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 first 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.
Feed levels of 1 mg/kg and 3 mg/kg of DON are possible daily doses for children. In Portugal, by using HPLC to analyse 307 samples of plant crops, the highest levels of DON concentration was found to be 17.9 mg/kg [36]. It was reported that the average content of 23,980 samples contaminated by DON was as follows: wheat, 9900 mg/kg; corn, 4772 mg/kg; rice, 183 mg/kg; barley, 6349 mg/kg; oats, 537 mg/kg; and rye, 190 mg/kg [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 inflammatory 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, ethoxyresorufin-O-deethylase and methoxyresorufin-O-demethylase were unaffected, whereas pentoxyresorufin-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 significantly 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. Aflatoxin 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 detoxification of DON [46]. However, various investigations into DON in piglet liver generated inconsistent results, with some showing hepatotoxicity of DON, and others not. For example, DON at 3.1 mg/kg feed for 37 days had no impact on pig liver [47]. Similar results were reported by Van Le Thanh et al. (2016), who found that the activity of other antioxidant enzymes or glutathione concentrations were not affected by DON (0.8 and 3.1 mg/kg feed) over 17 days of exposure [30]. Renner et al. (2017) did not find the liver histology activity index (HAI) in young pigs fed DON at 4.59 mg/kg feed for 27 days [48]. However, pigs given DON-contaminated feed (4 mg/kg for 15, 30 and 37 days) showed oxidative stress and lipid peroxidation [49]. Gerez et al. (2015b) found that pigs given DON-contaminated feed (1.5, 2 and 3 mg/kg for 28 days) showed significant histological changes in the liver [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 significantly increased the level of serum GLO, suggesting that DON may cause an inflammatory 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-CpG-binding 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 significantly increased the methylation level of the CYP2B22 gene promoter, but the expression of CYP2B22 was significantly 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 classified as a phase II metabolising enzyme [70, 71]. NNMT is essential for the biotransformation and detoxification 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 inflammation [74]. In our study, DON significantly 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 inflammation, energy metabolism and reproduction [75–77]. The expression of GALP can be detected in pituitary, brain, liver and testis tissue [52, 75, 78–80]. After fasting, the expression of GALP gene decreases significantly [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 significantly 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 significantly 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) significantly 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 first 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 significantly 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].