Dietary exposure of deoxynivalenol affected cytochrome P450 and growth related-gene expression via DNA methylation in piglet liver

Deoxynivalenol (DON) is an inevitable contaminant in animal feed and human food and can lead to decreased appetite and growth retardation in children and piglets, which are often used as models for children. Hepatotoxicity induced by DON is closely related to growth inhibition. Although many molecular mechanisms are related to the liver damage caused by DON, few studies have been done on cytochrome P450 (CYP450s) and DNA methylation, and the role of DNA methylation in growth inhibition of piglets was also unclear. Statistical analysis using SPSS, as mean ± SD. Difference between groups determined by a one -way ANOVA using a signicance level of P < 0.05. The BSP data were assessed by the Mann–Whitney non-parametric test for multiple comparisons.

Background Deoxynivalenol (DON), as a serious natural source of global contaminant, is one of the most common mycotoxins produced by several pathogenic fungi, such as Fusarium graminearum [1]. DON is generally stable and therefore not easily destroyed or removed from food even after processing [2]. DON The toxicity of DON-induced retardation of human growth has become a burning topic. Studies have found that DON can cause growth retardation and weight loss through protein synthesis inhibition and other phenotypic mechanisms [22]. Recently, it was found that DNA methylation was closely related to growth inhibition [23,24]. The expression of growth hormone (GH), growth hormone receptor (GHR) and insulin-like growth factor 1 (IGF-1) were in uenced by DNA methylation [25,26]. Another study revealed that 8 mg/kg of DON in the diet was responsible for signi cant decreases in the mRNA expression of nicotinamide N-methyltransferase (NNMT), galanin-like peptide (GALP) and IGF-1, but signi cantly increased the expression of insulin-like growth factor-binding protein 2 (IGFBP2) [16]. However, how DNA methylation affects the expression of these genes under DON exposure is still unknown.
On the basis of previous studies, we used piglets as models to study the role of CYP450s and DNA methylation in DON-induced growth inhibition in children, because piglets are used as models of human paediatric surrogacy and show similarities to observations in humans in studies of the expression of CYPs [27,28]. The dose used in this study was selected on the basis of previous research reports on DON in piglets [29][30][31], and piglets were exposed to chronic toxic levels of 1 mg/kg and 3 mg/kg for 4 weeks of treatment. The effect of DON on liver function was determined by analysing the changes of liver function indices in the piglets' blood. To check whether DON affected liver metabolising enzymes and DNA methylation, the expression of CYP450 and growth-and DNA methylation-related genes was detected by qPCR. Furthermore, the level of 5-mC in the whole genome was detected by colorimetry. The methylation level of CYP450 genes and growth-related gene promoter in the liver was detected by methylation-speci c PCR (MSP) to further study DNA methylation in liver injury caused by DON.

Methods
Reagents and chemicals DON

Experimental animals and design
A total of 15 crossbred newly weaned piglets (3 weeks of age) were selected and allotted into three dietary treatment groups and each group contained ve piglets. All diets were formulated to meet the amino acid requirements of NRC (1994). Treatment Group 1 received the basal diet (BD, NRC 2012, control), Group 2 received BD + 1.0 mg/kg DON and Group 3 received BD + 3.0 mg/kg DON. The piglets were immunised with the OVA (0.5 mg + Quil A adjuvant) pseudorabies (PS) vaccine, swine fever (SF) vaccine and porcine circoviruses (PC) vaccine at 4 (1 week into the feeding trial) and 5 weeks of age (2 weeks into the feeding trial). After 4 weeks of experiment, the piglets were humanely killed and the blood and liver collected for further assays.
Quantitative real-time PCR (qPCR) examination Total RNA from the liver was isolated using the TRIzol extraction method according to the manufacturer's instructions (Invitrogen Inc., Carlsbad, CA). The quality of RNA was veri ed by evaluating the optical density at 260 nm and 280 nm. The extracted RNA was reverse-transcribed into cDNA using a Prime Script reverse transcription-PCR kit (TaKara, Dalian, P.R. China) for qPCR.
The Primer Express software was applied to design the rat-speci c primers according to the software guidelines (Table 1). Each 10 µL reaction mixture consisted of 5 µL SYBR® Premix Ex Taq™, 0.4 µL of each primer (10 µM), 1 µL of cDNA and 3.2 µL RNase-free H 2 O. For all genes, the cycling conditions were as follows: step 1: 30 s at 95 °C; step 2: 45 cycles at 95 °C for 5 s, 50-60 °C for 30 s; step 3: dissociation stage. In this study, the housekeeping gene β-actin was used as an internal calibrator reference gene for expression pro ling of genes. Data were analysed and quanti ed using the 2 −ΔΔCt methods.

