
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00800-9
10.1016/j.psj.2024.104221
104221
METABOLISM AND NUTRITION
Zearalenone-induced hepatointestinal toxicity in laying hens: unveiling the role of gut microbiota and fecal metabolites
Wang Lingling *‡
Deng Zifeng *‡
Huang Jieying *‡
Li Tingyuan *‡
Jiang Jun *‡
Wang Wence §
Sun Yu *‡
Deng Yiqun yqdeng@scau.edu.cn
*†1
⁎ State Key Laboratory of Swine and Poultry Breeding Industry, South China Agricultural University, Guangzhou, Guangdong 510642, PR China
† Guangdong Academy of Agricultural Sciences, Guangzhou, Guangdong 510640, PR China
‡ Guangdong provincial key laboratory for the development biology and environmental adaptation of agricultural organisms, Guangzhou, Guangdong 510642, PR China
§ Guangdong Provincial Key Laboratory of Animal Nutrition and Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong 510642, PR China
1 Corresponding author: yqdeng@scau.edu.cn
16 8 2024
11 2024
16 8 2024
103 11 10422124 4 2024
11 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Zearalenone (ZEN), a mycotoxin produced by Fusarium species, is known for its reproductive toxicity as an estrogen analogue. However, there are limited knowledge about its hepatointestinal toxicity, as well as the role that gut microbiota and metabolites play in this process. In this study, a total of 24 thirty-week-old hens were fed to investigate the hepatointestinal toxicity subjected to long-term ZEN consumption at 2.0 mg/kg for 90 d. And we employed uncultured 16S rRNA sequencing for gut microbiota and untargeted metabolomics for fecal metabolites assessment. Notably, ZEN induced significant hepatic damage, as evidenced by hepatocyte necrosis, inflammatory cell infiltrate, increased liver lipopolysaccharide (LPS) and blood aspartate aminotransferase (AST) levels (P < 0.05). The decreased villus height, disruption of simple columnar epithelial cells, and exposure of the mucosal intrinsic layer were observed in the intestine. The gut microbial community composition and metabolites differed between ZEN group and control group. ZEN group exhibited higher gut microbial diversity (P < 0.05), lower Firmicutes/Bacteroidetes ratio and Lactobacillus abundance, and higher abundance in the genus such as Bacteroidetes, Parabacteroidetes and Desulfovibrio. Metabolomic analysis showed that ZEN treatment altered biosynthesis of siderophore group nonribosomal peptides and phenylpropanoids, metabolism of amino acid, digestion and absorption of vitamin and ABC transporters. Differential metabolites suggested that ZEN increase the risk of estrogen disorder, nucleic acid degradation, intestinal oxidative stress and inflammation. Neural network analysis showed that Ruminococcus was positively correlated with glyceric acid, and Prevotella was positively correlated with phenylacetylglycine. Both metabolites were positively correlated with blood AST level (P < 0.05), suggesting that intestinal microbe Ruminococcus and Prevotella might exacerbate liver damage by producing these harmful metabolites. Overall, we conclude that ZEN has damaged hepatointestinal system and the altered gut microbiota with resultant metabolite changes contribute to the adverse hepatointestinal effects of ZEN on laying hens. This study underscores the need for monitoring and mitigating ZEN exposure in poultry diets, highlighting its broader implications for animal health and food safety.

Key words

zearalenone
hepatointestinal damage
gut microbiota
fecal metabolite
correlation analysis
==== Body
pmcINTRODUCTION

Zearalenone (ZEN), a secondary metabolite produced by fungi Fusarium, is one of the most frequent contaminants in wheat, barley, maize and other crops (Stob et al., 1962). Zearalenone and its derivatives compete with 17 β-estradiol for binding to estrogen receptors (ER), leading to reproductive system injury in humans and animals as an estrogen analogue. Sensitivity to ZEN varies across species, with pigs showing high sensitivity, whereas chickens demonstrate a relatively higher tolerance (Jiang et al., 2010; Wang et al., 2022; Soffa et al., 2023). ZEN toxicological studies of chickens have mainly focused on the reproductive and immune systems. For example, diet containing 0.4 mg/kg ZEN significantly increased the weight of testes and combs in male chicks over a 10-d period (Sherwood and Peberdy, 2007). Degeneration and atrophy of ovarian tissues were also found in 70 day-old growing-laying hens which were fed with 0.4 mg/kg ZEN diet in a 49-d period (Cheng et al., 2017). Wu et al. reported 20 μmol/L ZEN triggered heterophil extracellular traps (HETs) formation, negatively affecting chicken innate immunity in vitro (Wu et al., 2023a). After intake, ZEN was absorbed in the small intestine and then metabolized in the liver (Biehl et al., 1993). ZEN can induce liver lesions, and gut microbiota is involved in the metabolic transformation of ZEN (Jia et al., 2022) and impair the intestinal structural integrity of juvenile grass carp (Wang et al., 2019). The live and gut damage caused by ZEN cannot be ignored. However, the current knowledge of ZEN hepatointestinal toxicity in chickens is very limited.

With advancements in multiomic technologies, researchers have demonstrated that the negative effects of mycotoxin are mediated not only directly but also through altering gut microbiota and their metabolites. For example, in a study on T-2 toxin in chicks, researchers pointed out an increase of lipids-phospholipid level and a decrease in mononucleotides were involved with the T-2 toxin-induced intestinal damage (Liu et al., 2023). Other studies have suggested that an increase of LPS producing bacteria and decrease of short-chain fatty acids (SCFA) producing bacteria aggravated the risk of mycotoxin-induced inflammation in the liver and mucosal barrier dysfunction in the intestine (Wang et al., 2019; Bai et al., 2023). However, multi-omic studies on ZEN toxicology are scarce. There are no reports on the alteration of gut microbiota and metabolite composition in chicken with ZEN exposure, and thus its implications for animal disease development remains unknown.

Laying hens, due to their long feeding cycle for optimal economic benefit, the risk of long-term ZEN exposure is much more significant than broiler chickens. Current understanding of long-term ZEN toxicity on laying hens is limited. In this study, we conducted a 90-day ZEN exposure experiment on laying hens to assess the hepatointestinal toxicity of ZEN. We detected the gut microbiome and metabolism through 16S rRNA gene sequencing and mass spectrometry techniques respectively, and proposed correlations by neural network co-occurrence analysis and Spearman's correlation analysis. The aim of the present work was to detect the hepatointestinal toxicity of ZEN on laying hens and to evaluate the role of the gut microbiota in this process.

