed that: (i) in nearly all intestinal segments (excluding the ileum), ZEN concentrations were highest on D1; (ii) ZEN concentrations were highest in both gyri of the ascending colon, the jejunum, and cecum; (iii) ZEN concentrations were low and similar on all exposure dates only in the ileum (3.13, 3.26, and 4.10 ng/g, respectively); (iv) interestingly, ZEN concentrations tended to decrease over time in all intestinal segments.

*Toxins* **2022**, *14*, 354

**Table 1.** The transfer coefficient and mean (±) concentration of ZEN (ng/g) in the intestines of gilts before puberty.

and **aa** ,

**cc** ,

**dd** *p* ≤ 0.01; **a**

,

difference between group ZEN5 and groups ZEN10 and ZEN15; **d**

,

**aa** notable difference between exposure date D1 and exposure dates D2 and D3; **b** notable difference between exposure date D2 and exposure date D3; **c**

**dd** notable difference between group ZEN10 and group ZEN15.

,

**cc** notable

The concentrations of ZEN differed in the ZEN10 group (see Table 1). In the duodenum, ZEN concentrations were very high and similar on all exposure dates (D1–D3) (64.36, 46.41, and 39.84 ng/g, respectively). In the remaining intestinal segments, ZEN concentrations were very low (3–20 ng/g) on D2 and D3, with a rising trend on successive dates of exposure.

In group ZEN15, ZEN concentrations were relatively high on D1 compared with the remaining dates of exposure (see Table 1). On D2, ZEN concentrations decreased in almost all sections of the intestine (excluding the ascending colon centrifugal gyrus—35.15 ng/g). On the last exposure date, ZEN concentrations increased in nearly all intestinal segments (excluding the centripetal gyri of the ascending colon—8.17 ng/g). The observed changes in ZEN values were relatively low in all intestinal segments, excluding the duodenum and the transverse colon.

In group ZEN5, significant differences in αZEL concentrations (see Table 2) were observed between D3 and D2 and between D2 and D1. In both cases, these differences were noted in the cecum and the centrifugal gyri of the ascending colon. In group ZEN10, significant differences were found in the cecum between D3 and D1, between D3 and D2, and in the centripetal gyri of the ascending colon between D3 and D1. In group ZEN 15, significant differences were noted in the duodenum, jejunum, cecum, and the centrifugal gyri of the ascending colon between D3 vs. D2 and D1 and between D2 and D1.

On D1 (see Table 2), significant differences in αZEL concentrations between groups ZEN10 and ZEN15 vs. group ZEN5 were found only in the cecum. On D2, the analyzed parameter differed significantly between group ZEN5 and group ZEN15 only in the centripetal gyri of the ascending colon and the transverse colon. On D3, significant differences between group ZEN5 and group ZEN15 were noted in the jejunum, cecum, and the centrifugal gyri of the ascending colon.

An analysis of αZEL concentrations (see Table 2) in different intestinal segments revealed that in group ZEN5, they were very low and similar (0.78 to 3.98 ng/g) on D1 and D3, whereas a wider range of values (1.07 ng/g on D1 to 6.69 ng/g on D3) was observed only in the ileum. On D2, αZEL levels were two or even three times higher than on D1 and D3, ranging from 7.00 ng/g in the descending colon to 49.47 ng/g in the cecum.

In group ZEN10, αZEL concentrations (see Table 2) were proportional to the time of exposure. However, on D2, αZEL concentrations in the ileum were below the values noted on D1 (difference of 2.00 ng/g) and D3 (difference of 4.03 ng/g).

In group ZEN15, αZEL concentrations were proportional in each intestinal segment. However, the analyzed parameter decreased in some segments (ileum) and increased rapidly in other parts of the intestinal tract (cecum). Alpha-ZEL levels were very low on exposure day 42, e.g., in the descending colon.

It should be noted that significant differences in *β*-ZEL concentrations were very rarely observed on exposure dates D1 and D2, and the highest number of significant differences was noted on D3 (see Table 3), in particular in group ZEN15. In group ZEN5, highly significant differences in *β*-ZEL concentrations were found only between D3 and D1 in the duodenum, jejunum, and cecum, and between D3 and D2 in the duodenum and jejunum. In group ZEN10, significant differences in the analyzed parameter were observed between D3 vs. D1 and D2 in the centrifugal gyri of the ascending colon. In group ZEN15, significant differences were noted between D3 and D1 in nearly all intestinal segments, excluding the ileum and the centripetal gyri of the ascending colon; significant differences were also noted between D3 and D2 in the jejunum, centripetal gyri of the ascending colon, transverse colon, and descending colon. Additionally, significant differences in *β*-ZEL levels were noted between D2 and D1 in the duodenum, jejunum, and cecum.

An analysis of *β*-ZEL concentrations (see Table 3) on different exposure dates revealed significant differences on D1 between group ZEN5 vs. groups ZEN10 and ZEN15 in the cecum and between group ZEN10 and group ZEN15 in the cecum. On D2, significant differences in *β*-ZEL concentrations were observed between group ZEN5 vs. groups ZEN10 and ZEN15 in the jejunum and cross member and between group ZEN10 and group ZEN15 in the jejunum. On D3, differences in the studied parameter were noted between group ZEN5 vs. groups ZEN10 and ZEN15 in the centrifugal gyri of the ascending colon and between group ZEN5 and group ZEN15 in the duodenum and descending colon.

**Table 2.** The transfer coefficient and mean (±) concentration of *α*ZEL (ng/g) in the intestines of gilts before puberty.

**Key**: CFG—Ascending colon Centrifugal gyrus; CPG—Ascending colon Centripetal gyrus; Exposure dates: D1—exposure day 7; D2—exposure day 21; D3—exposure day 42. Experimental groups: group ZEN5—5 µg ZEN/kg BW; group ZEN10—10 µg ZEN/kg BW; group ZEN15—15 µg ZEN/kg BW. LOD > values below the limit of detection were expressed as 0. The statistically notable differences were determined at **a** , **b** , **c** *p* ≤ 0.05 and **aa** , **bb** , **cc** *p* ≤ 0.01; **a** , **aa** notable difference between exposure date D1 and exposure date D2 and D3; **b** , **bb** notable difference between exposure date D2 and exposure date D3; **c** , **cc** notable difference between group ZEN5 and groups ZEN10 and ZEN15.

#### *2.4. Carry-Over Factor (CF)*

Natural contamination of cereals and feeds with mycotoxins is frequently reported. Mycotoxins ingested by livestock with feed are carried over to food products of animal origin [16]. Undesirable substances are transferred from contaminated feed to animal tissues. The CF and the resulting health risks remain insufficiently investigated in vivo, in particular during low-dose mycotoxicosis, which is why they were analyzed in this study.

The carry-over rate of ZEN from the intestinal lumen to the intestinal wall was affected by the dose and time of exposure in each group (see Table 1). The highest CF values were noted on D1 in groups ZEN5 (centrifugal gyri of the ascending colon) and ZEN10 (duodenum). In group ZEN15, the highest CF values were observed in six of the eight investigated intestinal segments (excluding the jejunum and ileum). In turn, the lowest CF values were noted on D3 in all groups: in the transverse colon in group ZEN5 (8 × 10−6 ), in the jejunum in group ZEN10 (6 × 10−6 ), and in the jejunum in group ZEN15 (3 × 10−6 ).

The CF of αZEL in the intestinal tract ranged from 10−4 to 10−6 (see Table 2). The lowest values of this parameter (10−6 ) were noted in group ZEN5 on D3 in the transverse colon and the descending colon; in group ZEN10 on D1 in the descending colon and on D3 in the transverse colon and the descending colon; in group ZEN15 on D1 in the ileum, centripetal gyri of the ascending colon, transverse colon, and descending colon; on D2 in all intestinal segments, excluding the cecum; and on D3 in the ileum, transverse colon, and

descending colon. Interestingly, CF values were lowest in the descending colon on all days of exposure. In turn, the highest values of CF (10−4 ) were found in group ZEN5 on D2 in the cecum; in group ZEN10 on D2 and D3 in the jejunum and cecum; and in group ZEN15 on D2 in the cecum. In 80% of the cases, the highest CF values were noted in the cecum.

**Table 3.** The transfer coefficient and mean (±) concentration of *β*ZEL (ng/g) in the intestines of gilts before puberty.

**Key**: CFG—ascending colon centrifugal gyrus; CPG—ascending colon centripetal gyrus; exposure dates: D1—exposure day 7; D2—exposure day 21; D3—exposure day 42. Experimental groups: group ZEN5—5 µg ZEN/kg BW; group ZEN10—10 µg ZEN/kg BW; group ZEN15—15 µg ZEN/kg BW. LOD > values below the limit of detection were expressed as 0. The statistically notable differences were determined at **a** , **b** , **c** *p* ≤ 0.05 and **aa** , **bb** , **cc** , **dd** *p* ≤ 0.01; **a** , **aa** notable difference between exposure date D1 and exposure dates D2 and D3; **b** , **bb** notable difference between exposure date D2 and exposure date D3; **c** , **cc** notable difference between group ZEN5 and groups ZEN10 and ZEN15; **dd** notable difference between group ZEN10 and group ZEN15.

The carry-over rates of β-ZEL (see Table 3) in different segments of the intestinal tract were determined within a narrower range of values (10−5 to 10−8 ) than the carry-over rates of ZEN and α-ZEL. The highest CF values were noted on D1 in group ZEN10 in the duodenum, jejunum, and centripetal gyri of the ascending colon; on D2 in group ZEN10 in the jejunum and cecum; and on D3 in group ZEN5 in the duodenum and jejunum. In group ZEN15, the highest CF values were observed on D3 in the ileum. The CF factor was calculated in 72 cases, and it reached 10−8 in 1 case (ileum, group ZEN15, D1), 10−7 in 17 cases, 10−6 in 50 cases, 10−5 in 3 cases, and 0 in 1 case (ileum, group ZEN5, D1).