Calculation of benchmark dose (BMD)
According to the expression of different metabolising enzymes in the liver of piglets, the BMD curves of gene expression were calculated and the corresponding BMD and lower con dence limit of the BMD (BMDL) values obtained using BMD software (https://benchmarkdose.org/). The BMD and BMDL values of metabolic enzymes are shown in Table 2.

MSP analysis
To determine the methylation level of gene promoter regions related to the metabolism and growth in liver tissue, the EZ DNA Methylation-Gold™ kit was used for bisul te conversion of GC-rich DNA according to the manufacturer's directions. Methylated and unmethylated primers for CYP450s and NNMT, GALP, IGF-1 and IGFBP2 genes were synthesised by Nanjing Genescript Co. Ltd., Nanjing, P.R.China (Table 3). MSP used the following cycle parameters: 95 °C for 5 min, followed by 20, 30 and 40 cycles, respectively, at 95 °C for 30 s, 50-60 °C for 30 s and 72 °C for 30 s, and a nal extension at 72 °C for 7 min. The PCR product (5 µl) was subjected to electrophoresis on 3% agarose gel and stained with 0.5 µg/ml ethidium bromide. Optical density values were measured using Quantity One 4.6.2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The following formula was used to calculate the results of methylation or unmethylation: The PCR product was joined to T vector and then cloned and the blue colonies of sequence selected. The bisul te sequence data on CpG site methylation analysis were aligned, visualised, and quanti ed by quantitative tool for methylation analysis (QUMA) software.

Statistical analysis
Statistical analysis was performed using SPSS, version 13 (Chicago, IL, USA). Data were presented as mean ± SD. Difference between groups was determined by a one -way ANOVA using a signi cance level of P < 0.05. The BSP data were assessed by the Mann-Whitney non-parametric test for multiple comparisons.

DON affected serum liver biochemical indices, causing liver damage
To observe the effect of DON toxin on piglet liver function, serum chemical parameters including AST, ALT, ALP, TP, GLO, ALB, TBIL, DBIL, IBIL, GGT, UREA and LDH were measured. According to the data presented in Fig. 1, serum ALT activity and GLO levels in the DON group signi cantly increased after DON administration (1 or 3 mg/kg) (p < 0.5). However, AST and LDH activity in the DON-treated group at 3 mg/kg exhibited lower levels compared to the control group. Additionally, a sharp increase was observed in GGT activity following DON treatment at 1 mg/kg when compared to the control group (p < 0.01). These results suggested that DON leads to liver dysfunction in piglets.