MATERIALS AND METHODS

Experimental Design, Animals, and Diet

The Institutional Animal Care Committee of South China Agricultural University approved the current animal experiments study. A total of 24 Sanhuang laying hens, aged 210 d with similar body weight, were randomly divided into 2 treatments with 12 replicates each. Hens in the 2 groups were fed either a basal diet (ZEN not detected, the detection limits was 0.1 mg/kg) as control group or a basal diet supplemented with 2.0 mg/kg ZEN (purity > 98%, Shanghai Aladdin Biochemical Technology Co., Ltd, Shanghai, China) as ZEN group for 90 d (including a 7-d adaptation period and an 83-d experimental phase). The composition and nutritional levels of the basal diet were shown in Table 1. The hens were fed 100 g per day and had free access to water during the experiment. Health status and mortality were daily monitored during the whole experimental period. The hens were housed in 3-stage stepped-stainless steel cages with one hen per cage and maintained under a 16 h light/8 h dark cycle. The weight was recorded at an individual level on d 0 and 90. And the egg production was recorded every day during the period. Fecal samples from 3 hens in each group were randomly selected for subsequent analysis, and were stored at -80°C on the final day of the experimental period. Blood samples were collected and kept at room temperature for 30 min before centrifugation at 4°C and 12,000 rpm for 10 min to obtain the serum sample. After 24 h of fasting, hens were humanely euthanized by venesection for morphological, morphometric and microbiological analyses of the liver and intestinal tract. The collected serum and tissues in the experiment were stored at -80°C.Table 1 Basic diet components and nutrition level.

Table 1Ingredients	Content(%)	Nutrition level	Content(%)	
Corn	58.8	Crude protein	13.00	
Soybean meal	16	Crude fiber	6.00	
Distillers Dried Grains with soluble	12.4	Crude ash	13.00	
Monocalcium phosphate	0.8	Calcium	2.50	
Limestone	7	Total phosphorus	0.40	
Premix1	5	Sodium chloride	0.8	
Total	100	Lysine	0.70	
		Methionine	0.52	
		Arginine	0.88	
1 Premixed supplied per kg of diet: vitamin A 6,500 IU, vitamin D3 2,560 IU, vitamin E 40 IU, vitamin K3 1.2 mg, vitamin B1 7.5 mg, vitamin B2 10.7 mg, vitamin B6 4.2 mg, vitamin B12 0.025 mg, folic acid 1 mg, Fe 100 mg, Mn 80 mg, Zn 80 mg.

Measurements of Serum Biochemical Indicators

Serum biochemical indicators including asparatate aminotransferase (AST) (rate method), alanine aminotransferase (ALT) (rate method), alkaline phosphatase (ALP) (end point method) and total protein (TP) (end point method) were measured by Automatic Biochemical Analyzer (Biobase Biodustry, Co., Ltd, Shandong, China). The contents of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH) and glutathione peroxidase (GSH-Px) in serum were measured by a Microplate Reader (BioTek Instruments, Inc, Vermont) with kit methods (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Lipopolysaccharide (LPS) in liver was analyzed by Microplate Reader (BioTek Instruments, Inc, Vermont) with ELISA kit (Uscn Life Science Inc, Wuhan, China).

Histopathological Examination

The collected liver and intestine tissue samples were fixed in 10% formalin. Subsequently, the fixed liver, duodenum, jejunum, ileum and cecum tissues were treated with a gradient series of ethanol and xylene solutions and were conventionally embedded in paraffin wax. Serial sections of 5 μm thickness were sliced, dewaxed in a gradient solution of xylene and ethanol after being dried at 37°C, and stained with hematoxylin and eosin. The intestinal tissue structure was observed under light microscopy. The intestinal villus height and crypt depth were carried out using Image J software (v1.54d).

DNA Extraction and 16S rRNA Gene Sequencing

Total genomic DNA samples were extracted from fecal samples using the OMEGA Soil DNA Kit (M5635-02) (Omega Bio-Tek, Norcross, GA), following the manufacturer's instructions, and stored at -20°C prior to further analysis. The quantity and quality of extracted DNAs were assessed using a NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and agarose gel electrophoresis. Amplicon sequencing was performed using Illumia MiSeq paired-end sequencing technology, targeting the V3-V4 regions of the bacterial 16S rRNA gene with the forward primer 338F (5’-ACTCCTACGGGAGGCAGCA-3’) and the reverse primer 806R (5’-GGACTACHVGGGTWTCTAAT-3’).

Microbiome Sequencing Analysis

Microbiome bioinformatics analysis were conducted utilizing QIIME2 2019.4 and R packages (v3.2.0). Raw sequence data underwent demultiplexed with the demux plugin, followed by primers trimming using cutadapt plugin. Subsequent processes included quality filtered, denoised, merged, and chimera removed by the DADA2 plugin. Non-singleton amplicon sequence variants (ASVs) were aligned using mafft. Taxonomic assignment of ASVs was performed using the classify-sklearn naive Bayes classifier in the feature-classifier plugin against the SILVA Release 138 Database. Taxonomic compositions and abundances were analyzed and visualized using MEGAN and GraPhlAn. Venn diagrams depicting shared and unique ASVs among samples or groups were created using the “VennDiagram” R package, based on the presence of ASVs across samples/groups regardless of relative abundance. Statistical comparisons of ASV-level taxa abundances among samples or groups were conducted using MetagenomeSeq. Alpha-diversity metrics were computed, including Chao1, Observed species, Shannon, Simpson, and Good's coverage. To investigate the structural variation in microbial communities across samples (beta-diversity), nonmetric multidimensional scaling (NMDS) was employed. The significance of microbial community differentiation among groups was assessed using permutational multivariate analysis of variance (PERMANOVA). Linear discriminant analysis effect size (LEfSe) was applied to identify differentially abundant taxa across groups, using default parameters.

Untargeted Metabolomics Analysis of Feces

Fecal samples were thawed, and an appropriate amount of each sample was accurately weighed into a 2 mL centrifuge tube. Then, 600 µL MeOH (stored at -20°C) (Containing 2-Amino-3-(2-chloro-phenyl) -propionic acid 4 μg/mL) was added and vortexed for 30 s. The samples were processed in a tissue grinder for 90 s at 60 Hz and room temperature ultrasound with 100 mg glass bead for 10 min. Finally, the samples were centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was filtered through a 0.22 μm membrane and transferred into a detection bottle for Liquid Chromatograph Mass Spectrometer (LC-MS) detection.