#### *2.5. Expression of CYP1A1 and GSTP1 Genes*

Cytochrome P450 1A1 (CYP1A1) participates in the phase I metabolism of xenobiotics and drugs. It can exert protective effects on DNA and does not contribute to potentially cancerogenic DNA modifications, which could be attributed to the fact that CYP1A1 is highly active in the intestinal mucosa and prevents xenobiotics and carcinogens from reaching the circulatory system [33]. In this experiment, the *CYP1A1 m*RNA gene was generally silenced in both segments of the large intestine in all groups (see Figure 2). In the ascending colon, on D1 and D3, *CYP1A1 m*RNA expression was most strongly silenced in group C in comparison with the experimental groups. Similar results were reported by

Sun et al. [6] in Leydig cells exposed in vitro to ZEN. In the ascending colon, on D1 and D3, *CYP1A1 m*RNA expression was most strongly silenced in group C (1.226 and 0.623, respectively). In contrast, on D2, *CYP1A1 m*RNA expression was more silenced in the experimental groups (1.103, 0.983, and 0.929, respectively) than in group C (1.313). The reverse was noted in the descending colon, where *CYP1A1 m*RNA expression was more silenced in the experimental groups on D1 (0.952, 0.613, and 0.676, respectively) and D3 (0.542, 0.480, and 0.462, respectively). On D2, *CYP1A1 m*RNA expression was most strongly silenced in groups C and ZEN5 (0.536 and 0.588, respectively). *Toxins* **2022**, *14*, 354 8 of 22

Glutathione S-transferases (GSTs) are detoxification enzymes that catalyze conjugation reactions between endogenous glutathione and electrophilic metabolites produced during phase I biotransformation [34]. These enzymes protect cells against the harmful effects of electrophilic chemical compounds and oxidation products. Glutathione S-transferases are a family of dimeric enzymes responsible for the conjugation of exogenous and endogenous compounds with glutathione. Glutathione protects DNA against damage by binding toxic compounds in the cytoplasm and preventing them from interacting with nucleic acid [35]. calculated in 72 cases, and it reached 10−8 in 1 case (ileum, group ZEN15, D1), 10−7 in 17 cases, 10−6 in 50 cases, 10−5 in 3 cases, and 0 in 1 case (ileum, group ZEN5, D1). *2.5. Expression of CYP1A1 and GSTP1 Genes* Cytochrome P450 1A1 (CYP1A1) participates in the phase I metabolism of xenobiot‐ ics and drugs. It can exert protective effects on DNA and does not contribute to potentially cancerogenic DNA modifications, which could be attributed to the fact that CYP1A1 is highly active in the intestinal mucosa and prevents xenobiotics and carcinogens from

In the present study, the expression of the GSTπ1 gene was silenced [34,36] as a result of low-dose zearalenone mycotoxicosis (see Figure 3). The suppression of gene expression was directly proportional to the zearalenone dose in the experimental groups. The expression of GSTP1 mRNA in both intestinal segments was highest in group C on all dates of exposure. In the ascending colon, significant differences were found between group C vs. groups ZEN10 and ZEN15 on all exposure dates (difference of 0.187 and 0.404 on D1; difference of 0.302 and 0.405 on D2; difference of 0.331 and 0.351 on D3, respectively). In group ZEN5, GSTP1 mRNA expression was also more strongly silenced than in group C, but the difference was not statistically notable. In the descending colon, notable differences were not observed on D1 and D2, whereas on D3, a significant difference (0.303) was noted between group C and group ZEN15. reaching the circulatory system [33]. In this experiment, the *CYP1A1 m*RNA gene was generally silenced in both segments of the large intestine in all groups (see Figure 2). In the ascending colon, on D1 and D3, *CYP1A1 m*RNA expression was most strongly silenced in group C in comparison with the experimental groups. Similar results were reported by Sun et al. [6] in Leydig cells exposed in vitro to ZEN. In the ascending colon, on D1 and D3, *CYP1A1 m*RNA expression was most strongly silenced in group C (1.226 and 0.623, respectively). In contrast, on D2, *CYP1A1 m*RNA expression was more silenced in the ex‐ perimental groups (1.103, 0.983, and 0.929, respectively) than in group C (1.313). The re‐ verse was noted in the descending colon, where *CYP1A1 m*RNA expression was more silenced in the experimental groups on D1 (0.952, 0.613, and 0.676, respectively) and D3 (0.542, 0.480, and 0.462, respectively). On D2, *CYP1A1 m*RNA expression was most strongly silenced in groups C and ZEN5 (0.536 and 0.588, respectively).

**Figure 2.** The expression of CYP1A1 in the ascending colon and descending colon at different dates of exposure. Bars represent the mean values (*n* = 5) of enzyme gene expression ratios (±) relative to the control sample at the beginning of the experiment (ER = 1.00; dashed line). Asterisks (\*) denote experimental groups (group ZEN5—5 μg ZEN/kg BW; group ZEN10—10 μg ZEN/kg BW; group ZEN15—15 μg ZEN/kg BW), which differed significantly in mRNA levels compared to the corre‐ sponding control (C) groups (\* *p* ≤ 0.05, \*\* *p* ≤ 0.01). **Figure 2.** The expression of CYP1A1 in the ascending colon and descending colon at different dates of exposure. Bars represent the mean values (*n* = 5) of enzyme gene expression ratios (±) relative to the control sample at the beginning of the experiment (ER = 1.00; dashed line). Asterisks (\*) denote experimental groups (group ZEN5—5 µg ZEN/kg BW; group ZEN10—10 µg ZEN/kg BW; group ZEN15—15 µg ZEN/kg BW), which differed significantly in mRNA levels compared to the corresponding control (C) groups (\* *p* ≤ 0.05, \*\* *p* ≤ 0.01).

Glutathione S‐transferases (GSTs) are detoxification enzymes that catalyze conjuga‐ tion reactions between endogenous glutathione and electrophilic metabolites produced

was noted between group C and group ZEN15.

nucleic acid [35].

during phase I biotransformation [34]. These enzymes protect cells against the harmful effects of electrophilic chemical compounds and oxidation products. Glutathione S‐trans‐ ferases are a family of dimeric enzymes responsible for the conjugation of exogenous and endogenous compounds with glutathione. Glutathione protects DNA against damage by binding toxic compounds in the cytoplasm and preventing them from interacting with

In the present study, the expression of the GST1 gene was silenced [34, 36] as a result of low‐dose zearalenone mycotoxicosis (see Figure 3). The suppression of gene expression was directly proportional to the zearalenone dose in the experimental groups. The expres‐ sion of GSTP1 mRNA in both intestinal segments was highest in group C on all dates of exposure. In the ascending colon, significant differences were found between group C vs. groups ZEN10 and ZEN15 on all exposure dates (difference of 0.187 and 0.404 on D1; difference of 0.302 and 0.405 on D2; difference of 0.331 and 0.351 on D3, respectively). In group ZEN5, GSTP1 mRNA expression was also more strongly silenced than in group C, but the difference was not statistically notable. In the descending colon, notable differ‐ ences were not observed on D1 and D2, whereas on D3, a significant difference (0.303)

**Figure 3.** The expression of GSTπ1 in the ascending colon and descending colon at different dates of exposure. Bars represent the mean values (*n* = 5) of enzyme gene expression ratios (±) relative to the control sample at the beginning of the experiment (ER = 1.00; dashed line). Asterisks (\*) denote experimental groups (group ZEN5—5 μg ZEN/kg BW; group ZEN10—10 μg ZEN/kg BW; group ZEN15—15 μg ZEN/kg BW), which differed significantly in mRNA levels compared to the corre‐ sponding control (C) groups (\* *p* ≤ 0.05, \*\* *p* ≤ 0.01). **Figure 3.** The expression of GSTπ1 in the ascending colon and descending colon at different dates of exposure. Bars represent the mean values (*n* = 5) of enzyme gene expression ratios (±) relative to the control sample at the beginning of the experiment (ER = 1.00; dashed line). Asterisks (\*) denote experimental groups (group ZEN5—5 µg ZEN/kg BW; group ZEN10—10 µg ZEN/kg BW; group ZEN15—15 µg ZEN/kg BW), which differed significantly in mRNA levels compared to the corresponding control (C) groups (\* *p* ≤ 0.05, \*\* *p* ≤ 0.01).

#### **3. Discussion**

metabolites in prepubertal gilts were highly individualized (see Tables 1, 2, and 3). A com‐ parison between the current and previous studies indicates that long‐term exposure to low ZEN doses stimulates proliferation processes and migration on all dates of exposure [5,7,8,12,13,20,31,37]. The question that remains to be answered is why ZEN metabolites were detected in intestinal tissues if their concentrations were considerably lower in the This study demonstrated that the absorption and biotransformation of ZEN and its metabolites in prepubertal gilts were highly individualized (see Tables 1–3). A comparison between the current and previous studies indicates that long-term exposure to low ZEN doses stimulates proliferation processes and migration on all dates of exposure [5,7,8,12,13,20,31,37]. The question that remains to be answered is why ZEN metabolites were detected in intestinal tissues if their concentrations were considerably lower in the blood [5] and the heart muscle [31] and if they were completely absent in the bone marrow microenvironment [12].