DON exposure caused changes in CYP450s expression in piglet liver
To check whether DON exposure cause abnormal expression of CYP450s, the mRNA level was examined.
As shown in Fig. 2 DON exposure caused abnormal expression of DNA methyltransferases and affected the genomic 5-mC level in piglet liver In DNA methylation analysis, we focused primarily on level of 5-mC, which is regulated by DNA methyltransferases (DNMT1, DNMT3A, DNMT3B). As shown in Fig. 3, the mRNA expression levels of DNMT1 and DNMT3B were signi cantly increased after DON toxin exposure compared to the control group (p < 0.05). However, the DNMT3B expression of DON groups showed no signi cant difference compared to the control group. We tested the genomic DNA methylation levels in pig livers, where our results revealed that 5-mC global levels were signi cantly higher in livers treated with DON toxin (1 or 3 mg/kg) than in the control (p < 0.05).
DON exposure caused DNA methylation changes in promoters of metabolic enzyme genes in piglet liver The MSP was used to measure the methylation levels of CpG sites in the CYP450 gene promoter region. DON exposure caused DNA methylation changes in promoters of growth-regulated genes in piglet liver To further determine whether DNA methylation was involved in the expression of NNMT, GALP, IGF-1 and IGFBP2 after exposure to DON toxin in pig liver, MSP and BS were applied. For these genes, the CpG sites of MSP assay were included in the sequence of BSP analysis. As shown in Fig. 6A-B, although only methylated primers ampli ed the bands of all groups, the methylated bands of the DON treatment group were stronger, suggesting an increased methylation level of NNMT to some extent. BSP results of NNMT gene also revealed that some CpG sites in DON-exposed groups were completely methylated compared to the control group, thus promoting NNMT transcription. For the GALP gene in Fig. 6C-D, the methylation level in one DON group (3 mg/kg) was lower than the control group and induced GALP expression. In Fig. 6E-F, the results showed that DON treatment could decrease the promoter methylation level of IGF-1, promoting IGF-1 expression. However, the IGFBP2 methylation level in DON groups exhibited no signi cant difference compared to the control group (Fig. 6G-H).