The LC analysis was performed on a Vanquish UHPLC System (Thermo Fisher Scientific, Waltham, MA). Chromatography was carried out with an ACQUITY UPLC HSS T3 (150 × 2.1 mm, 1.8 µm) (Waters, Milford, MA). The column maintained at 40°C. The flow rate and injection volume were set at 0.25 mL/min and 2 μL, respectively. The MS detection of metabolites was performed on Orbitrap Exploris 120 (Thermo Fisher Scientific, Waltham, MA) with ESI ion source. Simultaneous MS1 and MS/MS (Full MS-ddMS2 mode, data-dependent MS/MS) acquisition was used. The parameters were as follows: sheath gas pressure, 30 arb; aux gas flow, 10 arb; spray voltage, 3.50 kV and -2.50 kV for ESI(+) and ESI(-) respectively; capillary temperature, 325°C; MS1 range, m/z 100-1,000; MS1 resolving power, 60,000 FWHM; MS/MS resolving power, 15,000 FWHM; normalized collision energy, 30%; dynamic exclusion time, automatic.

The raw data were firstly converted to an mzXML format by MSConvert in the ProteoWizard software package (v3.0.8789) and processed using XCMS for feature detection, retention time correction and alignment. Metabolites were identified by accuracy mass (< 30 μg/mL) and MS/MS data, which were matched with HMDB (http://www.hmdb.ca), massbank (http://www.massbank.jp/), LipidMaps (http://www.lipidmaps.org), mzcloud (https://www.mzcloud.org) and KEGG (http://www.genome.jp/kegg/). Robust LOESS signal correction (QC-RLSC) was applied for data normalization to correct for any systematic bias. After normalization, only ion peaks with relative standard deviations (RSDs) less than 30% in QC were retained to ensure proper metabolite identification. The Ropls software was used for all multivariate data analyses and modelings. After scaling data, models were built on partial least-square discriminant analysis (OPLS-DA). The metabolic profiles could be visualized as score plot, where each point represents a sample. Corresponding loading plots and S-plots were generated to identify metabolites influencing sample clustering. All models evaluated were tested for overfitting using permutation tests. The descriptive performance of the models was determined by R2X (cumulative) (perfect model: R2X (cum) = 1) and R2Y (cumulative) (perfect model: R2Y (cum) = 1) values. The prediction performance was measured by Q2 (cumulative) (perfect model: Q2 (cum) = 1) and a permutation test. The permuted model should not be able to predict classes: R2 and Q2 values at the Y-axis intercept must be lower than those of Q2 and the R2 of the non-permuted model. OPLS-DA enabled the determination of discriminating metabolites using variable importance on projection (VIP). P-value, VIP from OPLS-DA and fold change (FC) were applied to identify contributory variables for classification. Finally, metabolites were considered statistically significant at P < 0.05, FC > 2 or FC < 0.5 and VIP > 1.

Neural Networks Analysis for Microbe-Metabolite Interactions and Spearman's Correlation

Microbiome-metabolite vectors (MMVEC) was used to learn co-occurrence probabilities between microbes and metabolites through iterative training (Morton et al., 2019). Co-occurrence probabilities refer to the conditional probability of observing a metabolite given that a microbe was observed, thereby allowing to identify the most likely microbe-metabolite interactions. Spearman's correlation was used to analyze the correlation between gut microbes and serum indices. All tests were 2-tailed and P < 0.05 was considered significant.

Statistical Analysis

All data were expressed as means and standard error of mean (SEM), and then analyzed by one-way ANOVA using IBM SPSS Statistics software (v26.0). A probability level of P < 0.05 was used to determine statistical significance.

RESULTS

Living Weight and Egg Production of Laying Hens After ZEN Exposure

The living weight and the average daily egg production of hens are summarized in Table 2. The hens readily accepted the experimental diets in both groups. Before the start of the trial, all hens were nearly the same weight and there was no significant difference between the groups. After 90 d of feeding, ZEN group has a tendency to increase body weight, but the difference is not significant compared with the control group. The average daily egg production was not significant between groups.Table 2 Effects of ZEN on weights of laying hens. The average daily egg production is equal to the total egg production per laying hen during the trial period divided by the number of days during the trial period.

Table 2Item	Diets	SEM	P value	
CK (n = 12)	ZEN (n = 12)	
Weight (0 d), g	1474.20	1417.50	26.46	0.294	
Weight (90 d), g	1545.80	1641.70	29.20	0.102	
Average daily
egg production, pcs	0.61	0.64	0.04	0.697	

The Negative Effects on the Liver and Intestine After ZEN Exposure

At a concentration of 2.0 mg/kg ZEN, hepatocytes necrosis was observed. The original cell structure in the necrotic foci was lost, replaced by unstructured powder material, with numerous inflammatory cells infiltrating the edge of the necrotic foci (Figure 1A). Serum biochemical analysis showed that the level of AST increased significantly in ZEN group (P < 0.05), while the levels of ALT and TP were increased and the level of ALP was decreased but all not significantly compared to control group (Figures 1B–1E). Oxidative stress index including MDA, GSH, SOD and GSH-PX showed no significant difference (Figures 1F–1I). In liver tissue, a significant increase of LPS was detected (P < 0.05) (Figure 1J). Morphological observation showed prominent differences in duodenum, ileum, jejunum and cecum (Figure 2A). In control group, intestinal villi appeared normal and arranged tightly and regularly. But in ZEN group, villi were generally shapeless and disorganized. Villus height significantly decreased compared to control group in each gut segments (P < 0.05) (Figure 2B), and the ratio of villus height to crypt depth significantly decreased in jejunum and ileum (P < 0.05) whereas the crypt depth increased and showed no significant difference compared to control group (Figures 2C and 2D). Additionally, simple columnar epithelial cells were widely separated or even broken, exposing the mucosal intrinsic layer. Damage in the cecum seemed more severe, the intrinsic layer was apparently decreased and ruffles formed by mucosal sublayer protrusions were unconsolidated and decreased in ZEN group. Our observation demonstrated that supplemented with 2.0 mg/kg ZEN led to significant injuries in the liver and intestine of laying hens.Figure 1 ZEN induced hepatic injury and disturbance in serum biochemical parameters. (A) Histopathological sections of liver. The black arow indicates necrotic hepatocytes that have lost their original cell structure in necrotic foci, replaced by a mass of unstructured powder material, and numerous inflammatory cell infiltration at the edge of necrotic foci. (B–E) Liver function indices. (F–I) Oxidative stress indices. (J) LPS level in liver. *P < 0.05, compared with control group.

Figure 1

Figure 2 ZEN disrupted the intestinal morphology. (A) Histopathological sections of duodenum, jejunum, ileum and caecum. The black arrow indicates damage to the normal structure of villi, with separation of simple columnar epithelial cells and exposure of the mucosal intrinsic layer. (B) Villus height. (C) Crypt depth. (D) The ratio of villus height to crypt depth.