This study demonstrated that the absorption and biotransformation of ZEN and its

#### *3.1. Zearalenone and its Metabolites*

#### 3.1.1. Zearalenone Concentrations

**3. Discussion**

An analysis of ZEN concentrations in gut tissue (see Table 1) revealed the highest values in the duodenum on exposure date D1 (ZEN levels were not always proportional to the ingested dose) in group ZEN10. This observation can be explained by the fact that in the initial stages of intestinal absorption, ZEN is most effectively absorbed in the duodenum, where it is hydroxylated to α-ZEL and β-ZEL [3]. This is because the duodenum and jejunum have firmly adherent mucus and polysaccharide layers [38], and prepubertal gilts have high requirements for estrogen and estrogenic compounds [5] due to supraphysiological hormonal levels [39]. In groups ZEN10 and ZEN15, higher ZEN concentrations could also be attributed to the presence of "free ZEN", which contributes to: (i) the inhibition of steroidogenesis [6]; (ii) the conversion of testosterone (ZEN suppresses testosterone levels) to estradiol (which inhibits maturation processes in prepubertal gilts, [40]); and (iii) increased feed intake and the accumulation of energy [7], supporting homeostasis and, at later procreation of the organism, reproduction in prepubertal gilts [41]. It should also be noted that the period of adaptation to an ongoing mycotoxicosis ends after seven dates of exposure (i.e., on D1) [42].

On D2 and D3, ZEN levels in intestinal tissues increased (see Figure 1) in all groups, but values recorded in groups ZEN10 and ZEN15 (excluding the centrifugal gyri of the

ascending colon) were proportionally lower than in group ZEN5. This is because the intestinal tract constitutes the first line of defense against the harmful effects of mycotoxins [14,43]. The intestines are covered by a layer of mucus and polysaccharides that adhere loosely or firmly to the intestinal wall [38] and determine the rate of absorption processes. Mycotoxins, including ZEN, exert negative effects mainly during prolonged exposure to high doses [14]. Zearalenone stimulated the proliferation of colonic cells in vitro [3]. Therefore, intestinal responses are determined mainly by the ZEN dose. Low doses promote proliferation and migration [44], while high doses have cytotoxic effects and inhibit biotransformation processes [3], which probably occurred in group ZEN15 in the early stages of exposure.

In view of the above, the present results suggest that the administered ZEN doses (LOAEL, NOAEL, and MABEL) were appropriate to reliably assess the carry-over of ZEN in the digestive tract of prepubertal gilts.

#### 3.1.2. Concentrations of ZEN and Its Metabolites

The levels of ZEN metabolites (α-ZEL and β-ZEL; see Tables 2 and 3, Figure 1) in intestinal tissues were proportional to the administered doses and time of exposure. The proportions of α-ZEL and β-ZEL were similar to those noted in other studies [3], which is natural in our opinion [5,45]. The bioavailability of ZEN metabolites in intestinal tissues was influenced by biotransformation processes during pubescence. The distribution of concentration values in intestinal tissues preceded the values obtained in blood [5]. Zearalenone metabolites were not found in the blood of ZEN5 animals on D1 due to a very low supply of endogenous steroid hormones [5] (supraphysiological hormonal levels [39]). Mycoestrogens supplementation [46] can modify the levels of estrogen hormones [7,37] and the anti-Müllerian hormone [47].

The following arguments should be also considered: (i) adaptive processes end on D1 [42]; and (ii) ZEN metabolites can be used as substrates that regulate the expression levels of genes encoding hydroxysteroid dehydrogenases [20] that act as molecular switches and allow for the modulation of steroid hormone pre-receptors. In this study, these processes were particularly noticeable in group ZEN5, where ZEN concentrations decreased and α-ZEL concentrations increased on successive dates of exposure (see Table 2). The above indicates that in prepubertal gilts, even the smallest amounts of estrogenic compounds are used by the body [48] to make up for the deficiency of endogenous estrogen [5]. In the remaining groups, metabolite levels increased proportionally to the ingested ZEN dose.

Based on previous research findings [49,50] and extrapolation, the above observations could be attributed to the activity of transport proteins in the intestinal wall. The activity of the antiporter plays a very important role in the initial stage stages of mycotoxin biotransformation. Processes with the participation of antiport proteins are influenced by the availability of energy compounds (which were depleted) [7], which affect active ion pumps that transport ZEN from and to the intestinal lumen, thus stabilizing ZEN concentrations inside cells [51]. In enterocytes, numerous detoxification enzymes are localized near the cell wall. When mycotoxins and their metabolites do not undergo further biotransformation, they reach the cytosol and, consequently, the circulatory system. To prevent this from happening, active ion pumps remove mycotoxins and their metabolites from cells. These toxins return to the intestinal lumen and are subsequently recirculated to enterocytes. The described mechanisms restore energy reserves in cells, and mycotoxins can be metabolized again before they reach the cytosol and cause pathological states. Antiporter activity in the intestines is also determined by phase I enzymes, including the cytochrome P450 3A4 isoenzyme, which plays a key role in detoxification [15] and energy supply [7].

These observations could suggest that α-ZEL contributes to the modulation of life processes by participating in feminization processes in prepubertal gilts.

#### 3.1.3. Carry-Over Factor

One of the main objectives of risk management in the food processing industry is to protect public health through the effective identification and control of known threats and selecting the most appropriate strategies for mitigating these risks. The significance of the CF has to be understood before a given strategy is selected and implemented [1]. The CF is the concentration ratio of the undesirable substances (mycotoxin) in contaminated digesta to the concentrations of ZEN and its metabolites in gut tissues, which marks the beginning of biotransformation processes. Our previous research demonstrated that even MABEL doses can induce specific changes in homeostasis [52], metabolic processes [7], endocrine processes [5,6], and gut microbiota [8] in prepubertal gilts.

The CF values noted in the current study suggest that the accumulation of the parent compound in the intestinal wall was low and stable on the first two dates of exposure (D1 and D2). A different trend was noted in group ZEN10 in the duodenum, where ZEN is most easily and rapidly absorbed [38]. These values were sufficient to induce considerable disruptions in metabolic processes [18] and, most importantly, steroidogenesis [6,20,53,54] via estrogen receptors [4,52]. These findings confirm that the effects exerted by mycotoxins applied at low doses have not been fully elucidated in prepubertal females. However, on D3, the CF values were higher in the ZEN15 group than in the other groups, which may suggest the rate of biotransformation processes decreases over time. These observations are difficult to interpret because little is known about the rate and course of biological processes in sexually immature pigs. It can only be speculated that estrogen supply and estrogen demand reach equilibrium and that the steroid hormone profile is modified when testosterone is converted to estradiol [5,42] or when detoxification processes begin to dominate [55].

The CF values of both ZEN metabolites were characterized by different trends (Tables 2 and 3). In general, this parameter was highest in group ZEN10 on all exposure dates, with a decreasing trend in the colon. On D3, the CF of α-ZEL in the ZEN10 group increased in all sections of the small intestine. The CF of β-ZEL was highest in group ZEN5 and also in the small intestine. Interestingly, a comparison of the CF values of both ZEN metabolites on all exposure dates revealed average values on D3. These observations suggest that after 21 days of exposure to the pure parent compound, life processes in prepubertal gilts are stabilized, and biotransformation processes are shifted towards detoxification [55,56]. This indicates that the macroorganism has developed tolerance to low ZEN doses due to the availability of "free ZEN" [20], its suppressive effects on testosterone and progesterone levels [6], and the negative feedback effect on FSH synthesis and secretion [5,57]. These effects could be ascribed to the fact that prepubertal gilts were fed a balanced diet [7,58], and the amount of gut microbiota was higher in the distal part of the intestinal tract [8], where mycotoxin concentrations were lower.

Contrary to the observations made in vitro by Alvarez-Ortega et al. [59], the present study did not provide any evidence to prove that exposure to low doses of ZEN, the determined concentrations of ZEN and its metabolites, and CF values enhanced proliferation processes in the analyzed tissues. It can be concluded that the noted CF values indicate that the tested ZEN doses did not exert harmful effects and that the macroorganism relatively easily adapted to these doses.

### *3.2. Expression of CYP1A1 and GSTπ1 Genes*

The biotransformation of xenobiotics, including ZEN, often alters the biological activity and chemical properties of these compounds. Phase I metabolic processes usually enhance the functionality of their molecules, while phase II is often considered the detoxification stage. However, the nature and extent of these processes are highly compound-specific and can vary between individuals [2]. During the biotransformation process, ZEN is not only eliminated from the body, but the parent compound is inactivated, and the products of phase I biotransformation are activated. Various types of enzymes participate in these processes, including cytochrome P450 isoenzymes and CYP1A family enzymes in phase I, in particular in intestinal tissues. Microsomal GST enzymes are involved in successive phases of biotransformation [3].

The cytochrome P450 1A1 enzyme adds hydroxyl groups in the phase I metabolism of xenobiotics. Hydroxyl groups increase the polarity of xenobiotics, thus facilitating their excretion via urine, and they also act as sites for further modification in phase II biotransformation processes. As a result of enzymatic modifications, ZEN is partly transformed to more active forms (such as α-ZEL; it should be noted that pure ZEN was administered per os in this study) [2] that more easily interact with the molecular targets in cells (such as estrogen receptors) [60] and effectively participate in phase II biotransformation. These processes occur in two mutually opposing directions.

Analysis of the expression of the CYP1A1 gene in the ascending and descending colon (Figure 2) on dates D1 and D2 showed that the absorption and accumulation of ZEN and its metabolites in the gut tissues of prepubertal gilts (Figure 1) corresponded to the CF values in the analyzed intestinal segments (see Table 1). The above could be attributed to physiological estrogen deficiency [6,39] as well as the fact that the parent compound (ZEN) absorbed in intestinal tissues was biotransformed to a more active form (α-ZEL). These processes promote effective modulation of molecular targets in cells, facilitate steroidogenesis [5], and induce cross-talk between the receptors activated by undesirable substances (the estrogen receptor and the aryl hydrocarbon receptor). The interactions between these receptors increase the expression of the respective target genes [60].