Discussion
Due to its high resistance to temperature, high toxicity levels and widespread occurrence in food, DON is considered an unavoidable contaminant in nature and to pose a serious threat to public health [35].
Although the liver toxicity of DON has received much attention, the role of DNA methylation and CYP450s on its deleterious toxicity is still poorly understood. In this study, DON exposure increased the serum ALT and GLO levels. Moreover, the present study proved for the rst time that DNA methylation regulated the expression of CYP450s in DON-treated piglet livers. DON exposure reduced the expression of NNMT and GALP, with decreases in feed intake and weight of piglets. It was worthy of note that DON could regulate the expression of NNMT, GALP and IGF-1 through DNA methylation and thus affect the growth of piglets.  [37]. DON was also detected in pasta with the highest level in the European Union of 3200 µg/kg [38]. Therefore, the dose used in this study was within the exposure dose range of children to DON.
Although CYP450 does not participate in the direct metabolism of DON, abnormal changes in CYP450s were the mechanism for DON-induced hepatotoxicity. Recent literature suggested that CYP450s are involved in oxidative stress, apoptosis and in ammatory response against foreign particles [39,40].
Different metabolic enzyme patterns also accompanied the pathological lesions. For example, the activities of liver microsomal mixed-function oxidase, ethoxyresoru n-O-deethylase and methoxyresoru n-O-demethylase were unaffected, whereas pentoxyresoru n-O-depentylase activity was increased. Protein levels of glutathione S-transferase α and π were increased, whereas CYP1A protein level was unchanged [41,42]. Another study reported that DON had no effect on the mRNA expression of different CYP450s (CYP1A4 and CYP3A37) in duodenum and liver [43]. However, in the present study, we found that 3 mg/kg DON could signi cantly increase the mRNA expression level of CYP450s (CYP1A1, CYP1A2, CYP2B22, CYP2C33, CYP2D25, CYP2E1, CYP3A22 and CYP3A39). The differences in the result may be related to the DON dose, the animal species or environmental conditions. A atoxin B1 (AFB1)induced generation of reactive oxygen species can lead to oxidative stress, potentially requiring the activation of CYP450s [44]. Similarly, the increase in CYP450s might be an important mechanism of liver injury under DON exposure. Importantly, increased CYP1A1 expression was the most sensitive metabolic enzyme in the assessment of DON-induced liver injury.
Piglets are one of the most sensitive species with regard to their response to DON-contaminated feed and are the best models for studying the toxic effect of DON on children [22,45]. The liver plays a key role in the metabolism and detoxi cation of DON [46].  [50]. The study by Pierron et al. (2018) found that piglets exposed by gavage to 1 and 0.5 nM DON/kg b.w/day for 3 weeks revealed a slight decrease in weight gain [51]. A diet containing 8 mg/kg DON fed for 4 weeks disrupted the immune-related processes in the liver of piglets [52]. Based on previous literature [53], in the present study we selected the administration dose of DON of 1 and 3 mg/kg feed and fed piglets for 4 weeks. Herein, DON at 1 mg/kg elevated the ALT and GGT levels, suggesting that DON may destroy the liver cell membrane, leading toleakage of enzymes from injured hepatocytes [54,55]. However, compared with the 1 mg/kg DON group, DON at 3 mg/kg decreased serum levels of ALT, AST and LDH, especially AST and LDH, suggesting that 3 mg/kg DON may lead to massive necrosis of liver cells or acute hepatitis, basically resulting in depletion of transaminase in the liver tissue [56,57]. In addition, DON signi cantly increased the level of serum GLO, suggesting that DON may cause an in ammatory response and immune system disorders in piglet liver [58].
DNA methylation could be used as a sensitive molecular indicator of DON-induced liver damage.
Regarding DNA methylation, DNMT1, DNMT3A and DNMT3B, maintain synergistically the stability of DNA methylation [20]. Most of the changes in DNA methylation are due to chemicals, including mycotoxins, in food and in the environment [20,21]. It was found that 10 mM DON increased the percentage of 5-methylcytosine in DNA from 4.5-9% in Caco-2 cells [59]. However, another study reported that DON at 3 mg/kg decreased the expression of methyltransferases and upregulated methyl-CpGbinding domain 2 (MBD2) expression in porcine splenic lymphocytes [60]. Abnormal changes in DNA methylation are common in tumorigenesis [61]. In the current study, DON exposure resulted in the increased expression of the DNMT1 and DNMT3B genes, and raised the genomic 5-mC level in piglet livers. We also observed that with the higher concentration of DON the effect was also greater. This might indicate that DON has a liver-cancer-promoting effect [62], and the content of genomic 5-mC may be a potential epigenetic biomarker for the hepatotoxicity of DON [63].