Figure 2

The Alteration of Gut Microbiota After ZEN Exposure

As shown in Wayne diagrams, 845 OTUs were identified in the control group, and 4,770 OTUs were identified in the ZEN group, and 404 OTUs were recognized as common core bacteria (Figure 3A). We conducted alpha-diversity analysis between control group and ZEN group. Good's coverage indexes in both groups were beyond 99.5%, indicating adequate sequencing depth and coverage (Figure 3B). Chao1 indexes indicated significantly higher species richness in ZEN group than in control group (P < 0.05) (Figure 3C). Similarly, Shannon and Simpson indexes were also significantly higher in ZEN group than in control group (P < 0.05) (Figures 3D and 3E). These results suggested that ZEN might increase the alpha-diversity of the gut microbiota in laying hens. The variation of beta-diversity between ZEN group and control group was analyzed based on Bray-Curtis distance and was visualized using non-dimensional multi-dimensional scaling (NMDS) analysis (Figure 3F). Between-group variation showed a trend of alteration between ZEN group and control group (R = 0.740741, P = 0.089) (Figure 3G).Figure 3 Alteration of gut microbiota after ZEN exposure. (A) Wayne diagram of OTU. (B–E) Alpha diversity indices including Goods converage index, Chao1, Shannon and Simpson. (F–G) Beta diversity analysis including NMDS and Anosim. (H–I) Relative abundance of gut microbiota at the phylum and genus levels. (J) Cladogram of the main microbiota taxa with significant differences, as determined by LEfSe analysis (LDA score > 3.5).

Figure 3

Relative abundance of bacteria was calculated using QIIME2 software. The top 3 phyla were Firmicutes (47.6%), Bacteroidetes (32.2%), and Proteobacteria (8.1%) in ZEN group, and Firmicutes (50.5%), Proteobacteria (42.3%) and Actinobacteria (5.8%) in control group (Figure 3H). The ratio of Firmicutes/ Bacteroidetes was decreased from 44.7 to 1.5 after ZEN exposure. The top 5 genera were Bacteroides for 10.8%, Gallibacterium for 5.4%, Lactobacillus for 5.1%, Lachnospiraceae Ruminococcus for 4.6% and Phascolarctobacterium for 4.2% in ZEN group, while Gallibacterium for 32.0%, Enterococcus for 25.8%, Lactobacillus for 15.1%, Shigella for 8.1% and Rothia for 2.3% in control group (Figure 3I). It could be seen that the relative abundance of Gallibacterium, Enterococcus and Lactobacillus was lower in control group compared with ZEN group, while the relative abundance of Bacteroides, Phascolarctobacterium and Lachnospiraceae Ruminococcus was higher in the group compared with ZEN group.

LEfSe analysis identified the marker microorganisms in ZEN group included Bacteroidetes, Candidatus Eremiobacterota and Synergistetes at the phylum level and Collinsella, Bacteroides, Parabacteroides, Prevotella, Anaerofustis, Ruminococcaceae Ruminococcus, Lachnospiraceae Ruminococcus, Faecalibacterium, Oscillospira, Subdoligranulum, Megamonas, Megasphaera, and Desulfovibrio at the genus level. The marker microorganisms in control group included Proteobacteria at the phylum and Avibacterium and Gallibacterium at the genus level (Figure 3J).

The Alteration of Microbe-Derived Metabolites After ZEN Exposure

LC-MS detected 946 metabolites in fecal samples, including 613 metabolites from positive ions and 333 metabolites from negative ions. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed to reveal metabolic differences between groups (Figures 4A–4D). Samples in control group were closely clustered that separated from samples in ZEN group, indicating the overall difference between groups. A total of 46 differential metabolites were identified (Figure 4E), and their relative abundance was illustrated (Figure 4F, Supplementary Table S1 and Table S2). 23 metabolites were upregulated and 23 metabolites were downregulated in ZEN group. Steroids and steroid derivatives like pregnenolone, sarsasapogenin and (24R) -24-Methylcycloarta-25-en-3-β-ol were upregulated. The 5 most significantly upregulated metabolites were glyceric acid, daidzin, 2-arachidonoylglycerol, phenylacetylglycine and N-acetyl-D-tryptophan, while the 5 most significantly downregulated metabolites were quinolinic acid, aesculetin, chalconaringenin, gabapentin and astragalin (P < 0.05).Figure 4 Alteration of microbe-derived metabolites after ZEN exposure. (A) PCA score plot for the positive ion model. (B) PCA score plot for the negative ion model. (C) OLPS-DA score plot for the positive ion model. (D) OLPS-DA score plot for the negative ion model. (E) Volcano plot showing differential metabolites. Each red dot represents a single differential upregulated metabolite and each blue plot represents a single differential downregulated metabolite. (F) Heatmap of differential metabolites. The relative level are shown by color intensity; red denotes higher expression levels and blue denotes lower expression levels.

Figure 4

KEGG pathway analysis showed that ZEN exposure might have affected 6 metabolic pathways including ABC transporters (mineral and organic ion transporters), biosynthesis of phenylpropanoids, biosynthesis of siderophore group nonribosomal peptides, arginine and proline metabolism, β-alanine metabolism and vitamin digestion and absorption. In this study, D-Fructose, L-Proline, Adenosine, Riboflavin, Spermidine and Nopaline were found to be involved in the ABC transporter metabolic pathway. These pathways involved twelve differential metabolites, including spermidine, L-proline, adenosine, nopaline, D-fructose, riboflavin, umbelliferone, chalconaringenin, aesculetin, 2-pyrocatechuic acid, quinolinic acid and pyridoxal phosphate.

Neural Network Co-Occurrence Analysis of Gut Microbiome-Metabolite and Spearman's Correlation Analysis of Serum Indices-Metabolite

The microbiota-metabolome interaction analysis using neural networks was displayed in Figure 5. Bacteroides had a high co-occurrence probability with tigogenin and 2-pyrocatechuic acid. Prevotella had a high co-occurrence probability with phenylacetylglycine. Clostridium had a high co-occurrence probability with tigogenin and 2-pyrocatechuic acid. Lachnospiraceae Ruminococcus had a high co-occurrence probability with glyceric acid and aesculetin. Megamonas had a high co-occurrence probability with pregnenolone, umbelliferone and 2-iminobutanoate. Spearman's correlation test between upregulated significant metabolites and serum indexes is showed in Figure 6. The AST level in serum was strongly positively correlated with the levels of 16 metabolites including pregnenolone, prostaglandin F3α, phenylacetylglycine, glyceric acid, etc (R > 0.8). The MDA level in serum was strongly positively correlated with the levels of 12 metabolites including isophorone, tigogenin, 2-iminobutanoate, etc. (R > 0.8). Total proteins (TP) which reflects the liver's capacity for storage, was strongly positively correlated with adenosine, N-a-acetylcitrulline, prostaglandin F3α and (24R)-24-methylcycloarta-25-en-3-β-ol (R > 0.8).Figure 5 Co-occurrence probability between microbes and metabolites. This heatmap displays the log conditional probabilities between microbes and metabolites. The color scale represents these probabilities, with red indicating higher positive log conditional probabilities, signifying a stronger likelihood of co-occurrence. Low and negative values indicate no significant relationship, not necessarily implying a negative correlation.