It should also be noted that various substrates can stimulate or inhibit the activity of proteins, which significantly influences the biotransformation of ZEN catalyzed by these enzymes. In addition to protein polymorphism, the induction of biotransforming enzymes is also responsible for differences in the susceptibility to ZEN and the mycotoxin's impact on gilts before puberty. Mean concentrations of ZEN and its metabolites in the intestinal wall (Figure 1) were inversely related to the expression of the CYP1A1 gene in two sections of the colon (see Figure 2), and these differences were more pronounced on successive dates of exposure [6]. The above can be attributed to the increasing accumulation of mycotoxins in the intestinal wall on day 42 of exposure (D3). In an in silico study, all mycotoxins (ZEN, α-ZEL, and β-ZEL) acted as substrates, inducers, and inhibitors ranging from 60% to 90%, 21% to 38%, and 23% to 32%, respectively, for the CYP1A1 isoform [15], which was not confirmed in vivo in this study. These observations could suggest that these mycotoxins suppress the expression of the CYP1A1 gene.

Glutathione S-transferases (GSTs) are a family of dimeric enzymes that conjugate exogenous and endogenous substances with glutathione. Glutathione prevents DNA damage by binding toxic compounds in the cytoplasm and preventing their interactions with nucleic acid. It should be noted that GSTs are a part of the unified cellular defense system [61]. In the GST family, the GSTπ1 subclass predominates in the colon [62]. The substrates for GSTs, in particular for the GSTπ1 subclass, include active metabolites of cyclophosphamide, platinum derivatives, and xenobiotics. GSTπ1 plays a special role in the conjugation of reactive cyclophosphamide metabolites with glutathione [63]. In the present study, a minor increase (non-significant) in GSTπ1 expression in the descending colon was noted only on D2 in groups ZEN10 and ZEN15 (Figure 3), which suggests that proliferation processes are somewhat dominant in enterocytes [64,65]. These results are difficult to interpret due to the scarcity of published data for comparisons. In most experimental weeks, GSTπ1 expression tended to be higher in the ascending colon and lower in the descending colon. Increased GSTπ1 expression in the ascending colon is attributable to hyperestrogenism in prepubertal gilts. Lower levels of GSTπ1 expression in the descending colon were also reported by Hokaiwado et al. [66], who concluded that GSTπ1 silencing decreases cell proliferation, promotes apoptosis, and contributes to controlled proliferation. According to other authors, GSTπ1 can be silenced in response to chemical stress [28,34], including exposure to ZEN. However, there are no published data that can be directly compared with our findings. Our previous studies revealed that both ZEN and deoxynivalenol (DON) could silence GSTπ1 expression [4,36].

As a result, long-term exposure to ZEN applied at low doses induced functional changes in the distal gastrointestinal tract in gilts before puberty. However, GSTπ1 expression in the descending colon was higher in group C on D2. This observation could be attributed to: (i) the balanced supply and demand (homeostasis) of GSTπ1 for the maintenance of cytoprotective and detoxification functions in phase II biotransformation processes; (ii) excess intracellular levels of GSTπ1 in the ascending and descending colon in response to high GSTπ1 expression [63]; or (iii) intensified apoptosis [66].

Exposure to low mycotoxin doses led to controlled silencing of GSTπ1 expression, thus promoting a balance between intestinal cells, the degree of exposure to toxic compounds, and the detoxifying effect.

#### **4. Summary and Conclusions**

The gut tissues are the first line of defense against any substances that enter the body. A healthy intestinal barrier guarantees homeostasis in the body. On the basis of the presented research, it could be hypothesized that the obtained results confirm the correctness of the adopted nomenclature of doses (LOAEL, NOAEL, and MABEL) used in the experience. Based on the observations made during 42 days of exposure to pure ZEN, it can be hypothesized that all mycotoxins (ZEN, α-ZEL, and β-ZEL) contribute to a balance between gut cells and the expression of genes that encode enzymes that participate in biotransformation processes in the large intestine, modulate feminization processes in prepubertal gilts, and elicit flexible, adaptive responses of the macroorganism to mycotoxin exposure at the analyzed doses.

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

### *5.1. General Information*

All experimental procedures on animals were carried out in accordance with the Polish regulations specifying the conditions for conducting experiments on animals (Opinions No. 12/2016 and 45/2016/DLZ issued by the Local Ethics Committee for Animal Experimentation of the University of Warmia and Mazury in Olsztyn, Poland, on 30 November 2016). This article is a continuation of the previously published study protocol [32].

### *5.2. Experimental Feed*

ZEN analytical samples were dissolved in 96 µL of 96% ethanol (SWW 2442-90, Polskie Odczynniki SA, Poland) in doses appropriate for various body weights (BWs). The food was placed in gel capsules saturated with the solution and kept at room temperature until the alcohol was evaporated. All groups of gilts received the same feed throughout the trial. The animals were weighed at weekly intervals, and the results were used to adjust the individual mycotoxin doses [8,13,67].

The feed given to all test animals was supplied by the same producer. The brittle feed was administered ad libitum twice a day at 8:00 a.m. and 5:00 p.m. throughout the experiment. The composition of the complete diet declared by the producer is presented in Table 4 [8,13,67]. Zearalenone analytical samples were dissolved in 96 µL of 96% ethanol (SWW 2442-90, Polskie Odczynniki SA, Poland) in appropriate weight doses. The feeds containing various amounts of ZEN in the alcoholic solution were placed in gel capsules. Prior to administration, the capsules were stored at room temperature until the alcohol evaporated. Pigs in the experimental groups received ZEN in gel capsules daily before morning feeding. The animals were weighed at weekly intervals to adjust the individual mycotoxin doses. The carrier was feed, and the gilts from group C received identical gel capsules without ZEN [7,8]. The feeds were supplied by the same manufacturer. During the experiment, the gilts were fed brittle fodder ad libitum twice a day (at 8:00 a.m. and 5:00 p.m.). The composition of the complete diets was specified by the producer, and it is presented in Table 4.

**Table 4.** Declared composition of the complete diet [12].

1 Composition of the vitamin-mineral premix per kg: vitamin A—500.000 IU; iron—5000 mg; vitamin D3—100.000 IU; zinc—5000 mg; vitamin E (alpha-tocopherol)—2000 mg; manganese—3000 mg; vitamin K—150 mg; copper (CuSO4·5H2O)—500 mg; vitamin B1—100 mg; cobalt—20 mg; vitamin B2—300 mg; iodine—40 mg; vitamin B6—150 mg; selenium—15 mg; vitamin B12—1500 µg; L-lysine—9.4 g; niacin—1200 mg; DL—methionine+cystine—3.7 g; pantothenic acid—600 mg; L-threonine—2.3 g; folic acid—50 mg; tryptophan—1.1 g; biotin—7500 µg; phytase + choline—10 g; ToyoCerin probiotic + calcium—250 g; antioxidant + mineral phosphorus and released phosphorus—60 g; magnesium—5 g; sodium and calcium—51 g.

The proximate chemical composition of the diets fed to pigs in groups C, ZEN5, ZEN10, and ZEN15 was determined using the NIRS™ DS2500 F feed analyzer (FOSS, Hillerød, Denmark), a monochromator-based NIR reflectance and transflectance analyzer with a scanning range of 850–2500 nm [32].

#### Toxicological Analysis of Feed

Feed was analyzed for the presence of mycotoxins and their metabolites: ZEN, α-ZEL, and deoxynivalenol (DON). Mycotoxin concentrations in feed were determined by separation in immunoaffinity columns (Zearala-TestTM Zearalenone Testing System, G1012, VICAM, Watertown, MA, USA; DON-TestTM DON Testing System, VICAM, Watertown, MA, USA). Feed samples were ground in a laboratory mill. Ground samples of 25 g each were eluted with 150 mL of acetonitrile (90%) to extract the mycotoxins. A total of 10 mL of the resulting solution was withdrawn and diluted with 40 mL of water. The obtained solution (10 mL) was collected and passed through the immunoaffinity column (VICAM). The immunoaffinity bed in the column was subsequently washed with demineralized water (Millipore Water Purification System, Millipore S.A., Molsheim, France). The column was eluted with 99.8% methanol (LIChrosolvTM, No. 1.06 007, Merck-Hitachi, Germany) to remove the bound mycotoxin. The obtained solutions were analyzed by high-performance liquid chromatography (HPLC system, Hewlett Packard type 1050 and 1100), coupled with a diode array detector (DAD), a fluorescence detector (FLD), and chromatography columns (Atlantis T3 3 µm 3.0 150 mm Column No. 186003723, Waters, AN Etten-Leur, Ireland). Mycotoxins were separated in a mobile phase of acetonitrile:water:methanol (46:46:8, *v*/*v*/*v*). The flow rate was 0.4 mL/min. The limit of detection was set at 5 µg/kg of feed for DON and 2 µg/kg of feed for ZEN, based on validation of chromatographic methods for the determination of ZEN and DON levels in feed materials and feeds. Chromatographic methods were validated at the Department of Veterinary Prevention and Feed Hygiene [68]; see File S1 in Supplementary Materials.

#### *5.3. Experimental Animals*

An in vivo experiment involving 60 clinically healthy prepubertal gilts with initial BW of 14.5 ± 2 kg was performed at the Department of Veterinary Prevention and Feed Hygiene of the Faculty of Veterinary Medicine at the University of Warmia and Mazury in Olsztyn, Poland [7,32]. During the experiment, the animals were housed in pens, fed identical diets, and provided with ad libitum access to water. The gilts were randomly divided into a control group (group C; *n* = 15) and three experimental groups (ZEN5, ZEN10, and ZEN15; *n* = 15 each) [69,70]. Groups ZEN5, ZEN10, and ZEN15 were administered ZEN (Sigma-Aldrich Z2125-26MG, St. Louis, MO, USA) per os at 5 µg/kg BW, 10 µg/kg BW, and 15 µg/kg BW, respectively. Each experimental group was kept in a separate pen in the same building. Pens had an area of 25 m2 each, which is consistent with the applicable

cross-compliance regulations (Regulation (EU) No 1306/2013 of the European Parliament and of the Council of 17 December 2013).