DNA methylation is a molecular switch that regulates CYP450 expression of DON-exposed piglet liver. Previous studies have shown that the expression of the CYP450 gene is related to the methylation of its promoter region, such as CYP1A1 [64], CYP1B1, CYP2E1 [65], CYP450 3A4 (CYP3A4) and CYP450 2D6 (CYP2D6) [66]. In the present study, it was found that DON at 3 mg/kg could reduce the methylation level of the promoter region of enzymes, including CYP1A1, CYP1A2, CYP2D25, CYP2E1 and CYP450 3A29 (CYP3A29), and thus increase their mRNA expression levels. Strangely, the results found that DON at3 mg/kg signi cantly increased the methylation level of the CYP2B22 gene promoter, but the expression of CYP2B22 was signi cantly increased, which suggested that DON also affected CYP2B22 expression through other transcriptional factors [67,68]. There were differences in the regulation of DNA methylation on the expression of different CYP450s, which may be related to the polymorphism of the CYP450 gene [69].
DNA methylation affects the expression of genes related to animal feeding and growth. As a key cytosolic methyltransferase in the liver, NNMT is classi ed as a phase II metabolising enzyme [70,71]. NNMT is essential for the biotransformation and detoxi cation of some heterogenous compounds, and plays a role in catalysing N-methylation of nicotinamide, pyridine and other structural analogues [72,73].
The abnormal expression of NNMT has been found in many diseases and pathophysiological processes, such as cancer, obesity and cirrhotic liver [72]. It was found that the inhibition of NNMT increases the level of S-adenosylmethionine (SAM) and nicotinamide adenine dinucleotide (NAD) in fat and consequently produces the effect of weight loss [72]. In addition, NNMT is closely related to ALT release and liver in ammation [74]. In our study, DON signi cantly reduced the expression of NNMT, which may be an important factor in the weight loss of piglets. The methylation level of several CpG sites in the NNMT promoter increased slightly, which may have partially reduced the expression of NNMT.
GALP, as a protein-coding gene, is involved in regulating appetite and in ammation, energy metabolism and reproduction [75][76][77]. The expression of GALP can be detected in pituitary, brain, liver and testis tissue [52,75,[78][79][80]. After fasting, the expression of GALP gene decreases signi cantly [81]. It has been proved that acute GALP treatment can change the food intake of primates, mice and rats. For example, GALP (1-10 ptg) was infused into the ventricles of rats with satiety and starvation at the same time and the feeding increased at 1 h after injection, but the feeding and weight decreased signi cantly 24 h after injection. The short-term and long-term effects of GALP on food intake may be achieved through different neural pathways [82]. In addition, the expression of GALP gene is controlled by a leptin signal [78]. Our study found that DON at 1 mg/kg can signi cantly reduce the expression of GALP in the liver, which may lead to decreased appetite and weight loss in piglets, as found in the previous study [83]. However, a higher dose of DON (3 mg/kg) signi cantly increased the expression of GALP in the liver, which may be due to the fact that the high dose of DON fed to piglets for a long time resulted in a sharp decrease in weight, malnutrition and an increase in the secretion of leptin in the animals, resulting in an increase in the expression of GALP [84]. Meanwhile, DON at 3 mg/kg could demethylate CpG sites in the promoter region of the GALP gene, leading to a sharp increase in the expression of GALP, which is the rst time it has been discovered.
IGF1 is the key mediator of GH. GH is synthesised in the anterior pituitary and then released into the blood, stimulating the liver to produce IGF1. In turn, IGF1 stimulates whole-body growth and plays a growth-promoting role in many cell types [85]. In addition, IGFBP2 affects the animal's immune response and cell proliferation [52]. In our study, it was found that DON could signi cantly increase the expression of IGF-1 and IGFBP2, which may be a compensatory response or negative feedback regulation of the GH reduction caused by DON [46]. Besides, it was found that DNA methylation of IGF-1 promoter decreased in DON-exposed piglet liver, which is consistent with existing research [86].

Conclusion
In this work, we found that DON could induce hepatotoxicity, increase the expression of CYP450s and

Declarations
Availability of data and materials The data sets used and analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The animal experiment was conducted according to the individual license (Aimei Liu, w20180312) obtained by the training base of experimental animal practitioners in Hubei Province, and animals were followed by proper veterinary surveillance throughout the experiment.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.       The methylation levels of CYP450 genes in the liver ofpiglets fed diets containing DON-contaminated corn (n = 5). Genomic DNA extracted from the liver was treated with bisul te and then subjected to methylation-speci c PCR (MSP) using the methylated DNA (m)-and unmethylated DNA (u)-speci c primer sets. In the gure, M represents methylation, U represents unmethylation. *p < 0.05, **p < 0.01, ***p < 0.001 versus 0 mg/kg group.

Figure 4
The methylation levels of CYP450 genes in the liver ofpiglets fed diets containing DON-contaminated corn (n = 5). Genomic DNA extracted from the liver was treated with bisul te and then subjected to methylation-speci c PCR (MSP) using the methylated DNA (m)-and unmethylated DNA (u)-speci c primer sets. In the gure, M represents methylation, U represents unmethylation. *p < 0.05, **p < 0.01, ***p < 0.001 versus 0 mg/kg group.