Figure 5

Figure 6 Spearman's correlation of serum indices and metabolites. The color scale represents spearman's correlation coefficients. Red denotes positive correlations and blue denotes negative correlation. Strong correlations with R > 0.8 or R < -0.8 were showed with a * symbol.

Figure 6

DISCUSSION

Hepatointestinal Toxicity of ZEN in Laying Hens

In this study, there were obviously damage in the liver and gut of laying hens after 90-d 2.0 mg/kg ZEN exposure. ZEN-induced liver inflammation is generally thought to be caused by oxidative damage on hepatocyte, with ERS and MAPK signaling pathways participating in this process (Chatopadhyay et al., 2012; Shi et al., 2017; Wu et al., 2023b). Interestingly, our findings first observed that ZEN accelerated LPS accumulation in liver, suggesting that LPS accumulation might be another contributor to liver inflammation. LPS is a crucial inflammatory factor, and its abundant accumulation might exacerbate the progression of the hepatic inflammation and injury (Wu et al., 2021a). Polystyrene nanoplastics damage intestinal barrier and cause LPS release. The high levels of LPS further activate TLR4/NF-κB/NLRP3/GSDMD pathways in the liver, inducing liver inflammation and hepatocyte pyroptosis in mice (Chen et al., 2024). Similarly, carbonate promoting LPS synthesis and absorption, which enter the liver through the gut-liver axis, increasing liver autophagy and iron death on crucian carp (Lei et al., 2024).

Consequently, we further investigated the health status of the intestine. As expected, intestinal damage was observed at different segments, including the decreased villus height, decreased intrinsic layer, separation of simple columnar epithelial cells, and exposure of the mucosal intrinsic layer. The integrity of the intestinal barrier determines whether harmful substances in the intestine can flow into other organ through the portal vein. To our knowledge, this is the first report of intestinal damage caused by ZEN exposure in poultry. Given that the digestive tract of the chicken is relatively shorter and the retention time of feed in the intestine is about 2 h, the toxicity research of ZEN on chicken intestines has been underestimated. Moreover, studies on ZEN intestinal toxicity in farm animals are very limited. Ma et al. reported ZEN exposure increased intestinal permeability and caused intestinal damage in weaned piglet, they speculated that inflammatory response and metabolic disorders induced might be contributed to the intestinal injury by quantitative proteomic analysis (Ma et al., 2022).

Although we observed significant liver and intestinal damage in laying hens, there were no significant changes in body weight. ZEN, as an estrogen compound, might possess an unknown growth promoting mechanism. Some studies have shown that ZEN consumption leads to weight gain (Rykaczewska et al., 2018; Chang et al., 2020). We speculate that the counteracting effects of growth promotion and digestive disorder result in the relative stability of weight. Additionally, average daily egg production was also not affected, despite a decrease in estradiol level and significant lesions in the oviduct tissue (see Supplementary Figure S1 and Figure S2). The observed decrease in egg production may require a longer period of ZEN consumption to manifest. Previous studied suggested egg production may be related to the feeding dose and feeding duration of ZEN exposure (Sypecka et al., 2004; Yuan et al., 2022).

The Alteration of Gut Microbiota After ZEN Exposure in Laying Hens

Although the laying hens have a more stable intestinal flora compared to young chickens, our study demonstrated that prolonged ZEN exposure significantly altered the intestinal microbiota, resulting in higher alpha-diversity indexes of species richness and diversity in ZEN group (P < 0.05). These findings are consistent with previous studies in rats and fish (Wu et al., 2021b; Zhang et al., 2020). However, some other studies showed different patterns. Studies on rabbits and pigs showed no significant change in alpha-diversity, while a significant decrease was observed in mice (Li et al., 2018; Reddy et al., 2018; Yan et al., 2022). The variation may probably result from the intake dose, exposure period and animal species. Growing evidence showed that gut microbiota played an important role in mycotoxins biotransformation including T-2, DON and ZEN (Guerre, 2020; Sun et al., 2022). It was reported that the gut microbiota in duodenum of broilers could degrade more than half the dose of ZEN in vivo, with partially degradation was found whereas the gut microbiota in ileum, cecum and colon (Jia et al., 2022). We conjectured that ZEN was converted into various metabolites, which led to an increase of substrates available to gut bacteria, resulting in a significant rise in microbial species.

In addition to microbial diversity, the microbial composition also showed significant differences in ZEN group. Bacteroidetes was significantly increased and the ratio of Firmicutes/Bacteroidetes was significantly decreased after ZEN exposure in our study. Bacteroidaceae were the colonizers in the later period of poultry production and the ratio of Firmicutes/Bacteroidetes often reflects intestinal health and related to aging in chickens (Rychlik, 2020; Yin et al., 2023). Additionally, Lactobacillus was also significant decreased, which has been observed in rabbits, piglets and mice (Li et al., 2018; Liu et al., 2018; Wang et al., 2018). Lactobacillus strains are thought to modulate the native gut microbiota and improve health via various mechanisms, such as mucus production and anti-microbial peptides/factors to prevent the impact of pathogenic bacteria (Dempsey and Corr, 2022). Several studies also reported that ZEN can directly bind to Lactobacillus, which may be an important reason for its reduced proportion in the intestine (El-Nezami et al., 2002; Long et al., 2012). Bacteroides, Parabacteroides and Desulfovibrio were identified as three of the marker microorganisms in ZEN group. Some studies have shown that the enrichment of Bacteroides and Parabacteroides was associated with the onset of IBS (Lo et al., 2019; Pittayanon et al., 2019). Desulfovibrio has been reported to be associated with the accumulation of sulfide which aligns with the gut inflammation observed in our study. The decrease of beneficial bacteria and the increase of these pro-inflammatory bacteria might increase the risk of pathological changes in intestinal tissue.