**7. Targeting DDC Is One of the Most E**ffi**cient Ways to Tackle Cancer Cells**

Cancer cells accumulate DNA damage in an increased fashion as compared to normal cells and rely on DDC to avoid that excessive damage is inherited after cell division, for this reason several inhibitors targeting this pathway have been developed or are in development (Table 1).

As previously mentioned, DNA damage determines cell cycle arrest before entering mitosis, if this arrest is prolonged enough, cells go through the apoptotic pathway. Sometimes, cancer cells can avoid mitotic arrest and inappropriately enter mitosis before completion of the S and G2 phase; this frequently determines an improper distribution of chromosomes leading to mitotic catastrophe, which is an onco-suppressive signaling cascade preceding cell death via apoptosis, senescence or necrosis, in the first or subsequent cell divisions [101]. Inhibition of proteins that control the S/G2 checkpoint promotes this pathway and in solid cancer this is the main cell death-leading pathway induced by IR treatment [102,103].

ATM mutations could induce synthetic lethality in association with other mutations or specific drugs; this could be exploited to develop more accurate therapies [22]. Since ATM inhibition determines uncontrolled cell entry into mitosis by removing several checkpoints control, the use of ATM inhibitors in cancer therapy has been reported to cause sensitization to radiotherapy. In addition, ATM inhibition enhances cell sensitivity to topoisomerase I and II inhibitors such as camptothecin, etoposide, and doxorubicin [104]. Several ATM inhibitors have been used in cancer therapy. AZD0156 is a strong radio-sensitizer that recently has been demonstrated to inhibit tumor growth after radiation treatment of lung xenograft [105]; in addition, a combinatory effect with PARP inhibitors (olaparib) is under study since sensitivity to these drugs is related to ATM deficiency [106]. Currently, AZD0156 is in phase 1 trial (NCT02588105). A phase I clinical trial with the inhibitor AZD1390 (NCT03423628) (Table 1), which has been proved to radio-sensitize brain tumor models in pre-clinical studies, has also been initiated [107]. Several other compounds have been produced such as KU-55933, KU-60019 and KU-59403 that are in pre-clinical studies, therefore targeting ATM is a promising strategy for cancer treatment [108].

DNA-PK inhibition promotes radio-sensitivity in gastric cancer [109]; accordingly, its overexpression is associated to radio-resistance in thyroid, cervix, and prostate cancer [110] (Table 1). DNA-PKs deficiency can sensitize cells to DNA-damaging agents such as topoisomerase I and II inhibitors. This has led to the development of several DNA-PK inhibitors such as M3814, which has been demonstrated to increase efficacy of topoisomerase II inhibition in ovarian cancer models [111]. A clinical trial is ongoing to study efficacy of this drug in combination with radiotherapy and capecitabine, a drug that interferes with DNA and RNA metabolism (NCT03770689). Similarly, the DNA-PK inhibitor VX-984 enhances radio-sensitivity of glioblastoma and a clinical trial has been completed to assess safety of this drug in combination with chemotherapy (NCT02644278) [112]. Other drugs, currently under study, are CC-115 and MSC2490484A (NCT02516813) (NCT02833883), whose efficacy has been suggested in ATM-deficient cells and in combination with radiotherapy [113] (Table 1).

Differently from the other kinases, ATR does not participate in the initial recognition phases of DNA damage, but its activity is essential to prevent replication fork collapse, thus ensuring faithful duplication by binding to ssDNA and to arrest cell cycle in G2/M phase by interacting with CHK1 [114]. Since cancer cells are often defective in G1 phase checkpoint due to mutation of p53 and Rb tumor suppressor genes, they arrive in S phase with replication stress that could stall replication fork; therefore, cells rely on S-G2 phase checkpoint to avoid cell death due to excessive DSBs. Thus, a deficiency of ATR activity determines sensitivity to DNA damage agents such as IR, DNA cross-linking agents, and topoisomerase poisons [115]; its inhibition combined with PARP inhibitors also reduces tumor growth in BRCAness models [116] (Table 1). Currently, the ATR inhibitors M6620, AZD6738, BAY1895344, and VE-821 have been shown to increase lethality of cancer cell lines ATM, defective such as gastric cancer, non-small cell lung cancer and chronic lymphocytic leukemia, strongly suggesting a synthetic lethality interaction between the ATM and ATR pathways [117,118] (Table 1). A synthetic lethality interaction has been observed in triple-negative breast cancer cell lines also by combining AZD6738 with WEE1 inhibition, and with CHK1 inhibitor AZD7762, since combination of ATR and CHK1 inhibition leads to ssDNA accumulation and replication stress [119,120] (Table 1). In addition, since ATR inhibition enhances replication stress, as anticipated in pre-clinical trials, several studies analyze cancer cell sensitization by these drugs to DNA damaging agents such as cisplatin, gemcitabine, and radiation. In particular, M6620 was studied in combination with radiation in patients with brain metastases (NCT02589522) and with cisplatin for head and neck squamous cell carcinoma (NCT02567422) (Table 1); AZD6738 in combination with radiotherapy in patients with solid tumors, VE-821 in combination with radiation and chemotherapeutics in pancreatic cancer patients [121,122]. The effect of BAY1895344 in combination with chemotherapeutics or radiotherapy on tumor cell growth and survival was studied in cancer xenograft models that carry DNA damage repair deficiencies [123].

Targeting DDC pathway is an attractive strategy in cancer therapy. In fact, it has proven to be particularly effective in combination with radiotherapy determining cell death by induction of mitotic catastrophe pathway. Moreover, as DDC is upstream to several DNA repair pathways, different synthetic lethality combinations, currently under study, could be exploited taking advantage of this network.

For each target it is reported the name of the drugs currently in analysis; combination of target inhibition and classic anti-cancer therapy under study; possible synthetic lethality interaction; ongoing clinical trials with the drugs; references.

### **8. Combination of DSB Inducers and DSB-Repair Proteins Inhibition Is Greatly E**ff**ective**

A large number of DNA damaging agents exert their activity by inducing DSBs; therefore, combining these agents with compounds inhibiting DSB repair-involved players produced promising results. Particularly, inhibition of HR-involved proteins combined with DNA damage induction forces cells to use more error-prone DNA repair pathways, thus determining accumulation of DNA aberrations. Nevertheless, upregulation of mutagenic repair pathways could promote therapy resistance ultimately leading to disease progression [125]; for this reason, compounds inhibiting NHEJ or MMEJ players are in development to help in overcoming this problem and improve clinical outcomes of classic therapies.

Regarding HR, several inhibitors have been developed, targeting MRN complex and among them, mirin is the most studied (Table 2).

Mirin inhibits MRE11-associated exonuclease activity preventing MRN-dependent ATM activation. This inhibitor potentially sensitizes different cancers to genotoxic agents, such as malignant glioma, prostate cancer, and neuroblastoma [24,126,127]. In addition, several studies suggest that MRN defects could synergize with PARP inhibition, indicating a possible synthetic lethality interaction that could be exploited in specific cancer therapy [128].

As overexpression of *RAD51* stimulates cancer aggressiveness, it has been shown that lowering its expression or activity (through inhibition) can sensitize cancer cells to chemotherapeutics [113]. To date, different classes of RAD51 inhibitors which take advantage of different inhibition mechanisms have

been developed (Table 2): some inhibitors interfere with RAD51 ssDNA binding ability and could lead to the formation of toxic RAD51 complexes; other inhibitors interfere with RAD51 ability to interact with other DNA repair factors such as BRCA2 and finally drugs that inhibit the RAD51 capacity to migrate through the DNA strand and form foci after DNA damage. B02 is a RAD51 inhibitor which interferes with ssDNA binding ability and has proved to sensitize cancer cells to several DNA damage agents in preclinical studies also in combination with PARP inhibitors [129–131]. The RAD51 inhibitor RI-1 destabilizes RAD51 oligomerization/filament formation on DNA strand leading to the formation of cytotoxic complexes. This compound specifically works on those cancer cells where *RAD51* is overexpressed, sensitizing them to chemo/radiotherapy and PARP inhibitors [132,133]. IBR120 is an inhibitor that disrupts RAD51 interaction with BRCA2 sensitizing cancer cells to IR [134,135]. Finally, CYT-0851 is a compound that reduced RAD51 migration to damaged DNA ends [125]. A clinical trial to test this inhibitor has already started, (NCT03997968) (Table 2). Anyhow, even if the other inhibitors are not in clinical trials, they proved to have great potential in pre-clinical studies.

Since BRCA1 and BRCA2 are large proteins that interact with several partners with their functional domains and act as hubs in DNA damage signaling/repair, it is difficult to design specific compounds to inhibit their activities [136]. Some BRCA1/BRCA2 inhibitors acting on the BRCT domain, which is essential for the interaction with DNA repair partners, such as RAD51, determine an increase of cell sensitivity to drugs such as PARP inhibitors [137,138]. Moreover, small molecules acting on the RING domain of BRCA1 have been developed and this could provide novel ways to inhibit HR [24].

**Table 2.** HR repair proteins targeted in cancer therapy.

For each target it is reported: name of the drug currently in analysis; combination of target inhibition and classic anti-cancer therapy under study; possible synthetic lethality interaction; ongoing clinical trials with the drug; references.