Microbe-Derived Metabolites Participate in ZEN Toxicity in Laying Hens

At present, there are limited studies on fecal metabolomics analysis of ZEN exposure in animals. Fecal metabolomics can be used to characterize not only the function of the gut microbiome, but also the interactions between the gut microbiome and the host, to reveal the effects of ZEN. In this study, 46 differential signature metabolites upon ZEN exposure were detected by LC-MS. According to KEGG's analysis, vitamin digestion and absorption pathway was downregulated. The riboflavin (vitamin B2) was downregulated in ZEN group in this study which might gain susceptibility to intestinal oxidative stress and inflammationin animals (Powers et al., 1983; Dringen, 2000). Besides, the levels of astragalin and artemisinin were also decreased in ZEN group, whose anti-inflammation activities and oxidative stress inhibition have been reported (Gu et al., 2023; Zhu et al., 2023). In a word, the decrease of anti-inflammatory factors mentioned above could be a contributing factor to the hepatointestinal damage observed in this study. Additionally, the progesterone level was upregulated in ZEN group, which might promote estrogen synthesis and increase the risk of estrogen disorder. Additionally, the progesterone had been associated with a higher risk of ovarian cancer as a pro-hormone (Trabert et al., 2021). Previous studies have shown that ZEN affects mitochondrial function through peroxide stress, induces lipid peroxidation, cell death, and inhibits protein and DNA synthesis (Kouadio et al., 2005). In this paper, we found that the level of adenosine in the gut of laying hens increased significantly after ZEN exposure, suggesting that ZEN exposure may lead to nucleic acid degradation or inhibit DNA and RNA synthesis. Such changes might possibly be explained by metabolic disorders and intestinal microbial alterations after ZEN exposure.

In this study, neural network analysis and Spearman's correlation tests jointly explained the toxicity of ZEN contributed from gut microbiota and the derived metabolite. We found that the glyceric acid has high co-occurrence probabilities with Lachnospiraceae Ruminococcus. Lachnospiraceae Ruminococcus can use mucin as a carbon source and directly cause damage to intestinal barrier (Png et al., 2010; Crost et al., 2013; Tailford et al., 2015). Meanwhile, the glyceric acid was strongly positively correlated with AST (R > 0.8). Glyceric acid is a product of triacylglycerol, and then enters into the glycolysis, involved in energy metabolism. High level of glyceric acid might be due to some acquired metabolic disease, especially to the liver and kidney deficiencies. The higher concentration of glyceric acid may indicate that the body metabolisms were under deterioration (Fontaine et al., 1989). In summary, our results suggest that the enrichment of Lachnospiraceae Ruminococcus was related to the increase of glyceric acid, which leads to the increase of AST and even liver injury, and relevant researches needs to be further studied. Aside from that, we also observed the high co-occurrence between Prevotella and phenylacetylglycine. This generally harmless commensal possesses virulence factors such as adhesins, hemolysins, secretion systems exopolysaccharide, LPS, proteases, quorum sensing molecules and antibiotic resistance to evolve into a well-adapted pathogen capable of causing successful infection and proliferation in the host tissue (Tett et al., 2021). In our study, unsatisfied and unidentified Prevotella spp. were enriched, suggesting that the body may be at risk of infection by pathogenic bacteria. Meanwhile, phenylacetylglycine was strongly positively correlated with AST (R > 0.8) by Spearman's correlation analysis. Phenylacetylglycine is mainly generated from bacterial producing phenylacetate, via the phase II detoxification mechanism in the liver (Akira et al., 2013). In previous study, phenylacetylglycine showed significant increase when liver injury was induced by dimethylnitrosamine (Ju et al., 2013). Thus, we suggested that the enrichment of Prevotella might eventually lead to a significant increase in phenylacetylglycine, and associated with liver injury. Currently, the understanding of the implication of gut microorganisms on chicken health is still very limited. Feng (2023) speculated the foregut microbiota had closer connections with chicken serum metabolites, and some microorganisms were related to chicken lipid and amino acid metabolism. Yin (2023) reported that Erysipelotrichaceae Clostridium and Shigella were positively correlated with serum levels of total cholesterol, tryglucerides, HDL-C and LDL-C by Spearman's correlation analysis. However, these researches were just based on association analysis and experimental validation works are necessary in future.

CONCLUSIONS

In this study, a 90-day 2.0 mg/kg ZEN exposure experiment on laying hens was conducted to assess the hepatointestinal toxicity of ZEN and the role that gut microbiota and metabolites play, we found that there was significant damage in the liver and different segments of the hens gut. Moreover, LPS, travelling to the liver through the damaged intestinal barrier, exacerbated liver inflammation. The altered gut microbiota and the microbial-derived metabolites were found to act significant roles in ZEN toxicity. These findings highlight the broader implications of ZEN hepatointestinal toxicity for animal health and food safety, emphasizing the critical importance of maintaining safe levels of ZEN in animal feed to prevent adverse health outcomes in poultry and potential risks in the food supply chain.

CRediT authorship contribution statement

Lingling Wang: Conceptualization, Methodology, Investigation, Writing – original draft. Zifeng Deng: Investigation, Visualization, Software, Data curation, Writing – original draft. Jieying Huang: Investigation, Software. Tingyuan Li: Investigation, Software. Jun Jiang: Data curation, Supervision. Wence Wang: Conceptualization, Writing – review & editing. Yu Sun: Supervision, Writing – review & editing. Yiqun Deng: Supervision, Writing – review & editing.

DISCLOSURES

The authors declare no conflicts of interest.

Appendix Supplementary materials

Image, application 1

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Acknowledgments

This study was supported by the National Key Research and Development Programs of China (2023YFD1301002 ) and Guangdong Basic and Applied Basic Research Foundation (2022B1515130003 ).

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.104221.
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REFERENCES