RAD52 is another therapeutic target in breast and ovarian cancer because its depletion is synthetically lethal to cells BRCA2 or BRCA1/PALB2 deficient [139,140]. Different groups have developed several RAD52 inhibitors (F79, 6-OH-dopa, A5MP, AICAR/ZMP, D-I03, NP-004255, F779-0434) that act either by inhibiting oligomerization or blocking ssDNA binding activities of the protein, similar to some RAD51 inhibitors [141] (Table 1). Recently, to potentiate the effect of RAD52 drugs and reduce resistance, a new strategy named "dual synthetic lethality" has been proposed with the idea to combine them with PARP inhibitors, in BRCA1-deficient cancer cells [142].

Regarding NHEJ, several chemical compounds have been developed for cancer therapy, but they are all in preclinical studies. These studies are analyzing how their inhibition could influence cancer development and drugs resistance (Table 3). It has been reported that Ku70/80 inhibition sensitizes cells to radiation treatment, XLF inhibition contributes to overcome chemoresistance in colorectal cancer cells, and LIG IV silencing results in increased cellular sensitivity to chemotherapeutic alkylating agents [143–145].

Some studies are developing small ligands able to inhibit TP53BP1 by binding to its interacting domain and competing with regular substrates, making it unable to coordinate downstream effectors [146,147], moreover a synthetic lethal interaction between TP53BP1 loss and treatment with ATR inhibitors and cisplatin has been demonstrated [148] (Table 3).

MMEJ pathway is still far from being fully characterized and several groups are working to identify drugs able to inhibit Pol-θ activity and to exploit this pathway in cancer therapy [149]. Recently, it has been discovered that Pol-θ inhibition sensitizes cells to replication stress caused by agents such as topoisomerases poisons or ATR inhibitors, suggesting novel cancer treatments [150] (Table 3). In addition, thanks to a CRISPR genetic screen, 140 genes whose deletion is synthetically lethal with *POLQ* mutations have been discovered. Among them, several HR and NHEJ-involved genes and components of the TP53BP1 pathway have been identified. Moreover, in the same study, it has been discovered that 30% of breast cancer cases deposited in TCGA (the Cancer Genome Atlas) have alteration in at least one of these 140 genes; the proteins encoded by these genes could be potential targets to develop new strategies for cancer patients who have Pol-θ alteration [151]. Moreover, knockdown of Pol-θ in ovarian cancer cell lines deficient for FA factor FANCD2 enhances cell death [152]. Finally, the antibiotic novobicin is now under study as specific Pol-θ inhibitor, and preliminary results showed that this drug is able to kill HR-deficient tumor cells due to accumulation of toxic RAD51 foci [153] (Table 3).

For each target it is reported: name of the drugs currently in analysis; combination of target inhibition and classic anti-cancer therapy under study; possible synthetic lethality interaction; ongoing clinical trials with the drug; references.

#### **9. Targeting DNA Helicases Increases Sensitivity to Several Treatments**

BLM is one of the helicases that have an essential role in HR pathway, promoting DNA end resection, RAD51 filament and D-loop formation [154]. The inhibitor ML216 targets this helicase (Table 4), by competing for ATP binding and for BLM binding to DNA; anyhow, this compound showed poor aqueous solubility and cell permeability, therefore it will require further optimization [155].

DNA2 helicase allows cancer cells to resist DNA replication stresses induced by chemotherapy or radiotherapy, by promoting flap removal during DNA replication, DNA resection during repair, and stabilization and restart of stalled replication forks [156]. C5, a potent DNA2 inhibitor, has been

identified by a high throughput screening; this inhibitor sensitizes cells to camptothecin and PARP inhibitors, making it interesting for cancer therapy [157] (Table 4).

*WRN* downregulation leads to mitotic catastrophe and cancer cell death [158]. Interestingly, evidences show that treatment with a WRN inhibitor (NSC 19630) induces apoptosis in leukemia cell lines [159]. NSC617145 is an improved derivative of the previous drug, which likely traps WRN on the DNA substrate, leading to sensitization of cancer cells to DNA-damaging agents. This drug sensitizes cells deficient of the FA and NHEJ pathways to mitomycin C, suggesting the possibility of a synthetic lethality approach [160] (Table 4). Recently, new small molecule WRN inhibitors, identified by a high throughput screen, have been shown to reduce proliferation of cancer cells [161].

These data suggest that DNA helicases are potential targets for cancer therapy to be exploited as novel synthetic lethal interactions. Several studies are now ongoing to identify new drugs that could provide alternative and useful strategies to sensitize several cancers to DNA damage agents [155,162] (Table 4).

**Table 4.** Helicases and nucleases targeted in cancer therapy.

For each target it is reported: name of the drugs currently in analysis; combination of target inhibition and classic anti-cancer therapy under study; possible synthetic lethality interaction; references.


**4.1 Curcumin-loaded nanoparticles and their antibacterial activities**

According to a study by Tyagi et al. [30], curcumin has greater effectiveness in controlling both gram-positive and gram-negative bacteria. Despite curcumin having potential antibacterial properties, its low solubility, low stability, and low bioavailability remain a debate [142]. Curcumin nanoparticles exhibit better biological activity, solubility, and stability than all other forms of curcumin [143]. When evaluated, it was found that the curcumin particles are smaller, which enhanced their toxicity and sensitization in bacterial cells, compared to curcumin alone [144].

Furthermore, an evaluation of curcumin nanoparticle's effectiveness in inhibiting bacteria like *Shigella dysenteriae*, *Staphylococcus aureus*, *Escherichia coli*, and *Streptococcus pneumonia* was observed to be greater than amoxicillin, a commercial

*Germicidal and Antineoplastic Activities of Curcumin and Curcumin-Derived Nanoparticles DOI: http://dx.doi.org/10.5772/intechopen.103076*

**Table 3.**

*Summary of the activities of curcumin-loaded nanoparticles for the treatment of different diseases.*

antibiotic [52]. In order to manifest antibacterial properties, curcumin-loaded nanoparticles attach to the cell wall of the bacterial cell, break it, and penetrate inside the cell, disrupting the structure of cellular organelles [143]. Since curcumin-loaded nanoparticles are effective on the broad spectrum of microorganisms and human cancer cell lines, therefore targeting curcumin-loaded nanoparticles for therapeutic purpose is a promising strategy.


**1. Introduction**

One of the main characteristics of yeast affecting their oenological use is their capacity to ferment sugars. Non-*Saccharomyces* yeasts have traditionally been considered bad fermenters. For that reason, selected strains of *S. cerevisiae* have been used in the oenological industry to ensure that complete fermentation occurs [1,2]. Recently, however, wineries have been encouraged to apply new, non*-Saccharomyces* species in winemaking processes to provide distinguishable flavours within wines [3–6]. Non-*Saccharomyces* species have been used to produce different aromas and flavours, compared with *Saccharomyces* strains [7,8]. Therefore, many efforts have been made to identify non-conventional yeast strains for oenological purposes [4,8,9].

The selection of oenological yeasts is commonly accomplished by identifying species from raw material. Spontaneously fermented grape musts are the niche that is most commonly used to identify novel strains that are both capable of fermenting sugars and confer desirable flavours to wines [10,11]. *Hanseniaspora* is the most abundant genus on grapes and grape juices. Studies have shown that up to 75% of the yeast population during the early stages of fermentation is made up of *Hanseniaspora* species [12,13]. After the first 48–72 h of spontaneous fermentation, the percentage of

> 19

*Hanseniaspora* species present decreases and the *S. cerevisiae* strains correspondingly increase. However, some *Hanseniaspora* species have been detected throughout the fermentative process [14].

Researchers have maintained that observed changes in yeast populations during fermentation occur, at least in part, because *Hanseniaspora* species are sensitive to ethanol [15]. *S. cerevisiae* is able to produce high quantities of ethanol rapidly. Therefore, this species dominates the fermentation process until sugar is completely depleted. Recent studies have shown the e ffects of antimicrobial peptides secreted by *S. cerevisiae* throughout the fermentation [16,17] inhibiting the growth of non-*Saccharomyces* yeasts. Therefore, the reputation of *Hanseniaspora* species as poor fermenters may be due to the presence of other inhibitors and not directly related to their reduced capacity to ferment sugars.

Not all the *Hanseniaspora* strains have the same properties. Species of the genus produce di fferent secondary metabolites and exhibit di fferent fermentative behaviours. In fact, di fferences could even be detected between strains belonging to the same species [18,19]. *H. vineae* is an epiphyte yeast that is not easily isolated from fruit, a feature it shares with all the *S. cerevisiae* strains [20]. *H. vineae* can be isolated from samples after one or two days of spontaneous fermentation in wines and other fruit beverages such as cider [19]. This highlights the distinct behaviour of the species, compared with the majority of *Hanseniaspora* apiculate yeasts, which are commonly isolated from the skin of grapes or grapevine soil. The ability of strains identified as *H. vineae* to complete grape juice fermentation has been demonstrated via single inoculation [7]. Moreover, selected strains of *H. vineae* contribute positively to wine aromas by providing floral and honey notes, even when sequential inoculation with *S. cerevisiae* strains was performed [21]. Our assessment of *H. vineae* showed that levels of phenylpropanoid flavour compounds synthetized from grape must were elevated compared with other yeasts. In *H. vineae*, the presence of metabolic pathways that actively transform aromatic amino acids explains the elevated phenyl acetate ester and benzenoid derived compounds synthesis compared to other yeasts and these flavour compounds provide fruity and flowery aromas [21–23].

Although several phenotypic studies have been carried out throughout wine fermentation using non-*Saccharomyces* species, there is a lack of information regarding the genetic basis of observed characteristics in non-*Saccharomyces* strains [8]. Due to the development of next generation sequencing, genomes of *Hanseniaspora* species from wine have been recently sequenced [24–27]. Further work will be needed to determine which genes are responsible for each function. In previous studies, the aromatic profile of *H. vineae* was correlated with genomics and transcriptomics data [22]. However, genes involved in glycolysis and fermentative behaviour in the species remain unknown. In *S. cerevisiae*, genes necessary for fermentation have been reported using mutant analysis. All of these, were grouped in a "fermentome" [28]. The genome of *H. guilliermondii* has been recently analysed [27] and the presence and absence of genes involved in the glycolytic and fermentative pathways compared with *S. cerevisiae* and other *Hanseniaspora* species were reported. Moreover, the *H. uvarum* glycolytic pathway has been assessed in a study that revealed the catalytic potentials of enzymes involved in the route [29]. The authors showed that the main glycolytic enzyme of *H. uvarum*, pyruvate kinase, had a 15-fold lower enzymatic activity than that of the *S. cerevisiae* enzyme.

The aim of this work is to establish the di fferences and similarities between *H. vineae*, other *Hanseniaspora* species, and *S. cerevisiae* regarding glycolytic and fermentative behaviour. In the present study, a comparative analysis of the fermentative capacity of *H. vineae* was performed using genetic and transcriptomic data. Characterization of the glycolytic and fermentative potential of *H. vineae* will enhance our understanding about the mechanisms and the regulation of the fermentative process in a non-*Saccharomyces* yeast. *Hanseniaspora* genus studies might help reveal new signs of *S. cerevisiae* domestication mechanisms for wine production.

#### **2. Materials and Methods**

#### *2.1. Yeast Strains*

Yeast strains used for this study are listed in Table 1.

#### *2.2. Fermentation in Natural Grape Must*

Chardonnay grape must containing 300 mg N/L and 200 g/L of sugars at pH 3.5 was treated with 200 mg/L dimethyldicarbonate to prevent microorganism growth. Pre-cultures of *H. vineae* T02/19AF, *H. vineae* T02/05AF, *H. uvarum* AWRI1280, *H. osmophila* AWRI3579, and *S. cerevisiae* ALG804 were isolated from the Chardonnay grape must and incubated at 25 ◦C for 12 h in a rotary shaker at 150 rpm. Then, 125-mL Erlenmeyer flasks closed with cotton plugs used to simulate microaerobic conditions were inoculated with 75 mL of must containing 1 × 105 cells/mL. Static batch fermentations were conducted at 20 ◦C to simulate winemaking conditions.

#### *2.3. Growth Kinetics in Di*ff*erent Types of Media*

Six types of growth media were prepared using yeast nitrogen base (YNB) (Difco, Detroit, MI, USA) as a sole nitrogen source (6.7 g/L). Media were supplemented with the following carbon sources: Glucose, fructose, sucrose, xylose, glycerol, and maltose (2% w/w). YNB that lacked a carbon source was used as a negative control.

Chardonnay must used in the fermentation analysis was also used to measure the growth kinetics of yeast strains tested. Moreover, synthetic media that mimicked must fermentations at pH adjusted to 3.5 and ethanol concentrations of either 5% or 10% were used (20 g/L glucose, 4 g/L tartaric acid; 0.134 g/L sodium acetate; 5 g/L glycerol; and 1.7 g/L YNB) (v/v).

Pre-cultures of *H. vineae* T02/19AF, *H. vineae* T02/05AF, *H. uvarum* AWRI1280, *H. osmophila* AWRI3579, and *S. cerevisiae* ALG804 were prepared in yeast extract peptone dextrose (YPD) media (1% yeast extract and 2% peptone, 2% glucose) via incubation for 12 h in a rotary shaker at 150 rpm and 25 ◦C. These pre-cultures were used to inoculate fermentations carried out in microtitler plates at a final volume of 250 μL. Inoculates producing 1 × 105 cells/mL in media were used for all strains and treatments. All conditions were tested in triplicate. Absorbance at 620 nm was measured at 30-min intervals for 48 h at 25 ◦C using an automatic plate reader (Tecan, Männedorf, Switzerland) and data were acquired with the Magellan software for further statistical analyses.

#### *2.4. Fermentation Ability in Di*ff*erent Carbon Sources*

The carbohydrate fermentation capacity was tested using Durham tubes immersed in media to detect gas production. Each type of medium tested was inoculated to produce a final concentration of 1 × 106 cells/mL in a final volume of 8 mL performed in triplicate. Results were visually assessed after a 48 and 96 h static incubation period at 28 ◦C.

## *2.5. Genomic Analysis*

Genomic DNA was obtained from *H. vineae* cultures grown in a YPD medium at 30 ◦C using the Wizard Genomic DNA Purification Kit (Promega, NY, USA), according to the manufacturer's instructions.

The Illumina Genome Analyzer Iix platform in paired end mode was used to perform genomic sequencing as described previously [22]. Gene prediction was carried out using Augustus [30] trained with *S. cerevisiae* gene models. Peptide predictions were then annotated using BLASTp (cuto ff for e-value 1−10) against *S. cerevisiae* proteins, obtained from the *Saccharomyces* Genome Database [31].

A dendrogram was constructed using the sequences of nine genes encoding components of pathways related to glycolysis and fermentation from the *Hanseniaspora* species and *S. cerevisiae*. The genes assessed were *CDC19*, *FBA1*, *PGI1*, *PFK1*, *PFK2*, *HXK2*, *ENO1*, *PGK1,* and *PDC1*. *Schyzosaccharomyces pombe* was used as an external group. Neighbour joining and Kimura 2-parameter methods were carried out using the MEGA version 4 software [32,33].

## *2.6. Transcriptomic Analysis*

Fermentations were performed in triplicate using chemically defined grape (CDG) must with a composition similar to that of natural grape juice, but devoid of grape precursors. Components of CDG must were defined as described in Carrau et al. [34], with modifications. Briefly, glucose and fructose were added in equimolar concentrations until a total sugar concentration of 200 g/L was reached. Vitamins and salts were added as previously described [35]. Yeast available nitrogen (YAN) content was adjusted to 100 mg N/L. Of this total, 50 mg N/L corresponded to amino acids and 50 mg N/L corresponded to diammonium phosphate (DAP) supplementation, as described previously [35]. The pH of the media was adjusted to 3.5 using HCl and a final concentration of 10 mg/L ergosterol was the only lipid provided.

Pre-cultures of *H. vineae* T02/19AF were prepared in a CDG medium and incubated 12 h in a rotary shaker at 150 rpm and 25 ◦C. The pre-cultures were subsequently used to inoculate fermentation reactions carried out in 250 mL Erlenmeyer flasks that were closed with cotton plugs to simulate microaerobic conditions. For all strains, fermentations were performed using 125 mL CDG and an inoculum to produce 1 × 105 cells/mL in the final medium. Static batch fermentations were conducted at 20 ◦C to simulate winemaking conditions.

Wine samples for transcriptomic analyses were taken during the fermentation process at day 1 (exponential growth), day 4 (end of exponential phase), and day 10 (stationary phase of fermentation). For transcriptomic studies, total RNA obtained from *H. vineae* T02/19AF isolated from three replicates sampled from three di fferent fermentation stages (days 1, 4, and 10) were analysed independently. The nine samples were paired-end sequenced using Illumina MySeq. Trinity software was used to assemble raw reads from transcriptomic analyses and further statistical analyses were performed as specified by Giorello et al. [22].

## *2.7. Statistical Analysis*

All the treatments were performed in triplicate and the statistical error was calculated as the standard deviation of all data analysed. To compare growth and fermentation kinetics, variance comparison was performed by the ANOVA test carried out with STATISTICA 7.0 software. Di fferences in the mean absorbance or weight loss were evaluated using the Tukey test.

#### **3. Results and Discussion**

#### *3.1. Fermentative Capacity of H. vineae in Di*ff*erent Media*

*Hanseniaspora* species used a limited number of carbon sources, which may have been related to the reduced competitiveness of the species throughout fermentations [27]. Regarding growth in di fferent carbon sources (Figure 1A), growth of all the *Hanseniaspora* strains tested on both glucose and fructose had kinetics similar to that of *S. cerevisiae* ALG804. The media supplemented with sucrose was fermented by *S. cerevisiae* in a similar manner as that of media containing simple hexose. *H. uvarum* AWRI1280, however, did not grow on media containing sucrose. *H. vineae* T02/05AF, *H. vineae* T02/19AF, and *H. osmophila* AWRI3579 were able to grow on and ferment sucrose to an extent. Invertase gene (*SUC2*) is present in the genome of *H. vineae. SUC2* is highly expressed on day 4 of fermentation reactions, but not day 1 or 10. However, other invertase homologs were not observed in

the genomes of any other *Hanseniaspora* species except *H. osmophila* [25]. Recently, Steenwyk et al. [36] grouped *H. vineae* and *H. osmophila* within the slower-evolving linage of *Hanseniaspora*. In this branch, the *SUC2* gene is present. This is different in species of the fast evolving linage including *H. uvarum*, *H. opuntiae, H. valbyensis,* and *H. guilliermondii,* which might have lost the gene as a result of rapid mutation rates [36]. The same fact was detected with another key gene that show *Saccharomyces* wine yeast adaptations. Increased sulfite tolerance conferred by *SSU1* (Table 2) is present in *H. vineae* and *H. osmophila* and it is absent in the other *Hanseniaspora* species. The presence of *SUC2* and *SSU1* genes are indicators of adaptations to alcoholic fermentation in yeast [37].

Glycerol was not used as a unique carbon source for *H. vineae* in accordance with data reported by Albertin et al. [38]. However, Hv*GUT1* and Hv*GUT2* genes were present in the genomes of both *H. vineae* strains analysed. In addition, xylose was not used by the *H. vineae* strains as expected. A finding that was likely due to the lack of enzymes needed to carry out the xylose conversion. The group of genes were also determined to be absent in *H. guilliermondii*, *H. uvarum,* and *H. opuntiae* [27]. However, *H. vineae* T02/05AF and T02/19AF have the ability to grow weakly when maltose is provided as a sole carbon source, despite the fact that they were not able to ferment the sugar. The same behaviour was also observed for *H. jakobsenii* [36].