Akira K. Hichiya H. Morita M. Shimizu A. Mitome H. Metabonomic study on the biochemical response of spontaneously hypertensive rats to chronic taurine supplementation using (1)H NMR spectroscopic urinalysis J. Pharm. Biomed. Anal 85 2013 155 161 23954436
Bai Y. Meng Q. Wang C. Ma K. Li J. Li J. Shan A. Gut microbiota mediates lactobacillus rhamnosus GG alleviation of deoxynivalenol-induced anorexia J. Agr. Food Chem 71 2023 8164 8181 37199714
Biehl M.L. Prelusky D.B. Koritz G.D. Hartin K.E. Buck W.B. Trenholm H.L. Biliary excretion and enterohepatic cycling of zearalenone in immature pigs Toxicol. Appl. Pharmacol 121 1993 152 159 8337696
Chang J. Wang T. Wang P. Yin Q. Liu C. Zhu Q. Lu F. Gao T. Compound probiotics alleviating aflatoxin B(1) and zearalenone toxic effects on broiler production performance and gut microbiota Ecotoxicol. Environ. Saf 194 2020 110420
Chatopadhyay P. Pandey A. Chaurasia A.K. Upadhyay A. Karmakar S. Singh L. Hepatic hyperplasia and damages induces by zearalenone Fusarium mycotoxins in BALB/c mice Arq. Gastroenterol 49 2012 77 81 22481690
Chen X. Xuan Y. Chen Y. Yang F. Zhu M. Xu J. Chen J. Polystyrene nanoplastics induce intestinal and hepatic inflammation through activation of NF-kappaB/NLRP3 pathways and related gut-liver axis in mice Sci. Total. Environ 935 2024 173458
Cheng Q. Jiang S.Z. Li S.Q. Wang Y.X. Zhang C.Y. Yang W.R. Effects of low-dose zearalenone-contaminated diets with or without montmorillonite clay adsorbent on nutrient metabolic rates, serum enzyme activities, and genital organs of growing-laying hens J. Appl. Poult Res 26 2017 367 375
Crost E.H. Tailford L.E. Le Gall G. Fons M. Henrissat B. Juge N. Utilisation of mucin glycans by the human gut symbiont Ruminococcus gnavus is strain-dependent PLoS One 8 2013 e76341 24204617
Dempsey E. Corr S.C. Lactobacillus spp. for Gastrointestinal Health: Current and Future Perspectives Front Immunol 13 2022 840245
Dringen R. Metabolism and functions of glutathione in brain Prog Neurobiol 62 2000 649 671 10880854
El-Nezami H. Polychronaki N. Salminen S. Mykkanen H. Binding rather than metabolism may explain the interaction of two food-Grade Lactobacillus strains with zearalenone and its derivative (')alpha-earalenol Appl Environ Microbiol 68 2002 3545 3549 12089040
Feng Y. Zhang M. Liu Y. Yang X. Wei F. Jin X. Liu D. Guo Y. Hu Y. Quantitative microbiome profiling reveals the developmental trajectory of the chicken gut microbiota and its connection to host metabolism Imeta 2 2023 e105 38868437
Fontaine M. Porchet N. Largilliere C. Marrakchi S. Lhermitte M. Aubert J.P. Degand P. Biochemical contribution to diagnosis and study of a new case of D-glyceric acidemia/aciduria Clin Chem 35 1989 2148 2151 2551543
Gu Y. Wang W. Zhan Y. Wei X. Shi Y. Cui D. Peng T. Han J. Li X. Chen Y. Xue Z. Wang W. Dietary artemisinin boosts intestinal immunity and healthy in fat greenling (Hexagrammos otakii) Front. Immunol 14 2023 1198902
Guerre P. Mycotoxin and Gut Microbiota Interactions Toxins (Basel) 12 2020 769 33291716
Jia S. Ren C. Yang P. Qi D. Effects of intestinal microorganisms on metabolism and toxicity mitigation of Zearalenone in broilers Animals (Basel) 12 2022 1962 35953951
Jiang S. Yang Z. Yang W. J.Gao F.Liu Chen C. Chi F. Physiopathological effects of zearalenone in post-weaning female piglets with or without montmorillonite clay adsorbent Livest Sci 131 2010 130 136
Ju H.K. Chung H.W. Lee H. Lim J. Park J.H. Lim S.C. Kim J.M. Hong Soon-Sun. Kwon S.W. Investigation of metabolite alteration in dimethylnitrosamine-induced liver fibrosis by GC-MS Bioanalysis 5 2013 41 51 23256471
Kouadio J.H. Mobio T.A. Baudrimont I. Moukha S. Dano S.D. Creppy E.E. Comparative study of cytotoxicity and oxidative stress induced by deoxynivalenol, zearalenone or fumonisin B1 in human intestinal cell line Caco-2 Toxicology 213 2005 56 65 16019124
Lei X. Cao X. Sun J. Bi C. Wang X. Li Y. Effect of different concentrations of carbonate on growth performance, intestinal health and hepatic lipid metabolism of Crucian carp Aquaculture 589 2024 740990
Liu M. Zhu D. Guo T. Zhang Y. Shi B. Shan A. Chen Z. Toxicity of zearalenone on the intestines of pregnant sows and their offspring and alleviation with modified halloysite nanotubes J. Sci. Food Agric 98 2018 698 706 28671336
Li P. Yang S. Zhang X. Huang S. Wang N. Wang M. Long M. He J. Zearalenone changes the diversity and composition of caecum microbiota in weaned rabbit Biomed Res Int 2018 2018 3623274
Liu M. Zhao L. Wei J.T. Huang Y.X. Khalil M.M. Wu W.D. Kuca K. Sun Lv-Hui. T-2 toxin-induced intestinal damage with dysregulation of metabolism, redox homeostasis, inflammation, and apoptosis in chicks Arch. Toxicol 97 2023 805 817 36695871
Lo P.A. Zorzi F. Del C.F. Altomare A. Cocca S. Avola A. Cocca S. Avola A. De Biasio F. Russo A. Cella E. Reddel S. Calabrese E. Biancone L. Monteleone G. Cicala M. Angeletti S. Ciccozzi M. Putignani L. Guarino M.P.L. Fecal and mucosal microbiota profiling in irritable bowel syndrome and inflammatory bowel disease Front. Microbiol 10 2019 1655 31379797
Long M. Li P. Zhang W. Li X. Zhang Y. Wang Z. Liu G. Removal of Zearalenone by strains of Lactobacillus sp. isolated from rumen in vitro J. Anim. Vet. Adv. 11 14 2012 2417 2422
Ma L. Jiang Y. Lu F. Wang S. Liu M. Liu F. Huang L. Li Y. Jiao N. Jiang S. Yuan X. Yang W. Quantitative Proteomic Analysis of Zearalenone-Induced Intestinal Damage in Weaned Piglets Toxins (Basel) 14 2022 702 36287972
Morton J.T. Aksenov A.A. Nothias L.F. Foulds J.R. Quinn R.A. Badri M.H. Swenson T.L. Van Goethem M.W. Northen T.R. Vazquez-Baeza Y. Wang M. Bokulich N.A. Watters A. Song S.J. Bonneau R. Dorrestein P.C. Knight R. Learning representations of microbe–metabolite interactions Nat Methods 16 2019 1306 1314 31686038