**Figure 1.** Capacity of *Hanseniaspora vineae* and *Saccharomyces cerevisiae* to grow and ferment under varied conditions. (**A**) Growth of *H. vineae* and *S. cerevisiae* (G) and the capacity of the species ferment (F) when six different carbon sources (2% w/w) were provided. Yeast nitrogen base (YNB) that lacked a carbohydrate was used as a negative control. Black filled circles indicate that full growth and fermentation were observed, grey circles indicate the moderate capacity of species to grow and ferment, and white circles indicate that the species was not able to grow or ferment. (**B**) Growth kinetics of *Hanseniaspora spp.* and *S. cerevisiae* on the Chardonnay grape juice measure as increased absorbance over a period of 48 h. (**C**) Fermentation kinetics of the three strains in the Chardonnay grape juice after 12 days are shown. Growth and fermentation experiments were performed using independent triplicate samples and error bars express standard deviation.

**Table 2.** Genes involved in sugar transport, glycolysis, and alcoholic fermentation from *S. cerevisiae* and *H. vineae*. Gene copy numbers are detailed in brackets.

As expected, *Saccharomyces* was able to grow and ferment sugars faster than *Hanseniaspora* species and significant differences between the species occurred after 16 h. The growth kinetics of the three *Hanseniaspora* strains tested were similar on grape must (Figure 1B), however fermentation kinetics of *H. vineae* and *H. osmophila* revealed that these species consume sugars significantly faster than *H. uvarum* (Figure 1C).


*2.3.6 Ground attenuation* Agr

The attenuation Agr is mostly the result of the interference between direct and reflected sound waves. This attenuation is mainly determined by the ground surface near the source and receiver. The calculation method proposed in [5] is only applicable if the terrain is flat, either horizontal or with a constant slope.

In order to calculate the attenuation due to ground absorption, three regions are defined (see **Figure 3**).

The total ground attenuation is obtained for each octave band, by adding the attenuations occurring in the three abovementioned zones. See Eq. (11)

**Figure 3.**

*Regions to calculate the attenuation due to ground absorption (based on [5]).*

$$\mathbf{A\_{gr}} = \mathbf{A\_s} + \mathbf{A\_m} + \mathbf{A\_r} \tag{11}$$

Where:

Agr: Total sound absorption due to ground effects (dB)

As: Sound absorption due to ground effects at the source region (dB)

Am Sound absorption due to ground effects at the middle region (dB)

Ar: Sound absorption due to ground effects at the receiver region (dB)

ISO Standard 9613-2 [5] explains in detail how to calculate the values of As, Am, and Ar. In the expressions for calculating the attenuation, the acoustic properties of each of these zones are taken into account through the so-called "G factor." When the sound is expected to propagate over hard ground: G = 0; for porous or soft ground: G = 1; and for mixed soil along the sound path, G should take a value between 0 and 1.


*3.3. Di*ff*erential Expression of Chemokines and Collagens by Activated Myofibroblast Subsets*

Upregulated expression of chemokines, such as CCL2, CXCL1, or CXCL12, and collagens, particularly COL1A2, COL3A1, or COL5A2, are often named attributes for activated MFB [13]. We herein wanted to analyze whether different subsets of either chemokine or collagen-producing MFB sub-clusters exist in fibrotic livers in vivo (Figure 3A). By scRNASeq analysis, the expression of collagens is homogenously upregulated in activated MFB, while chemokines show a more restricted pattern. While the monocyte recruiting chemokine *Ccl2* is expressed by both resting HSCs and MFB, neutrophil recruiting *Cxcl1* is strongly associated with activated MFB only. *Cxcl12,* on the other hand, is highly expressed by both HSCs and MFB, except for cluster MFB II, representing myeloid myofibroblasts (Figure 3A,B and Supplementary Table S1). By correlating the expression of the chemokine *Ccl2* with *Col3a1* on a single-cell level, resting HSCs comprised cells that express only *Col3a1*, cells co-expressing *Col3a1*, or cells that only express *Ccl2*. Interestingly, activated MFB could be differentiated by their expression of *Ccl2*, while all cells highly expressed *Col3a1* (Figure 3C). These data confirm the universal importance of an upregulated collagen expression during the activation of HSCs to MFB, while resting HSCs and selected MFB clusters demonstrate the capacity to secrete chemokines and thereby modulate the inflammatory environment in their surroundings.

**Figure 3.** Differential chemokine and collagen gene expression patterns in hepatic stellate cells and myofibroblasts. (**A**) Feature plots showing the relative gene expression strength of selected marker genes. (**B**) Violin plots showing the relative gene expression of activation markers. (**C**) Gene plots for the normalized gene expression of *Col3a1* and *Ccl2*. *n* = 4 with an average of 5000 cells per condition and ~60,000 reads per cell.


*3.4. Synthesis of Cu/Cu3P Nanoarrays*

To prepare Cu/Cu3P nanoarrays, NaH2PO2 was placed at the center of the tube furnace and the Cu/CuO nanoarray was placed at the downstream side of the furnace at carefully adjusted locations to set the temperature, and the distance between them was measured to be approximately 24 cm. After flushing with Ar, the center of the furnace was elevated to 270 ◦C with a heating rate of 2 ◦C/min and held at this temperature for 60 min. For comparison, samples at different phosphating temperatures were synthesized by the same process.

#### *3.5. Material Characterization*

XRD patterns were collected using an M21X diffractometer (MAC Science Co. Ltd., Japan) with high-intensity Cu Kα radiation (λ = 1.541Å). The morphology of the products was characterized via scanning electron microscopy (SEM, ZEISS SUPRA55). The HRTEM images were collected on a FEI Tecnai F20 electron microscope operated at 200 kV. The elemental compositions and valence states of the samples were determined by XPS. XPS measurements were performed using a Thermo Fisher Scientific, Escalab-250Xi spectrometer with an Al K*α* X-ray resource. The C 1s contamination peak was used for charge correction (284.8 eV).

#### *3.6. Electrochemical Measurements*

The electrochemical performances of Cu/Cu3P nanoarrays were evaluated with the CHI 660D electrochemical workstation. All the electrochemical measurements were conducted in a typical three-electrode setup with an electrolyte solution of 0.5 M H2SO4 using Cu/Cu3P nanoarrays as the working electrode, a graphite plate as the counter electrode and Ag/AgCl as the reference electrode. In all measurements, the Ag/AgCl reference electrode was calibrated with respect to a reversible hydrogen electrode (RHE). Linear sweep voltammetry (LSV) measurements were conducted in 0.5 M H2SO4 with a scan rate of 2 mV s−1 . All the potentials reported in our work were versus the RHE according to E vs. RHE = E vs. Ag/AgCl + Eθ vs. Ag/AgCl + 0.059 pH. Impedance measurements were carried out with a frequency range from 0.1 Hz to 10 kHz at the open-circuit potentials.

#### **4. Conclusions**

In summary, we have successfully prepared Cu/Cu3P nanoarrays via a facile two-step synthetic strategy, including the hydrothermal synthesis of a Cu/CuO nanoarray precursor and a low-temperature phosphorization process in an Ar atmosphere. The as-prepared Cu/Cu3P nanoarrays, as electrocatalysts, display excellent catalytic performance and durability for electrochemical hydrogen generation. Specifically, the Cu/Cu3P nanoarray-270 exhibits a low onset overpotential (96 mV) and a small Tafel slope (131 mV dec−1 ). The excellent electrocatalytic efficiency of the Cu/Cu3P nanoarray catalyst for the HER

can be attributed to its unique architecture. The nanoarray structure provides more active sites and contributes to the diffusion of the products. The Cu/Cu3P nanoarrays show good electrical conductivity, which is favorable to a faster transfer rate of electrons. This strategy provides an efficient technique that can be extended to other metal phosphides and metal-based nanostructures, thus creating a new opportunity in hydrogen production.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/catal12070762/s1, Figure S1: Characterization of the morphology and structure of samples: (a) SEM image of Cu nanosheet, (b) XRD patterns of Cu nanosheets, and (c) XRD patterns of Cu/CuO nanoarrays; Figure S2: The high-magnification SEM images of samples: (a,b) SEM image of Cu/CuO nanoarrays; Figure S3: The high-magnification SEM images of (a) CuO nanoplate, (b) Cu3P nanoplate; Figure S4: XPS survey spectrum of Cu /Cu3P nanoarrays; Figure S5: The SEM images and XRD patterns of samples at different phosphating time: (a,b) 1 h; (c,d) 1.5 h; Figure S6: The pure Cu nanosheet was directly phosphated and calcined: (a) SEM image; (b) XRD pattern; Table S1: Comparison of HER catalytic performance of Cu/Cu3P nanoarray-270 and other non-noble-metal electrocatalysts in acidic media.

**Author Contributions:** The manuscript was written with contributions from all authors. Conceptualization, R.D. and J.L.; methodology and investigation, R.D. and X.X.; data curation, M.X.; formal analysis, R.D. and X.X.; writing—original draft preparation, R.D.; writing—review and editing, R.D. and J.L.; visualization, M.X.; project administration, R.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (Program No. 21905232) and Natural Science Basic Research Plan in Shaanxi Province of China (Program No. 2022JM-236).

**Data Availability Statement:** The data are available upon request from the corresponding author.

**Acknowledgments:** The authors thank G. Wang and H.Y. Gao (University of Science and Technology Beijing, Beijing 100083, China) and X.W. Zhang and M.Y. Han (Beijing Normal University, 100088, China) for their help.

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

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