Pittayanon R. Lau J.T. Yuan Y. Leontiadis G.I. Tse F. Surette M. Moayyedi P. Gut microbiota in patients with irritable bowel syndrome-a systematic review Gastroenterology 157 2019 97 108 30940523
Png C.W. Linden S.K. Gilshenan K.S. Zoetendal E.G. McSweeney C.S Sly L.I. McGuckin M.A. Florin T.H.J. Mucolytic bacteria with increased prevalence in IBD mucosa augment in vitro utilization of mucin by other bacteria Am J Gastroenterol 105 2010 2420 2428 20648002
Powers H.J. Bates C.J. Prentice A.M. Lamb W.H. Jepson M. Bowman H. The relative effectiveness of iron and iron with riboflavin in correcting a microcytic anaemia in men and children in rural Gambia Hum Nutr Clin Nutr 37 1983 413 425 6668226
Reddy K.E. Jeong J.Y. Song J. Lee Y. Lee H.J. Kim D.W. Jung H.J. Kim K.H. Kim M. Oh Y.K. Lee S.D. Kim M. Colon microbiome of pigs fed diet contaminated with commercial purified Deoxynivalenol and Zearalenone Toxins (Basel) 10 2018 347 30158450
Rychlik I. Composition and function of chicken gut microbiota Animals (Basel) 10 2020 103 31936291
Rykaczewska A. Gajecka M. Dabrowski M. Wisniewska A. Szczesniewska J. Gajecki M.T. Zielonka L. Growth performance, selected blood biochemical parameters and body weights of pre-pubertal gilts fed diets supplemented with different doses of zearalenone (ZEN) Toxicon 152 2018 84 94 30055259
Sherwood R.F. Peberdy J.F. Effects of Zearalenone on the developing male chick Br. Poult. Sci 14 2007 127 129
Shi B. Su Y. Chang S. Sun Y. Meng X. Shan A. Vitamin C protects piglet liver against Zearalenone-induced oxidative stress by modulating expression of nuclear receptors PXR and CAR and their target genes Food Funct 8 2017 3675 3687 28920616
Soffa D.R. Stewart J.W. Pack E.D. Arneson A.G. De Vita R. Knight J.W. Fausnacht D.W. Rhoads R.P. Clark S.G. Schamle D.G. 3rd Rhoads M.L. Short-term consumption of the mycotoxin zearalenone by pubertal gilts causes persistent changes in the histoarchitecture of reproductive tissues J. Anim. Sci 101 2023 skac421 36574505
Stob M. Baldwin R.S. Tuite J. Andrews F.N. Gillette K.G. Isolation of an anabolic, uterotrophic compound from corn infected with Gibberella zeae Nature 196 1962 1318 13984207
Sun Y. Jiang J. Mu P. Lin R. Wen J. Deng Y. Toxicokinetics and metabolism of deoxynivalenol in animals and humans Arch. Toxicol 96 2022 2639 2654 35900469
Sypecka Z. Kelly M. Brereton P. Deoxynivalenol and zearalenone residues in eggs of laying hens fed with a naturally contaminated diet: effects on egg production and estimation of transmission rates from feed to eggs J. Agric. Food Chem 52 2004 5463 5471 15315386
Tailford L.E. Owen C.D. Walshaw J. Crost E.H. Hardy-Goddard J. Le Gall G. Vos W.M.de Taylor G.L. Juge N. Discovery of intramolecular trans-sialidases in human gut microbiota suggests novel mechanisms of mucosal adaptation Nat. Commun 6 2015 7624 26154892
Tett A. Pasolli E. Masetti G. Ercolini D. Segata N. Prevotella diversity, niches and interactions with the human host Nat. Rev. Microbiol 19 2021 585 599 34050328
Trabert B. Geczik A.M. Bauer D.C. Buist D. Cauley J.A. Falk R.T. Gierach G.L. Hue T.F. Lacey Jr J.V. LaCroix A.Z. Michels K.A. Tice J.A. Xu X. Brinton L.A. Dallal C.M. Association of endogenous pregnenolone, progesterone, and related metabolites with risk of endometrial and ovarian cancers in postmenopausal women: the B approximately FIT cohort Cancer Epidemiol Biomarkers Prev 30 2021 2030 2037 34465588
Wang S. Fu W. Zhao X. Chang X. Liu H. Zhou L. Li J. Cheng R. Wu X. Li X. Sun C. Zearalenone disturbs the reproductive-immune axis in pigs: the role of gut microbial metabolites Microbiome. 10 2022 234 36536466
Wang W. Zhai S. Xia Y. Wang H. Ruan D. Zhou T. Zhu Y. Zhang H. Zhang M. Ye H. Ren W. Yang L. Ochratoxin A induces liver inflammation: involvement of intestinal microbiota Microbiome 7 2019 151 31779704
Wang X. Yu H. Shan A. Jin Y. Fang H. Zhao Y. Shen J. Zhou C. Zhou Y. Fu Y. Wang J. Zhang J. Toxic effects of Zearalenone on intestinal microflora and intestinal mucosal immunity in mice Food Agric. Immunol 29 2018 1002 1011
Wu J. Du H. Wu J. Luo J. Fu P. Qiao X. Wei Q. Effects of dietary zearalenone on the serum biochemistry, hepatic and intestinal histology, and intestinal microbiota of juvenile Dabry’s sturgeon (Acipenser dabryanus) J. Appl. Ichthyol 37 2021 932 941
Wu J. Li J. Wu Y. Yang M. Chen Y. Wang N. Wang J. Yuan Z. Yi J. Yang C. Betulinic acid mitigates zearalenone-induced liver injury by ERS/MAPK/Nrf2 signaling pathways in mice Food Chem Toxicol 177 2023 113811
Wu H. Li X. Zhang Z. Ye Y. Chen Y. Wang J. Yang Z. Zhou E. The release of Zearalenone-induced heterophil extracellular traps in chickens is associated with autophagy, glycolysis, PAD enzyme, and P2×1 receptor Poult. Sci. 102 2023 102946
Wu H. Wang Y. Yao Q. Fan L. Meng L. Zheng N. Li H. Wang J. Alkaline phosphatase attenuates LPS-induced liver injury by regulating the miR-146a-related inflammatory pathway Int. Immunopharmacol 101 2021 108149
Yan J. Kong L. Zhang X. Yu M. Zhu K. Zhao A. Shi D. Sun Y. Wang J. Shen W. Li L. Maternal Zearalenone exposure affects gut microbiota and follicular development in suckled offspring J. Agric. Food Chem 70 2022 15570 15582 36514903
Yin Z. Ji S. Yang J. Guo W. Li Y. Ren Z. Yang X. Cecal microbial succession and its apparent association with nutrient metabolism in broiler chickens mSphere 8 2023 e61422
Yuan T. Li J. Wang Y. Li M. Yang A. Ren C. Qi D. Zhang N. Effects of Zearalenone on production performance, egg quality, ovarian function and gut microbiota of laying hens Toxins (Basel) 14 2022 653 36287922
Zhang W. Zhang S. Wang J. Shan A. Xu L. Changes in intestinal barrier functions and gut microbiota in rats exposed to zearalenone Ecotoxicol. Environ. Saf 204 2020 111072
Zhu K. Zhang W. Wu Q. Yang Q. Gong Z. Shao S. Zhang W. Astragalin protects the liver from oxidative damage by modulating the lnc XIST/miR-155-5p/Nrf2 axis J. Funct. Foods 108 2023 105769
